HK40010503B - Sorting of t lymphocytes in a microfluidic device - Google Patents

Description

Sorting T lymphocytes in a microfluidic device

Cross Reference to Related Applications

Priority of U.S. patent application No. 62/365,372 filed 2016, 7, 21, is hereby incorporated by reference herein in its entirety, pursuant to 35 u.s.c. § 119 requirements 2016.

Technical Field

The art relates generally to methods, systems, and devices for sorting T lymphocytes, particularly activated T lymphocytes, in a microfluidic environment.

Background

Immunotherapy is an area where the patient's own immune system is used to help combat the rapid development of cancer. Various immunotherapy strategies have been evaluated, including stimulation of the patient's own immune system to attack cancer cells or administration of immune system components from external sources. For example, monoclonal antibodies designed to attack cancer cells in vivo have been administered alone or in genetically engineered constructs. In addition, various T cell therapies have been investigated. Autologous T cell therapy involves obtaining T cells from a subject, expanding the T cells ex vivo, and reintroducing the expanded T cells into the subject. Chimeric antigen receptor T cell (CAR-T) therapy involves genetically engineering T cells to express on their surface fusion proteins containing chimeric antibodies that target the cancer in question and allow the T cells to kill the cancer cells. Both types of T cell therapy have advantages. However, further improvements in therapy are still needed.

One of the key issues in autologous T cell therapy and CAR-T therapy is the lack of methods to select T cells ex vivo in a way that yields T cell populations with the highest tumor killing potential. Embodiments of the present invention provide solutions for sorting T cells ex vivo to obtain populations enriched for T cells having a desired phenotype. Embodiments of the invention also provide microfluidic devices and compositions obtained therefrom that facilitate such sorting.

Disclosure of Invention

In one aspect, a method of sorting T lymphocytes in a microfluidic device based on the size of the T lymphocytes is provided. The method can include generating a sample enriched in activated T lymphocytes that specifically recognize the antigen of interest. The microfluidic device may include a flow path having a first region including a first array of pillars. The first region may be a channel (e.g., a main channel), and the first array of pillars may extend over the entire width of the channel. The method includes flowing a fluid sample containing T lymphocytes through a first region of a flow path (or channel) of a microfluidic device, and thus through a first column array.

The first array is characterized by a critical dimension (D) _c ) From about 4 microns to about 10 microns. The pillars of the first array may be arranged in rows and columns, wherein the rows of pillars define a first array direction which differs from a first direction of the first areas by an inclination angle (epsilon), wherein the first direction of the first areas is defined by the overall direction of fluid flow through the first areas. The columns in the first array may be repeated periodically with a period equal to 1/epsilon, where epsilon is measured in radians. Adjacent columns in each respective column in the first array define gaps through which fluid can flow, generally transverse to the column. Of columns The columns may be arranged substantially transversely with respect to the first direction of the first region (e.g., each column of pillars may be arranged along an axis oriented about 80 ° to about 100 ° with respect to the first direction of the first region), or more generally, the columns of pillars may be arranged along an axis oriented about 45 ° to about 135 ° with respect to the first direction of the first region.

The fluid sample containing T lymphocytes may include, for example, CD8 ⁺ T lymphocytes. The fluid sample may be derived from a starting population of T lymphocytes that have been incubated with an activator comprising an antigen of interest. The activator may be, for example, a Dendritic Cell (DC) or an artificial antigen presenting cell (aAPC). The starting population of T lymphocytes can be obtained from, for example, peripheral blood or PBMCs. Optionally, the starting population of T lymphocytes can be enriched for non-immunized T cells (e.g., CD 8) ⁺ Non-immunized T cells). For example, the starting population of T lymphocytes can comprise 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of non-immunized T cells (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of non-immunized CD 8) ⁺ T cells).

The method can be used, for example, to identify T cells having a TCR specific for an antigen of interest. The antigen of interest may be a peptide sequence derived from a pathogen, such as a bacterial pathogen, a fungal pathogen, a parasitic pathogen, or a viral pathogen. Alternatively, the antigen of interest may be a peptide sequence that is a tumor associated antigen. The identified T cells can be cloned, and a subset of cells from one or more such clones can be used for TCR sequencing analysis. Alternatively or additionally, the method may be used to isolate a population of activated T cells suitable (or suitable when expanded) for use as an endogenous T cell therapeutic.

In another aspect, a microfluidic device suitable for sorting T lymphocytes is provided. The microfluidic device may include a flow path having a first region including a first array of pillars, the first region of the flow path having a first direction corresponding to a general direction of fluid flow through the first region. The first region may be a channel (e.g., a main channel), and the first array of pillars may extend over the entire width of the channel. The first array is characterized by a critical dimension (Dc) of from about 4 microns to about 7 microns, or from about 7 microns to about 10 microns. The pillars of the first array may be arranged in rows and columns, the rows of pillars in the first array defining a first array direction which differs from the first direction of the first areas by a tilt angle (epsilon). The columns in the first array may be repeated periodically with a period equal to 1/epsilon, where epsilon is measured in radians. Adjacent columns in each respective column in the first array define gaps through which fluid can flow, generally transverse to the column. The columns of posts may be arranged substantially transversely with respect to the first direction of the first region (e.g., each column of posts may be arranged along an axis oriented from about 80 ° to about 100 ° with respect to the first direction of the first region), or more generally, the columns of posts may be arranged along an axis oriented from about 45 ° to about 135 ° with respect to the first direction of the first region.

The flow path of the microfluidic device may include a second region that receives fluid through the first region, and the second region may include a divider that divides the second region into a first channel and a second channel. The first channel may receive a first portion of any fluid passing through the first zone, and the second channel may receive a second portion of any fluid passing through the first zone. The first channel or the second channel may comprise an array of second pillars. The microfluidic device may further comprise one or more isolation docks, each of which may have an opening to the first channel or the second channel.

In another aspect, compositions are provided comprising T lymphocytes, particularly T lymphocytes that have been sorted/enriched according to any one of the methods disclosed herein. As noted above, such compositions may be suitable for use as endogenous T cell therapeutics, or as starting materials for the production of such therapeutics.

Additional aspects, objects, and advantages will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice. The aspects, objects, and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiment(s) and together with the description, serve to explain the principles described herein.

Brief description of the drawings

Fig. 1 illustrates an example of a system for operating and monitoring a microfluidic device according to certain embodiments.

Fig. 2A and 2B illustrate a microfluidic device according to certain embodiments.

Fig. 2C and 2D illustrate an isolation dock according to some embodiments.

Figure 2E illustrates a detailed isolating dock according to some embodiments.

Fig. 2F illustrates a microfluidic device according to certain embodiments.

Fig. 3 illustrates a nest according to certain embodiments, which can be part of a system for operating and monitoring a microfluidic device.

Fig. 4 illustrates an imaging device according to some embodiments, which may be part of a system for operating and monitoring a microfluidic device.

Fig. 5 illustrates a microfluidic device having a coating material covalently bonded to the inner surface of the substrate and device cover, according to certain embodiments.

Fig. 6 is a schematic diagram of an array of pillars that may be included in a microfluidic device, according to some embodiments.

Fig. 7A and 7B illustrate a microfluidic device having an array of pillars, according to some embodiments.

Fig. 8 is an image of a portion of a microfluidic device having a column array configured to separate activated "large" T lymphocytes from activated "small" T lymphocytes, and the T lymphocytes are fluorescently labeled to allow their detection within the array.

Figures 9A-9D depict cytometric maps generated by FACS analysis of T lymphocyte populations (including activated T lymphocytes and resting T lymphocytes) before and after sorting using a microfluidic device with an array of pillars.

Fig. 10 is an image of a portion of a microfluidic device having first and second channels located downstream of a pillar array configured to separate cells larger than a critical dimension (Dc) of the pillar array into the second channel, wherein the microfluidic device further comprises a isolation dock to the second channel.

Fig. 11 is a flow chart summarizing methods of enriching for T lymphocytes according to certain embodiments.

Detailed Description

This specification describes exemplary embodiments and applications of the invention. However, the invention is not limited to these exemplary embodiments and applications, nor to the manner in which the exemplary embodiments and applications operate or are described herein. Further, the figures may show simplified or partial views, and the sizes of elements in the figures may be exaggerated or not in proportion. In addition, as the terms "on," "attached," "connected," "coupled" or similar terms are used herein, an element (e.g., a material, a layer, a substrate, etc.) can be "on," "attached to," "connected to" or "coupled to" another element, whether or not the element is directly on, attached to, connected to or coupled to the other element, or one or more intervening elements may be present between the element and the other element. Where a list of elements (e.g., elements a, b, c) is referred to, such reference is intended to include any one of the listed elements themselves, any combination of fewer than all of the listed elements, and/or combinations of all of the listed elements. The division of sections in the specification is for ease of review only and does not limit any combination of the elements discussed.

As used herein, "substantially" means sufficient for the intended purpose. Thus, the term "substantially" allows for minor, insignificant variations from absolute or perfect states, dimensions, measurements, results, etc., such as would be expected by one of ordinary skill in the art without significantly affecting overall performance. "substantially" when used in relation to a numerical value or a parameter or characteristic that may be expressed as a numerical value means within ten percent.

As used herein, the terms "a" and "an" mean more than one. As used herein, the term "plurality" can be 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

As used herein, the term "disposed" includes within its meaning "located".

As used herein, a "microfluidic device" or "microfluidic apparatus" is a device that: comprising one or more separate microfluidic conduits configured to contain a fluid, each microfluidic conduit comprising fluidly interconnected conduit elements including, but not limited to, regions, flow paths, channels, chambers, and/or docks; and at least two ports configured to allow fluid (and optionally micro-objects suspended in the fluid) to flow into and/or out of the microfluidic device. Typically, the microfluidic circuit of a microfluidic device will comprise at least one microfluidic channel and at least one chamber, and will accommodate a fluid volume of less than about 1mL (e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 μ Ι _). In certain embodiments, the microfluidic circuit contains about 1-2, 1-3, 1-4, 1-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-30, 5-40, 5-50, 10-75, 10-100, 20-150, 20-200, 50-250, or 50-300 μ L.

As used herein, a "nanofluidic device" or "nanofluidic apparatus" is a microfluidic device having microfluidic tubing containing at least one tubing element configured to accommodate a fluid volume of less than about 1 μ L, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1nL or less. Typically, the nanofluidic device will include a plurality of piping elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000, or more). In certain embodiments, one or more (e.g., all) of the at least one piping element is configured to hold a fluid volume of about 100pL to 1nL, 100pL to 2nL, 100pL to 5nL, 250pL to 2nL, 250pL to 5nL, 250pL to 10nL, 500pL to 5nL, 500pL to 10nL, 500pL to 15nL, 750pL to 10nL, 750pL to 15nL, 750pL to 20nL, 1 to 10nL, 1 to 15nL, 1 to 20nL, 1 to 25nL, or 1 to 50 nL. In other embodiments, one or more (e.g., all) of the at least one piping element is configured to hold a fluid volume of 100 to 200nL, 100 to 300nL, 100 to 400nL, 100 to 500nL, 200 to 300nL, 200 to 400nL, 200 to 500nL, 200 to 600nL, 200 to 700nL, 250 to 400nL, 250 to 500nL, 250 to 600nL, or 250 to 750 nL.

As used herein, "microfluidic channel" or "channel" refers to a flow region of a microfluidic device that is significantly longer than the horizontal and vertical dimensions. The length of the channel is generally defined by the flow path of the channel. In the case of a straight channel, the length will be the "longitudinal axis" of the channel. The "horizontal dimension" or "width" of a channel is the horizontal dimension as viewed in cross-section oriented perpendicular to the longitudinal axis of the channel (or, if the channel is curved, perpendicular to an axis tangential to the flow path of the channel in the plane of the cross-section). The "vertical dimension" or "height" of a channel is the vertical dimension as viewed in cross-section oriented perpendicular to the longitudinal axis of the channel (or, if the channel is curved, perpendicular to an axis tangent to the flow path of the channel in the plane of the cross-section). For example, the flow channel can be at least 5 times the length of the horizontal or vertical dimension, e.g., at least 10 times the length, at least 25 times the length, at least 100 times the length, at least 200 times the length, at least 500 times the length, at least 1,000 times the length, at least 5,000 times the length, or longer. In some embodiments, the length of the flow channel is in the range of about 100,000 micrometers to about 500,000 micrometers, including any range therebetween. In some embodiments, the horizontal dimension (or width) is in the range of about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns) and the vertical dimension (or height) is in the range of about 25 microns to about 200 microns, e.g., about 40 to about 150 microns. It should be noted that the flow channels may have a variety of different spatial configurations in the microfluidic device and are therefore not limited to perfectly linear elements. For example, the flow channel may be or include one or more portions having the following configurations: curved, bent, spiral, inclined, descending, forked (e.g., multiple distinct flow paths), and any combination thereof. In addition, the flow channel may have different cross-sectional areas along its path, widening and narrowing to provide the desired fluid flow therein.

As used herein, the term "obstruction" generally refers to a protrusion or similar type of structure that is large enough to partially (but not completely) impede movement of a target micro-object between two different regions or conduit elements in a microfluidic device. The two different regions/pipe elements may for example be a microfluidic isolation dock and a microfluidic channel, or a connection region and a separation region of a microfluidic isolation dock.

As used herein, the term "constriction" generally refers to a narrowing of the width of a conduit element (or the interface between two conduit elements) in a microfluidic device. The constriction may be located, for example, at the interface between the microfluidic isolation dock and the microfluidic channel, or at the interface between the separation region and the connection region of the microfluidic isolation dock.

As used herein, the term "transparent" refers to a material that allows the passage of visible light without substantially changing the light when passed.

As used herein, the term "micro-object" generally refers to any microscopic object that can be separated and collected according to the present disclosure. Non-limiting examples of micro-objects include: inanimate micro-objects such as microparticles; microbeads (e.g., polystyrene beads, Luminex) ^TM Beads, etc.); magnetic beads; a micron rod; microfilaments; quantum dots, and the like; biological micro-objects, such as cells (e.g., embryos, oocytes, sperm cells, cells dissociated from a tissue, eukaryotic cells, protist cells, animal cells, mammalian cells, human cells, immune cells, hybridomas, cultured cells, cells from a cell line, cancer cells, infected cells, transfected and/or transformed cells, reporter cells, prokaryotic cells, etc.); a biological organelle; vesicles or complexes (ii) a Synthesizing vesicles; liposomes (e.g., synthetic or derived from membrane preparations); lipid nanorafts (nanorafts) (as described in Ritchie et al (2009) "Reconstation of Membrane Proteins in Phospholipid Bilayer Nanodiscs," Methods enzymol.,464: 211-231) and the like; or a combination of inanimate micro-objects and biological micro-objects (e.g., cell-attached microbeads, liposome-coated magnetic beads, etc.). The beads may further have other moieties/molecules, such as fluorescent markers, proteins, small molecule signaling moieties, antigens, or chemical/biological substances that can be used for assays, covalently or non-covalently attached.

As used herein, the terms "T lymphocyte" and "T cell" are used interchangeably.

As used herein, the term "maintaining the cell(s)" refers to providing an environment comprising fluid and gas components and optionally surfaces that provide the conditions necessary to keep the cells viable and/or expanded.

A "component" of a fluid medium is any chemical or biochemical molecule present in the medium, including solvent molecules, ions, small molecules, antibiotics, nucleotides and nucleosides, nucleic acids, amino acids, peptides, proteins, sugars, carbohydrates, lipids, fatty acids, cholesterol, metabolites, and the like.

As used herein with respect to a fluid medium, "diffusion" and "diffusion" refer to the thermodynamic movement of components of the fluid medium down a concentration gradient.

The phrase "flow of the medium" means that the fluid medium is moved as a whole primarily due to any mechanism other than diffusion. For example, the flow of the medium may include the fluid medium moving from one point to another due to a pressure difference between the points. Such flow may include continuous, pulsed, periodic, random, intermittent, or reciprocating flow of liquid, or any combination thereof. When one fluid medium flows into another fluid medium, turbulence and mixing of the media may result.

The phrase "substantially no flow" refers to a flow rate of the fluid medium that, on average over time, is less than the rate at which a component of the material (e.g., the analyte of interest) diffuses into or within the fluid medium. The diffusion rate of the components of such materials may depend on, for example, the temperature, the size of the components, and the strength of the interaction between the components and the fluid medium.

As used herein with respect to different regions within a microfluidic device, the phrase "fluidically connected" refers to the fluids in each region being connected to form a single liquid when the different regions are substantially filled with a fluid (e.g., a fluidic medium). This does not mean that the fluids (or fluid media) in the different regions are necessarily identical in composition. In contrast, fluids in different fluidically connected regions of a microfluidic device can have different compositions (e.g., different concentrations of solutes, such as proteins, sugars, ions, or other molecules) that are in change as the solutes move down their respective concentration gradients and/or the fluid flows through the device.

Microfluidic (or nanofluidic) devices may include "swept" areas and "unswept" areas. As used herein, a "swept-out" area includes one or more fluidically interconnected tubing elements of a microfluidic tubing, each tubing element being subjected to a flow of a medium as fluid flows through the microfluidic tubing. The conduit elements that sweep the area may include, for example, areas, channels, and all or part of the chamber. As used herein, an "unswept" region includes one or more fluidically interconnected conduit elements of a microfluidic conduit, each conduit element being substantially free of the flow of fluid as the fluid flows through the microfluidic conduit. The unswept region may be fluidly connected to the swept region, provided that the fluid connection is configured to enable diffusion but substantially no media flow between the swept region and the unswept region. Thus, the microfluidic device may be configured to substantially separate the unswept region from the flow of the medium in the swept region, while substantially only diffusive fluid communication is enabled between the swept region and the unswept region. For example, the flow channel of a microfluidic device is an example of a swept area, while the separation area of a microfluidic device (described in further detail below) is an example of an unswept area.

As used herein, "flow path" refers to one or more fluidly connected conduit elements (e.g., channels, regions, chambers, etc.) that define and are constrained by the trajectory of the media flow. Thus, the flow path is an example of a swept (swept) region of a microfluidic device. Other conduit elements (e.g., unswept areas) may be in fluid connection with the conduit elements comprising the flow path, independent of the flow of the medium in the flow path.

The ability of a biological micro-object (e.g., a biological cell) to produce a particular biological material (e.g., a protein, such as an antibody) can be determined in such a microfluidic device. In one particular embodiment of an assay, sample material comprising biological micro-objects (e.g., cells) to be assayed for the production of target analytes can be loaded into a swept-out region of a microfluidic device. Those biological micro-objects (e.g., mammalian, e.g., human cells) having particular characteristics can be selected and placed in unswept areas. The remaining sample material can then be flowed out of the swept area and the assay material flowed into the swept area. Because the selected biological micro-objects are in the unswept region, the selected biological micro-objects are substantially unaffected by the outflow of residual sample material or the inflow of assay material. The selected biological micro-objects may be allowed to produce target analytes that may diffuse from unswept regions into swept regions, where the target analytes may react with the assay material to produce locally detectable reactions, each reaction may be associated with a particular unswept region. Any unswept areas associated with the detected reaction can be analyzed to determine which, if any, biological micro-objects in the unswept areas are sufficient producers of the target analyte.

T lymphocytes are cultured, selected and expanded in a microfluidic/nanofluidic device. Methods for selecting and expanding biological cells, including T lymphocytes, within a microfluidic device have been described, for example, in U.S. patent application No. 15/135,707 filed on 2016, 4, 22, which is incorporated herein by reference in its entirety. Methods of activating and expanding T lymphocytes within a microfluidic device are described in international application number PCT/US 17/22846 filed 3, 16, 2017, which is incorporated herein by reference in its entirety.

Microfluidic devices and systems for operating and viewing such devices. Fig. 1 shows a generalized example of a microfluidic device 100 and system 150 that may be used to operate and view the microfluidic device. A perspective view of the microfluidic device 100 is shown with the cover 110 partially cut away to provide a partial view into the microfluidic device 100. The microfluidic device 100 generally includes a microfluidic circuit 120 having a flow path 106, and a fluidic medium 180 may optionally carry one or more micro-objects (not shown) into and/or through the microfluidic circuit 120 via the flow path 106. Although a single microfluidic circuit 120 is shown in fig. 1, a suitable microfluidic device may include a plurality (e.g., 2 or 3) of such microfluidic circuits. Regardless, the microfluidic device 100 may be configured as a nanofluidic device. In the embodiment shown in fig. 1, microfluidic circuit 120 includes a plurality of microfluidic isolation stations 124, 126, 128, and 130, each of which has a single opening in fluid communication with flow path 106. As discussed further below, the microfluidic sequestration dock includes various features and structures that have been optimized for retaining micro-objects in a microfluidic device (e.g., microfluidic device 100) even as the medium 180 flows through the flow path 106. However, before the above is described, a brief description of the microfluidic device 100 and system 150 is provided.

As shown generally in fig. 1, microfluidic circuit 120 is defined by housing 102. Although the housing 102 may be physically configured in different configurations, in the example shown in fig. 1, the housing 102 is depicted as including a support structure 104 (e.g., a base), a microfluidic conduit structure 108, and a cover 110. The support structure 104, the microfluidic circuit structure 108 and the cover 110 may be attached to each other. For example, the microfluidic circuit structure 108 may be arranged on an inner surface 109 of the support structure 104, and the cover 110 may be arranged over the microfluidic circuit structure 108. The microfluidic circuit structure 108, together with the support structure 104 and the cover 110, may define elements of a microfluidic circuit 120.

As shown in fig. 1, the support structure 104 may be located at the bottom of the microfluidic circuit 120 and the lid 110 may be located at the top of the microfluidic circuit 120. Alternatively, the support structure 104 and the cover 110 may be configured in other orientations. For example, the support structure 104 may be located at the top of the microfluidic circuit 120 and the lid 110 may be located at the bottom of the microfluidic circuit 120. In any event, there may be one or more ports 107, each of which includes access into or out of the housing 102. Examples of passageways include valves, gates, through-holes, and the like. As shown, the port 107 is a through hole created by a gap in the microfluidic conduit structure 108. However, the port 107 may be located in other components of the housing 102 (e.g., the cover 110). Only one port 107 is shown in fig. 1, but the microfluidic circuit 120 may have two or more ports 107. For example, there may be a first port 107 that serves as an inlet for fluid into the microfluidic circuit 120, and there may be a second port 107 that serves as an outlet for fluid out of the microfluidic circuit 120. Whether the port 107 serves as an inlet or an outlet may depend on the direction of fluid flow through the flow path 106.

The support structure 104 may comprise a rigid material. The rigid material may be glass or a material with similar properties. In some embodiments, the support structure 104 may comprise a deformable material. The deformable material may be a polymer, such as PDMS. In some embodiments, the support structure 104 may include rigid and deformable materials.

The support structure 104 may include one or more electrodes (not shown) and a substrate or a plurality of interconnected substrates. For example, the support structure 104 may include one or more semiconductor substrates, each semiconductor substrate being electrically connected to an electrode (e.g., all or a portion of a semiconductor substrate may be electrically connected to a single electrode). The support structure 104 may further include a printed circuit board assembly ("PCBA"). For example, the semiconductor substrate may be mounted on a PCBA. However, the support structure 104 need not contain any electrodes or semiconductor substrates.

The microfluidic circuit structure 108 may define a circuit element of a microfluidic circuit 120. When microfluidic circuit 120 is filled with a fluid, such circuit elements may include spaces or regions that may be fluidically interconnected, such as flow channels or chambers, any of which may include an array of pillars (e.g., formed by microfluidic circuit structure 108), docks (pen), wells (trap), and the like. In the microfluidic circuit 120 shown in fig. 1, the microfluidic circuit structure 108 includes a frame 114 and a microfluidic circuit material 116. The frame 114 may partially or completely surround the microfluidic circuit material 116. The frame 114 may be, for example, a relatively rigid structure that substantially encloses the microfluidic circuit material 116. For example, the frame 114 may comprise a metallic material.

The microfluidic circuit material 116 may be patterned with cavities or the like to define circuit elements (including arrays of pillars, not shown) and interconnects of the microfluidic circuit 120. The microfluidic circuit material 116 may include a flexible material, such as a flexible polymer (e.g., rubber, plastic, elastomer, silicone, polydimethylsiloxane ("PDMS"), etc.), which may be gas permeable. Other examples of materials from which the microfluidic circuit material 116 may be constructed include molded glass; etchable materials such as silicone (e.g., photo-patternable silicone or "PPS"), photoresist (e.g., SU8), and the like. In some embodiments, such materials (and thus the microfluidic circuit material 116) may be rigid and/or substantially gas impermeable. Regardless, the microfluidic circuit material 116 may be disposed on the support structure 104 and within the frame 114.

The cover 110 may be an integral (integral) component of the frame 114 and/or the microfluidic circuit material 116. Alternatively, the cover 110 may be a structurally different element, as shown in FIG. 1. The cover 110 may comprise the same or different material as the frame 114 and/or the microfluidic circuit material 116. Similarly, the support structure 104 may be a separate structure from the frame 114 or the microfluidic circuit material 116 (as shown), or an integral component of the frame 114 or the microfluidic circuit material 116. Likewise, the frame 114 and the microfluidic circuit material 116 may be separate structures as shown in fig. 1 or integral components of the same structure.

In some embodiments, the cover 110 may comprise a rigid material. The rigid material may be glass or a material with similar properties. In some embodiments, the cover 110 may include a deformable material. The deformable material may be a polymer, such as PDMS. In some embodiments, the cover 110 may include both a rigid material and a deformable material. For example, one or more portions of the cover 110 (e.g., one or more portions located above the isolation docks 124, 126, 128, 130) may include a deformable material that interfaces with the rigid material of the cover 110. In some embodiments, the cover 110 may further include one or more electrodes. The one or more electrodes may comprise a conductive oxide, such as Indium Tin Oxide (ITO), which may be coated on glass or similar insulating material. Alternatively, the one or more electrodes may be flexible electrodes, such as single-walled nanotubes, multi-walled nanotubes, nanowires, clusters of conductive nanoparticles embedded in a deformable material such as a polymer (e.g., PDMS), or a combination thereof. Flexible electrodes that may be used in microfluidic devices have been described, for example, in US 2012/0325665(Chiou et al), the contents of which are incorporated herein by reference. In some embodiments, the cover 110 may be modified (e.g., by adjusting all or a portion of the surface facing inward toward the microfluidic circuit 120) to support cell adhesion, viability, and/or growth. The modification may include a coating of a synthetic or natural polymer. In some embodiments, the cover 110 and/or the support structure 104 may be optically transparent. The cap 110 may also include at least one gas permeable material (e.g., PDMS or PPS).

The flow path of the microfluidic device may include a first region including an array of pillars. The first region is bounded by a pair of walls (e.g., a first sidewall and a second sidewall) that together define a first direction that corresponds to a direction of fluid flow in the first region of the intended flow path. The pillars in the pillar array may be arranged in rows and columns, as generally shown in FIG. 6. The rows of pillars may define a first array direction differing from the first direction of the first area by an inclination angle (epsilon), the columns of pillars in the first array repeating periodically with a period equal to 1/epsilon, where epsilon is measured in radians. Adjacent columns in each respective column in the first array define gaps through which fluid can flow, generally transverse to the column. Typically, the gaps between adjacent pillars in a column of the pillar array will have a characteristic dimension. As used herein, the term "feature size" with respect to the gaps between adjacent pillars in a column of an array of pillars refers to a size that is the same (+/-5%) for most gaps in the array of pillars. In other words, at least 50% of the gaps between adjacent pillars in a column of the array of pillars may have a characteristic dimension. More typically, at least 60%, 70%, 80%, 90%, 95%, or more of the gaps between adjacent pillars in a column of the array of pillars may have a characteristic dimension.

The pillars of the array may be generally referred to as obstacles. Barrier/post arrays have been described, for example, in U.S. patent nos. 7,150,812 and 8,783,467, the contents of which are incorporated herein by reference in their entirety.

The array of pillars will typically extend over the entire width of the first region of the flow path. Microfluidic devices may have one or more ports for inlets and one or more ports for outlets. For example, as shown in fig. 7B, a port upstream of first region 706 may be used as inlet 702, while a port downstream of first region 706 may be used as outlet 708/710. Alternatively, as shown in fig. 7A, a pair of ports located upstream of the first region may be used as inlets 702 and 704 of the microfluidic device (e.g., the first upstream port 702 may provide a fluid sample stream containing cells (e.g., T lymphocytes) while the second upstream port 704 may provide a medium or buffer stream devoid of cells). Similarly, a pair of ports 708 and 710 located downstream may allow fluid to flow out of the microfluidic device. For example, the first downstream port 708 may provide an outlet for fluid enrichment of a desired cell (particularly activated T lymphocytes) population, and the second downstream port 710 may provide a waste outlet. In some embodiments, the waste stream may come from a bypass channel.

The pillar array may be generally characterized by a critical dimension (D) _c ) It can be from about 3 microns to about 15 microns (e.g., from about 4 microns to about 10 microns or from about 7 microns to about 12 microns). In some embodiments, the array is characterized by a Dc of about 4 microns to about 7 microns (e.g., about 4 microns to about 5 microns, about 4.5 microns to about 5.5 microns, about 5 microns to about 6 microns, about 5.5 microns to about 6.5 microns, about 6 microns to about 7 microns, or any range defined by the foregoing endpoints). In other embodiments, the array is characterized by D _c From about 7 microns to about 10 microns (e.g., from about 7 microns to about 8 microns, from about 7.5 microns to about 8.5 microns, from about 8 microns to about 9 microns, from about 8.5 microns to 9.5 microns, from about 9 microns to about 10 microns, orAny range defined by the aforementioned endpoints). Importantly, D can be selected _c Such that non-immunized T lymphocytes, which are generally smaller in diameter than activated T lymphocytes, will flow through the column array primarily in the general direction of fluid flow, while activated T lymphocytes will travel in the direction of the first array defined by the rows of the first array. In this manner, fluid flowing through the column array may be enriched for activated T lymphocytes. As used herein, "enriched" means that the proportion of target cells in a portion of the fluid is increased as a result of moving through the column array as compared to the proportion of such target cells in the portion of the fluid before the fluid moves through the column array. The enrichment can be calculated in different ways. For example, one simple measure is the percentage of activated T lymphocytes in the fluid portion after moving through the column array divided by the percentage of activated T lymphocytes in the fluid portion immediately before entering the column array. Alternatively, enrichment may be calculated as (N) ⁺ _{Leave from} /N ^- _{Leave from} )/(N ⁺ _{Enter into} /N ^- _{Enter into} ) In which N is ⁺ _{Leave from} Is the number of target cells detected in the fluid portion after moving through the column array, N ^- _{Leave from} Is the number of cells other than the target cells detected in the fluid portion after moving through the column array, N ⁺ _{Enter into} Is the number of target cells detected in the fluid portion prior to moving through the column array, N ^- _{Enter into} Is the number of cells other than the target cells detected in the fluid portion prior to moving through the column array. The exact calculation of the enrichment is not important. For example, any of the foregoing definitions may be used, and as long as at least one calculation indicates enrichment, the portion of fluid that has moved through the column array will be considered enriched.

In certain embodiments, the array has an inclination angle ε of about 1/3 radians to about 1/100 radians (e.g., about 1/5 radians to about 1/20 radians, or about 1/10 radians to about 1/16 radians).

The gaps between adjacent pillars in each column of the first array can be about 15 microns to about 100 microns (e.g., about 20 microns to about 30 microns, about 25 microns to about 35 microns, about 30 microns to about 40 microns, about 35 microns to about 45 microns, about 40 microns to about 50 microns, about 45 microns to about 55 microns, about 50 microns to about 60 microns, about 55 microns to about 65 microns, about 60 microns to about 70 microns, about 65 microns to about 75 microns, about 70 microns to about 90 microns, about 80 microns to about 100 microns, or any range defined by the foregoing endpoints). In certain particular embodiments, the gap may be from about 15 microns to about 30 microns, from about 20 microns to about 35 microns, or from about 25 microns to about 40 microns.

Typically, the gaps between adjacent pillars in the same column of the first array are substantially equal in size, equal in size to the feature size. However, exceptions are allowed. In particular, the gap size between adjacent pillars (in the same column) closest to the sidewalls defining the area containing the array of pillars may deviate from the feature size. As will be appreciated by those skilled in the art, such deviations in gap size may be designed to reduce boundary irregularities in fluid flow through the array caused by the spacing between the side walls and the posts immediately adjacent these walls.

In certain embodiments, the pillars of the array have a circular cross-section. Alternatively, the cross-section of the pillars of the first array is polygonal, such as triangular, square, diamond, parallelogram, pentagonal, hexagonal, etc., or even irregularly shaped. Typically, the pillars in the array will have the same orientation (when viewed in cross-section relative to the first orientation of the array). In certain embodiments, the polygonal/irregularly shaped posts are asymmetrically oriented with respect to an axis defined by the first direction. In this way, the pillars may be oriented such that no axis of symmetry in the cross-sectional shape of the pillars is parallel to an axis defined by the first direction of the array.

The pillars of the first array may have a diameter of about 30 microns to about 100 microns (e.g., about 30 microns to about 50 microns, about 30 microns to about 60 microns, about 30 microns to about 70 microns, about 40 microns to about 60 microns, about 40 microns to about 70 microns, about 40 microns to about 80 microns, about 40 microns to about 90 microns, about 50 microns to about 70 microns, about 50 microns to about 80 microns, about 50 microns to about 90 microns, about 50 microns to about 100 microns, about 60 to about 80 microns, about 60 microns to about 90 microns, about 60 microns to about 100 microns, about 70 microns to about 90 microns, about 70 microns to about 100 microns, about 80 microns to about 100 microns, or any range defined by the foregoing endpoints). For a polygonal or irregularly shaped column, the "diameter" of the column is the maximum cross-sectional width measured along an axis perpendicular to the direction of fluid flow (i.e., the first direction).

Table 1 provides various designs of exemplary pillar arrays and their corresponding critical dimensions D _c Either can be used in the methods of the present disclosure, depending on the size separation desired.

Table 1: exemplary pillar array design

Pillar shape	Size of column	Size of gap	Array tilt angle	Critical dimension
					Triangle shape	50	30	1/12	7.2
Circular shape	50	30	1/15	8.8
					Triangle shape	50	15	1/16	3
Triangle shape	50	20	1/16	4
					Triangle shape	50	20	1/12	5
Triangle shape	50	25	1/12	6
					Triangle shape	50	25	1/10	7
Triangle shape	50	29	1/10	8
					Triangle shape	50	33	1/10	9
Triangle shape	50	37	1/10	10
					Triangle shape	50	15	1/16	3
Triangle shape	50	25	1/12	6
					Diamond shape	70	21	1/12	6
Diamond shape	70	17.5	1/12	5
					Diamond shape	70	21	1/10	7

The pillars of the first array may be formed from any of a variety of materials, including any of the materials described herein for use in constructing microfluidic devices, such as microfluidic tubing material 116. Thus, for example, the posts may be made of a silicone polymer (e.g., PDMS, PPS, etc.).

A microfluidic device having an array of pillars as described above will typically have a flow path comprising a first region having an array of pillars and a second region configured to receive a flow of fluid after the fluid passes through the first region. The length of the first region can be about 5mm to about 15mm (e.g., about 5mm to about 10mm, about 6mm to about 11mm, about 7mm to about 12mm, about 8mm to about 13mm, about 9mm to about 14mm, about 10mm to about 15mm, or any range defined by the foregoing endpoints), wherein the length is measured along an axis defined by the first direction. The second region may be separated by a divider (or wall) that divides the second region into, for example, a first channel and a second channel. Other arrangements are also possible, such as multiple dividers (or walls), as will be apparent from the discussion below. The second region may be configured relative to the first region such that the diameter is less than feature D of the pillar array _c Predominantly into the first channel and have a diameter greater than the array characteristic D _c Mainly into the second channel (e.g., T lymphocytes). As used herein, the "diameter" of a particle/cell (e.g., a T lymphocyte) is the effective size of the particle/cell as it passes through the column array. The effective diameter can be affected by a number of factors, including the health of the cells, the stage of the cell cycle, the composition of the pillars in the array, the coating on the pillars of the array, and the like. In certain embodiments, the first channel may be a "bypass channel" directly to the outlet/waste, and the second channel may be a selection and/or assay channel into which the most desirable particles/cells (e.g., T lymphocytes) are directed.

In some embodiments, the first channel is configured to receive at least 50% (e.g., to) of the fluid flowing out of the first region (and the array of pillars)60%, 70%, 75%, 80%, 85%, 90% or more less). In some embodiments, the first channel is configured to receive about 85% to about 95% (e.g., about 87% to about 93%, about 88% to about 92%, about 89% to about 91%, about 90%, or any range defined by the foregoing endpoints) of the fluid exiting the first region (and the array of pillars). In such embodiments, the remaining fluid flowing into the second channel (which may be, for example, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less of the fluid flowing out of the first region) may include all or most of the particles/cells having an effective diameter greater than the Dc of the column array. Thus, for example, when the diameter is greater than D _c The T lymphocytes of (a) can be effectively concentrated at least about 2-fold, about 2.5-fold, about 3-fold, about 3.5-fold, about 4-fold, about 4.5-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 15-fold, about 20-fold, or more upon exiting the first region and entering the second channel of the second region.

In certain embodiments, the flow path of the microfluidic device can include a second region that is divided into a first channel and a second channel, the first and second channels configured such that a pressure differential across the first channel is equal to a pressure differential across the second channel. This equal pressure may be achieved, for example, if the channels are reconnected (e.g., before reaching the outlet port) or if they lead to a different outlet port. To ensure that the pressure in each channel is substantially equal, Q may be determined according to the formula P _CH1 *R _CH1 ＝Q _CH2 *R _CH2 Matching resistance in the channel, wherein Q _CH1 Is the volume flow in the first channel per unit time, Q _CH2 Is the volume flow in the second channel per unit time, R _CH1 And R _CH2 Are the respective fluid resistances in the first and second channels. For channels with rectangular cross-section, R L/(W d) ³ ) Where L is the length of the channel, W is a cross-sectional dimension of the channel, and d is the minimum cross-sectional dimension of the channel.

In certain embodiments, the first channel comprises a length and the second channel comprises a length, and the length of the second channel is greater than the length of the first channel (e.g., at least 6, 7, 8, 9, 10, 11, 12, 15, or 20 times greater). The microfluidic device may comprise at least one sequestration dock as described herein that leads to the second channel of the second area and has a volume large enough to accommodate at least one T lymphocyte. The volume of the isolation dock can be about 250pL to about 3nL (e.g., about 250pL to about 1nL, about 375pL to about 1nL, about 500pL to about 1nL, about 750pL to about 1nL, about 250pL to about 1.25nL, about 500pL to about 1.25nL, about 750pL to about 1.25nL, about 1nL to about 1.25nL, about 500pL to about 1.5nL, about 750pL to about 1.5nL, about 1nL to about 1.5nL, about 500pL to about 2nL, about 750pL to about 2nL, about 1nL to about 2nL, about 1.25nL to about 2nL, about 1.5nL to about 2nL, about 1nL, 5nL to about 2nL, about 1nL to about 2nL, about 1.5nL to about 2nL, about 1nL to about 2.5nL, about 2 to about 2.5nL, about 3 to about 3.5 nL, about 3nL to about 3 to about 3.5 nL).

The microfluidic device may comprise more than one array of pillars. For example, the second channel may comprise a first sub-region comprising an array of second pillars. The second channel may be configured such that a portion of the fluid flowing through the first region of the flow path will enter the second channel and that portion of the fluid and any cells contained therein will pass through the second array. The second array may be similar to the first array. E.g. having the same critical dimension D _c (or similar critical dimensions, e.g. +/-0.5 microns) may facilitate removal of unwanted cells/micro-objects and further enrichment of the sample. In some embodiments, the second array may have a different critical dimension (Dc). For example, the first array may have a critical dimension of about 4 microns to about 7 microns (e.g., about 6 microns), and the second array may have a critical dimension of about 7 microns to about 10 microns (e.g., about 9 microns). The larger critical dimension of the second array can remove unwanted cells larger than the desired micro-objects, e.g., cells that are about to divide.

In some embodiments, the second channel may comprise a first sub-region comprising a second column array and a second sub-region. The second sub-zone may be configured to receive the fluid flow after it passes through the first sub-zone (and second column array). For example, the second sub-region may comprise a divider, such as a wall, which divides the second channel into a third channel and a fourth channel. In this way, cells (T lymphocytes) in a sample can be more finely sorted by passing the sample through a series of column arrays. In addition, the isolation docks may be positioned such that they open into the third channel or the fourth channel, allowing the liquid flow to stop and the target cells to be enclosed and optionally cultured on the chip.

An exemplary design of a microfluidic chip with an array of pillars is shown in fig. 7A and 7B (i.e., microfluidic devices 700 and 715), and described in connection with examples.

Fig. 1 also shows a system 150 for operating and controlling a microfluidic device, such as the microfluidic device 100. As shown, the system 150 includes a power source 192, an imaging device 194, and a tilting device 190.

The power source 192 may provide power to the microfluidic device 100 and/or the tilting device 190 to provide a bias voltage or current as desired. The power supply 192 may, for example, include one or more Alternating Current (AC) and/or Direct Current (DC) voltage or current sources. The imaging device 194 may include a device for capturing images within the microfluidic circuit 120, such as a digital camera. In some cases, the imaging device 194 further includes a detector with a fast frame rate and/or high sensitivity (e.g., for low light applications). The imaging device 194 may also include a mechanism for directing stimulating radiation and/or light beams into the microfluidic circuit 120 and collecting radiation and/or light beams reflected or emitted from the microfluidic circuit 120 (or micro-objects contained therein). The emitted light beam may be in the visible spectrum and may, for example, include fluorescent emissions. The reflected light beam may comprise reflected emissions from an LED or a broad spectrum lamp such as a mercury lamp (e.g. a high pressure mercury lamp) or a xenon arc lamp. As discussed with respect to fig. 4, the imaging device 194 may further include a microscope (or optical train), which may or may not include an eyepiece.

The system 150 can further include a tilting device 190 configured to rotate the microfluidic device 100 about one or more axes of rotation. In some embodiments, the tilting device 190 is configured to support and/or hold the housing 102 including the microfluidic circuit 120 about at least one axis such that the microfluidic device 100 (and thus the microfluidic circuit 120) can be held in a horizontal orientation (i.e., 0 ° with respect to the x-axis and y-axis), a vertical orientation (i.e., 90 ° with respect to the x-axis and/or y-axis), or any orientation therebetween. The orientation of the microfluidic device 100 (and microfluidic circuit 120) relative to the axis is referred to herein as the "tilt" of the microfluidic device 100 (and microfluidic circuit 120). For example, the tilting device 190 may tilt the microfluidic device 100 relative to the x-axis by 0.1 °, 0.2 °, 0.3 °, 0.4 °, 0.5 °, 0.6 °, 0.7 °, 0.8 °, 0.9 °, 1 °, 2 °, 3 °, 4 °, 5 °, 10 °, 15 °, 20 °, 25 °, 30 °, 35 °, 40 °, 45 °, 50 °, 55 °, 60 °, 65 °, 70 °, 75 °, 80 °, 90 °, or any angle therebetween. The horizontal orientation (and thus the x-axis and y-axis) is defined as being perpendicular to the vertical axis defined by gravity. The tilting device may also tilt the microfluidic device 100 (and the microfluidic circuit 120) by any degree greater than 90 ° with respect to the x-axis and/or the y-axis, or tilt the microfluidic device 100 (and the microfluidic circuit 120) by 180 ° with respect to the x-axis or the y-axis, to completely invert the microfluidic device 100 (and the microfluidic circuit 120). Similarly, in some embodiments, the tilting device 190 tilts the microfluidic device 100 (and the microfluidic circuit 120) about an axis of rotation defined by the flow path 106 or some other portion of the microfluidic circuit 120.

In some cases, the microfluidic device 100 is tilted into a vertical orientation such that the flow path 106 is located above or below one or more isolation docks. The term "above" as used herein means that the flow path 106 is positioned higher than the one or more isolation docks on a vertical axis defined by gravity (i.e., an object in an isolation dock above the flow path 106 will have a higher gravitational potential energy than an object in the flow path). The term "below" as used herein means that the flow path 106 is positioned below the one or more isolation docks on a vertical axis defined by gravity (i.e., an object in an isolation dock below the flow path 106 will have a lower gravitational potential energy than an object in the flow path).

In some cases, the tilting device 190 tilts the microfluidic device 100 about an axis parallel to the flow path 106. Furthermore, the microfluidic device 100 may be tilted to an angle of less than 90 ° such that the flow path 106 is located above or below one or more isolation docks, rather than directly above or below the isolation docks. In other cases, the tilting device 190 tilts the microfluidic device 100 about an axis perpendicular to the flow path 106. In still other cases, the tilting device 190 tilts the microfluidic device 100 about an axis that is neither parallel nor perpendicular to the flow path 106.

The system 150 may further include a media source 178. The media source 178 (e.g., container, reservoir, etc.) may include multiple portions or containers, each portion or container for holding a different fluid media 180. Thus, the media source 178 can be a device that is external to and separate from the microfluidic device 100, as shown in fig. 1. Alternatively, the media source 178 can be located wholly or partially within the housing 102 of the microfluidic device 100. For example, the media source 178 can include a reservoir that is part of the microfluidic device 100.

Fig. 1 also shows a simplified block diagram depicting an example of a control and monitoring apparatus 152 that forms part of the system 150 and that may be used in conjunction with the microfluidic device 100. As shown, examples of such control and monitoring devices 152 include a master controller 154 including a media module 160 for controlling a media source 178; a motion module 162 for controlling movement and/or selection of micro-objects (not shown) and/or media (e.g., media droplets) in the microfluidic circuit 120; an imaging module 164 for controlling an imaging device 194 (e.g., a camera, a microscope, a light source, or any combination thereof) to capture an image (e.g., a digital image); and a tilt module 166 for controlling the tilt device 190. The control apparatus 152 may also include other modules 168 for controlling, monitoring, or performing other functions with respect to the microfluidic device 100. As shown, the device 152 may further include a display 170 and an input/output device 172.

The main controller 154 may include a control module 156 and digital memory 158. The control module 156 may include, for example, a digital processor configured to operate in accordance with machine-executable instructions (e.g., software, firmware, source code, etc.) stored as non-transitory data or signals in the memory 158. Alternatively or additionally, the control module 156 may include hard-wired digital circuitry and/or analog circuitry. Media module 160, motion module 162, imaging module 164, tilt module 166, and/or other modules 168 may be similarly configured. Accordingly, the functions, processes, actions, acts, or steps of the processes discussed herein as being performed with respect to the microfluidic device 100 or any other microfluidic apparatus may be implemented by any one or more of the master controller 154, the substrate module 160, the motion module 162, the imaging module 164, the tilt module 166, and/or the other modules 168 configured as discussed above. Similarly, the master controller 154, media module 160, motion module 162, imaging module 164, tilt module 166, and/or other module 168 may be communicatively coupled to send and receive data used in any of the functions, processes, actions, acts, or steps discussed herein.

The media module 160 controls the media source 178. For example, the media module 160 may control the media source 178 to input a selected fluid media 180 into the housing 102 (e.g., through the inlet port 107). The media module 160 may also control the removal of media from the housing 102 (e.g., through an outlet port (not shown)). Thus, one or more media may be selectively input into and removed from the microfluidic circuit 120. The media module 160 may also control the flow of fluidic media 180 in the flow path 106 within the microfluidic circuit 120. For example, in some embodiments, the media module 160 stops the flow of the media 180 in the flow path 106 and through the housing 102 before the tilt module 166 causes the tilt device 190 to tilt the microfluidic device 100 to a desired tilt angle.

The motion module 162 may be configured to control the selection, trapping, and movement of micro-objects (not shown) in the microfluidic circuit 120. As discussed below with reference to fig. 2A and 2B, the enclosure 102 may include a Dielectrophoresis (DEP), optoelectronic tweezers (OET), and/or optoelectronic wetting (OEW) configuration (not shown in fig. 1), and the motion module 162 may control activation of electrodes and/or transistors (e.g., phototransistors) to select and move micro-objects (not shown) and/or media droplets (not shown) in the flow path 106 and/or isolation docks 124, 126, 128, 130.

The imaging module 164 may control an imaging device 194. For example, the imaging module 164 may receive and process image data from the imaging device 194. The image data from the imaging device may include any type of information captured by the imaging device 194 (e.g., the presence or absence of micro-objects, drops of media, accumulation of markers (e.g., fluorescent markers), etc.). Using the information captured by imaging device 194, imaging module 164 may further calculate the locations of objects (e.g., micro-objects, media drops) within microfluidic device 100 and/or the rates of motion of these objects.

The tilt module 166 may control the tilting motion of the tilting device 190. Alternatively or additionally, the tilting module 166 may control the tilting rate and timing to optimize the transfer of micro-objects to one or more isolation docks via gravity. Tilt module 166 is communicatively coupled with imaging module 164 to receive data describing the movement of micro-objects and/or media drops in microfluidic circuit 120. Using this data, tilt module 166 can adjust the tilt of microfluidic circuit 120 in order to adjust the rate at which micro-objects and/or media droplets move in microfluidic circuit 120. Tilt module 166 may also use this data to iteratively adjust the position of micro-objects and/or media drops in microfluidic circuit 120.

In the example shown in fig. 1, microfluidic circuit 120 is shown to include microfluidic channel 122 and isolation docks 124, 126, 128, 130. Each dock includes an opening to the channel 122, but otherwise is enclosed so that the dock can substantially separate micro-objects within the dock from the fluid medium 180 and/or micro-objects in the flow path 106 of the channel 122 or other dock. In some cases, the docks 124, 126, 128, 130 are configured to physically enclose one or more micro-objects within the microfluidic circuit 120. An isolating dock according to the present invention may include various shapes, surfaces and features, which are optimized for use with DEP, OET, OEW and/or gravity, as will be discussed and illustrated in detail below.

The microfluidic circuit 120 may include any number of microfluidic isolation docks. Although five isolation docks are shown, microfluidic circuit 120 may have fewer or more isolation docks. The sequestration dock according to the present disclosure is used to culture, select and expand T cells. As shown, the microfluidic isolation docks 124, 126, 128, and 130 of the microfluidic circuit 120 each include different features and shapes that may provide one or more benefits for culturing, selecting, and expanding T cells. In some embodiments, microfluidic circuit 120 includes a plurality of identical microfluidic isolation docks. In some embodiments, microfluidic circuit 120 includes a plurality of isolation docks, wherein two or more isolation docks include different structures and/or features that provide different benefits in culturing, selecting, and expanding T cells.

In the embodiment shown in fig. 1, a single channel 122 and flow path 106 are shown. However, other embodiments may contain multiple channels 122, each configured to include a flow path 106. The microfluidic circuit 120 further includes an inlet valve or port 107 in fluid communication with the flow path 106 and the fluidic medium 180, whereby the fluidic medium 180 may enter the channel 122 via the inlet port 107. In some cases, the flow path 106 comprises a single path. In some cases, the single paths are arranged in a zigzag pattern whereby the flow paths 106 pass through the microfluidic device 100 in alternating directions two or more times.

In some cases, microfluidic circuit 120 includes a plurality of parallel channels 122 and flow paths 106, wherein fluidic medium 180 within each flow path 106 flows in the same direction. In some cases, the fluid medium within each flow path 106 flows in at least one of a forward direction or a reverse direction. In some cases, the plurality of isolated docks are configured (e.g., relative to channel 122) so that they can be loaded with the target micro-object in parallel.

In some embodiments, microfluidic circuit 120 further comprises one or more micro-object wells 132. The wells 132 are generally formed in the walls that border the channel 122 and may be disposed opposite the openings of one or more of the microfluidic isolation stations 124, 126, 128, 130. In some embodiments, the trap 132 is configured to receive or capture a single micro-object from the flow path 106. In some embodiments, the trap 132 is configured to receive or capture a plurality of micro-objects from the flow path 106. In some cases, well 132 includes a volume approximately equal to the volume of a single target micro-object.

The well 132 may also include an opening configured to assist the flow of the target micro-object into the well 132. In some cases, well 132 includes an opening having a height and width approximately equal to the dimensions of a single target micro-object, thereby preventing larger micro-objects from entering the micro-object well. The well 132 may further include other features configured to help retain the target micro-object within the well 132. In some cases, trap 132 is aligned with respect to the opening of the microfluidic isolation dock and is located on the opposite side of channel 122 such that when microfluidic device 100 is tilted about an axis parallel to channel 122, the trapped micro-objects exit trap 132 with a trajectory that causes the micro-objects to fall into the opening of the isolation dock. In some cases, well 132 includes side channels 134 that are smaller than the target micro-object in order to facilitate flow through well 132, thereby increasing the likelihood of micro-objects being trapped in well 132.

In some embodiments, Dielectrophoretic (DEP) forces are applied to the fluidic medium 180 (e.g., in the flow path and/or in the isolation dock) via one or more electrodes (not shown) to manipulate, transport, separate, and sort micro-objects located therein. For example, in some embodiments, DEP forces are applied to one or more portions of the microfluidic circuit 120 in order to transfer individual micro-objects from the flow path 106 into a desired microfluidic isolation dock. In some embodiments, DEP forces are used to prevent micro-objects within an isolation dock (e.g., isolation dock 124, 126, 128, or 130) from being replaced therefrom. Further, in some embodiments, DEP forces are used to selectively remove micro-objects previously collected according to the teachings of the present disclosure from the isolation dock. In some embodiments, the DEP force comprises an optoelectronic tweezers (OET) force.

In other embodiments, an electro-optical wetting (OEW) force is applied to one or more locations (e.g., locations that help define a flow path and/or a plurality of isolation docks) in the support structure 104 (and/or lid 110) of the microfluidic device 100 by one or more electrodes (not shown) to manipulate, transport, separate, and sort droplets located in the microfluidic circuit 120. For example, in some embodiments, OEW forces are applied to one or more locations in the support structure 104 (and/or the lid 110) to transfer individual droplets from the flow path 106 into a desired microfluidic isolation dock. In some embodiments, OEW forces are used to prevent droplets within an isolation dock (e.g., isolation dock 124, 126, 128, or 130) from being replaced therefrom. Further, in some embodiments, OEW forces are used to selectively remove droplets previously collected according to the teachings of the present disclosure from the isolation dock.

In some embodiments, DEP and/or OEW forces are combined with other forces (e.g., flow and/or gravity) in order to manipulate, transport, separate, and sort micro-objects and/or droplets within the microfluidic circuit 120. For example, the housing 102 can be tilted (e.g., by the tilting device 190) to position the flow path 106 and micro-objects located therein above the microfluidic isolation dock, and gravity can transport the micro-objects and/or droplets into the dock. In some embodiments, DEP and/or OEW forces may be applied before other forces are applied. In other embodiments, DEP and/or OEW forces may be applied after other forces are applied. In other cases, DEP and/or OEW forces may be applied simultaneously with or alternating with other forces.

Fig. 2A-2F illustrate various embodiments that can be used to implement the microfluidic devices of the present disclosure. Fig. 2A depicts an embodiment of an electrokinetic device in which the microfluidic device 200 is configured to be optically actuated. A variety of optically actuated electrokinetic devices are known in the art, including devices having an opto-electronic tweezers (OET) configuration and devices having an opto-electronic wetting (OEW) configuration. Examples of suitable OET configurations are shown in the following U.S. patent documents, all of which are incorporated herein by reference in their entirety: U.S. Pat. No. RE 44,711(Wu et al) (originally issued in U.S. Pat. No. 7,612,355); and U.S. Pat. No. 7,956,339(Ohta et al). Examples of OEW configurations are shown in U.S. patent No. 6,958,132(Chiou et al) and U.S. patent application publication No. 2012/0024708(Chiou et al), both of which are incorporated herein by reference in their entirety. Another example of a light actuated electrodynamic device includes a combined OET/OEW configuration, examples of which are shown in U.S. patent publication nos. 20150306598 (Khandros et al) and 20150306599(Khandros et al) and their corresponding PCT publications WO2015/164846 and WO2015/164847, both of which are incorporated herein by reference in their entirety.

A microfluidic device motion configuration. As mentioned above, the control and monitoring device of the system may comprise a motion module for selecting and moving objects, such as micro-objects or droplets, in the microfluidic circuit of the microfluidic device. Microfluidic devices may have various motion configurations depending on the type of object being moved and other considerations. For example, a Dielectrophoresis (DEP) configuration can be used to select and move micro-objects in a microfluidic circuit. Accordingly, the support structure 104 and/or the lid 110 of the microfluidic device 100 may comprise a DEP configuration for selectively inducing DEP forces on micro-objects in the fluidic medium 180 in the microfluidic circuit 120 to select, capture and/or move individual micro-objects or groups of micro-objects. Alternatively, the support structure 104 and/or the cover 110 of the microfluidic device 100 can comprise an Electrowetting (EW) configuration for selectively inducing EW forces on droplets in the fluidic medium 180 in the microfluidic circuit 120 to select, capture, and/or move individual droplets or groups of droplets.

One example of a microfluidic device 200 containing a DEP configuration is shown in fig. 2A and 2B. While fig. 2A and 2B show a side cross-sectional view and a top cross-sectional view, respectively, of a portion of the housing 102 of the microfluidic device 200 having a region/chamber 202 for purposes of simplicity, it is understood that the open region/chamber 202 may be part of a fluid conduit element having a more detailed structure, such as a growth chamber, an isolation dock, a flow region, or a flow channel. In addition, the microfluidic device 200 may include other fluid conduit elements. For example, the microfluidic device 200 may include multiple growth chambers or isolation docks and/or one or more flow regions or flow channels, such as those described herein with respect to the microfluidic device 100. The DEP configuration can be incorporated into any such fluid conduit element of the microfluidic device 200, or selected portions thereof. It should also be understood that any of the microfluidic device components and system components described above or below may be incorporated into the microfluidic device 200 and/or used in combination with the microfluidic device 200. For example, the system 150 including the control and monitoring device 152 described above may be used with a microfluidic device 200, the microfluidic device 200 including one or more of a media module 160, a motion module 162, an imaging module 164, a tilt module 166, and other modules 168.

As shown in fig. 2A, the microfluidic device 200 includes a support structure 104 having a bottom electrode 204 and an electrode activation substrate 206 covering the bottom electrode 204, and a lid 110 having a top electrode 210, the top electrode 210 being spaced apart from the bottom electrode 204. The top electrode 210 and the electrode activation substrate 206 define opposing surfaces of the region/chamber 202. Thus, the dielectric 180 contained in the region/chamber 202 provides a resistive connection between the top electrode 210 and the electrode activation substrate 206. Also shown is a power supply 212 configured to connect to the bottom electrode 204 and the top electrode 210 and generate a bias voltage between these electrodes as required to generate DEP forces in the region/chamber 202. The power source 212 may be, for example, an Alternating Current (AC) power source.

In certain embodiments, the microfluidic device 200 shown in fig. 2A and 2B can have a light-actuated DEP configuration. Thus, changing the pattern of light 222 from the light source 220 (which may be controlled by the motion module 162) may selectively activate and deactivate the changing pattern of DEP electrodes at the regions 214 of the inner surface 208 of the electrode activation substrate 206. (hereinafter, the region 214 of the microfluidic device having the DEP configuration is referred to as the "DEP electrode region") as shown in fig. 2B, a light pattern directed at the inner surface 208 of the electrode activation substrate 206 can illuminate a selected DEP electrode region 214a (shown in white) in a pattern such as a square light pattern 224. The non-illuminated DEP electrode regions 214 (cross-hatched) are referred to hereinafter as "dark" DEP electrode regions 214. The relative electrical impedance through the DEP electrode activation substrate 206 (i.e., from the bottom electrode 204 up to the inner surface 208 of the electrode activation substrate 206 interfacing with the medium 180 in the flow region 106) is greater than the relative electrical impedance through the medium 180 in the region/chamber 202 at each dark DEP electrode region 214 (i.e., from the inner surface 208 of the electrode activation substrate 206 to the top electrode 210 of the lid 110). However, the illuminated DEP electrode regions 214a exhibit a reduced relative impedance through the electrode activation substrate 206 that is less than the relative impedance through the medium 180 in the region/chamber 202 at each illuminated DEP electrode region 214 a.

With the power supply 212 activated, the aforementioned DEP configuration creates an electric field gradient in the fluid medium 180 between the illuminated DEP electrode region 214a and the adjacent dark DEP electrode region 214, which in turn creates a local DEP force that attracts or repels nearby micro-objects (not shown) in the fluid medium 180. Thus, by varying the light pattern 222 projected from the light source 220 into the microfluidic device 200, DEP electrodes that attract or repel micro-objects in the fluid medium 180 can be selectively activated and deactivated at many different such DEP electrode regions 214 at the inner surface 208 of the region/chamber 202. Whether the DEP force attracts or repels nearby micro-objects may depend on parameters such as the frequency of the power source 212 and the dielectric properties of the medium 180 and/or micro-objects (not shown).

The square pattern 224 of the illuminated DEP electrode regions 214a shown in fig. 2B is merely an example. Any pattern of DEP electrode regions 214 may be illuminated (and thus activated) by a light pattern 222 projected into the device 200, and the pattern of illuminated/activated DEP electrode regions 214 may be repeatedly changed by changing or moving the light pattern 222.

In some embodiments, the electrode activation substrate 206 may include or consist of a photoconductive material. In such embodiments, the inner surface 208 of the electrode activation substrate 206 may be featureless. For example, the electrode activation substrate 206 may include or consist of a hydrogenated amorphous silicon (a-Si: H) layer. H may contain, for example, about 8% to 40% hydrogen (calculated as 100 x the number of hydrogen atoms/total number of hydrogen and silicon atoms). The a-Si: H layer may have a thickness of about 500nm to about 2.0 μm. In such embodiments, DEP electrode regions 214 may be formed in any pattern anywhere on the inner surface 208 of the electrode activation substrate 206, according to the light pattern 222. Thus, the number and pattern of DEP electrode regions 214 need not be fixed, but may be made to correspond to the light pattern 222. Examples of microfluidic devices having DEP configurations comprising a photoconductive layer (such as those described above) have been described, for example, in U.S. patent No. RE 44,711(Wu et al) (originally issued as U.S. patent No. 7,612,355), the entire contents of which are incorporated herein by reference.

In other embodiments, the electrode activation substrate 206 may comprise a substrate comprising a plurality of doped layers, electrically insulating layers (or regions), and electrically conductive layers forming a semiconductor integrated circuit, such as is known in the semiconductor arts. For example, the electrode activation substrate 206 may include a plurality of phototransistors, including, for example, lateral bipolar phototransistors, each corresponding to a DEP electrode region 214. Alternatively, the electrode activation substrate 206 can include electrodes (e.g., conductive metal electrodes) controlled by the phototransistor switches, wherein each such electrode corresponds to a DEP electrode region 214. The electrode activation substrate 206 may include a pattern of such phototransistors or phototransistor-controlled electrodes. For example, the pattern may be an array of substantially square phototransistors or phototransistor-controlled electrodes arranged in rows and columns, as shown in fig. 2B. Alternatively, the pattern may be an array of substantially hexagonal phototransistors or phototransistor-controlled electrodes forming a hexagonal lattice. Regardless of the pattern, the circuit elements can form electrical connections between the DEP electrode regions 214 and the bottom electrodes 210 at the inner surface 208 of the electrode activation substrate 206, and those electrical connections (i.e., phototransistors or electrodes) can be selectively activated and deactivated by the light pattern 222. When not activated, each electrical connection can have a high impedance such that the relative impedance through the electrode activation substrate 206 (i.e., from the bottom electrode 204 to the inner surface 208 of the electrode activation substrate 206 interfacing with the dielectric 180 in the region/chamber 202) is greater than the relative impedance through the dielectric 180 at the corresponding DEP electrode region 214 (i.e., from the inner surface 208 of the electrode activation substrate 206 to the top electrode 210 of the cover 110). However, when activated by light in the light pattern 222, the relative impedance through the electrode activation substrate 206 is less than the relative impedance through the medium 180 at each illuminated DEP electrode region 214, thereby activating the DEP electrode at the respective DEP electrode region 214, as described above. Thus, DEP electrodes that attract or repel micro-objects (not shown) in the medium 180 can be selectively activated and deactivated at a number of different DEP electrode regions 214 at the inner surface 208 of the electrode activation substrate 206 in the region/chamber 202, in a manner determined by the light pattern 222.

Examples of microfluidic devices having electrode-activated substrates including phototransistors have been described, for example, in U.S. Pat. No. 7,956,339(Ohta et al) (see, e.g., device 300 shown in fig. 21 and 22 and the description thereof), the entire contents of which are incorporated herein by reference. Examples of microfluidic devices having electrode activated substrates including electrodes controlled by phototransistor switches have been described, for example, in U.S. patent publication No. 2014/0124370(Short et al) (see, e.g., devices 200, 400, 500, 600, and 900 and the description thereof shown throughout the figures), the entire contents of which are incorporated herein by reference.

In some embodiments of DEP configured microfluidic devices, the top electrode 210 is part of a first wall (or lid 110) of the housing 102, and the electrode activation substrate 206 and the bottom electrode 204 are part of a second wall (or support structure 104) of the housing 102. The region/chamber 202 may be located between the first wall and the second wall. In other embodiments, the electrode 210 is part of the second wall (or support structure 104) and one or both of the electrode activation substrate 206 and/or the electrode 210 is part of the first wall (or cover 110). Further, the light source 220 may alternatively be used to illuminate the housing 102 from below.

With the microfluidic device 200 of fig. 2A-2B having a DEP configuration, the motion module 162 can select micro-objects (not shown) in the medium 180 in the region/chamber 202 by projecting a light pattern 222 into the device 200 to activate a first set of one or more DEP electrodes at the DEP electrode region 214a of the inner surface 208 of the electrode activation substrate 206 in a pattern (e.g., a square pattern 224) that surrounds and captures the micro-objects. The motion module 162 can then move the captured micro-object by moving the light pattern 222 relative to the device 200 to activate the second set of one or more DEP electrodes at the DEP electrode region 214. Alternatively, the microfluidic device 200 may be moved relative to the light pattern 222.

In other embodiments, the microfluidic device 200 can have a DEP configuration that does not rely on photo-activation of DEP electrodes at the inner surface 208 of the electrode activation substrate 206. For example, the electrode activation substrate 206 can include selectively addressable and energizable electrodes located opposite a surface (e.g., the lid 110) that includes at least one electrode. A switch (e.g., a transistor switch in a semiconductor substrate) can be selectively opened and closed to activate or deactivate the DEP electrode at the DEP electrode region 214, thereby creating a net DEP force on a micro-object (not shown) in the region/chamber 202 near the activated DEP electrode. Depending on characteristics such as the frequency of the power source 212 and the dielectric properties of the medium (not shown) and/or micro-objects in the region/chamber 202, DEP forces may attract or repel nearby micro-objects. One or more micro-objects in the region/chamber 202 can be captured and moved in the region/chamber 202 by selectively activating and deactivating a set of DEP electrodes (e.g., at a set of DEP electrode regions 214 forming a square pattern 224). The motion module 162 of fig. 1 can control such switches to activate and deactivate the various DEP electrodes to select, capture, and move specific micro-objects (not shown) around the region/chamber 202. Microfluidic devices having DEP structures comprising selectively addressable and excitable electrodes are known in the art and have been described, for example, in U.S. patent nos. 6,294,063(Becker et al) and 6,942,776(Medoro), the entire contents of which are incorporated herein by reference.

As yet another example, the microfluidic device 200 may have: an Electrowetting (EW) configuration, which may replace the DEP configuration, or may be located in a section of the microfluidic device 200 separate from the section having the DEP configuration. The EW configuration can be an electro-wetting configuration or an electro-wetting on dielectric (EWOD) configuration, both of which are known in the art. In some EW configurations, the support structure 104 has an electrode activation substrate 206 sandwiched between a dielectric layer (not shown) and the bottom electrode 204. The dielectric layer may comprise a hydrophobic material and/or may be coated with a hydrophobic material. For microfluidic devices 200 having an EW configuration, the inner surface 208 of the support structure 104 is an inner surface of a dielectric layer or hydrophobic coating thereof.

The dielectric layer (not shown) may include one or more oxide layers and may have a thickness of about 50nm to about 250nm (e.g., about 125nm to about 175 nm). In certain embodiments, the dielectric layer may include an oxide layer, such as a metal oxide (e.g., aluminum oxide or hafnium oxide) layer. In certain embodiments, the dielectric layer may comprise a dielectric material other than a metal oxide, such as silicon oxide or nitride. Regardless of the exact composition and thickness, the dielectric layer may have an impedance of about 10kOhms to about 50 kOhms.

In some embodiments, the surface of the dielectric layer facing inward toward the region/chamber 202 is coated with a hydrophobic material. The hydrophobic material may include, for example, a fluorocarbon molecule. Examples of fluorocarbon molecules include perfluoropolymers, such as polytetrafluoroethylene (e.g.,) Or poly (2, 3-difluoromethylene-perfluorotetrahydrofuran) (e.g., CYTOP) ^TM ). Molecules constituting the hydrophobic material may be covalently bonded to the surface of the dielectric layer. For example, molecules of the hydrophobic material may be covalently bonded to the surface of the dielectric layer by means of a linking group such as a siloxane group, a phosphonic acid group or a thiol group. Thus, in some embodiments, the hydrophobic material may comprise an alkyl-terminated siloxane, an alkyl-terminated phosphonic acid, or an alkyl-terminated thiol. The alkyl group can be a long chain hydrocarbon (e.g., a chain having at least 10 carbons or at least 16, 18, 20, 22 or more carbons). Alternatively, a fluoro (or perfluoro) carbon chain may be used in place of the alkyl group. Thus, for example, the hydrophobic material may comprise a fluoroalkyl terminated siloxane, a fluoroalkyl terminated phosphonic acid, or a fluoroalkyl terminated thiol. In some embodiments, the hydrophobic coating has a thickness of about 10nm to about 50 nm. In other embodiments, the hydrophobic coating has a thickness of less than 10nm (e.g., less than 5nm, or about 1.5nm to 3.0 nm).

In some embodiments, the cover 110 of the microfluidic device 200 having an electrowetting configuration is also coated with a hydrophobic material (not shown). The hydrophobic material may be the same hydrophobic material used to coat the dielectric layer of the support structure 104, and the hydrophobic coating may have a thickness that is substantially the same as the thickness of the hydrophobic coating on the dielectric layer of the support structure 104. In addition, the lid 110 may include an electrode activation substrate 206 sandwiched between a dielectric layer and a top electrode 210 in the manner of the support structure 104. The dielectric layers of the electrode activation substrate 206 and the cap 110 may have the same composition and/or dimensions as the dielectric layers of the electrode activation substrate 206 and the support structure 104. Thus, the microfluidic device 200 may have two electrowetting surfaces.

In some embodiments, the electrode activation substrate 206 may include a photoconductive material, such as those described above. Thus, in certain embodiments, the electrode activation substrate 206 may comprise or consist of a hydrogenated amorphous silicon layer (a-Si: H). H may contain, for example, about 8% to 40% hydrogen (calculated as 100 x the number of hydrogen atoms/total number of hydrogen and silicon atoms). The a-Si: H layer may have a thickness of about 500nm to about 2.0 microns. Alternatively, as described above, the electrode activation substrate 206 may include an electrode (e.g., a conductive metal electrode) controlled by a phototransistor switch. Microfluidic devices having electro-optical wetting configurations are known in the art and/or may be constructed with electrode-activated substrates known in the art. For example, U.S. Pat. No. 6,958,132(Chiou et al), the entire contents of which are incorporated herein by reference, discloses a electrowetting configuration having a photoconductive material such as a-Si: H, while U.S. Pat. No. 2014/0124370(Short et al), cited above, discloses an electrode activated substrate having electrodes controlled by phototransistor switches.

Thus, microfluidic device 200 can have a photo-electrowetting configuration, and light pattern 222 can be used to activate a photoconductive EW region or a photo-responsive EW electrode in electrode activation substrate 206. Such activated EW regions or EW electrodes of the electrode activation substrate 206 can generate electrowetting forces at the inner surface 208 of the support structure 104 (i.e., the inner surface that covers the dielectric layer or hydrophobic coating thereof). By varying the light pattern 222 incident on the electrode-activated substrate 206 (or moving the microfluidic device 200 relative to the light source 220), droplets (e.g., containing an aqueous medium, solution, or solvent) in contact with the inner surface 208 of the support structure 104 can be moved through an immiscible fluid (e.g., an oil medium) present in the region/chamber 202.

In other embodiments, the microfluidic device 200 may have an EWOD configuration, and the electrode activation substrate 206 may include selectively addressable and excitable electrodes that do not rely on light for activation. Thus, the electrode activation substrate 206 can include a pattern of such Electrowetting (EW) electrodes. For example, the pattern can be an array of substantially square EW electrodes arranged in rows and columns, as shown in figure 2B. Alternatively, the pattern can be an array of substantially hexagonal EW electrodes forming a hexagonal lattice of dots. Regardless of the pattern, the EW electrode can be selectively activated (or deactivated) by an electrical switch (e.g., a transistor switch in a semiconductor substrate). By selectively activating and deactivating the EW electrodes in the electrode activation substrate 206, droplets (not shown) in contact with the inner surface 208 of the covered dielectric layer or hydrophobic coating thereof can be moved within the region/chamber 202. The motion module 162 in figure 1 can control such switches to activate and deactivate individual EW electrodes to select and move specific droplets around the region/chamber 202. Microfluidic devices having EWOD configurations with selectively addressable and excitable electrodes are known in the art and have been described, for example, in U.S. patent No. 8,685,344(Sundarsan et al), the entire contents of which are incorporated herein by reference.

Regardless of the configuration of the microfluidic device 200, the power supply 212 may be used to provide a potential (e.g., an AC voltage potential) that powers the circuitry of the microfluidic device 200. The power supply 212 may be the same as or a component of the power supply 192 referenced in FIG. 1. The power supply 212 may be configured to provide an AC voltage and/or current to the top electrode 210 and the bottom electrode 204. For AC voltages, the power supply 212 may provide a range of frequencies and a range of average or peak powers (e.g., voltages or currents): which, as described above, is sufficient to generate a net DEP force (or electrowetting force) strong enough to capture and move individual micro-objects (not shown) in the region/chamber 202, and/or which, as also described above, is sufficient to alter the wetting properties of the inner surface 208 of the support structure 104 (i.e., the dielectric layer and/or the hydrophobic coating on the dielectric layer) in the region/chamber 202. Such frequency ranges and average or peak power ranges are known in the art. See, for example, U.S. Pat. No. 6,958,132(Chiou et al), U.S. Pat. No. RE44,711(Wu et al) (originally issued as U.S. Pat. No. 7,612,355), and U.S. patent application publication Nos. US2014/0124370(Short et al), US2015/0306598(Khandros et al), and US2015/0306599(Khandros et al).

Isolating the dock. Non-limiting examples of general isolation docks 244, 246 and 248 are shown within the microfluidic device 240 depicted in fig. 2C-2D. Each isolation dock 244, 246, and 248 may include a separation structure 250 defining a separation region 258 and a connection region 254 fluidly connecting the separation region 258 to the channel 122. The connecting region 254 may include a proximal opening 252 to the passage 122 and a distal opening 256 to a separating region 258. The connection region 254 may be configured such that a maximum penetration depth of a flow of fluid medium (not shown) from the channel 122 into the isolation docks 244, 246, 248 does not extend into the separation region 258. Thus, due to the connection region 254, micro-objects (not shown) or other materials (not shown) disposed in the separation region 258 of the isolation docks 244, 246, 248 may be separated from and substantially unaffected by the flow of the medium 180 in the channel 122.

Thus, microfluidic channel 122 may be an example of a swept area, and separation region 258 of isolation docks 244, 246, 248 may be an example of an unswept area. It should be noted that the channel 122 and spacers 244, 246, 248 may be configured to contain one or more fluid mediums 180. In the example shown in fig. 2C-2D, port 242 is connected to channel 122 and allows for the introduction or removal of fluidic medium 180 into or from microfluidic device 240. The microfluidic device may be loaded with a gas, such as carbon dioxide gas, prior to introduction of the fluid medium 180. Once microfluidic device 240 contains fluidic medium 180, flow 242 of fluidic medium 180 in channel 122 may be selectively generated and stopped. For example, as shown, the ports 260 may be arranged at different locations (e.g., opposite ends) of the channel 122, and a flow 260 of the medium may be formed from one port 242 serving as an inlet to another port 242 serving as an outlet.

Fig. 2E shows a detailed view of an example of an isolating dock 244 according to the present disclosure. An example of a micro-object 270 is also shown.

As is known, the flow 260 of fluidic medium 180 in the microfluidic channel 122 through the proximal opening 252 of the isolation dock 244 may cause a secondary flow 262 of medium 180 to enter and/or exit the isolation dock 244. In order to separate the separated area 25 of the dock 244Micro-objects 270 in 8 separate from the secondary flow 262, isolating the length L of the connection region 254 of the dock 244 _con (i.e., from proximal opening 252 to distal opening 256) should be greater than the penetration depth D of secondary flow 262 into junction region 254 _p . Depth of penetration D of secondary flow 262 _p Depending on the velocity of the fluid medium 180 flowing in the channel 122 and various parameters related to the configuration of the channel 122 and the proximal opening 252 to the connection region 254 of the channel 122. For a given microfluidic device, the configuration of channel 122 and opening 252 will be fixed, while the rate of flow 260 of fluidic media 180 in channel 122 will be variable. Thus, for each isolation dock 244, the maximum velocity V of the flow 260 of the fluid medium 180 in the channel 122 may be identified _max Ensuring the penetration depth D of the secondary flow 262 _p Not exceeding the length L of the connecting region 254 _con . As long as the velocity of the flow 260 of fluid medium 180 in the passage 122 does not exceed the maximum velocity V _max The resulting secondary flow 262 may be confined to the passage 122 and the connecting region 254 and remain outside of the separation region 258. Thus, the flow 260 of medium 180 in the channel 122 will not drag the micro-objects 270 out of the separation region 258. In contrast, micro-objects 270 located in the separation region 258 will stay in the separation region 258 regardless of the flow 260 of fluid medium 180 in the channel 122.

Furthermore, as long as the velocity of the flow 260 of the medium 180 in the channel 122 does not exceed V _max The flow 260 of the fluid medium 180 in the channel 122 does not move the intermixed particles (e.g., microparticles and/or nanoparticles) from the channel 122 into the separation region 258 of the isolation dock 244. Thus, the length L of the connecting region 254 is made _con Greater than the maximum penetration depth D of the secondary flow 262 _p One isolation dock 244 may be prevented from being contaminated by miscellaneous particles from channel 122 or another isolation dock (e.g., isolation docks 246, 248 in fig. 2D).

Because the channel 122 and the connection region 254 of the isolation docks 244, 246, 248 may be affected by the flow 260 of the medium 180 in the channel 122, the channel 122 and the connection region 254 may be considered a swept (or flow) region of the microfluidic device 240. On the other hand, the separation region 258 of the isolation docks 244, 246, 248 may be considered an unswept (or no flow) region. For example, a component (not shown) in first fluid medium 180 in channel 122 may mix with second fluid medium 280 in separation region 258 substantially only by diffusion of the component of first medium 180 from channel 122 through connection region 254 and into second fluid medium 280 in separation region 258. Similarly, the components (not shown) of the second media 280 in the separation region 258 may mix with the first media 180 in the channel 122 substantially only by diffusion of the components of the second media 280 from the separation region 258, through the connection region 254, and into the first media 180 in the channel 122. The first medium 180 may be the same medium as the second medium 280 or a different medium. In addition, the first medium 180 and the second medium 280 may be initially the same and then become different (e.g., by conditioning the second medium 280 by separating one or more cells in the region 258, or by altering the medium 180 flowing through the channel 122).

As described above, the maximum penetration depth D of the secondary flow 262 caused by the flow 260 of the fluid medium 180 in the channel 122 _p May depend on a number of parameters. Examples of such parameters include: the shape of the channel 122 (e.g., the channel may direct media into the connection region 254, divert media from the connection region 254, or direct media in a direction substantially perpendicular to the proximal opening 252 to the connection region 254 of the microfluidic channel 122); width W of channel 122 at proximal opening 252 _ch (or cross-sectional area); and the width W of the connecting region 254 at the proximal opening 252 _con (or cross-sectional area); velocity V of flow 260 of fluid medium 180 in passage 122; viscosity of the first medium 180 and/or the second medium 280, and so on.

In some embodiments, the dimensions of the channel 122 and isolation docks 244, 246, 248 may be oriented relative to the vector of the flow 260 of the fluid medium 180 in the channel 122 as follows: width W of channel _ch (or cross-sectional area of the channel 122) may be substantially perpendicular to the flow 260 of the medium 180; width W of connecting region 254 at opening 252 _con (or cross-sectional area) may be substantially parallel to the flow 260 of the medium 180 in the channel 122; and/or length L of the connecting region _con May be substantially perpendicular to the channel 122 Of the medium 180. The foregoing are examples only, and the relative positions of the channel 122 and isolation docks 244, 246, 248 may be in other orientations relative to one another.

As shown in FIG. 2E, the width W of the connecting region 254 _con May be uniform from proximal opening 252 to distal opening 256. Thus, the width W of the connecting region 254 at the distal opening 256 _con May be referred to herein as the width W of the connecting region 254 at the proximal opening 252 _con Any range identified. Alternatively, the width W of the connecting region 254 at the distal opening 256 _con May be greater than the width W of the connecting region 254 at the proximal opening 252 _con 。

As shown in FIG. 2E, the width of the separation region 258 at the distal opening 256 may be the same as the width W of the connection region 254 at the proximal opening 252 _con Are substantially the same. Thus, the width of the separation region 258 at the distal opening 256 may be referred to herein as the width W of the connection region 254 at the proximal opening 252 _con Any range identified. Alternatively, the width of the separation region 258 at the distal opening 256 may be greater than or less than the width W of the connection region 254 at the proximal opening 252 _con . Further, distal opening 256 may be smaller than proximal opening 252, and connecting region 254 has a width W _con May narrow between proximal opening 252 and distal opening 256. For example, using a variety of different geometries (e.g., beveling, etc.) the connection region 254 may narrow between the proximal and distal openings. Further, any portion or sub-portion of the connection region 254 may be narrowed (e.g., a portion of the connection region adjacent the proximal opening 252).

In various embodiments of the isolation dock (e.g., 124, 126, 128, 130, 244, 246, or 248), the isolation region (e.g., 258) is configured to contain a plurality of micro-objects. In other embodiments, the separation region may be configured to contain only one, two, three, four, five, or a similar relatively small number of micro-objects. Thus, the volume of the separation zone may be, for example, at least 3 × 10 ³ 、6×10 ³ 、9×10 ³ 、1×10 ⁴ 、2×10 ⁴ 、4×10 ⁴ 、8×10 ⁴ 、1×10 ⁵ 、2×10 ⁵ 、4×10 ⁵ 、8×10 ⁵ 、1×10 ⁶ 、2×10 ⁶ 、4×10 ⁶ 、6×10 ⁶ Cubic microns or larger.

In various embodiments of the isolation dock, the width W of channel 122 at the proximal opening (e.g., 252) _ch May be in any of the following ranges: about 50-1000 microns, 50-500 microns, 50-400 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 70-500 microns, 70-400 microns, 70-300 microns, 70-250 microns, 70-200 microns, 70-150 microns, 90-400 microns, 90-300 microns, 90-250 microns, 90-200 microns, 90-150 microns, 100-300 microns, 100-250 microns, 100-200 microns, 100-150 microns, 100-120 microns. The above are examples only, and the width W of the channel 122 _ch May be within other ranges (e.g., within a range defined by any of the endpoints listed above). Further, W of channel 122 is in the area of the channel other than the proximal opening that isolates the dock _ch May be selected to be within any of these ranges.

In some embodiments, the cross-sectional height of the isolating dock is from about 30 to about 200 microns or from about 50 to about 150 microns. In some embodiments, the cross-sectional area of the isolation dock is about 100,000 to about 2,500,000 square microns or about 200,000 to about 2,000,000 square microns. In some embodiments, the cross-sectional height of the connection region matches the cross-sectional height of the corresponding isolation dock. In some embodiments, the cross-sectional width of the attachment region is from about 50 to about 500 microns or from about 100 to about 300 microns.

In various embodiments of the isolation dock, the height H of channel 122 at proximal opening 252 _ch May be in any of the following ranges: 20-100 microns, 20-90 microns, 20-80 microns, 20-70 microns, 20-60 microns, 20-50 microns, 30-100 microns, 30-90 microns, 30-80 microns, 30-70 microns, 30-60 microns, 30-50 microns, 40-100 microns, 40-90 microns, 40-80 microns, 40-70 microns, 40-60 microns, or 40-50 microns. The foregoing are examples only, and the height H of the channel 122 _ch May be within other ranges (e.g., by any of the ranges listed above)The range defined by the endpoints). Height H of channel 122 in the area of the channel other than the proximal opening that isolates the dock _ch May be selected to be within any of these ranges.

In various embodiments of the isolation dock, the cross-sectional area of channel 122 at proximal opening 252 may be in any of the following ranges: 500-50,000 square micron, 500-40,000 square micron, 500-30,000 square micron, 500-25,000 square micron, 500-20,000 square micron, 500-15,000 square micron, 500-10,000 square micron, 500-7,500 square micron, 500-5,000 square micron, 1,000-25,000 square micron, 1,000-20,000 square micron, 1,000-15,000 square micron, 1,000-10,000 square micron, 1,000-7,500 square micron, 1,000-5,000 square micron, 2,000-20,000 square micron, 2,000-15,000 square micron, 2,000-10,000 square micron, 2,000-7,500 square micron, 2,000-6,000 square micron, 3,000-20,000 micron, 3,000 square micron, 10,000 square micron, 3,000-3,000 square micron, 3,000 square micron. The foregoing are examples only, and the cross-sectional area of the passage 122 at the proximal opening 252 may be within other ranges (e.g., within a range defined by any of the endpoints listed above).

In various embodiments of the isolation dock, the length L of the connection region 254 _con May be in any of the following ranges: 1-200 microns, 5-150 microns, 10-100 microns, 15-80 microns, 20-60 microns, 20-500 microns, 40-400 microns, 60-300 microns, 80-200 microns, and 100-150 microns. The foregoing are examples only, and the length L of the connection region 254 _con May be in a different range than the preceding examples (e.g., within a range defined by any of the endpoints listed above).

In various embodiments of the isolation dock, the width W of the connection region 254 at the proximal opening 252 _con May be in any of the following ranges: 20-500 microns, 20-400 microns, 20-300 microns, 20-200 microns, 20-150 microns, 20-100 microns, 20-80 microns, 20-60 microns, 30-400 microns, 30-300 microns, 30-200 microns, 30-150 microns, 30-100 microns, 30-80 microns, 30-60 microns, 40-300 microns, 40-200 microns, 40-150 microns, 40-10 microns0 micron, 40-80 microns, 40-60 microns, 50-250 microns, 50-200 microns, 50-150 microns, 50-100 microns, 50-80 microns, 60-200 microns, 60-150 microns, 60-100 microns, 60-80 microns, 70-150 microns, 70-100 microns, and 80-100 microns. The foregoing are examples only, and the width W of the connection region 254 at the proximal opening 252 _con May be different from the foregoing examples (e.g., within a range defined by any of the endpoints listed above).

In various embodiments of the isolation dock, the width W of the connection region 254 at the proximal opening 252 _con Can be any of the following ranges: 2-35 microns, 2-25 microns, 2-20 microns, 2-15 microns, 2-10 microns, 2-7 microns, 2-5 microns, 2-3 microns, 3-25 microns, 3-20 microns, 3-15 microns, 3-10 microns, 3-7 microns, 3-5 microns, 3-4 microns, 4-20 microns, 4-15 microns, 4-10 microns, 4-7 microns, 4-5 microns, 5-15 microns, 5-10 microns, 5-7 microns, 6-15 microns, 6-10 microns, 6-7 microns, 7-15 microns, 7-10 microns, 8-15 microns, and 8-10 microns. The foregoing are examples only, and the width W of the connection region 254 at the proximal opening 252 _con May be different from the foregoing examples (e.g., within a range defined by any of the endpoints listed above).

In various embodiments of the isolation dock, the length L of the connection region 254 _con And the width W of the connection region 254 at the proximal opening 252 _con The ratio of (d) may be greater than or equal to any of the following ratios: 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, or more. The above are examples only, and the length L of the connection region 254 _con And the width W of the connection region 254 at the proximal opening 252 _con The ratio of (d) may be different from the previous examples.

In various embodiments of the microfluidic devices 100, 200, 240, 290, 500, 700, 715, V _max Can be set to about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 mul/sec.

In various embodiments of microfluidic devices with isolation docks, the volume of isolation region 258 of the isolation dock can be, for example, at least 3 x10 ³ 、6×10 ³ 、9×10 ³ 、1×10 ⁴ 、2×10 ⁴ 、4×10 ⁴ 、8×10 ⁴ 、1×10 ⁵ 、2×10 ⁵ 、4×10 ⁵ 、8×10 ⁵ 、1×10 ⁶ 、2×10 ⁶ 、4×10 ⁶ 、6×10 ⁶ Cubic microns or larger. In various embodiments of microfluidic devices with isolation docks, the volume of the isolation dock may be about 5x10 ³ 、7×10 ³ 、1×10 ⁴ 、3×10 ⁴ 、5×10 ⁴ 、8×10 ⁴ 、1×10 ⁵ 、2×10 ⁵ 、4×10 ⁵ 、6×10 ⁵ 、8×10 ⁵ 、1×10 ⁶ 、2×10 ⁶ 、4×10 ⁶ 、8×10 ⁶ 、1×10 ⁷ 、3×10 ⁷ 、5×10 ⁷ Or about 8X 10 ⁷ Cubic microns or larger. In some embodiments, the microfluidic device has a separate dock, wherein no more than 1 x10 can be maintained ² Individual biological cells, and the volume of the isolation dock may not exceed 2 x10 ⁶ Cubic microns. In some embodiments, the microfluidic device has a separate dock, wherein no more than 1 x10 can be maintained ² Individual biological cell, and the isolation dock may not exceed 4 × 10 ⁵ Cubic microns. In further embodiments, the microfluidic device has an isolation dock, wherein no more than 50 biological cells can be held, and the isolation dock can be no more than 4 x10 ⁵ Cubic microns.

In various embodiments, the microfluidic device has isolated docks configured as in any of the embodiments discussed herein, wherein the microfluidic device has from about 100 to about 500 isolated docks; about 200 to about 1000 isolated docks, about 500 to about 1500 isolated docks, about 1000 to about 2000 isolated docks, or about 1000 to about 3500 isolated docks.

In some other embodiments, the isolation dock of the microfluidic device is configured as any of the embodiments discussed herein, wherein the microfluidic device has from about 1500 to about 3000 isolation docks, from about 2000 to about 3500 isolation docks, from about 2500 to about 4000 isolation docks, from about 3000 to about 4500 isolation docks, from about 3500 to about 5000 isolation docks, from about 4000 to about 5500 isolation docks, from about 4500 to about 6000 isolation docks, from about 5000 to about 6500 isolation docks, from about 5500 to about 7000 isolation docks, from about 6000 to about 7500 isolation docks, from about 6500 to about 8000 isolation docks, from about 7000 to about 8500 isolation docks, from about 7500 to about 9000 isolation docks, from about 8000 to about 9500 isolation docks, from about 8500 to about 10,000 isolation docks, from about 9000 to about 10,500 isolation docks, from about 11,000 isolation docks, from about 9500 to about 9500 isolation docks, from about 11,000 to about 500,000 isolation docks, from about 11,000 isolation docks, from about 11,500 to about 11,000 isolation docks, from about 11,12 to about 500,13, from about 500,000 isolation docks, from about 11,000 isolation docks, from about 500,13, from about 500,000 isolation docks, and about 500,000 isolation docks, About 12,500 to about 14,000 isolated docks, about 13,000 to about 14,500 isolated docks, about 13,500 to about 15,000 isolated docks, about 14,000 to about 15,500 isolated docks, about 14,500 to about 16,000 isolated docks, about 15,000 to about 16,500 isolated docks, about 15,500 to about 17,000 isolated docks, about 16,000 to about 17,500 isolated docks, about 16,500 to about 18,000 isolated docks, about 17,000 to about 18,500 isolated docks, about 17,500 to about 19,000 isolated docks, about 18,000 to about 19,500 isolated docks, about 18,500 to about 20,000 isolated docks, about 19,000 to about 20,500 isolated docks, about 19,500 to about 21,000 isolated docks, or about 20,000 to about 21,500 isolated docks.

Fig. 2F shows a microfluidic device 290 according to one embodiment. The microfluidic device 290 shown in fig. 2F is a stylized schematic diagram of the microfluidic device 100. In implementation, the microfluidic device 290 and its constituent plumbing components (e.g., the channel 122 and the isolation dock 128) will have the dimensions discussed herein. The microfluidic circuit 120 shown in fig. 2F has two ports 107, four different channels 122, and four different flow paths 106. The microfluidic device 290 further includes a plurality of isolation docks opening into each channel 122. In the microfluidic device shown in fig. 2F, the isolation dock has a similar geometry to the dock shown in fig. 2E, and thus has both a connection region and a separation region. Accordingly, microfluidic circuit 120 includes a swept region (e.g., channel 122 and connecting region 254 at maximum penetration depth D of secondary flow 262) _p Inner portion) and non-swept areas (e.g., separation area 258 and junction area 254 are not at the maximum penetration depth D of the secondary flow 262 _p Inner portion).

Fig. 3-4 illustrate various embodiments of a system 150 that can be used to operate and view microfluidic devices (e.g., 100, 200, 240, 290, 500, 700, 715) according to the present disclosure. As shown in fig. 3, the system 150 may include a structure ("nest") 300 configured to hold the microfluidic device 100 (not shown) or any other microfluidic device described herein. Nest 300 can include a socket 302 that can interface with a microfluidic device 360 (e.g., a light-actuated electrokinetic device 100) and provide an electrical connection from power source 192 to microfluidic device 360. Nest 300 may also include an integrated electrical signal generation subsystem 304. The electrical signal generation subsystem 304 may be configured to provide a bias voltage to the receptacle 302 such that when the receptacle 302 is holding the microfluidic device 360, a bias voltage is applied across a pair of electrodes in the microfluidic device 360. Thus, the electrical signal generation subsystem 304 may be part of the power supply 192. The ability to apply a bias voltage to the microfluidic device 360 does not mean that the bias voltage is always applied when the receptacle 302 is holding the microfluidic device 360. In contrast, in most cases, the bias voltage will be applied intermittently, e.g., only when needed to facilitate generation of an electrokinetic force (e.g., dielectrophoresis or electrowetting) in the microfluidic device 360.

As shown in fig. 3, nest 300 may include a Printed Circuit Board Assembly (PCBA) 320. The electrical signal generation subsystem 304 may be mounted on the PCBA 320 and electrically integrated therein. The exemplary support also includes a socket 302 mounted on the PCBA 320.

Typically, electrical signal generation subsystem 304 will include a waveform generator (not shown). The electrical signal generation subsystem 304 can also include an oscilloscope (not shown) and/or a waveform amplification circuit (not shown) configured to amplify waveforms received from the waveform generator. The oscilloscope (if any) may be configured to measure the waveform supplied to the microfluidic device 360 held by the receptacle 302. In certain embodiments, the oscilloscope measures the waveform at a location near the microfluidic device 360 (and away from the waveform generator), thereby ensuring a more accurate measurement of the waveform actually applied to the device. Data obtained from oscilloscope measurements may be provided, for example, as feedback to a waveform generatorAnd the waveform generator may be configured to adjust its output based on such feedback. An example of a suitable combined waveform generator and oscilloscope is Red Pitaya ^TM 。

In certain embodiments, nest 300 further includes a controller 308, such as a microprocessor for detecting and/or controlling electrical signal generating subsystem 304. Examples of suitable microprocessors include Arduino ^TM Microprocessors, e.g. Arduino Nano ^TM . The controller 308 may be used to perform functions and analyses or may communicate with the external master controller 154 (shown in FIG. 1) to perform functions and analyses. In the embodiment shown in fig. 3, the controller 308 communicates with the master controller 154 via an interface 310 (e.g., a plug or connector).

In some embodiments, nest 300 may include an electrical signal generation subsystem 304, which includes Red Pitaya ^TM A waveform generator/oscilloscope cell ("Red Pitaya cell") and a waveform amplification circuit that amplifies the waveform generated by the Red Pitaya cell and transmits the amplified voltage to the microfluidic device 100. In some embodiments, the Red Pitaya cell is configured to measure the amplified voltage at the microfluidic device 360 and then adjust its own output voltage as needed so that the measured voltage at the microfluidic device 360 is a desired value. In some embodiments, the waveform amplification circuit may have a +6.5V to-6.5V power supply generated by a pair of DC-DC converters mounted on PCBA 320, thereby generating up to 13Vpp of signals at microfluidic device 100.

As shown in fig. 3, the support structure 300 may further include a thermal control subsystem 306. The thermal control subsystem 306 may be configured to regulate the temperature of the microfluidic device 360 held by the support structure 300. For example, the thermal control subsystem 306 may include a Peltier thermoelectric device (not shown) and a cooling unit (not shown). The Peltier thermoelectric device may have a first surface configured to interface with at least one surface of the microfluidic device 360. The cooling unit may be, for example, a cooling block (not shown), such as a liquid cooled aluminum block. A second surface (e.g., a surface opposite the first surface) of the Peltier thermoelectric device may be configured as Interfacing with the surface of such a cooling block. The cooling block may be connected to a fluid path 330, the fluid path 330 being configured to circulate a cooled fluid through the cooling block. In the embodiment shown in fig. 3, the support structure 300 includes an inlet 332 and an outlet 334 to receive cooled fluid from an external reservoir (not shown), introduce the cooled fluid into the fluid path 330 and through the cooling block, and return the cooled fluid to the external reservoir. In some embodiments, the Peltier thermoelectric device, the cooling unit, and/or the fluid path 330 may be mounted on a housing 340 of the support structure 300. In some embodiments, the thermal control subsystem 306 is configured to regulate the temperature of the Peltier thermoelectric device to achieve a target temperature for the microfluidic device 360. Temperature regulation of the Peltier thermoelectric device can be effected, for example, by a thermoelectric power supply, for example, by Pololu ^TM Thermoelectric power supply (Pololu semiconductors and Electronics Corp.) was implemented. Thermal control subsystem 306 may include feedback circuitry, such as temperature values provided by analog circuitry. Alternatively, the feedback circuit may be provided by a digital circuit.

In some embodiments, nest 300 may include a thermal control subsystem 306 having a feedback circuit that is an analog voltage divider circuit including a resistor (e.g., having an impedance of 1kOhm +/-0.1% +/-0.02ppm/C0 temperature coefficient) and an NTC thermistor (e.g., having a nominal impedance of 1kOhm +/-0.01%). In some cases, thermal control subsystem 306 measures the voltage from the feedback circuit and then uses the calculated temperature value as an input to the on-board PID control loop algorithm. The output from the PID control loop algorithm may drive, for example, Pololu ^TM Directional and pulse width modulated signal pins on a motor driver (not shown) to actuate the thermoelectric power supply to control the Peltier thermoelectric device.

Nest 300 may include a serial port 350 that allows the microprocessor of controller 308 to communicate with external master controller 154 via interface 310. Additionally, the microprocessor of the controller 308 may be in communication with the electrical signal generation subsystem 304 and the thermal control subsystem 306 (e.g., via a Plink tool (not shown)). Thus, the electrical signal generation subsystem 308 and the thermal control subsystem 306 may communicate with the external master controller 154 via a combination of the controller 308, the interface 310, and the serial port 350. In this manner, the main controller 154 may assist the electrical signal generation subsystem 308 by performing, among other things, scaling calculations for output voltage regulation. A Graphical User Interface (GUI) provided via a display device 170 coupled to the external master controller 154 may be configured to plot temperature and waveform data obtained from the thermal control subsystem 306 and the electrical signal generation subsystem 308, respectively. Alternatively or additionally, the GUI may allow for updating the controller 308, the thermal control subsystem 306, and the electrical signal generation subsystem 308.

As discussed above, the system 150 may include an imaging device 194. In some embodiments, the imaging device 194 includes a light modulation subsystem 404. The light modulation subsystem 404 may include a Digital Mirror Device (DMD) or a micro-shutter array system (MSA), either of which may be configured to receive light from the light source 402 and transmit a portion of the received light into the optical train of the microscope 400. Alternatively, light modulation subsystem 404 may include a device that generates its own light (and thus does not require light source 402), such as an organic light emitting diode display (OLED), a Liquid Crystal On Silicon (LCOS) device, a ferroelectric liquid crystal on silicon device (FLCOS), or a transmissive Liquid Crystal Display (LCD). The light modulation subsystem 404 may be, for example, a projector. Thus, the light modulation subsystem 404 is capable of emitting structured light and unstructured light. One example of a suitable light modulation subsystem 404 is from Andor Technologies ^TM Mosaic of ^TM Provided is a system. In certain embodiments, the imaging module 164 and/or the motion module 162 of the system 150 may control the light modulation subsystem 404.

In certain embodiments, the imaging device 194 further comprises a microscope 400. In such embodiments, the nest 300 and the light modulation subsystem 404 may be separately configured to be mounted on the microscope 400. Microscope 400 may be, for example, a standard research grade optical microscope or a fluorescent microscope. Thus, the nest 300 can be configured to mount on the stage 410 of the microscope 400 and/or the light modulation subsystem 404 can be configured to mount on a port of the microscope 400. In other embodiments, the nest 300 and the light modulation subsystem 404 described herein may be integrated components of the microscope 400.

In certain embodiments, the microscope 400 may further include one or more detectors 422. In some embodiments, the detector 422 is controlled by the imaging module 164. The detector 422 may include an eyepiece, a Charge Coupled Device (CCD), a camera (e.g., a digital camera), or any combination thereof. If there are at least two detectors 422, one detector may be, for example, a fast frame rate camera and the other detector may be a high sensitivity camera. Further, the microscope 400 may include an optical train configured to receive light reflected and/or emitted from the microfluidic device 360 and focus at least a portion of the reflected and/or emitted light onto the one or more detectors 422. The optical train of the microscope may also include different tube lenses (not shown) for different detectors so that the final magnification on each detector may be different.

In certain embodiments, the imaging device 194 is configured to use at least two light sources. For example, a first light source 402 may be used to generate structured light (e.g., via the light modulation subsystem 404), and a second light source 432 may be used to provide unstructured light. The first light source 402 may generate structured light for optically driven electrical motion and/or fluorescence excitation, and the second light source 432 may be used to provide bright field illumination. In these embodiments, the motion module 162 may be used to control the first light source 404 and the imaging module 164 may be used to control the second light source 432. The optical train of microscope 400 may be configured to (1) receive structured light from light modulation subsystem 404 and focus the structured light on at least a first area in a microfluidic device (e.g., a light-actuated electro-mechanical device) when the device is held by support structure 200, and (2) receive light reflected and/or emitted from the microfluidic device and focus at least a portion of such reflected and/or emitted light onto detector 422. The optical train may be further configured to receive unstructured light from the second light source and focus the unstructured light on at least a second area of the microfluidic device when the microfluidic device is held by the support structure 300. In certain embodiments, the first and second regions of the microfluidic device may be overlapping regions. For example, the first region may be a subset of the second region.

In fig. 3B, a first light source 402 is shown providing light to a light modulation subsystem 404, which provides structured light to an optical train of the microscope 400. Second light source 432 is shown providing unstructured light to the optical train via beam splitter 436. The structured light from light modulation subsystem 404 and the unstructured light from second light source 432 travel together through an optical train from beam splitter 436 to a second beam splitter (or dichroic filter 406, depending on the light provided by light modulation subsystem 404), where the light is reflected down through objective lens 408 to sample plane 412. Light reflected and/or emitted from sample plane 412 then returns back up through objective lens 408, through beam splitter and/or dichroic filter 406, and back to dichroic filter 424. Only a portion of the light that reaches the dichroic filter 424 passes through to the detector 422.

In some embodiments, the second light source 422 emits blue light. With an appropriate dichroic filter 424, blue light reflected from the sample plane 412 can pass through the dichroic filter 424 and reach the detector 422. In contrast, structured light from the light modulation subsystem 404 reflects from the sample plane 412 but does not pass through the dichroic filter 424. In this example, the dichroic filter 424 filters out visible light having a wavelength longer than 495 nm. This filtering of light from the light modulation subsystem 404 is accomplished (as shown) only if the light emitted from the light modulation subsystem does not include any wavelengths shorter than 495 nm. In an implementation, if the light from the light modulation subsystem 404 includes a wavelength shorter than 495nm (e.g., a blue wavelength), some of the light from the light modulation subsystem may pass through the filter 424 to the detector 422. In such embodiments, the filter 424 acts to change the balance between the amount of light reaching the detector 422 from the first and second light sources 402, 432. This may be beneficial if the first light source 402 is significantly stronger than the second light source 432. In other embodiments, the second light source 432 may emit red light, and the dichroic filter 424 may filter out visible light other than red light (e.g., visible light having a wavelength shorter than 650 nm).

Blocking solutions and blocking agents. Without wishing to be bound by theory, when one or more internal surfaces of the microfluidic device have been conditioned or coated so as to present a layer of organic and/or hydrophilic molecules that provides a primary interface between the microfluidic device and the T cells grown therein, culture and expansion of the T cells within the microfluidic device may be facilitated (i.e., the T cells exhibit increased viability and greater expansion). In some embodiments, one or more interior surfaces of the microfluidic device (e.g., an interior surface of an electrode-activated substrate of the DEP-configured microfluidic device, a surface of a lid and/or tubing material of the microfluidic device) may be treated with a coating solution and/or a coating agent to produce a desired layer of organic and/or hydrophilic molecules. In some embodiments, T cells cultured and optionally expanded in a microfluidic device are infused into a coating solution comprising one or more coating agents.

In other embodiments, prior to introducing T cells into the microfluidic device, the interior surface of the microfluidic device (e.g., a DEP configured microfluidic device) is treated or "primed" with a coating solution comprising a coating agent. Any convenient coating agent/solution may be used, including but not limited to: serum or serum factors, Bovine Serum Albumin (BSA), polymers, detergents, enzymes, and any combination thereof. In some specific embodiments, the coating agent will be used to treat the interior surfaces of the microfluidic device. In one example, a polymer containing alkylene ether moieties is included in the coating solution as a coating agent. Many alkylene ether-containing polymers may be suitable. One non-limiting exemplary class of alkylene ether-containing polymers is the amphoteric nonionic block copolymers, which comprise blocks of Polyoxyethylene (PEO) and polyoxypropylene (PPO) subunits having different proportions and positions within the polymer chain. Polymers (BASF) are such block copolymers and are known in the art to be suitable for use when in contact with living cells. Average molecular weight M of the Polymer _w And may range from about 2000Da to about 20 KDa. In some embodiments, the PEO-PPO block copolymer may have a large sizeA hydrophilic-lipophilic balance (HLB) of at about 10 (e.g., 12-18). Specific for producing coated surfacesThe polymer comprisesL44, L64, P85 and F127 (including F127 NF). Another class of alkylene ether-containing polymers is polyethylene glycol (PEG M) _w <100,000Da) or alternatively, polyoxyethylene (PEO, M) _w >100,000). In some embodiments, the PEG can have an M of about 1000Da, 5000Da, 10,000Da, or 20,000Da _w 。

In some embodiments, the coating solution may comprise a plurality of proteins and/or peptides as coating agents. In a particular embodiment, the coating solution used in the present disclosure comprises as a coating agent a protein such as Bovine Serum Albumin (BSA) and/or a serum comprising albumin (or a combination of different sera) and/or one or more other similar proteins. The serum may be from any convenient source, including but not limited to fetal bovine serum, sheep serum, goat serum, horse serum, and the like. In certain embodiments, BSA is present in the coating solution at a concentration ranging from about 1mg/mL to about 100mg/mL, including 5mg/mL, 10mg/mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, or any value therebetween. In certain embodiments, serum may be present in the coating solution at a concentration ranging from about 20% (v/v) to about 50% v/v, including 25%, 30%, 35%, 40%, 45%, or higher or any value in between. In some embodiments, BSA may be present as a coating agent in the coating solution at 5mg/mL, while in other embodiments, BSA may be present as a coating agent in the coating solution at 70 mg/mL. In certain embodiments, serum is present at 30% in the coating solution as a coating agent.

And (3) coating materials. Depending on the embodiment, any of the above coating agents/coating solutions may be replaced by, or used in combination with, a variety of coating materials for coating one or more interior surfaces of a microfluidic device (e.g., a DEP-configured and/or EW-configured microfluidic device). In some embodiments, at least one surface of the microfluidic device comprises a coating material that provides a layer of organic and/or hydrophilic molecules suitable for T cell culture and expansion. In some embodiments, substantially all of the interior surfaces of the microfluidic device comprise a coating material. The coated interior surface may include a flow region (e.g., a channel), a surface of a chamber or isolation dock, or a combination thereof. In some embodiments, each of the plurality of isolated docks has at least one interior surface coated with a coating material. In other embodiments, each of the plurality of flow regions or channels has at least one interior surface coated with a coating material. In some embodiments, at least one interior surface of each of the plurality of isolated docks and each of the plurality of channels is coated with a coating material.

A polymer-based coating material. At least one of the inner surfaces may include a coating material comprising a polymer. The polymer may be covalently or non-covalently bound (or may be linked) to at least one surface. The polymers can have a variety of structural motifs such as found in block (and copolymers), star (star copolymers), and graft or comb (graft copolymers), all of which can be adapted for use in the methods disclosed herein.

The polymer may comprise a polymer comprising alkylene ether moieties. A wide variety of alkylene ether-containing polymers may be suitable for use in the microfluidic devices described herein. One non-limiting exemplary class of alkylene ether-containing polymers is the amphoteric nonionic block copolymers, which comprise blocks of Polyoxyethylene (PEO) and polyoxypropylene (PPO) subunits having different proportions and positions within the polymer chain.Polymers (BASF) are such block copolymers and are known in the art to be suitable for use when in contact with living cells. Average molecular weight M of the Polymer _w From about 2000Da to about 20 kDa. In some embodiments, the PEO-PPO block copolymer may have greater than about 10 (e.g., 12-18)) Hydrophilic-lipophilic balance (HLB). Specific for producing coated surfacesThe polymer comprisesL44, L64, P85 and F127 (including F127 NF). Another class of alkylene ether-containing polymers is polyethylene glycol (PEG M) _w <100,000Da) or alternatively, polyoxyethylene (PEO, M) _w >100,000). In some embodiments, the PEG can have an M of about 1000Da, 5000Da, 10,000Da, or 20,000Da _w 。

In other embodiments, the coating material may include a polymer containing carboxylic acid moieties. The carboxylic acid subunits may be subunits containing alkyl, alkenyl or aromatic moieties. One non-limiting example is polylactic acid (PLA).

In other embodiments, the coating material may include a polymer containing sulfonic acid moieties. The sulfonate subunits may be subunits containing alkyl, alkenyl or aromatic moieties. One non-limiting example is polystyrene sulfonic acid (PSSA) or polyanetholesulfonic acid. These latter exemplary polymers are polyelectrolytes and may alter the characteristics of the surface to provide a layer of organic and/or hydrophilic molecules suitable for culturing and expanding T cells.

In some embodiments, the coating material may include a urethane moiety-containing polymer, such as, but not limited to, a polyurethane.

In other embodiments, the coating material may include a polymer that includes a phosphate moiety at a terminus of or pendant from the backbone of the polymer.

In other embodiments, the coating material may include a polymer containing sugar moieties. In one non-limiting example, a polysaccharide (e.g., from a seaweed or fungal polysaccharide, such as xanthan or dextran) may be suitable for forming a material that can reduce or prevent cell adhesion in a microfluidic device. For example, dextran polymers having a size of about 3kDa may be used to provide a coating material for surfaces within a microfluidic device.

In other embodiments, the coating material may comprise a polymer containing nucleotide moieties, i.e., nucleic acids, which may have ribonucleotide moieties or deoxyribonucleotide moieties. Nucleic acids may contain only natural nucleotide moieties or may contain non-natural nucleotide moieties that comprise nucleobase, ribose, or phosphate moiety analogs, such as, but not limited to, 7-deazaadenine, pentose, methylphosphonate, or phosphorothioate moieties. The nucleic acid-containing polymer may include an electrolyte, which may provide a layer of organic and/or hydrophilic molecules suitable for T cell culture and expansion.

In other embodiments, the coating material may include a polymer containing amino acid moieties. Polymers containing amino acid moieties may include polymers containing natural amino acids or polymers containing unnatural amino acids, which may each include peptides, polypeptides, or proteins. In one non-limiting example, the protein can be Bovine Serum Albumin (BSA). In some embodiments, extracellular matrix (ECM) proteins may be provided within the coating material for obtaining optimized cell adhesion to promote cell growth. Cell matrix proteins that may be included in the coating material may include, but are not limited to, collagen, elastin, RGD-containing peptides (e.g., fibronectin), or laminin. In other embodiments, growth factors, cytokines, hormones, or other cell signaling substances may be provided within the coating material of the microfluidic device.

In further embodiments, the coating material can include a polymer containing amine moieties. The polyamino polymer may comprise a natural polyamine polymer or a synthetic polyamine polymer. Examples of natural polyamines include spermine, spermidine, and putrescine.

In some embodiments, the coating material can include a polymer that includes more than one of an alkylene oxide moiety, a carboxylic acid moiety, a sulfonic acid moiety, a phosphate moiety, a sugar moiety, a nucleotide moiety, or an amino acid moiety. In other embodiments, the polymer conditioned surface may comprise a mixture of more than one polymer, each polymer having alkylene oxide moieties, carboxylic acid moieties, sulfonic acid moieties, phosphate moieties, sugar moieties, nucleotide moieties, and/or amino acid moieties, which may be incorporated into the coating material independently or simultaneously.

A covalently linked coating material. In some embodiments, at least one internal surface comprises covalently attached molecules that provide a layer of organic and/or hydrophilic molecules suitable for culturing and expanding T cells within a microfluidic device. The covalently linked molecules include a linking group, wherein the linking group is covalently linked to one or more surfaces of the microfluidic device. The linking group is also covalently attached to a moiety configured to provide an organic and/or hydrophilic molecular layer suitable for T cell culture and expansion. The surface to which the linking group is attached can comprise a substrate surface of a microfluidic device, and for embodiments in which the microfluidic device comprises a DEP configuration, can comprise silicon and/or silicon dioxide. In some embodiments, the covalently attached coating material coats substantially all of the interior surfaces of the microfluidic device.

In some embodiments, the covalently linked moieties configured to provide an organic and/or hydrophilic molecular layer suitable for T cell culture and expansion may comprise alkyl or fluoroalkyl (including perfluoroalkyl) moieties; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, including but not limited to polyvinyl alcohol; alkylene ethers including, but not limited to, polyethylene glycol; polyelectrolytes (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amino groups (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino groups, guanidinium salts, and heterocyclic groups containing a non-aromatic nitrogen ring atom such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynyl phosphonic acid (which can provide a phosphonate anionic surface); a sulfonate anion; a carboxybetaine; a sulfobetaine; sulfamic acid; or an amino acid.

The covalently linked moieties configured to provide a layer of organic and/or hydrophilic molecules suitable for culturing and expanding T cells in a microfluidic device can be any of the polymers described herein, and can include polymers comprising alkylene oxide moieties, carboxylic acid moieties, sugar moieties, sulfonic acid moieties, phosphate moieties, amino acid moieties, nucleotide moieties, or amino moieties.

In other embodiments, the covalently linked moieties configured to provide a layer of organic and/or hydrophilic molecules suitable for culturing and expanding T cells in a microfluidic device can include non-polymeric moieties such as alkyl moieties, substituted alkyl moieties (e.g., fluoroalkyl moieties (including but not limited to perfluoroalkyl moieties)), amino acid moieties, alcohol moieties, amino moieties, carboxylic acid moieties, phosphonic acid moieties, sulfonic acid moieties, sulfamic acid moieties, or sugar moieties.

In some embodiments, the covalently linked moiety can be an alkyl group comprising carbon atoms that form a straight chain (e.g., a straight chain of at least 10 carbons or at least 14, 16, 18, 20, 22 or more carbons). Thus, the alkyl group may be an unbranched alkyl group. In some embodiments, alkyl groups may include substituted alkyl groups (e.g., some carbons in an alkyl group may be fluorinated or perfluorinated). The alkyl group can include a linear chain of substituted (e.g., fluorinated or perfluorinated) carbons bonded to an unsubstituted carbon. For example, an alkyl group can include a first segment (which can include a perfluoroalkyl group) that is linked to a second segment (which can include an unsubstituted alkyl group). The first and second segments may be linked together directly or indirectly (e.g., via an ether linkage). The first segment of the alkyl group may be located distal to the linking group and the second segment of the alkyl group may be located proximal to the linking group. In other embodiments, the alkyl group may include branched alkyl groups, and may also have one or more arylene groups interrupting the alkyl backbone of the alkyl group. In some embodiments, the branched or arylene interrupted portion of the alkyl or fluoroalkyl group is located distal to the linking group to the surface and the covalent bond.

In other embodiments, the covalently linked moiety may comprise at least one amino acid, which may comprise more than one type of amino acid. Thus, the covalently linked moiety may comprise a peptide or a protein. In some embodiments, the covalently linked moieties may include amino acids, which may provide a zwitterionic surface to support cell growth, viability, portability (portability), or any combination thereof.

The covalently linked moiety may comprise one or more sugars. The covalently linked saccharide may be a monosaccharide, disaccharide or polysaccharide. The covalently linked sugar may be modified to introduce reactive pairing moieties that allow coupling or processing for attachment of the surface. Exemplary reactive partner moieties may include aldehyde, alkyne, or halogen moieties. The polysaccharide may be modified in a random manner, wherein each saccharide monomer or only a portion of the saccharide monomers within the polysaccharide may be modified to provide reactive partner moieties that may be coupled directly or indirectly to a surface. One example may include dextran polysaccharides, which may be indirectly coupled to a surface via an unbranched linker moiety.

The covalently linked moiety may include one or more amino groups. The amino group can be a substituted amine moiety, guanidine moiety, nitrogen-containing heterocyclic moiety, or heteroaryl moiety. The amino-containing moiety can have a structure that allows for pH modification of the environment within the microfluidic device and optionally within the sequestration dock and/or flow region (e.g., channel).

The coating material may comprise only one type of covalently linked moiety, or may comprise more than one different type of covalently linked moiety. For example, a fluoroalkyl-conditioned surface (including perfluoroalkyl) may have a plurality of covalently attached moieties that are all the same, e.g., having the same attachment group and covalent attachment to the surface, the same total length, and the same number of fluoromethylene units, including fluoroalkyl moieties. Alternatively, the coating material may have more than one type of covalently linked moiety attached to the surface. For example, the coating material may include molecules having covalently linked alkyl or fluoroalkyl moieties having a specified number of methylene or fluoromethylene units, and may also include another group of molecules having charged moieties covalently linked to alkyl or fluoroalkyl chains having a greater number of methylene or fluoromethylene units. In some embodiments, a coating material having more than one covalently attached moiety can be designed such that a first set of molecules having a greater number of backbone atoms and thus a longer distance from covalent attachment to the surface can provide the ability to present a larger portion on the coated surface, while a second set of molecules having different, less spatially demanding ends and fewer backbone atoms can help to functionalize the entire substrate surface, preventing undesired adhesion or contact with the silicon or aluminum oxide comprising the substrate itself. In another example, the covalently linked moieties can provide a zwitterionic surface that exhibits alternating charges on the surface in a random manner.

Conditioned surface properties. In some embodiments, the covalently linked moieties can form a monolayer when covalently linked to a surface of the microfluidic device (e.g., a substrate surface of a DEP configuration). In some embodiments, the conditioned surface formed by covalently linked moieties can have a thickness of less than 10nm (e.g., less than 5nm, or about 1.5 to 3.0 nm). In other embodiments, the conditioned surface formed by covalently linked moieties may have a thickness of about 10nm to about 50 nm. In some embodiments, the conditioned surface does not require a perfectly formed monolayer to function properly for operation within a DEP configured microfluidic device.

In various embodiments, the coating material of the microfluidic device can provide desired electrical properties. Without wishing to be bound by theory, one factor that affects the robustness of a surface coated with a particular coating material is inherent charge trapping. Different coating materials may trap electrons, which may lead to destruction of the coating material. Defects in the coating material may increase charge trapping and lead to further damage of the coating material. Similarly, different coating materials have different dielectric strengths (i.e., minimum applied electric field that results in dielectric breakdown), which may affect charge trapping. In certain embodiments, the coating material can have a bulk structure (e.g., a close-packed monolayer structure) that reduces or limits the amount of charge trapping.

In addition to the composition of the coating material, other factors, such as the physical (and electrical) thickness of the coating material, can influence the generation of DEP and electrowetting forces by the substrate of the microfluidic device. Various factors may alter the physical and electrical thickness of the coating material, including the manner in which the coating material is deposited on the substrate (e.g., vapor deposition, liquid deposition, spin coating, and electrostatic coating). The physical thickness and uniformity of the coating material can be measured using an ellipsometer.

In addition to its electrical properties, the coating material may have properties that are beneficial for use with biomolecules. For example, coating materials containing fluoro (or perfluoro) alkyl groups may provide benefits in reducing the amount of surface fouling relative to unsubstituted alkyl groups. Surface fouling, as used herein, refers to the amount of any substance deposited on the surface of a microfluidic device, which may include permanent or semi-permanent deposition of biological substances (e.g., proteins and their degradation products, nucleic acids, and respective degradation products). Such fouling can increase the amount of adhesion of biological micro-objects to the surface.

Various electrical and functional characteristics of the different coating materials that may be used in the microfluidic device are included in the table below.

In addition to the composition of the conditioned surface, other factors (e.g., the physical thickness of the hydrophobic material) may affect the DEP force. Various factors may alter the physical thickness of the conditioned surface, such as the manner in which the conditioned surface is formed on the substrate (e.g., vapor deposition, liquid deposition, spin coating, flooding, and electrostatic coating). The physical thickness and uniformity of the conditioned surface can be measured using an ellipsometer.

In addition to its electrical properties, the conditioned surface may also have properties that are beneficial for use with biomolecules. For example, a conditioned surface containing fluorinated (or perfluorinated) carbon chains may provide benefits in reducing the amount of surface fouling relative to alkyl terminated chains. Surface fouling, as used herein, refers to the amount of any substance deposited on the surface of a microfluidic device, which may include permanent or semi-permanent deposition of biological materials (e.g., proteins and their degradation products, nucleic acids and respective degradation products, and the like).

A surface linking group. The covalent linking moieties forming the coating material are attached to the surface via a linking group. The linking group can be a siloxy linking group formed by reaction of a siloxane-containing reagent with an oxide of the substrate surface, which can include silicon oxide (e.g., a substrate for DEP configuration) or aluminum oxide or hafnium oxide (e.g., a substrate for EW configuration). In some other embodiments, the linking group can be a phosphate ester formed by reacting a phosphonic acid-containing reagent with an oxide of the substrate surface.

A multi-part conditioned surface. As described below, the covalently linked coating material may be formed by the reaction of molecules (e.g., alkylsiloxane reagents or fluoro-substituted alkylsiloxane reagents, which may include perfluoroalkylsiloxane reagents) that already contain moieties configured to provide a layer of organic and/or hydrophilic molecules suitable for culturing and expanding T cells in a microfluidic device. Alternatively, the covalently linked coating material may be formed by coupling a moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for T cell culture and expansion to a surface modifying ligand (which itself is covalently linked to the surface).

A method of making a covalently linked coating material. In some embodiments, a coating material covalently attached to a surface of a microfluidic device (e.g., at least one surface comprising an isolation dock and/or a flow region) has the structure of formula 1.

Formula 1

The coating material may be covalently attached to the oxide on the surface of the DEP configured substrate. The DEP configured substrate may comprise silicon or aluminum oxide or hafnium oxide, and the oxide may be present as part of the initial chemical structure of the substrate, or may be introduced as discussed below.

The coating material may be attached to the oxide via a linking group ("LG"), which may be a siloxy or phosphonate group formed from the reaction of a siloxane or phosphonate group with the oxide. The portion configured to provide a layer of organic and/or hydrophilic molecules suitable for culturing and expanding T cells in a microfluidic device may be any portion described herein. The linking group LG may be directly or indirectly attached to a moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for culturing and expanding T cells in a microfluidic device. When the linking group LG is directly linked to the moiety, there is no optional linking moiety ("L") and n is 0. When the linking group LG is indirectly linked to the moiety, there is a linking moiety L and n is 1. The linking moiety L may have a linear portion, wherein the backbone of the linear portion may include 1 to 200 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, subject to chemical bonding limitations known in the art. In some non-limiting examples, it may be interrupted by any combination of one or more moieties that may be selected from ether, amino, carbonyl, amido, or phosphonate groups. In addition, the linking moiety L may have one or more arylene, heteroarylene, or heterocyclyl groups interrupting the backbone of the linking group. In some embodiments, the backbone of the linking moiety L may comprise 10 to 20 atoms. In other embodiments, the backbone of the linking moiety L may comprise from about 5 atoms to about 200 atoms; about 10 atoms to about 80 atoms; about 10 atoms to about 50 atoms; or from about 10 atoms to about 40 atoms. In some embodiments, the backbone atoms are all carbon atoms. In other embodiments, the backbone atoms are not all carbon, and may include any possible combination of silicon, carbon, nitrogen, oxygen, sulfur, and phosphorus atoms, subject to the limitations of chemical bonding known in the art.

When a moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for culturing and expanding T cells in a microfluidic device is added to the substrate surface in a one-step process, the molecules of formula 2 can be used to introduce a coating material:

part- (L) n-LG.

Formula 2

In some embodiments, the portion of the layer of organic and/or hydrophilic molecules configured to provide a suitable for culturing and expanding T cells in a microfluidic device may be added to the surface of the substrate in a multi-step process. When the moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for T cell culture and expansion is coupled to the surface in a stepwise manner, the linking moiety L may further comprise a coupling group CG, as shown in formula 3.

Formula 3

In some embodiments, the coupling group CG is represented by a reactive moiety R _x And a reactive partner R _px (i.e., configured to react with the reactive moiety R) _x Part of the reaction). For example, a typical coupling group CG may include a carboxamide group that is the result of the reaction of an amino group with a carboxylic acid derivative (e.g., an activated ester, an acid chloride, etc.). Other CGs may include triazolylene, carboxamido, thioamido, oxime, mercapto, disulfide, ether or alkenyl groups, or any other suitable group that may be formed upon reaction of a reactive moiety with its corresponding reactive partner moiety. The coupling group CG may be located at the second end of the linking group L (i.e. adjacent to the end of the portion configured to provide a layer of organic and/or hydrophilic molecules suitable for culturing and expanding T cells in a microfluidic device). In some other embodiments, the coupling group CG may interrupt the backbone of the linking group L. In some embodiments, the coupling group CG is a triazolylene group, which may be obtained from the reaction of an alkyne group and an azide group, any of which may be a reactive moiety R _x And a reactive partner R _px As known in the art for Click coupling reactions. For example, the dibenzocyclooctenyl-fused triazolylene group may be conjugated to a dibenzocyclooctynyl reactive partner R _px (ii) an azido-reactive moiety R with a surface-modifying molecule _x The reaction of (a), which is described in more detail in the following paragraphs. A variety of dibenzocyclooctynyl-modified molecules are known in the art, or can be synthesized to incorporate moieties configured to provide organic and/or hydrophilic molecular layers suitable for T cell culture and expansion.

When the coating material is formed in a multi-step process, moieties configured to provide a layer of organic and/or hydrophilic molecules suitable for culturing and expanding T cells in a microfluidic device may be introduced by reacting a reagent containing the moiety (formula 5) with a substrate having a surface modifying ligand covalently attached thereto (formula 6).

The modified surface of formula 4 has attached thereto a surface modifying ligand having the formula-LG- (L') j-R _x Which is connected to the oxide of the substrate and is formed similarly as described above for the conditioned surface of equation 1. The surface of the substrate may be a substrate surface of the DEP configuration as described above, and may comprise the substrate itself or an oxide incorporated therein. The linking group LG is as described above. The linking moiety L "may be present (j ═ 1) or absent (j ═ 0). The linking moiety L "may have a linear portion, wherein the backbone of the linear portion may comprise from 1 to 100 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, subject to chemical bonding limitations known in the art. In some non-limiting examples, it may be interrupted by any combination of ether, amino, carbonyl, amido, or phosphonate groups. In addition, the linking moiety L "may have one or more arylene, heteroarylene, or heterocyclic groups that interrupt the backbone of the linking moiety. In some embodiments, the backbone of the linking moiety L "may comprise 10 to 20 carbon atoms. In other embodiments, the backbone of the linking moiety L "can comprise from about 5 atoms to about 100 atoms; from about 10 atoms to about 80 atoms, from about 10 atoms to about 50 atoms, or from about 10 atoms to about 40 atoms. In some embodiments, all of the backbone atoms are carbon atoms. In other embodiments, the backbone atoms are not all carbon, and may include any possible combination of silicon, carbon, nitrogen, oxygen, sulfur, or phosphorus atoms, subject to the limitations of chemical bonding known in the art.

Reactive moiety R _x Is present at the end of the surface modifying ligand remote from the covalent attachment of the surface modifying ligand to the surface. Reactive moiety R _x Are useful for coupling reactions to introduce organic compounds providing suitable for culturing and expanding T cells in microfluidic devicesAnd/or any suitable reactive moiety of a portion of a layer of hydrophilic molecules. In some embodiments, the reactive moiety R _x Can be an azido, amino, bromo, thiol, activated ester, succinimidyl, or alkynyl moiety.

A reagent containing a moiety. The reagent containing moieties (formula 5) is configured to supply moieties configured to provide a layer of organic and/or hydrophilic molecules suitable for culturing and expanding T cells in a microfluidic device.

Moiety- (L') _m -R _px

Formula 5

A moiety for providing a layer of organic and/or hydrophilic molecules suitable for T cell culture and expansion in a reagent containing the moiety is provided by a reactive pairing moiety R _px With reactive moieties R _x Is linked to a surface modifying ligand. Reactive partner R _px Is any suitable reactive group configured to react with a corresponding reactive moiety R _x And (4) reacting. In a non-limiting example, a suitable reactive partner R _px Can be an alkyne, a reactive moiety R _x May be an azide. Reactive partner R _px May alternatively be an azide moiety, the corresponding reactive moiety R _x May be an alkyne. In other embodiments, the reactive partner R _px Can be an active ester functional group, a reactive moiety R _x May be an amino group. In other embodiments, the reactive partner moiety R _px May be an aldehyde, a reactive moiety R _x May be an amino group. Other reactive moiety-reactive partner combination are possible, and these examples are in no way limiting.

The moiety of the moiety-containing reagent of formula 5 configured to provide an organic and/or hydrophilic molecular layer suitable for T cell culture and expansion may include any of the moieties described herein, including alkyl or fluoroalkyl (including perfluoroalkyl) moieties; mono-or polysaccharides (which may include, but are not limited to, dextran); alcohols (including but not limited to propargyl alcohol); polyols, including but not limited to polyvinyl alcohol; alkylene ethers including, but not limited to, polyethylene glycol; polyelectrolytes (including but not limited to polyacrylic acid or polyvinylphosphonic acid); amino (including derivatives thereof such as, but not limited to, alkylated amines, hydroxyalkylated amino, guanidinium salts, and heterocyclic groups containing a nitrogen ring atom that is not aromatic, such as, but not limited to, morpholinyl or piperazinyl); carboxylic acids, including but not limited to propiolic acid (which may provide a carboxylate anionic surface); phosphonic acids, including but not limited to ethynylphosphonic acid (which can provide a phosphonic acid anionic surface); a sulfonate anion; a carboxybetaine; a sulfobetaine; (ii) sulfamic acid; or an amino acid.

The moiety of the moiety-containing reagent of formula 5 configured to provide an organic and/or hydrophilic molecular layer suitable for T cell culture and expansion may be directly linked (i.e., L', wherein m ═ 0) or indirectly linked to a reactive partner moiety R _px . When the reactive partner R is _px Reactive partner moieties R when indirectly attached to a moiety configured to provide a layer of organic and/or hydrophilic molecules suitable for T cell culture and expansion _px May be connected to the connecting portion L' (m ═ 1). Reactive partner R _px A moiety that can be attached to a first end of the linking moiety L 'and that is configured to reduce surface fouling and/or prevent or reduce cell adhesion can be attached to a second end of the linking moiety L'. The linking moiety L' may have a linear portion, wherein the backbone of the linear portion comprises 1 to 100 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms, subject to chemical bonding limitations known in the art. In some non-limiting examples, it may be interrupted by any combination of ether, amino, carbonyl, amido, or phosphonate groups. In addition, linker L 'may have one or more arylene, heteroarylene, or heterocyclic groups that interrupt the backbone of linker L'. In some embodiments, the backbone of the linking moiety L' may comprise 10 to 20 carbon atoms. In other embodiments, the backbone of the linking moiety L' may comprise from about 5 atoms to about 100 atoms; about 10 atoms to about 80 atoms; about 10 atoms to about 50 atoms; or from about 10 atoms to about 40 atoms. In some embodiments, all of the backbone atoms are carbon atoms. In other embodiments, the backbone atoms are not all carbon, and may include silicon, carbon, nitrogen, oxygen, sulfur Or any possible combination of phosphorus atoms, subject to the chemical bonding known in the art.

When the reagent containing moieties (formula 5) reacts with the surface having surface modifying ligands (formula 3), a substrate having a conditioned surface of formula 2 is formed. The linking moiety L 'and linking moiety L' are then formally part of the linking moiety L, and the reactive partner R _px With a reactive moiety R _x The reaction of (a) gives the coupling group CG of formula 2.

A surface modifier. The surface modifier is of the structure LG- (L') _j -R _x A compound of (formula 4). The linking group LG is covalently linked to the oxide on the surface of the substrate. The substrate may be a DEP configured substrate and may comprise silicon or aluminum oxide or hafnium oxide, and the oxide may be present as part of the native chemical structure of the substrate or may be incorporated as discussed herein. The linking group LG can be any linking group described herein, such as a siloxy or phosphonate group, formed from the reaction of a siloxane or phosphonate group with an oxide on the surface of a substrate. Reactive moiety R _x As described above. Reactive moiety R _x May be directly linked to (L ", j ═ 0) or indirectly linked to the linking group LG through a linking moiety L" (j ═ 1). The linking group LG may be linked to a first end of the linking moiety L' and the reactive moiety R _x Can be attached to the second end of the linking moiety L', the reactive moiety R once the surface modifying reagent has been attached to the surface as shown in formula 6 _x Will be located distally of the substrate surface.

The linking moiety L "may have a linear portion, wherein the backbone of the linear portion comprises 1 to 100 non-hydrogen atoms selected from any combination of silicon, carbon, nitrogen, oxygen, sulfur and phosphorus atoms. In some non-limiting examples, it may be interrupted by any combination of ether, amino, carbonyl, amido, or phosphonate groups. In addition, the linking moiety L "may have one or more arylene, heteroarylene, or heterocyclic groups that interrupt the backbone of the linking moiety L". In some embodiments, the backbone of the linking moiety L "may comprise 10 to 20 carbon atoms. In other embodiments, the backbone of the linking moiety L "can comprise from about 5 atoms to about 100 atoms; from about 10 atoms to about 80 atoms, from about 10 atoms to about 50 atoms, or from about 10 atoms to about 40 atoms. In some embodiments, all of the backbone atoms are carbon atoms. In other embodiments, the backbone atoms are not all carbon, and may include any possible combination of silicon, carbon, nitrogen, oxygen, sulfur, or phosphorus atoms, subject to the limitations of chemical bonding known in the art.

In some embodiments, the coating material (or surface-modified ligand) is deposited on the inner surface of the microfluidic device using chemical vapor deposition. By chemical vapor deposition, the coating material may achieve a close-packed monolayer, wherein molecules comprising the coating material are covalently bonded to molecules of the inner surface of the microfluidic device. To achieve the desired packing density, molecules comprising, for example, alkyl-terminated siloxanes can be vapor deposited at a temperature of at least 110 ℃ (e.g., at least 120 ℃, 130 ℃, 140 ℃, 150 ℃, 160 ℃, etc.) for at least 15 hours (e.g., at least 20, 25, 30, 35, 40, 45, or more hours). Such vapor deposition is typically carried out under vacuum and in a water source (e.g., hydrated sulfate salts (e.g., MgSO4 & 7H) ₂ O)) in the presence of oxygen. Generally, increasing the temperature and duration of the vapor deposition results in improved properties of the hydrophobic coating material.

The vapor deposition process can optionally be modified, for example, by pre-cleaning the lid 110, the microfluidic conduit material 116, and/or the substrate (e.g., the inner surface 208 of the electrode activation substrate 206 of a DEP configured substrate, or the dielectric layer of the support structure 104 of an EW configured substrate). For example, the pre-clean may include a solvent bath, such as an acetone bath, an ethanol bath, or a combination thereof. The solvent bath may include sonication. Alternatively or additionally, such pre-cleaning may include treating the cover 110, the microfluidic circuit material 116, and/or the substrate in an oxygen plasma cleaner, which may remove various impurities while introducing an oxidized surface (e.g., an oxide on the surface, which may be covalently modified as described herein). The oxygen plasma cleaner may be operated, for example, at 100W under vacuum for 60 seconds. Alternatively, a liquid phase treatment, which includes an oxidizing agent (e.g., hydrogen peroxide) to oxidize the surface, may be used in place of the oxygen plasma cleaner. For example, a mixture of hydrochloric acid and hydrogen peroxide or a mixture of sulfuric acid and hydrogen peroxide (e.g., a piranha solution, which may have a ratio range of sulfuric acid to hydrogen peroxide of about 3:1 to about 7: 1) is substituted for the oxygen plasma cleaner.

In some embodiments, vapor deposition is used to coat the inner surfaces of the microfluidic device 200 after the microfluidic device 200 has been assembled to form the housing 102 defining the microfluidic circuit 120. Depositing a coating material comprising a tightly packed monolayer on a fully assembled microfluidic circuit 120 may be beneficial in providing a variety of functional properties. Without wishing to be bound by theory, depositing such a coating material on the fully assembled microfluidic circuit 120 may be beneficial to prevent delamination caused by weakened bonding between the microfluidic circuit material 116 and the electrode activation substrate 206 dielectric layer and/or the cap 110.

Fig. 5 depicts a cross-sectional view of a microfluidic device 500, the microfluidic device 500 including an example class of coating materials. As shown, coating material 529 (shown schematically) may comprise a tightly packed molecular monolayer covalently bonded to both the inner surface 508 of the substrate 504 and the inner surface 509 of the cover 510 of the microfluidic device 500. Coating material 529 can be disposed on all interior surfaces 508, 509 of housing 502 adjacent and inwardly facing microfluidic device 500, in some embodiments and as discussed above, including surfaces of microfluidic tubing material (not shown) that are used to define tubing elements and/or structures within microfluidic device 500. In alternative embodiments, coating material 529 may be disposed only on one or some of the interior surfaces of microfluidic device 500.

In the embodiment shown in fig. 5, coating material 529 comprises a monolayer of alkyl-terminated siloxane molecules, each molecule covalently bonded to an interior surface 508, 509 of microfluidic device 500 via a siloxy group. However, any of the above-described coating materials 529 (e.g., alkyl-terminated phosphonate molecules) can be used. More specifically, the alkyl group can comprise a straight chain of at least 10 carbon atoms (e.g., 10, 12, 14, 16, 18, 20, 22, or more carbon atoms) and, optionally, can be a substituted alkyl group. As described above, the coating material 529 comprising the close-packed molecular monolayer may have beneficial functional characteristics for the DEP configured microfluidic device 500, such as minimal charge trapping, reduced physical/electrical thickness, and a substantially uniform surface.

In another particular embodiment, coating material 529 can include a fluoroalkyl group (e.g., fluoroalkyl or perfluoroalkyl) at its end facing the enclosure (i.e., the portion of the monolayer of coating material 529 that is not bonded to the interior surfaces 508, 509 and is proximate to the enclosure 502). As described above, coating material 529 may include a fluoroalkyl terminated siloxane or fluoroalkyl terminated phosphonate monolayer, where the fluoroalkyl groups are present at the end of coating material 529 that faces the enclosure. Such coating material 529 provides the functional benefit of improved T cell culture and expansion by separating or "shielding" the T cells from non-biological molecules (e.g., silicon and/or silicon oxide of the substrate).

In other particular embodiments, the coating material 529 used to coat the interior surfaces 508, 509 of the microfluidic device 500 may include anionic, cationic, or zwitterionic moieties, or any combination thereof. Without wishing to be bound by theory, by providing cationic, anionic, and/or zwitterionic moieties on the interior surface of the housing 502 of the microfluidic circuit 500, the coating material 529 can form strong hydrogen bonds with water molecules, such that the resulting hydrated water acts as a layer (or "shield") separating the core (nucleoei) from interactions with non-biological molecules (e.g., silicon and/or silicon oxide of the substrate). Additionally, in embodiments where coating material 529 is used in combination with a blocking agent, the anions, cations, and/or zwitterions of coating material 529 can form ionic bonds with charged portions of the blocking agent (e.g., a protein in solution) in medium 180 (e.g., a blocking solution) present in housing 502.

In another specific embodiment, the coating material may comprise or be chemically modified to provide a hydrophilic coating agent at its end facing the housing. In some embodiments, the coating agent may be an alkylene ether containing polymer, such as PEG. In some embodiments, the coating agent may be a polysaccharide, such as dextran. As with the charged moieties discussed above (e.g., anionic, cationic, and zwitterionic moieties), the hydrophilic coating agent can form strong hydrogen bonds with water molecules, such that the resulting water of hydration acts as a layer (or "shield") separating the core from interactions with non-biological molecules (e.g., silicon and/or silicon oxide of the substrate).

An enrichment method. The devices disclosed herein can be used to sort T lymphocytes and, for example, provide an enriched population of T lymphocytes, particularly activated T lymphocytes that have a functional response to an antigen of interest. Fig. 11 provides an overview of one such method 1100.

At step 1110, a peripheral blood sample is obtained from the subject. The subject may be a human donor or some other type of animal, such as a mammal (e.g., mouse, rat, guinea pig, rabbit, sheep, pig, cow, horse, primate, etc.), avian, reptile, amphibian, etc. The subject may be healthy, or the subject may have a disorder. For example, for human subjects, the condition can be caused by a pathogenic organism, such as a bacterial pathogen, a fungal pathogen, a parasitic pathogen, or a viral pathogen. Alternatively, the condition may be a form of cancer. For non-human animals, the animal can have any of the foregoing conditions (i.e., infection by a pathogen or cancer), and/or the animal can have a condition that is a model of a corresponding human condition.

Peripheral blood samples can be processed by leukapheresis to obtain Peripheral Blood Mononuclear Cells (PBMCs). PBMCs can be washed and frozen for later use. Alternatively, PBMCs may be washed and immediately further processed.

At step 1120, CD8 may be isolated from PBMCs ⁺ T lymphocytes. A number of different commercial kits are available for the isolation of CD8 from PBMCs ⁺ T lymphocytes. Examples include bead-based purification kits, e.g. EasySep ^TM Human CD8 ⁺ T Cell enrichment kit (Stem Cell Technologies) and EasySep ^TM Human Naive CD8 ⁺ T is thinCell enrichment kits (Stem Cell Technologies). All CDs 8 may be selected according to whether they are needed ⁺ T cells or only certain subpopulations (e.g., unimmunized CD 8) ⁺ T cells) to select different kits. In some embodiments, the CD8 is not immunized ⁺ T cells may provide good starting material for step 1130 (antigen-specific activation).

As an alternative to bead-based purification, FACS cell sorting can be used to obtain CD8 ⁺ T cells and optionally CD8 ⁺ Non-immunized T cells. For example, fluorescently labeled anti-CD 8 antibodies can be used for FACS sorting. As will be appreciated by those skilled in the art, many different antibodies and antibody combinations may be used to obtain the desired CD8 ⁺ A population of T cells. For example, a combination of anti-CD 45RO (for negative selection) and anti-CCR 7 antibody (for positive selection) antibodies can be used to isolate CD8 ⁺ A population of non-immunized T cells. In addition, anti-CD 45RA and/or anti-CD 62L antibodies may be used. In place of CD8 ⁺ Non-immunized T cells, central memory T cells (T) can be purified using the same antibodies described above _CM ) Although different (e.g., anti-CD 45RO for positive selection, anti-CD 45RA for negative selection, anti-CCR 7 for positive selection, etc.) were used.

At step 1130, the CD8+ T cell sample is contacted with a known antigen to stimulate antigen-specific activation. It is known that antigens may be part of artificial antigen presenting cells (aapcs). Aapcs can be designed to present MHC class I molecules bound to antigenic peptides. MHC class I molecules can be linked as tetramers, as described in U.S. patent No. 5,635,363, which is incorporated herein by reference in its entirety. For example, aapcs are described in PCT applications WO2013/086500 published on day 13, 6, 2013, WO2014/160132 published on day 2, 10, 2014, and WO2016/044530 published on day 24, 3, 2016, the entire contents of which are incorporated herein by reference. Contacted CD8 ⁺ The number of T cells may be about 1X 10 ⁵ To about 1X 10 ⁷ (e.g., about 5X 10) ⁵ To about 5X 10 ⁶ Or about 1X 10 ⁶ )。aAPC:CD8 ⁺ The ratio of T cells may vary. For example, the ratio may be 5:1, 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, or 1: 5.

The incubation can be performed, for example, in the wells of a microtiter plate. The length of incubation can be at least about 12 hours (e.g., about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 108 hours, about 120 hours, or any range defined by the foregoing two values). The incubation can be carried out in T cell culture medium, examples of which are well known in the art. See, e.g., Ho et al (2006), J.immunological Methods 310:40-52 and International application No. PCT/US17/22846 filed 3, 16.2017. Cell culture media can be supplemented with a CD28 agonist (e.g., 2 micrograms/mL of anti-CD 28 agonist antibody) or a CD28 agonist can bind to aapcs. The culture can be maintained under standard conditions (e.g., 5% CO at 37 ℃) ₂ Below).

As an alternative to using aapcs, DCs that have been challenged with an antigenic peptide containing the antigen of interest may be used. For example, the use of DC is described in International application No. PCT/US17/22846, filed 3, 16, 2017.

Many different antigenic peptides are known in the art. Examples include: the M27L peptide of Melan-a (Ho et al (2006), supra); WT1 of Wilms tumor protein ₁₂₆ Peptides (also described in Ho et al (2006)); and NY-ESO-1(Pollack et al (2014), J.Immunother Cancer 2: 36). The choice of peptide may depend on the type of MHC class I molecule present on the aapcs or in the DCs.

At step 1140, CD8 that was expected to have been contacted with the activator at step 1130 ⁺ The T lymphocyte sample comprises a number of antigen-specific expanded activated T cells. Such T cells can be selectively enriched by flowing the sample through a microfluidic device having a column array configured to separate activated T cells from resting/unimmunized T cells. The microfluidic device (and the array of pillars contained therein) may be as described in various embodiments herein. Accordingly, the microfluidic device may have a configuration generally as shown in microfluidic device 700 in fig. 7A. For example, the pillar array may have a predicted critical dimension (Dc) of about 6 microns, the tilt angle e is 1/12 radians, the gap between pillars in the same column is 25 microns, and the pillars are triangular and have a shape with About 50 microns in diameter. Alternatively, the microfluidic device may have a configuration generally as shown in microfluidic device 715 in fig. 7B. The pillar array may have a predicted critical dimension (D) of about 6 microns _c ) The tilt angle epsilon is 1/12 radians, the gap between pillars in the same column is 25 microns, the pillars are triangular and have a diameter of about 50 microns, and the microfluidic device comprises a DEP configuration.

The buffer can be flowed through the device rapidly (e.g., 10 to 100 microliters/second) to eliminate air bubbles before flowing the sample through the microfluidic device. Next, the contacted/activated T cell sample from step 1140 can be flowed through the column array of the microfluidic device at a rate of about 1.0 to about 10 microliters/second or using a pressure of about 10psi to about 30psi (e.g., about 20 psi).

Prior to step 1140, the incubated sample obtained from step 1130 can be labeled to identify individual T cells that specifically bind to the antigen. For example, the sample from step 1130 may be contacted with a fluorescently labeled soluble MHC class I tetramer that is bound to an antigenic peptide (as appropriate) to facilitate labeling and identification of antigen-specific T lymphocytes. Such labeling and identification can be used to select individual T lymphocytes for movement into isolated docks 725 on a microfluidic device (e.g., device 715 of fig. 7B), and subsequent cloning. Alternatively, after cloning individual T lymphocytes in the sequestration dock 725, fluorescently labeled soluble MHC class I tetramers bound to an antigenic peptide (as appropriate) can flow into the sorting channel 720 of the microfluidic device 715 and allowed to diffuse into the sequestration dock 725, whereby antigen-specific T lymphocyte clones can be labeled and identified. Such markers and identifications can be used to select T lymphocyte clones for export and subsequent analysis, as described below.

At step 1150, the enriched sample of activated CD8+ T lymphocytes may optionally be expanded within a microfluidic device. For example, after a sample passes through an array of pillars in a microfluidic device, the flow of fluid through the device may be stopped. If the microfluidic device includes a sequestration dock, such as shown in microfluidic device 715 of fig. 7B, individual T lymphocytes may be selected and moved to the corresponding sequestration dock, and the isolated T lymphocytes may be grown into a clonal population of cells. Cloning of activated T lymphocytes in a microfluidic device in this manner has been described, for example, in the above-mentioned international application No. PCT/US17/22846, filed 3, 16, 2017.

At step 1160, T cells activated by contact with an activating agent may be output from the microfluidic device. For devices such as the microfluidic device 700 of fig. 7A, this output occurs immediately after sorting, and activated T cells are collected from the sorted outlet 708. For a device such as microfluidic device 715 of fig. 7B, T cell clones that successfully expanded in isolation dock 725 may move back into channel 720 (e.g., the second channel) and use the fluid flow output through outlet 708/710. The movement of the activated T cell clones away from the dock can be accomplished, for example, using DEP forces, which can be optically driven, for example, with OEP.

Regardless of the exact configuration and output time of the microfluidic device, activated T cells can be collected and, optionally, further expanded upon exiting the chip or tested in various assays. For example, selecting T cells may be subjected to TCR sequencing to identify antigen-specific TCRs. Alternatively, an enriched sample of activated CD8+ T lymphocytes collected from the outlet of the microfluidic device 700 may be expanded using a Rapid Expansion Protocol (REP) prior to being introduced into a patient with melanoma. REP is known in the art. See, e.g., Ho et al (2006) as described above.

Examples

Example 1: column array based activation of human T lymphocytes and resting human T lymphocytes in microfluidic devices Separation of

By contact with anti-CD 3/anti-CD 28 magnetic beads (DYNABEADS) ^TM Thermo Fisher Scientific, Inc.) was mixed at a ratio of 1 bead/1 cell to activate CD3 isolated from peripheral blood ⁺ Human T lymphocytes. Mixing the mixture in 5% CO ₂ Incubate at 37 ℃ for 5 hours in an incubator. After incubation, the activated T cell/bead mixture was resuspended and applied to a CellTracker ^TM Fluorescent markers (Thermo Fisher Scientific, Inc.). The labeled T cells are then flowed through a microfluidic device having an array of pillars with about 9 microns Predicted critical dimension of meter (D) _c ) The flow rate was about 0.1. mu.l/sec. The pillar array is characterized by an inclination angle of 1/15 radians with 30 micron gaps between pillars in the same column. The pillars have a circular shape with a diameter of about 50 microns.

The labeled T cells are imaged as they flow through the column array. As shown in fig. 8, activated T cells having a "larger" size pass through the column array in the "sorting direction" of the array (i.e., generally along the axis defined by the rows of columns), while activated T cells having a "smaller" size pass through the column array in the direction of fluid flow through the array (i.e., generally along the direction of the flow path defined by the region of the microfluidic device containing the column array). Resting T cells also typically pass through the column array in the direction of fluid flow through the array.

This experiment demonstrates that T lymphocytes are of different sizes and can be sorted based on this size difference by flowing them through an appropriately configured array of pillars.

Example 2: activated human T lymphocytes following treatment of mixed populations of activated and resting T lymphocytes in a microfluidic device Enrichment of cells

Generally as shown in example 1, by interaction with anti-CD 3/anti-CD 28 magnetic beads (DYNABEADS) ^TM Thermo Fisher Scientific, Inc.) was mixed at a ratio of 1 bead/1 cell to activate CD3 isolated from peripheral blood ⁺ Human T lymphocytes. After incubation, the activated population of T lymphocytes was resuspended and labeled with red fluorescent CellTracker ^TM Reagents (Thermo Fisher Scientific, Inc.) were labeled. At the same time, CellTracker with green fluorescence label is used ^TM The reagents label an inactive population of CD3+ T lymphocytes isolated from peripheral blood (i.e., a "resting" population). The activated population of T lymphocytes is then mixed with the resting population of T lymphocytes to produce a density of about 1.2 x 10 ⁶ Individual cells/mL of T lymphocyte mixture, wherein about 5% of the T lymphocytes are from the activated population.

Flowing 400 microliters of a T lymphocyte mixture through a microfluidic device having a configuration generally as shown in microfluidic device 700 in figure 7A,there are two inlets 702/704, a column array 706 located in a first region of the flow path of the device, and two outlets 708/710. The T lymphocyte mixture is flowed into the sample inlet 702 while a buffer (DPBS, 5mM EDTA, 10mM Hepes, 2% FBS) is flowed into the second inlet 704. A lymphocyte mixture, supplied with a pressure of 28psi from a pressurized reservoir, and a buffer, supplied with a pressure of 30psi from a pressurized reservoir, were co-flowed through the microfluidic device. Predicted critical dimension (D) of pillar array _c ) About 5 microns, with an inclination angle e of 1/12 radians, the gap between pillars in the same column is 17.5 microns, and the pillars are diamond shaped and about 70 microns in diameter. The processed cell sample is collected from a collection outlet 708 ("sorted sample") and a waste outlet 710 ("waste sample").

In BD FACSAria ^TM A portion of the starting T lymphocyte mixture and each sorted and waste sample were analyzed on a cell sorter (Becton Dickinson). As shown in fig. 9A, forward scatter analysis of the starting T lymphocyte mixture identified two major peaks, one representing smaller resting T lymphocytes, and not all the second representing larger activated T lymphocytes. Based on CellTracker ^TM Green/CellTracker ^TM Red-labeled assay samples (fig. 9B-9D) also identified two major types of cells. As expected, in the starting T lymphocyte mixture (fig. 9B), 93.5% of the cells were identified as originating from the resting T lymphocyte population (i.e., green) ⁺⁺ Red color ^- ) And 4.9% of the cells were identified as originating from the activated T lymphocyte population (i.e., green) ^- Red color ⁺⁺ ). The waste sample was similar to the starting T lymphocyte sample, although some cells were depleted from the activated T lymphocyte population (fig. 9C), 97.1% of the cells were identified as originating from the resting T lymphocyte population and 1.54% of the cells were identified as originating from the activated T lymphocyte population. In contrast, in the sorted population (fig. 9D), 1.06% of the cells were identified as originating from the resting T lymphocyte population and 97.9% of the cells were identified as originating from the activated T lymphocyte population. A total of 8738 cells in the sorted sample were identified as originating from the activated T lymphocyte population, corresponding to a yield of 59% and an enrichment of 914%. In this embodiment of the present invention, The enrichment is calculated as (N) ⁺ _{Leave from} /N ^- _{Leave from} )/(N ⁺ _{Enter into} /N ^- _{Enter into} ) In which N is ⁺ _{Leave from} Is the number of activated T lymphocytes detected in the sorted sample, N ^- _{Leave from} Is the number of resting T lymphocytes detected in the sorted sample, N ⁺ _{Enter into} Is the number of activated T lymphocytes detected in the starting mixture, and N ^- _{Enter into} Is the number of resting lymphocytes detected in the starting mixture.

This experiment demonstrates that the disclosed microfluidic device is capable of processing a mixture of activated and quiescent T lymphocytes, thereby producing a cell population substantially enriched for activated T lymphocytes. Many variants of the column array used to produce these results can be produced, having a critical diameter Dc of about 6 microns, and any such variant is expected to produce an enriched sample of activated T lymphocytes substantially as shown above.

Example 3: activated in microfluidic devices with bypass channels and sorting channels characterised by an isolating dock Enrichment of human T lymphocytes

Generally as shown in example 1, by interaction with anti-CD 3/anti-CD 28 magnetic beads (DYNABEADS) ^TM Thermo Fisher Scientific, Inc.) was mixed at a ratio of 1 bead/1 cell to activate CD3 isolated from peripheral blood ⁺ Human T lymphocytes. After incubation, the activated population of T lymphocytes was resuspended and labeled with red fluorescent CellTracker ^TM Reagents (Thermo Fisher Scientific, Inc.) were labeled. At the same time, CellTracker with green fluorescence label is used ^TM The reagents label an inactive population of CD3+ T lymphocytes isolated from peripheral blood (i.e., a "resting" population). The activated population of T lymphocytes is then mixed with the resting population of T lymphocytes to produce a density of about 1.0X 10 ⁶ Individual cells/mL of T lymphocyte mixture, wherein about 50% of the T lymphocytes are from the activated population.

Flowing a T lymphocyte mixture through a microfluidic device having a structure generally asThe configuration shown for microfluidic device 715 in fig. 7B has a single inlet 702, an array of pillars 706 located in a first region of the main channel of the device, a first channel 730 that serves as a bypass channel, a second channel 720 that serves as a sorting channel 720, and a single outlet 708/710 located immediately downstream of where the first and second channels 730, 720 join together. The plurality of isolation docks 725 (see fig. 10) have connection areas that lead to the second channel. The T lymphocyte mixture flows into the sample inlet 702 and through the column array 706 at a rate of about 1.0 microliter/second. Predicted critical dimension (D) of pillar array _c ) About 6 microns, with an inclination angle e of 1/12 radians, the gap between pillars in the same column being 25 microns, and the pillars being triangular and about 50 microns in diameter (in this case, defined as 50 microns high and 50 microns low).

After the T lymphocyte mixture was flowed through the column array 706, the flow rate was reduced to zero, and an image of the microfluidic chip as shown in fig. 10 was taken. Analysis of the images showed that the density of activated T lymphocytes (stained red) in the second channel 720 was about 4.1X 10 ⁶ Individual cells/mL +/-2.6%. Since the starting mixture has a size of about 5X 10 ⁵ Activated T lymphocyte density of individual cells/mL, which represents about 8-fold enrichment. In this example, enrichment is calculated as P ⁺ _{Leave from} /P ⁺ _{Enter into} In which P is ⁺ _{Leave from} Is the concentration of activated T lymphocytes detected in the second channel, P ⁺ _{Enter into} Is the concentration of activated T lymphocytes detected in the starting mixture.

Example 4: antigen-specific activation of human T lymphocytes followed by enrichment in microfluidic devices

Step 1: peripheral blood samples were obtained from healthy human donors and Peripheral Blood Mononuclear Cells (PBMCs) were harvested from the samples by leukapheresis. PBMCs can be washed and frozen for later use, or processed immediately.

Step 2: using EasySep ^TM Separation of CD8 from PBMCs by Human CD8+ T Cell enrichment kit (Stem Cell Technologies) ⁺ T lymphocytes.

And step 3: tong (Chinese character of 'tong')Over 5X 10 ⁶ Individual cell/mL CD8 ⁺ T lymphocytes were contacted with artificial antigen presenting cells (aAPCs) presenting MHC class I tetramers bound to the M27L peptide of Melan-A at a ratio of 1T cell to l aAPCs to activate 5X 10 in an antigen-specific manner ⁶ Individual cell/mL CD8 ⁺ T lymphocytes. For example, aapcs are described in PCT applications WO2013/086500 published on 6/13/2013, WO2014/160132 published on 10/2/2014, and WO2016 published on 24/3/044530, which are incorporated herein by reference in their entirety. Tetramers of MHC class I molecules are described in U.S. patent No. 5,635,363, which is incorporated herein by reference in its entirety. For example, the M27L peptide, which is a tumor antigen associated with melanoma, has been described in Ho et al (2006), Journal of Immunological Methods 310:40-52, the entire contents of which are incorporated herein by reference.

Contacting CD8 in T cell culture medium ⁺ T lymphocytes, T cell culture medium containing 2ug/mL soluble functional anti-CD 28 antibody (clone 15E8, Miltenyi 130-093-375). Various T cell culture media are known in the art. See, for example, Ho et al (2006) cited above and International application number PCT/US17/22846, filed 3, 16, 2017. The contacting step is 5% CO off-chip ₂ Incubators were performed in 96-well plates at 37 ℃ for 3 to 4 days.

And 4, step 4: the already activated CD8+ T lymphocytes are expected to be expanded, and thus the already activated CD8 is selectively enriched by flowing the sample obtained at the end of the incubation in step 3 through a microfluidic device ⁺ T lymphocytes, the microfluidic device having a configuration generally as shown in microfluidic device 700 in fig. 7A. Predicted critical dimension (D) of pillar array _c ) About 6 microns, with an inclination angle of e 1/12 radians, the gap between pillars in the same column is 25 microns, and the pillars are triangular and about 50 microns in diameter (as described in example 3 above). The incubated sample from step 3 flows through the column array of the microfluidic device at a rate of about 1.0 to about 10 microliters/second.

And 5: an enriched sample of activated CD8+ T lymphocytes is collected from the outlet of the microfluidic device, at which time CD8+ T lymphocytes can be further expanded off-chip or tested in various immunological assays.

Variations that may be incorporated into the foregoing methods include:

in step 1, peripheral blood may be obtained from human donors suffering from melanoma, or from human donors suffering from different corresponding cancers depending on the antigenic peptide used in step 3.

In step 2, EasySep may be used ^TM Human Naive CD8 ⁺ T Cell enrichment kit (Stem Cell Technologies) for isolation of CD8 from PBMC ⁺ Non-immunized T lymphocytes.

In step 2, CD8 can be isolated from PBMC using FACS in combination with anti-CD 45RO antibody (for negative selection) and anti-CCR 7 antibody (for positive selection) ⁺ Non-immunized T lymphocytes; can be used for positive selection of CD8 ⁺ Other antibodies to non-immunized T lymphocytes include anti-CD 45RA and/or anti-CD 62L antibodies.

In step 2, CD8+ T lymphocytes may be isolated from PBMCs using FACS and an anti-CD 8 antibody.

In step 3, aAPC presents WT1 in association with Wilms' tumor protein ₁₂₆ Peptide-bound MHC class I tetramers, which are widely expressed in a broad spectrum of leukemias, lymphomas, and solid tumors. WT1 was used in step 3 ₁₂₆ The peptide can be bound to the peripheral blood of human donors obtained in step 1 with any Wilms tumor-associated cancer.

In step 3, the anti-CD 28 antibody may be conjugated to the aapcs, rather than being provided in culture.

In step 3, the aAPC can be replaced with Dendritic Cells (DCs) that have been challenged with an antigenic tumor-associated peptide, which can be the M27L peptide of Melan-A, WT1 of Wilms tumor protein ₁₂₆ A peptide or some other tumor-associated peptide. Methods of making such DCs and their use in stimulating CD8+ T lymphocytes are known in the art. See, for example, Ho et al (2006) and International application number PCT/US17/22846, filed 3/16.2017, both of which are mentioned above.

At step 4, the sample obtained after the incubation of step 3 is flowed through a column array of a microfluidic device having a microfluidic device 715 generally as in fig. 7BThe configuration shown. The pillar array has a predicted critical dimension (D) of about 6 microns _c ) With an inclination angle of 1/12 radians, the gap between pillars in the same column is 25 microns, and the pillars are triangular and about 50 microns in diameter (as described in example 3 above), the microfluidic device comprises a DEP configuration. The incubated sample of step 3 was flowed through the column array at a rate of about 0.1 microliters/second until the sorting channel 720 was filled with sorted T lymphocytes, at which time the flow was stopped. Individual T lymphocytes are selected and transferred to the corresponding sequestration dock 725 whereby the isolated T lymphocytes are cultured as a clonal population of cells. Cloning of activated T lymphocytes in a microfluidic device in this manner has been described, for example, in the above-mentioned international application No. PCT/US17/22846 filed 3, 16, 2017. After cloning, one or more cells from the selected T cell clone can be exported from the microfluidic device for subsequent analysis, which can include TCR sequencing to identify antigen-specific TCRs.

Just prior to step 4, the incubated sample of step 3 may be contacted with a fluorescently labeled soluble MHC class I tetramer bound to M27L peptide of Melan-a (or other antigenic peptide, as appropriate) to facilitate labeling and identification of antigen-specific T lymphocytes in the sorting channel 720 of the microfluidic device 715. Such labeling and identification can be used to select individual T lymphocytes for movement into the sequestration dock 725 and subsequent cloning.

Alternatively, after cloning of individual T lymphocytes in the isolation dock 725, fluorescently labeled soluble MHC class I tetramers bound to the M27L peptide of Melan-a (or other antigenic peptide, as appropriate) can be flowed into the sorting channel 720 of the microfluidic device 715 and allowed to diffuse into the isolation dock 725, whereby antigen-specific T lymphocyte clones can be labeled and identified. As described above, such labeling and identification can be used to select T lymphocyte clones for export and subsequent analysis.

At step 5, an enriched sample of activated CD8+ T lymphocytes collected from the outlet of the microfluidic device 700 can be used to treat a patient having melanoma. Such a change may be effected by a change of step 1, wherein the subject suffering from melanoma is a peripheral blood donor. The subject as a donor of peripheral blood may also be livingMethylated CD8 ⁺ An enriched sample of T lymphocytes treated patient. Optionally, the enriched sample of activated CD8+ T lymphocytes collected from the outlet of the microfluidic device 700 may be expanded using a Rapid Expansion Protocol (REP) prior to being introduced into a patient with melanoma. REP is known in the art. See, e.g., Ho et al (2006) cited above.

List of embodiments

Embodiment 1 a method of producing a sample enriched for activated T lymphocytes using a microfluidic device comprising a flow path having a first region comprising a first array of pillars, the method comprising: flowing a fluid sample comprising a mixture of activated and resting T lymphocytes through a first region of a flow path of a microfluidic device, wherein: the direction of fluid flow in the first region of the flow path defines a first direction; the pillars in the first array are arranged in rows and columns; the rows of pillars in the first array define a first array direction which differs from the first direction of the first area by an inclination angle (epsilon), and the pillars in the first array repeat periodically with a period equal to l/epsilon, where epsilon is measured in radians; adjacent pillars in each respective column in the first array are separated by a gap through which a fluid of the fluid sample can flow laterally with respect to the columns as a whole, wherein a majority of the gaps have a characteristic dimension corresponding to a major gap dimension of the first array, and the first array is characterized by a critical dimension (D) _c ) From about 4 microns to about 10 microns.

Embodiment 2. the method of embodiment 1, wherein the first region of the flow path is defined by a main channel having a width, and wherein the first array of pillars extends across the entire width of the main channel.

Embodiment 3. the method of embodiment 1 or 2, wherein the first array is characterized by D _c From about 4 microns to about 5 microns, from about 4.5 microns to about 5.5 microns, from about 5 microns to about 6 microns, from about 5.5 microns to about 6.5 microns, from about 6 microns to about 7 microns, from about 6.5 microns to about 7.5 microns, from about 7 microns to about 8 microns, from about 7.5 microns to about 8.5 microns, from about 8 microns to about 9 microns, from about 8.5 microns to about 9.5 microns, from about 9 microns to about 9 micronsAbout 10 microns, or any range defined by the two aforementioned endpoints.

Embodiment 4. the method of embodiment 1 or 2, wherein the first array is characterized by D _c From about 4 microns to about 7 microns.

Embodiment 5. the method of embodiment 1 or 2, wherein the first array is characterized by D _c From about 7 microns to about 10 microns.

Embodiment 6. the method of embodiment 1 or 5, wherein the first array has an inclination angle, epsilon, of about 1/3 radians to about 1/100 radians.

Embodiment 7 the method of any one of embodiments 1 to 5, wherein the tilt angle e of the first array is from about 1/5 radians to about 1/30 radians.

Embodiment 8 the method of any one of embodiments 1 to 5, wherein the tilt angle e of the first array is from about 1/10 radians to about 1/16 radians.

Embodiment 9 the method of any one of embodiments 1 to 8, wherein the primary gap dimension of the first array is about 15 microns to about 25 microns.

Embodiment 10 the method of any one of embodiments 1 to 8, wherein the primary gap dimension of the first array is about 25 microns to about 40 microns.

Embodiment 11 the method of any one of embodiments 1 to 10, wherein the first array of pillars has a diameter of about 30 microns to about 100 microns (e.g., about 40 microns to about 85 microns, or about 50 microns to about 70 microns).

Embodiment 12. the method of embodiment 10 or 11, wherein the diameter of the pillars of the first array is larger (e.g., 1.5 to 5 times larger) than the major gap dimension.

Embodiment 13 the method of embodiment 10 or 11, wherein the posts of the first array have a diameter two to four times greater than the major gap dimension.

Embodiment 14 the method of any one of embodiments 1 to 13, wherein the columns of the first array are arranged laterally with respect to the first direction of the first region.

Embodiment 15 the method of any one of embodiments 1 to 14, wherein the cross-section of the pillars of the first array is circular (e.g., circular or oval).

Embodiment 16 the method of any one of embodiments 1 to 14, wherein the cross-section of the pillars of the first array is polygonal (e.g., triangular, square, diamond, or parallelogram shaped).

Embodiment 17 the method of embodiment 15 or 16, wherein the pillars of the first array all have the same orientation, and wherein the orientation is such that no axis of symmetry in the cross-sectional shape of the pillars is parallel to the axis defined by the first direction.

Embodiment 18 the method of any one of embodiments 1 to 17, wherein the first array of pillars comprises a siloxane polymer.

Embodiment 19 the method of any one of embodiments 1-18, wherein the first region of the flow path comprises a first sidewall and a second sidewall that together define the first direction, wherein all gaps between adjacent pillars in a column of the first array are equal to a major gap dimension of the first array, except that a gap dimension between adjacent pillars of the same column that are closest to the first or second sidewall may deviate from the major gap dimension, and wherein the deviation in the gap dimension between the pillars in the first array reduces boundary irregularities otherwise caused by the first and second sidewalls as the fluid sample flows through the first array.

Embodiment 20 the method of any one of embodiments 1 to 19, wherein the flow path of the microfluidic device comprises a second region configured to receive the fluid sample after the fluid sample passes through the first region of the microfluidic device, the second region having a divider that divides the second region into a first channel that receives a first portion of the fluid sample and a second channel that receives a second portion of the fluid sample, and wherein the divider of the second region is positioned such that relative to the fluid sample, a diameter greater than D is enriched in the second portion of the fluid sample _c The T lymphocyte of (1).

Embodiment 21 the method of embodiment 20, wherein the diameter is less than D _c The T lymphocytes of (2) are predominantly located in the fluid sampleIn the first section.

Embodiment 22 the method of embodiment 20 or 21, wherein the first portion of the fluid sample comprises at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) of the fluid sample.

Embodiment 23 the method of embodiment 20 or 21, wherein the first portion of the fluid sample comprises about 85% to about 95% of the fluid sample.

Embodiment 24 the method of any one of embodiments 20 to 23, wherein the first and second channels are configured such that the pressure differential across the first channel is equal to the pressure differential across the second channel.

Embodiment 25 the method of any one of embodiments 20 to 24, wherein the first channel comprises a first length and the second channel comprises a second length, and wherein the second length is greater than the first length (e.g., wherein the second length of the second channel is at least 5 times longer than the first length of the first channel).

Embodiment 26 the method of any one of embodiments 20 to 25, wherein the microfluidic device comprises at least one isolation dock having a connection region with a proximal opening to the second channel, and further wherein the at least one isolation dock has an isolation region with a volume large enough to accommodate at least one T lymphocyte (e.g., a plurality of T lymphocytes).

Embodiment 27. the method of embodiment 26, wherein the microfluidic device comprises a plurality of isolation docks, each isolation dock having a connection region with a proximal opening to the second channel of the second region, and each isolation dock having an isolation region with a volume large enough to accommodate at least one T lymphocyte (e.g., a plurality of T lymphocytes).

Embodiment 28 the method of embodiment 26 or 27, wherein the volume of the isolation dock or each isolation dock of the plurality of isolation docks is about 250pL to about 3nL (e.g., about 250pL to about 750pL, about 400pL to about 900pL, about 500 μ L to about 1.5nL, about 1nL to about 2nL, about 1.5nL to about 2.5nL, about 2nL to about 3nL, or any range defined by the two aforementioned endpoints).

Embodiment 29 the method of any one of embodiments 20 to 28, wherein there is CD8 ⁺ 、CD45RO ⁺ /RA ^- 、CCR7 ^- 、CD62L ^- The T lymphocytes of the phenotype are enriched in a second portion of the fluid sample.

Embodiment 30 the method of any one of embodiments 20 to 28, wherein there is CD8 ⁺ 、CD45RO ⁺ /RA ^- 、CCR7 ⁺ 、CD62L ^- The T lymphocytes of the phenotype are enriched in a second portion of the fluid sample.

Embodiment 31 the method of any one of embodiments 1 to 30, wherein the length of the first region is about 5mm to about 15 mm.

Embodiment 32 the method of any one of embodiments 1 to 31, wherein the rate of fluid sample flow through the first region of the flow path is about 0.01 microliters/second to about 10 microliters/second (e.g., about 0.001 to about 0.01 microliters/second, about 0.005 to about 0.05 microliters/second, about 0.01 to about 0.1 microliters/second, about 0.05 to about 0.5 microliters/second, about 0.1 to about 1.0 microliters/second, about 0.5 to about 5 microliters/second, about 1.0 to about 10 microliters/second, about 5 to about 50 microliters/second, about 10 to about 100 microliters/second, about 15 to about 50 microliters/second, about 25 to about 75 microliters/second, about 50 to about 100 microliters/second, or any range defined by two of the foregoing endpoints).

Embodiment 33 the method of any one of embodiments 20 to 32, wherein the second channel comprises a first subregion comprising a second column array, wherein flowing the fluid sample through the first region of the flow path causes a second portion of the fluid sample, along with any cells contained therein, to flow through the second column array in the first subregion, and further wherein: the direction of fluid flow in the first sub-region of the second channel defines a second direction; the pillars in the second array are arranged in rows and columns; the rows of pillars in the second array define a second array direction, the second array direction differing from the second direction by an inclination angle (ε '), and the columns of pillars in the second array repeat periodically with a period equal to 1/ε ', where ε ' is measured in radians; in the second array Adjacent columns in each respective column are separated by a gap through which a second portion of the fluid sample can flow laterally with respect to the columns as a whole, wherein a majority of the gaps have a characteristic dimension corresponding to a minor gap dimension of the second array, and the second array is characterized by a critical dimension (D) _c ) From about 4 microns to about 10 microns.

Embodiment 34 the method of embodiment 33, wherein the second channel has a width, and wherein the second column array extends across the width of the second channel.

Embodiment 35 the method of embodiment 33 or 34, wherein the second array is characterized by D _c From about 4 microns to about 5 microns, from about 4.5 microns to about 5.5 microns, from about 5 microns to about 6 microns, from about 5.5 microns to about 6.5 microns, from about 6 microns to about 7 microns, from about 6.5 microns to about 7.5 microns, from about 7 microns to about 8 microns, from about 7.5 microns to about 8.5 microns, from about 8 microns to about 9 microns, from about 8.5 microns to about 9.5 microns, from about 9 microns to about 10 microns, or any range defined by both of the foregoing endpoints.

Embodiment 36 the method of embodiment 35, wherein the second column array is characterized by D _c From about 4 microns to about 7 microns.

Embodiment 37 the method of embodiment 35, wherein the second column array is characterized by D _c From about 7 microns to about 10 microns.

Embodiment 38 the method of any one of embodiments 33 to 37, wherein the second array has an inclination angle e of about 1/3 radians to about 1/100 radians.

Embodiment 39 the method of any one of embodiments 33 to 37, wherein the inclination angle e' of the second array is from about 1/5 radians to about 1/30 radians.

Embodiment 40 the method of any one of embodiments 33 to 37, wherein the inclination angle e' of the second array is from about 1/10 radians to about 1/16 radians.

Embodiment 41 the method of any one of embodiments 33 to 40, wherein the secondary gap size of the second array is about 15 microns to about 25 microns.

Embodiment 42 the method of any one of embodiments 33 to 40, wherein the secondary gap size of the second array is about 25 microns to about 40 microns.

Embodiment 43 the method of any one of embodiments 33 to 42, wherein the second array of pillars has a diameter of about 30 microns to about 100 microns (e.g., about 40 microns to about 85 microns or about 50 microns to about 70 microns).

Embodiment 44 the method of embodiment 41 or 42, wherein the diameter of the pillars of the second array is greater (e.g., 1.5 to 5 times greater) than the minor gap dimension.

Embodiment 45 the method of embodiment 41 or 42, wherein the diameter of the pillars of the second array is two to four times larger than the minor gap dimension.

Embodiment 46 the method of any one of embodiments 33 to 45, wherein the columns of the second array are arranged laterally with respect to the second direction of the first sub-regions of the second channels.

Embodiment 47 the method of any one of embodiments 33 to 46, wherein the cross-section of the pillars of the second array is circular (e.g., circular or oval).

Embodiment 48 the method of any one of embodiments 33 to 46, wherein the cross-section of the pillars of the second array is polygonal (e.g., triangular, square, diamond, or parallelogram shaped).

Embodiment 49 the method of embodiment 47 or 48, wherein the pillars of the second array all have the same orientation, and wherein the orientation is such that no axis of symmetry in the cross-sectional shape of the pillars is parallel to the axis defined by the second direction.

Embodiment 50 the method of any one of embodiments 33 to 49, wherein the second array of pillars comprises a siloxane polymer.

Embodiment 51 the method of any one of embodiments 33 to 50, wherein the first subregion of the second channel comprises a third sidewall and a fourth sidewall that together define the second direction, wherein all gaps between adjacent pillars in a column of the second array are equal to the minor gap dimension of the second array, except that the gap dimension between adjacent pillars of the same column that are closest to the third or fourth sidewall may deviate from the minor gap dimension, and wherein the deviation in the gap dimension between the pillars in the second array reduces boundary irregularities otherwise caused by the third and fourth sidewalls as the second portion of the fluid sample flows through the second array.

Embodiment 52. the method of any of embodiments 33 to 51, wherein the second channel comprises a second sub-region configured to receive the second portion of the fluid sample after the second portion of the fluid sample passes through the first sub-region, the second sub-region having a divider that divides the second channel into a third channel that receives the first sub-portion of the fluid from the second portion of the fluid sample and a fourth channel that receives the second sub-portion of the fluid from the second portion of the fluid sample, and wherein the divider of the second sub-region is positioned such that the second sub-portion of the fluid is enriched in the second sub-portion of the fluid with a diameter greater than D relative to the second portion of the fluid sample _c The T lymphocyte of (1).

Embodiment 53 the method of embodiment 52, wherein the diameter is less than D _c Is predominantly located in the first sub-portion of the fluid.

Embodiment 54 the method of embodiment 52, wherein the first sub-portion of the fluid comprises at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more) of the second portion of the fluid sample.

Embodiment 55 the method of embodiment 52, wherein the first sub-portion of the fluid comprises about 85% to about 95% of the second portion of the fluid sample.

Embodiment 56 the method of any one of embodiments 52 to 55, wherein the third and fourth channels are configured such that the pressure differential across the third channel is equal to the pressure differential across the fourth channel.

Embodiment 57 the method of embodiment 56, wherein the third channel comprises a third length and the fourth channel comprises a fourth length, and wherein the fourth length is greater than the third length.

Embodiment 58 the method of embodiment 57, wherein the fourth length is at least 5 times longer than the third length.

Embodiment 59 the method of any one of embodiments 52 to 58, wherein the microfluidic device comprises at least one isolation dock having a connection region with a proximal opening to the third channel or the fourth channel, and further wherein the at least one isolation dock has an isolation region with a volume large enough to accommodate at least one T lymphocyte (e.g., a plurality of T lymphocytes).

Embodiment 60 the method of any one of embodiments 42 to 45, wherein the microfluidic device comprises a plurality of isolation docks, each isolation dock of the plurality of isolation docks having a connection region with a proximal opening to the third channel (or fourth channel) and an isolation region having a volume large enough to accommodate at least one T lymphocyte (e.g., a plurality of T lymphocytes).

Embodiment 61 the method of embodiment 59 or 60, wherein the volume of the isolation dock or each isolation dock of the plurality of isolation docks is about 250pL to about 3nL (e.g., about 250pL to about 750pL, about 400pL to about 900pL, about 500 μ L to about 1.5nL, about 1nL to about 2nL, about 1.5nL to about 2.5nL, about 2nL to about 3nL, or any range defined by the two aforementioned endpoints).

Embodiment 62 the method of any one of embodiments 1 to 61, wherein the fluid sample is a peripheral blood sample obtained from the subject or a sample derived therefrom (e.g., PBMCs).

Embodiment 63 the method of embodiment 62, wherein the fluid sample is a peripheral blood sample that has been depleted of at least one non-T lymphocyte type (e.g., bone marrow cells, such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, megakaryocytes and platelets, B lymphocytes, Natural Killer (NK) cells, stem cells, or any combination thereof).

Embodiment 64 the method of embodiment 62 or 63, wherein the fluid sample is already enriched in CD8 ⁺ Peripheral blood samples of T lymphocytes.

Embodiment 65 the method of embodiment 64, wherein the fluid sample has been depleted of effector T lymphocytes (T) _EFF ) And/or memory T lymphocytes (T) _CM ). (e.g., a cell having CD45RO ⁺ Phenotype, optionally with PD-1 ⁺ 、PD-L1 ⁺ 、CD137 ⁺ Or any combination thereof, or, alternatively, with CCR7 ⁺ And/or CD62L ⁺ In combination).

Embodiment 66. the method of embodiment 64 or 65, wherein the fluid sample has been enriched for non-immunized T lymphocytes (T) _{Is not immunized} ) Or with CD45RA ⁺ Phenotype (optionally with CCR7 ⁺ And/or CD62L ⁺ Combination) of cells.

Embodiment 67. the method of embodiment 64 or 65, wherein the fluid sample has been enriched for central memory T lymphocytes (T) _CM ) Or with CD45RO ⁺ Phenotype and CCR7 ⁺ And/or CD62L ⁺ Cells with a combination of phenotypes.

Embodiment 68 the method of embodiment 62 or 63, further comprising: obtaining a peripheral blood sample or a sample derived therefrom, wherein the peripheral blood is derived from a human subject; and generating a fluid sample from the peripheral blood sample or a sample derived therefrom.

Embodiment 69 the method of embodiment 68, wherein generating a fluid sample comprises: depleting at least one non-T lymphocyte type of the peripheral blood sample or a sample derived therefrom; and/or enriching CD8 of a peripheral blood sample or a sample derived therefrom ⁺ T lymphocytes.

Embodiment 70 the method of embodiment 69, wherein the enriching step comprises enriching the CD8 of the peripheral blood sample or a sample derived therefrom ⁺ Non-immunized T lymphocytes or with CD45RA ⁺ Phenotype (optionally with CCR7 ⁺ And/or CD62L ⁺ Combination) of cells.

Embodiment 71 the method of any one of embodiments 1 to 61, wherein the fluid sample comprises cells isolated from a solid tumor sample of the subject.

Embodiment 72 the method of embodiment 71, wherein the solid tumor sample is Fine Needle Aspirate (FNA).

Embodiment 73 the method of embodiment 71, wherein the solid tumor sample is a biopsy sample.

Embodiment 74 the method of any one of embodiments 71 to 73, wherein the solid tumor is breast cancer, genitourinary cancer (e.g., a cancer derived from the urinary tract, such as a cancer derived from the kidney (e.g., renal cell carcinoma), ureter, bladder, or urethra; male genital tract cancer (e.g., testicular cancer, prostate cancer, seminal vesicle cancer, seminal duct cancer, or penile cancer), or female genital tract cancer (e.g., ovarian cancer, uterine cancer, cervical cancer, vaginal cancer, or fallopian tube cancer)), nervous system cancer (e.g., neuroblastoma), intestinal cancer (e.g., colorectal cancer), lung cancer, melanoma, or other type of cancer.

Embodiment 75 the method of any one of embodiments 71 to 73, wherein the solid tumor is myeloid breast cancer.

Embodiment 76 the method of any one of embodiments 71 to 73, wherein the solid tumor is mesothelioma.

Embodiment 77 the method of any one of embodiments 71 to 73, wherein the solid tumor is melanoma.

Embodiment 78 the method of any one of embodiments 71 to 77, wherein the cells isolated from the solid tumor sample have been depleted of at least one non-T lymphocyte type (e.g., bone marrow cells, such as monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, megakaryocytes, and platelets, B lymphocytes, Natural Killer (NK) cells, stem cells, or any combination thereof).

Embodiment 79 the method of any one of embodiments 71 to 78, wherein the cells isolated from the solid tumor sample are enriched for CD8 ⁺ T lymphocytes.

Embodiment 80 the method of embodiment 79, wherein the cells isolated from the solid tumor sample have been depleted of T lymphocytes having the phenotype CD4+ and/or have CD45RA ⁺ Phenotype (optionally with CCR7 ⁺ And/or CD62L + phenotype).

Embodiment 81 the method of any one of embodiments 1 to 80, further comprising: contacting the T lymphocytes in the fluid sample with an activating agent.

Embodiment 82 the method of embodiment 81, wherein the T lymphocytes are contacted with the activating agent at least prior to flowing the fluid sample through the first region of the flow path of the microfluidic device.

Embodiment 83 the method of embodiment 81, wherein after the fluid sample flows through the first region of the flow path of the microfluidic device, the T lymphocytes in the fluid sample are contacted with the activating agent.

Embodiment 84. the method of embodiment 83, wherein the T lymphocytes are contacted with the activating agent after the T lymphocytes are engrafted into a isolation dock (e.g., a isolation dock having a connecting region that opens to the proximal end of the second, third, or fourth channel).

Embodiment 85 the method of any one of embodiments 81 to 84, wherein the T lymphocytes in the fluid sample are contacted with the activating agent for a period of at least one hour prior to flowing the fluid sample through the first region of the flow path of the microfluidic device.

Embodiment 86 the method of any one of embodiments 81 to 85, wherein the T lymphocytes in the fluid sample are contacted with the activating agent for a time of at least 24 hours (e.g., at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 108 hours, at least 120 hours, or any time range defined by two of the foregoing values) prior to flowing the fluid sample through the first region of the flow path of the microfluidic device.

Embodiment 87 the method of any one of embodiments 81 to 86, wherein the activating agent comprises an artificial antigen presenting cell (aAPC), and wherein the aAPC comprises an MHC class I molecule complexed with an antigenic peptide (e.g., a tumor associated peptide).

Embodiment 88 the method of embodiment 87, wherein the aapcs further comprise a CD28 agonist (e.g., an anti-CD 28 agonist antibody).

Embodiment 89 the method of any one of embodiments 81 to 86, wherein the activator comprises Dendritic Cells (DCs).

Embodiment 90 the method of embodiment 89, wherein the DCs and T lymphocytes of the fluid sample are autologous cells.

Embodiment 91 the method of embodiment 89 or 90, wherein the DCs are challenged with the antigenic peptide prior to contact with the T lymphocytes in the fluid sample.

Embodiment 92 the method of embodiment 87, 88 or 91, wherein the antigenic peptide is identified or isolated in tumor cells autologous to the T lymphocytes of the fluid sample. (alternatively, the antigenic peptide may be identified or isolated in a pathogen, such as a bacterial, fungal, parasitic or viral pathogen).

Embodiment 93 the method of embodiment 87, 88 or 91, wherein the antigenic peptide is identified by genomic analysis of tumor cells (e.g., tumor cells autologous to the T lymphocytes of the fluid sample).

Embodiment 94 the method of any one of embodiments 20 to 94, further comprising: after the fluid sample has passed through the first region of the flow path and into the second channel of the microfluidic device (or the third or fourth channel of the microfluidic device), the flow of the fluid sample through the flow path of the microfluidic device is stopped.

Embodiment 95 the method of embodiment 94, further comprising: introducing at least one activated T lymphocyte into the isolation dock.

Embodiment 96 the method of embodiment 94, further comprising: introducing at least one activated T lymphocyte into each of the plurality of isolated docks.

Embodiment 97 the method of embodiment 95 or 96, wherein the isolation dock or plurality of isolation docks each have a connection region with a proximal opening to the second channel of the flow path to the microfluidic device (or to the third or fourth channel of the flow path to the microfluidic device).

Embodiment 98. the method of any one of embodiments 94 to 97, wherein the microfluidic device comprises a substrate having a Dielectrophoresis (DEP) configuration, and wherein introducing the at least one T-lymphocyte into the isolation dock (or each of the plurality of isolation docks) comprises selecting the at least one T-lymphocyte using a DEP force, and moving the at least one T-lymphocyte into the isolation dock (or each of the plurality of isolation docks).

Embodiment 99 the method of embodiment 98, wherein the at least one T lymphocyte is selected, at least in part, because its cell surface is CD8 ⁺ (and/or a TCR with specificity for detecting an antigen of interest).

Embodiment 100 the method of any one of embodiments 94 to 97, wherein introducing at least one T lymphocyte into the isolation dock (or each isolation dock of the plurality of isolation docks) comprises tilting the microfluidic device such that gravity pulls the at least one T lymphocyte into the isolation dock (or each isolation dock of the plurality of isolation docks).

Embodiment 101 the method of any one of embodiments 94 to 100, wherein the culture medium is perfused through the flow path of the microfluidic device for a period of at least 24 hours (e.g., at least 48 hours, at least 72 hours, at least 96 hours, or longer) after the at least one T lymphocyte is introduced into the isolation dock (or each of the plurality of isolation docks).

Embodiment 102 the method of any one of embodiments 94 to 101, wherein the at least one T lymphocyte is contacted with the activating agent after introduction of the isolated dock (or each isolated dock of the plurality of isolated docks).

Embodiment 103 the method of any one of embodiments 20 to 102, further comprising: selectively outputting a population of T lymphocytes from a second channel (or a third or fourth channel) of the flow path of the microfluidic device, wherein the population of T lymphocytes is output separately from any cells or T lymphocytes that have flowed through the first channel of the second region of the flow path of the microfluidic device.

Embodiment 104 the method of embodiment 103, wherein the population of T lymphocytes is output from a third channel of a second sub-region of a second channel of a second region of the flow path of the microfluidic device, and wherein the population of T lymphocytes is output separately from any cells or T lymphocytes that have flowed through a fourth channel of the second sub-region of the second channel.

Embodiment 105 the method of any one of embodiments 1 to 104, wherein prior to flowing the fluid through the first region of the flow path of the microfluidic device, the flow region of the microfluidic device is treated with a blocking solution comprising a blocking agent bound to an interior surface of the microfluidic device (e.g., a surface of the channel and/or any isolation dock).

Embodiment 106 the method of embodiment 105, wherein the blocking solution comprises serum, BSA, or a polymer (e.g., a polymer comprising polyethylene glycol (PEG) and/or polypropylene glycol (PPG)).

Embodiment 107 the method of any one of embodiments 1 to 106, wherein the microfluidic device comprises an interior surface comprising a coating material.

Embodiment 108 the method of embodiment 107, wherein the coating material comprises a fluoroalkane moiety.

Embodiment 109 the method of embodiment 107, wherein the coating material comprises a carboxylic acid moiety, a sugar moiety (e.g., dextran), or a polyethylene glycol (PEG) moiety.

Embodiment 110 the method of any one of embodiments 1 to 109, wherein flowing the fluid sample through the first region of the flow path of the microfluidic device produces a sorted sample enriched in activated T lymphocytes, and wherein the enrichment is at least 2-fold (e.g., at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or greater).

Embodiment 111 a microfluidic device comprising: a flow path having a first region comprising a first array of pillars, wherein: the first region includes a first sidewall and a second sidewall that together define a general direction of fluid flow in the first region of the fluid path, the general direction corresponding to a first direction of the first region; the pillars of the first array are arranged in rows and columns; the rows of columns in the first array define a first array direction which differs from the first direction of the first area by a tilt angle (epsilon), and the first arrayThe columns of columns in the column repeat periodically with a period equal to l/epsilon, where epsilon is measured in radians; adjacent pillars in each respective column in the first array are separated by a gap through which a fluid of the fluid sample can flow laterally with respect to the columns as a whole, wherein a majority of the gaps have a characteristic dimension corresponding to a major gap dimension of the first array, and the first array is characterized by a critical dimension (D) _c ) From about 4 microns to about 10 microns.

Embodiment 112 the device of embodiment 111, wherein the first region of the flow path is a main channel having a width defined by first and second sidewalls, and wherein the first array of pillars extends across the entire width of the main channel.

Embodiment 113 the device of embodiment 111 or 112, wherein the first array is characterized by D _c From about 4 microns to about 7 microns.

Embodiment 114 the device of embodiment 111 or 112, wherein the first array is characterized by D _c From about 7 microns to about 10 microns.

Embodiment 115 the device of any one of embodiments 111 to 114, wherein the first array has an inclination angle e of about 1/3 radians to about 1/100 radians.

Embodiment 116 the device of any one of embodiments 111 to 114, wherein the first array has an inclination angle e of about 1/5 radians to about 1/30 radians.

Embodiment 117 the device of any one of embodiments 111 to 114, wherein the first array has an inclination angle e of about 1/10 radians to about 1/16 radians.

Embodiment 118 the device of any one of embodiments 111 to 117, wherein the primary gap dimension of the first array is about 15 microns to about 25 microns.

Embodiment 119 the device of any one of embodiments 111 to 117, wherein the major gap dimension of the first array is about 25 microns to about 40 microns.

Embodiment 120 the device of any one of embodiments 111 to 119, wherein the first array of pillars has a diameter of about 30 microns to about 100 microns (e.g., about 40 microns to about 85 microns, or about 50 microns to about 70 microns).

Embodiment 121. the device of embodiment 118 or 119, wherein the diameter of the pillars of the first array is greater (e.g., 1.5 to 5 times greater) than the major gap dimension.

Embodiment 122 the device of embodiment 118 or 119, wherein the diameter of the pillars of the first array is two to four times greater than the major gap dimension.

Embodiment 123 the device of any one of embodiments 111 to 122, wherein the columns of the first array are arranged laterally with respect to the first direction of the first region.

Embodiment 124 the device of any one of embodiments 111 to 123, wherein the cross-section of the pillars of the first array is circular (e.g., circular or oval).

Embodiment 125 the device of any one of embodiments 111 to 123, wherein the cross-section of the pillars of the first array is polygonal (e.g., triangular, square, diamond, or parallelogram shaped).

Embodiment 126 the device of embodiment 124 or 125, wherein the pillars of the first array all have the same orientation, and wherein the orientation is such that no axis of symmetry in the cross-sectional shape of the pillars is parallel to the axis defined by the first direction.

Embodiment 127 the device of any one of embodiments 111 to 126, wherein the first array of pillars comprises a siloxane polymer.

Embodiment 128 the device of any one of embodiments 111 to 126, wherein all gaps between adjacent pillars in a column of the first array are equal to the major gap dimension of the first array, except that the gap dimension between adjacent pillars of the same column that are closest to the first or second sidewall can deviate from the major gap dimension, and wherein the deviation in gap dimension between the pillars in the first array reduces boundary irregularities otherwise caused by the first and second sidewalls when the fluid sample flows through the first array.

Embodiment 129 the device of any one of embodiments 111-128, wherein the flow path of the microfluidic device comprises a second region configured to receive the fluid sample after the fluid sample passes through the first region of the microfluidic device, the second region having a divider that divides the second region into a first channel that receives a first portion of the fluid sample and a second channel that receives a second portion of the fluid sample.

Embodiment 130 the device of embodiment 129, wherein the separator of the second region is positioned such that the enriched diameter is greater than D in the second portion of the fluid sample relative to the fluid sample _c T lymphocytes of (a); and a diameter less than D _c The T lymphocytes of (a) are predominantly located in the first portion of the fluid sample.

Embodiment 131 the device of embodiment 129 or 130, wherein the first portion of the fluid sample comprises at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) of the fluid sample.

Embodiment 132 the device of embodiment 129 or 130, wherein the first portion of the fluid sample comprises about 85% to about 95% of the fluid sample.

Embodiment 133 the device of any one of embodiments 129 to 132, wherein the first channel and the second channel are configured such that the pressure differential across the first channel is equal to the pressure differential across the second channel.

Embodiment 134 the device of any one of embodiments 129 to 133, wherein the first channel comprises a first length and the second channel comprises a second length, and wherein the second length is greater than the first length (e.g., wherein the second length of the second channel is at least 5 times greater than the first length of the first channel).

Embodiment 135 the device of any one of embodiments 129 to 134, wherein the microfluidic device comprises at least one isolation dock having a connection region with a proximal opening to the second channel, and further wherein the at least one isolation dock has an isolation region with a volume large enough to accommodate at least one T lymphocyte (e.g., a plurality of T lymphocytes).

Embodiment 136 the device of any one of embodiments 129 to 134, wherein the microfluidic device comprises a plurality of isolated docks, each isolated dock having a connection region with a proximal opening to the second channel of the second region, and each isolated dock having an isolation region with a volume large enough to accommodate at least one T lymphocyte (e.g., a plurality of T lymphocytes).

Embodiment 137. the device of embodiments 135 or 136, wherein the volume of the isolation dock or each isolation dock of the plurality of isolation docks is about 250pL to about 3nL (e.g., about 250pL to about 750pL, about 400pL to about 900pL, about 500 μ L to about 1.5nL, about 1nL to about 2nL, about 1.5nL to about 2.5nL, about 2nL to about 3nL, or any range defined by the two aforementioned endpoints).

Embodiment 138 the device of any one of embodiments 111 to 137, wherein the length of the first region is about 5mm to about 15 mm.

Embodiment 139 the device of any one of embodiments 129 to 138, wherein the second channel comprises a first subregion comprising a second column array, wherein flowing the fluid sample through the first region of the flow path causes a second portion of the fluid sample, along with any cells contained therein, to flow through the second column array in the first subregion of the second channel, and further wherein: the general direction of fluid flow in the first sub-region of the second channel defines a second direction; the pillars in the second array are arranged in rows and columns; the rows of pillars in the second array define a second array direction, the second array direction differing from the second direction by an inclination angle (ε '), and the columns of pillars in the second array repeat periodically with a period equal to 1/ε ', where ε ' is measured in radians; adjacent pillars in each respective column in the second array are separated by a gap through which a second portion of the fluid sample can flow laterally with respect to the column as a whole, wherein a majority of the gaps have a characteristic dimension corresponding to a minor gap dimension of the second array, and the second array is characterized by a critical dimension (D) _c ) From about 4 microns to about 10 microns.

Embodiment 140 the device of embodiment 139, wherein the second channel has a width, and wherein the second column array extends the entire width of the second channel.

Embodiment 141 the device of embodiment 139 or 140, wherein the first array is characterized by D _c From about 4 microns to about 5 microns, from about 4.5 microns to about 5.5 microns, from about 5 microns to about 6 microns, from about 5.5 microns to about 6.5 microns, from about 6 microns to about 7 microns, from about 6.5 microns to about 7.5 microns, from about 7 microns to about 8 microns, from about 7.5 microns to about 8.5 microns, from about 8 microns to about 9 microns, from about 8.5 microns to about 9.5 microns, from about 9 microns to about 10 microns, or any range defined by both of the foregoing endpoints.

Embodiment 142 the device of embodiment 139 or 140, wherein the second column array is characterized by D _c From about 4 microns to about 7 microns, or wherein the second column array is characterized by D _c From about 7 microns to about 10 microns.

Embodiment 143 the device of any one of embodiments 139 to 142, wherein the second array has an inclination angle e of about 1/3 radians to about 1/100 radians.

Embodiment 144 the device of any one of embodiments 139 to 142, wherein the second array has an inclination angle e' of about 1/5 radians to about 1/30 radians.

Embodiment 145 the device of any one of embodiments 139 to 144, wherein the secondary gap size of the second array is about 15 microns to about 25 microns.

Embodiment 146 the device of any one of embodiments 139 to 144, wherein the secondary gap size of the second array is about 25 microns to about 40 microns.

Embodiment 147 the device of any of embodiments 139 to 146, wherein the second array of pillars has a diameter of about 30 microns to about 100 microns (e.g., about 40 microns to about 85 microns or about 50 microns to about 70 microns).

Embodiment 148 the device of embodiment 145 or 146, wherein the diameter of the pillars of the second array is greater (e.g., 1.5 to 5 times greater) than the minor gap dimension.

Embodiment 149 the device of embodiment 145 or 146, wherein the diameter of the pillars of the second array is two to four times larger than the minor gap dimension.

Embodiment 150 the device of any one of embodiments 139 to 149, wherein the columns of the second array are arranged laterally with respect to the second direction of the first sub-regions of the second channels.

Embodiment 151 the device of any one of embodiments 139 to 150, wherein the cross-section of the pillars of the second array is circular (e.g., circular or oval).

Embodiment 152 the device of any one of embodiments 139 to 150, wherein the cross-section of the pillars of the second array is polygonal (e.g., triangular, square, diamond, or parallelogram shaped).

Embodiment 153 the device of embodiment 151 or 152, wherein the pillars of the second array all have the same orientation, and wherein the orientation is such that no axis of symmetry in the cross-sectional shape of the pillars is parallel to the axis defined by the second direction.

Embodiment 154 the device of any one of embodiments 139 to 153, wherein the second array of columns comprises a siloxane polymer.

Embodiment 155 the device of any one of embodiments 139 to 154, wherein the first subregion of the second channel comprises a third sidewall and a fourth sidewall that together define the second direction, wherein all gaps between adjacent pillars in a column of the second array are equal to the minor gap dimension of the second array, except that the gap dimension between adjacent pillars of the same column that are closest to the third or fourth sidewall may deviate from the minor gap dimension, and wherein the deviation in the gap dimension between the pillars in the second array reduces boundary irregularities otherwise caused by the third and fourth sidewalls as the second portion of the fluid sample flows through the second array.

Embodiment 156 the device of any one of embodiments 139 to 155, wherein the second channel comprises a second sub-region configured to receive the second portion of the fluid sample after the second portion of the fluid sample passes through the first sub-region, the second sub-region having a divider dividing the second channel into a third channel and a fourth channel, the third channel receiving a first sub-portion of the fluid from the second portion of the fluid sample, the fourth channel receiving a second sub-portion of the fluid from the second portion of the fluid sample.

Embodiment 157 the device of embodiment 156, wherein the divider of the second sub-region is positioned such that the enriched diameter in the second sub-portion of the fluid is greater than D relative to the second portion of the fluid sample _c And wherein the diameter is less than D _c Is predominantly located in the first sub-portion of the fluid.

Embodiment 158 the device of embodiment 156 or 157, wherein the first sub-portion of the fluid comprises at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more) of the second portion of the fluid sample.

Embodiment 159 the device of embodiment 156 or 157, wherein the first subportion of the fluid comprises from about 85% to about 95% of the second portion of the fluid sample.

Embodiment 160 the device of any one of embodiments 156-159, wherein the third channel and the fourth channel are configured such that a pressure differential across the third channel is equal to a pressure differential across the fourth channel.

Embodiment 161 the device of embodiment 160, wherein the third channel comprises a third length and the fourth channel comprises a fourth length, and wherein the fourth length is greater than the third length (e.g., wherein the fourth length is at least 5 times greater than the third length).

Embodiment 162 the device of any one of embodiments 156-161, wherein the microfluidic device comprises at least one isolation dock having a connection region with a proximal opening to the third channel or the fourth channel, and further wherein the at least one isolation dock has an isolation region with a volume large enough to accommodate at least one T lymphocyte (e.g., a plurality of T lymphocytes).

Embodiment 163 the device of any one of embodiments 156 to 161, wherein the microfluidic device comprises a plurality of isolation docks, each isolation dock of the plurality of isolation docks having a connection region with a proximal opening to the third channel (or fourth channel) and an isolation region having a volume large enough to accommodate at least one T lymphocyte (e.g., a plurality of T lymphocytes).

Embodiment 164. the device of embodiment 162 or 163, wherein the volume of the isolation dock or each isolation dock of the plurality of isolation docks is about 250pL to about 3nL (e.g., about 250pL to about 750pL, about 400pL to about 900pL, about 500 μ L to about 1.5nL, about 1nL to about 2nL, about 1.5nL to about 2.5nL, about 2nL to about 3nL, or any range defined by the two aforementioned endpoints.

Embodiment 165 the device of any one of embodiments 111 to 164, wherein the microfluidic device comprises an interior surface (e.g., at least one interior surface of the first region, the second region, the first sub-region, the second sub-region, the first channel, the second channel, the third channel, the fourth channel) comprising a coating material.

Embodiment 166. the device of embodiment 165, wherein the coating material comprises a fluoroalkane moiety.

Embodiment 167 the device of embodiment 165, wherein the coating material comprises a carboxylic acid moiety, a sugar moiety (e.g., dextran), or a polyethylene glycol (PEG) moiety.

Embodiment 168 a composition comprising T lymphocytes sorted according to the method of any one of embodiments 20 to 51 and 62 to 110, wherein the T lymphocytes are obtained by exporting cells from a second channel of a flow path of a microfluidic device, wherein the population of T lymphocytes is exported separately from any cells or T lymphocytes that have flowed through a first channel of the flow path of the microfluidic device.

Embodiment 169 a composition comprising T lymphocytes sorted according to the method of any one of embodiments 52 to 110, wherein the T lymphocytes are obtained by exporting cells from a third channel (or fourth channel) of the flow path of the microfluidic device, and wherein the population of T lymphocytes is exported separately from any cells or T lymphocytes that have flowed through the fourth channel (or third channel) of the flow path of the microfluidic device.

Embodiment 170 the composition of embodiment 168 or 169, further comprising a pharmaceutically acceptable carrier.

Embodiment 171 a method of treating a subject having a pathogenic disorder or cancer, the method comprising administering a composition of embodiment 170.

Identity of

The foregoing written description is considered to be sufficient to enable those skilled in the art to practice the embodiments. The foregoing description and examples detail certain embodiments and describe the best mode contemplated. It should be understood, however, that no matter how detailed the foregoing appears in text, the embodiments may be practiced in many ways and should be construed in accordance with the appended claims and any equivalents thereof.

Claims (92)

1. A method of producing a sample enriched for activated T lymphocytes using a microfluidic device comprising a flow path having a first region comprising a first array of pillars, the method comprising:

Flowing a fluid sample comprising a mixture of activated and resting T lymphocytes through a first region of a flow path of a microfluidic device, wherein:

the direction of fluid flow in the first region of the flow path defines a first direction;

the pillars in the first array are arranged in rows and columns;

the rows of pillars in the first array define a first array direction which differs from the first direction of the first area by an inclination angle (epsilon), and the pillars in the first array repeat periodically with a period equal to l/epsilon, where epsilon is measured in radians;

adjacent columns in each respective column in the first array are separated by a gap through which fluid of the fluid sample can flow laterally with respect to the columns as a whole, wherein a majority of the gaps have a characteristic dimension corresponding to a major gap dimension of the first array, and

the first array is characterized by a critical dimension (D) _c ) From 4 microns to 10 microns.

2. The method of claim 1, wherein the first region of the flow path is defined by a primary channel having a width, and wherein the first array of pillars extends across the entire width of the primary channel.

3. The method of claim 1, wherein the first array is characterized by D _c From 4 microns to 7 microns.

4. The method of claim 1, wherein the first array has an inclination angle e of 1/5 radians to 1/30 radians.

5. The method of claim 1, wherein the first array has a major gap dimension of 15 microns to 25 microns.

6. The method of claim 1, wherein the first array has a major gap dimension of 25 microns to 40 microns.

7. The method of claim 1, wherein the first array of pillars has a diameter of 30 microns to 100 microns.

8. The method of claim 1, wherein the columns of the first array are arranged laterally with respect to a first direction of the first region.

9. The method of claim 1, wherein the first array of pillars is circular or polygonal in cross-section.

10. The method of claim 9, wherein the pillars of the first array all have the same orientation, and wherein the orientation is such that no axis of symmetry in the cross-sectional shape of the pillars is parallel to an axis defined by the first direction.

11. The method of claim 1, wherein the flow path of the microfluidic device comprises a second region configured to receive the fluid sample after the fluid sample passes through the first region of the microfluidic device, the second region having a divider that divides the second region into a first channel that receives a first portion of the fluid sample and a second channel that receives a second portion of the fluid sample, and

Wherein the divider of the second region is positioned such that the enriched diameter is greater than D in the second portion of the fluid sample relative to the fluid sample _c The T lymphocyte of (1).

12. The method of claim 11, wherein the first portion of the fluid sample comprises 85% to 95% of the fluid sample.

13. The method of claim 11, wherein the first channel comprises a first length and the second channel comprises a second length, and wherein the second length of the second channel is at least 5 times longer than the first length of the first channel.

14. The method of claim 11, wherein the microfluidic device comprises at least one isolation dock having a connection region with a proximal opening to the second channel, and wherein the at least one isolation dock has an isolation region with a volume to accommodate the at least one T lymphocyte.

15. The method of claim 14, wherein the microfluidic device comprises a plurality of such isolated docks.

16. The method of claim 14, wherein the volume of the isolation dock is 250pL to 3 nL.

17. The method of claim 11, wherein there is CD8 ⁺ 、CD45 RO ⁺ /RA ^- 、CCR7 ^- 、CD62L ^- The T lymphocytes of the phenotype are enriched in a second portion of the fluid sample.

18. The method of claim 11, wherein there is CD8 ⁺ 、CD45 RO ⁺ /RA ^- 、CCR7 ⁺ 、CD62L ^- The T lymphocytes of the phenotype are enriched in a second portion of the fluid sample.

19. The method of claim 11, wherein the first region has a length of 5mm to 15 mm.

20. The method of claim 11, wherein the rate of fluid sample flow through the first region of the flow path is from 0.01 microliters/second to 10 microliters/second.

21. The method of claim 11, wherein the second channel comprises a first sub-region comprising a second column array, wherein flowing the fluid sample through the first region of the flow path causes a second portion of the fluid sample, along with any cells contained therein, to flow through the second column array in the first sub-region, and wherein:

the direction of fluid flow in the first sub-region of the second channel defines a second direction;

the pillars in the second array are arranged in rows and columns;

the rows of pillars in the second array define a second array direction, the second array direction differing from the second direction by an inclination angle (ε '), and the columns of pillars in the second array repeat periodically with a period equal to 1/ε ', where ε ' is measured in radians;

adjacent columns in each respective column in the second array are separated by a gap through which a second portion of the fluid sample can flow laterally relative to the columns as a whole, wherein a majority of the gaps have a characteristic dimension corresponding to a minor gap dimension of the second array, and

The second array is characterized by a critical dimension (D) _c ) From 4 microns to 10 microns.

22. The method of claim 21, wherein the second channel has a width, and wherein the second column array extends across the width of the second channel.

23. The method of claim 21, wherein the second column array is characterized by D _c From 4 microns to 7 microns.

24. The method of claim 21, wherein the second column array is characterized by D _c From 7 microns to 10 microns.

25. The method of claim 21, wherein the second array has an inclination angle e' of 1/5 radians to 1/30 radians.

26. The method of claim 21, wherein the second array has a minor gap dimension of 15 microns to 25 microns.

27. The method of claim 21, wherein the second array has a minor gap dimension of 25 microns to 40 microns.

28. The method of claim 21, wherein the second array of pillars has a diameter of 30 microns to 100 microns.

29. The method of claim 21, wherein the columns of the second array are arranged laterally with respect to the second direction of the first sub-region of the second channel.

30. The method of claim 21, wherein the second array of pillars is circular or polygonal in cross-section.

31. The method of claim 30, wherein the pillars of the second array all have the same orientation, and wherein the orientation is such that no axis of symmetry in the cross-sectional shape of the pillars is parallel to the axis defined by the second direction.

32. The method of claim 21, wherein the second channel comprises a second sub-region configured to receive the second portion of the fluid sample after the second portion of the fluid sample passes through the first sub-region, the second sub-region having a divider that divides the second channel into a third channel that receives the first sub-portion of the fluid from the second portion of the fluid sample and a fourth channel that receives the second sub-portion of the fluid from the second portion of the fluid sample, and

wherein the divider of the second sub-region is positioned such that the enriched diameter is greater than D in a second sub-portion of the fluid relative to a second portion of the fluid sample _c The T lymphocyte of (1).

33. The method of claim 32, wherein the first sub-portion of the fluid comprises at least 50% of the second portion of the fluid sample.

34. The method of claim 32, the third channel comprising a third length and the fourth channel comprising a fourth length, and wherein the fourth length is greater than the third length.

35. The method of claim 32, wherein the microfluidic device comprises at least one isolation dock having a connection region with a proximal opening to the third channel or the fourth channel, and wherein the at least one isolation dock has an isolation region with a volume to accommodate the at least one T lymphocyte.

36. The method of claim 35, wherein the microfluidic device comprises a plurality of such isolated docks.

37. The method of claim 32, wherein the volume of the isolation dock is 250pL to 3 nL.

38. The method of any one of claims 1 to 37, wherein the fluid sample is a peripheral blood sample obtained from a subject or a sample derived therefrom.

39. The method of claim 38, wherein the fluid sample is a peripheral blood sample that has been depleted of at least one non-T lymphocyte type.

40. The method of claim 38, wherein the fluid sample has been enriched for CD8 ⁺ Peripheral blood samples of T lymphocytes.

41. The method of claim 40, wherein the fluid sample has been depleted of effector T lymphocytes and/or memory T lymphocytes.

42. The method of claim 40, wherein the fluid sample has been enriched for non-immunized T lymphocytes or has CD45RA ⁺ A phenotypic cell.

43. The method of claim 40, wherein the fluid sample has been enriched for central memory T lymphocytes or has CD45RO ⁺ Phenotype and CCR7 ⁺ And/or CD62L ⁺ Cells with a combination of phenotypes.

44. The method of claim 38, further comprising:

obtaining a peripheral blood sample or a sample derived therefrom, wherein the peripheral blood is derived from a human subject; and is

A fluid sample is generated from a peripheral blood sample or a sample derived therefrom.

45. The method of claim 44, wherein generating a fluid sample comprises:

depleting at least one non-T lymphocyte type of the peripheral blood sample or a sample derived therefrom; and/or

CD8 enriched in peripheral blood samples or samples derived therefrom ⁺ T lymphocytes.

46. The method of claim 45, wherein the enriching step comprises enriching the peripheral blood sample or a sample derived therefrom for CD8 ⁺ Non-immunized T lymphocytes or with CD45RA ⁺ A phenotypic cell.

47. The method of claim 38, further comprising:

contacting the T lymphocytes in the fluid sample with an activating agent.

48. The method of claim 47, wherein the T lymphocytes are contacted with the activating agent at least prior to flowing the fluid sample through the first region of the flow path of the microfluidic device.

49. The method of claim 48, wherein the T lymphocytes in the fluid sample are contacted with the activating agent for a period of at least 48 hours prior to flowing the fluid sample through the first region of the flow path of the microfluidic device.

50. The method of claim 48, wherein the activating agent comprises an artificial antigen presenting cell (aAPC), and wherein the aAPC comprises an MHC class I molecule complexed with an antigenic peptide.

51. The method of claim 50, wherein the aAPCs further comprise a CD28 agonist.

52. The method of claim 48, wherein the activator comprises Dendritic Cells (DCs).

53. The method of claim 52, wherein the DC and T lymphocytes of the fluid sample are autologous cells.

54. The method of claim 50, wherein the antigenic peptide is identified or isolated in a bacterial pathogen, a fungal pathogen, a parasitic pathogen, a viral pathogen, or a tumor cell.

55. The method of claim 54, wherein the antigenic peptide is identified or isolated in cancer cells autologous to the T lymphocytes of the fluid sample.

56. The method of claim 15, further comprising:

stopping the flow of the fluid sample through the flow path of the microfluidic device after the fluid sample has passed through the first region of the flow path and into the second channel of the microfluidic device; and is

Introducing at least one activated T lymphocyte into one or more of the plurality of isolation docks.

57. The method of claim 56, wherein the microfluidic device comprises a substrate having a Dielectrophoresis (DEP) configuration, and wherein introducing the at least one activated T lymphocyte into the one or more sequestration docks comprises selecting the at least one T lymphocyte using a DEP force and moving the at least one T lymphocyte into each of the one or more sequestration docks.

58. The method of claim 57, wherein at least one T lymphocyte is selected, at least in part, for its cell surface being CD8 ⁺ 。

59. The method of claim 58, wherein at least one T lymphocyte is selected, at least in part, for its cell surface having a TCR that specifically detects an antigen of interest.

60. The method of claim 56, wherein the media is perfused through the flow path of the microfluidic device for a period of at least 24 hours after the at least one T lymphocyte is introduced into each of the one or more isolation docks.

61. The method of claim 11, further comprising:

selectively outputting a population of T lymphocytes from a second channel of the flow path of the microfluidic device, wherein the population of T lymphocytes is output separately from any cells or T lymphocytes that have flowed through the first channel of the flow path of the microfluidic device.

62. The method of any one of claims 1 to 37 or 56 to 61, wherein prior to flowing the fluid through the first region of the flow path of the microfluidic device, the flow region of the microfluidic device is treated with a blocking solution comprising a blocking agent bound to at least one internal surface of the microfluidic device, wherein the blocking solution comprises serum, BSA, or a polymer comprising polyethylene glycol and/or polypropylene glycol.

63. The method of any one of claims 1 to 37 and 56 to 61, wherein the microfluidic device comprises an inner surface comprising a coating material.

64. The method of claim 63, wherein the coating material comprises fluoroalkane moieties.

65. The method of claim 63, wherein the coating material comprises a carboxylic acid moiety, a sugar moiety, or a polyethylene glycol moiety.

66. The method of any one of claims 1 to 37, wherein flowing the fluid sample through the first region of the flow path of the microfluidic device produces a sorted sample enriched for activated T lymphocytes, and wherein the enrichment is at least 2-fold.

67. A microfluidic device comprising:

a flow path having a first region comprising a first array of pillars, wherein:

the first region includes a first sidewall and a second sidewall that together define a general direction of fluid flow in the first region of the fluid path, the general direction corresponding to a first direction of the first region;

The pillars of the first array are arranged in rows and columns;

the rows of pillars in the first array define a first array direction which differs from the first direction of the first area by an inclination angle (epsilon), and the pillars in the first array repeat periodically with a period equal to l/epsilon, where epsilon is measured in radians;

adjacent columns in each respective column in the first array are separated by a gap through which fluid of the fluid sample can flow laterally with respect to the columns as a whole, wherein a majority of the gaps have a characteristic dimension corresponding to a major gap dimension of the first array, and

first arrayThe columns are characterized by a critical dimension (D) _c ) From 4 microns to 10 microns.

68. The device of claim 67, wherein the first region of the flow path is a main channel having a width defined by the first and second sidewalls, and wherein the first array of posts extends across the entire width of the main channel,

wherein the first array is characterized by D _c Is in the range of 4 microns to 7 microns,

wherein the first array has an inclination angle epsilon of 1/5 radians to 1/30 radians,

wherein the first array has a major gap dimension of 15 to 40 microns, and

wherein the first array of pillars has a diameter of 30 to 100 microns.

69. The device of claim 67, wherein the columns of the first array are arranged laterally with respect to the first direction of the first region.

70. The apparatus of claim 67, wherein the first array of pillars is circular or polygonal in cross-section.

71. The device of claim 70, wherein the pillars of the first array all have the same orientation, and wherein the orientation is such that no axis of symmetry in the cross-sectional shape of the pillars is parallel to the axis defined by the first direction.

72. The device of claim 67, wherein all gaps between adjacent pillars in a column of the first array are equal to a major gap dimension of the first array, except that a gap dimension between adjacent pillars of the same column closest to the first or second sidewall can deviate from the major gap dimension, and wherein the deviation in gap dimension between the pillars in the first array reduces boundary irregularities otherwise caused by the first and second sidewalls as the fluid sample flows across the first array.

73. The device of claim 67, wherein the flow path of the microfluidic device comprises a second region configured to receive the fluid sample after the fluid sample passes through the first region of the microfluidic device, the second region having a divider that divides the second region into a first channel that receives a first portion of the fluid sample and a second channel that receives a second portion of the fluid sample.

74. The apparatus of claim 73, wherein the first portion of the fluid sample comprises 85% to 95% of the fluid sample.

75. The device of claim 73, wherein the first channel comprises a first length and the second channel comprises a second length, and wherein the second length of the second channel is at least 5 times longer than the first length of the first channel.

76. The device of claim 73, wherein the microfluidic device comprises a plurality of isolation docks, each isolation dock having a connection region with a proximal opening to the second channel of the flow path, and each isolation dock having an isolation region with a volume to accommodate a plurality of T lymphocytes.

77. The apparatus of claim 76, wherein each isolation dock of the plurality of isolation docks has a volume of 250pL to 3 nL.

78. The device of claim 67, wherein the first region has a length of 5mm to 15 mm.

79. The device of claim 73, wherein the second channel comprises a first sub-region comprising a second column array, wherein flowing the fluid sample through the first region of the flow path causes a second portion of the fluid sample, along with any cells contained therein, to flow through the second column array in the first sub-region of the second channel, and wherein:

The general direction of fluid flow in the first sub-region of the second channel defines a second direction;

the pillars in the second array are arranged in rows and columns;

the rows of pillars in the second array define a second array direction, the second array direction differing from the second direction by an inclination angle (ε '), and the columns of pillars in the second array repeat periodically with a period equal to 1/ε ', where ε ' is measured in radians;

adjacent columns in each respective column in the second array are separated by a gap through which a second portion of the fluid sample can flow laterally relative to the columns as a whole, wherein a majority of the gaps have a characteristic dimension corresponding to a minor gap dimension of the second array, and

the second array is characterized by a critical dimension (D) _c ) From 4 microns to 10 microns.

80. The apparatus of claim 79, wherein the second channel has a width, and wherein the second column array extends the entire width of the second channel,

wherein the second array has an inclination angle e' of 1/5 radians to 1/30 radians,

wherein the secondary gap dimension of the second array is from 15 microns to 40 microns, and

wherein the second array of pillars has a diameter of 30 to 100 microns.

81. The apparatus of claim 80, wherein the second column array is characterized by D _c From 4 microns to 7 microns.

82. The apparatus of claim 80, wherein the second column array is characterized by D _c From 7 microns to 10 microns.

83. The apparatus of claim 80, wherein the columns of the second array are arranged laterally with respect to the second direction of the first sub-region of the second channel.

84. The device of claim 80, wherein the second array of pillars is circular or polygonal in cross-section.

85. The apparatus of claim 84, wherein the pillars of the second array all have the same orientation, and wherein the orientation is such that no axis of symmetry in the cross-sectional shape of the pillars is parallel to an axis defined by the second direction.

86. The apparatus of claim 80, wherein the second channel comprises a second sub-region configured to receive the second portion of the fluid sample after the second portion of the fluid sample passes through the first sub-region, the second sub-region having a divider that divides the second channel into a third channel that receives the first sub-portion of the fluid from the second portion of the fluid sample and a fourth channel that receives the second sub-portion of the fluid from the second portion of the fluid sample,

wherein the divider of the second sub-region is positioned such that the enriched diameter is greater than D in a second sub-portion of the fluid relative to a second portion of the fluid sample _c The T lymphocyte of (1).

87. The device of claim 86, wherein the first sub-portion of the fluid comprises at least 50% of the second portion of the fluid sample.

88. The device of claim 86, wherein the microfluidic device comprises a plurality of isolation docks, each isolation dock of the plurality of isolation docks having a connection region and an isolation region, the connection region having a proximal opening to the third channel, the isolation region having a volume to accommodate the plurality of T-lymphocytes.

89. The apparatus of claim 88, wherein each isolation dock of the plurality of isolation docks has a volume of 250pL to 3 nL.

90. The device of any one of claims 67 to 89, wherein microfluidic device comprises at least one internal surface comprising a coating material.

91. The device of claim 90, wherein the coating material comprises fluoroalkane moieties.

92. The device of claim 90, wherein the coating material comprises a carboxylic acid moiety, a sugar moiety, or a polyethylene glycol moiety.