WO2015134831A1

WO2015134831A1 - Acoustic control apparatus, process, and fabrication thereof

Info

Publication number: WO2015134831A1
Application number: PCT/US2015/019093
Authority: WO
Inventors: Feng Guo; Peng Li; Tony Jun Huang; Stephen J. Benkovic; James R. Fick
Original assignee: The Penn State Research Foundation
Priority date: 2014-03-07
Filing date: 2015-03-06
Publication date: 2015-09-11

Abstract

Acoustic control apparatuses, acoustic control processes, and processes of fabricating acoustic control apparatuses are disclosed. The acoustic control apparatus includes a first interdigital transducer arrangement positioned to direct a first acoustic wave, a second interdigital transducer arrangement positioned to direct a second acoustic wave in a non-parallel direction relative to the first acoustic wave, and a manipulation region at least partially defined by an interference pattern at least partially formed by interaction between the first acoustic wave and the second acoustic wave. The acoustic control process includes directing the first acoustic wave, directing the second acoustic wave, and at least partially defining the manipulation region. The acoustic control apparatus fabrication process includes depositing metal on a photoresist-patterned substrate, performing a lift-off process, aligning one or more microfluidic channels, and maintaining a temperature range of between 0°C and 90°C.

Description

ACOUSTIC CONTROL APPARATUS, PROCESS, AND FABRICATION

THEREOF

GOVERNMENT CONTRACT

[0001] This invention was made with government support under Grant No. ECCS0801922, awarded by the National Science Foundation and Grant No. OD007209, awarded by the National Institutes of Health. The Government has certain rights in the invention.

PRIORITY

[0002] This application is an international Patent Cooperation Treaty (PCT) patent application claiming priority and benefit of U.S. Provisional Patent Application No. 61/949,538, filed on March 7, 2014, and titled Acoustic Control of Cell Interactions, the entirety of which is incorporated by references.

FIELD OF THE INVENTION

[0003] The present invention is directed to acoustic control. More particularly, the present invention is directed to an acoustic control apparatus, an acoustic control process, and an acoustic control apparatus fabrication process, where the acoustic control is due to interference patterns formed from two or more acoustic waves generated by interdigital transducer arrangements.

BACKGROUND OF THE INVENTION

[0004] Multicellular systems rely on the interaction between cells to coordinate cell signaling and regulate cell functions. Understanding the mechanisms and processes of cell-cell interactions is valuable to many physiological and pathological processes, such as embryogenesis, differentiation, cancer metastasis, immunological interactions, and diabetes. Despite significant advances in this field, to further understand how cells interact and communicate with each other, a means to precisely control the spatial and temporal association of cells, and to create defined cellular assemblies is urgently needed. [0005] While several methods have been employed to pattern cells, limitations still exist for the demonstrated technologies such as optical, electrical, magnetic, hydrodynamic, contact printing and so on.

[0006] Firstly, most of the methods require modification of the cell's active state. Dielectrophoresis (DEP), for example, requires the use of a relatively non-conductive medium that may affect cell normal physiology due to the lack of nutrients and low osmolality. The surface modification methods (for example, contact printing) restrict the space available for the cells to grow, while the magnetic assembly method requires cells to be labeled with magnetic beads. Use of optical tweezers is label-free and without contact, but it requires very high laser power to manipulate cells, leading to a high risk of cell damage.

[0007] Secondly, the working principles of the existing technologies mostly preclude the combination of high precision and high-throughput into a single device. It is difficult for high- throughput methods (such as magnetic assemblies) to achieve single-cell level precision, whereas the high-precision methods (such as optical tweezers) suffer from an inability to manipulate multiple cells simultaneously.

[0008] Thirdly, most of the existing methodologies lack the ability to maintain cell assemblies in suspension, thereby limiting the application of these methods for the study of cell-cell and cell- matrix interactions.

[0009] In addition to using optical, electrical, and magnetic forces, it has been demonstrated that cells can be manipulated using acoustic radiation force. Standing surface acoustic waves (SSAW) have recently been employed to manipulate cells through the formation of pressure nodes. This technology, however, has not yet been exploited as a tool for the study of cell-cell interactions. This is primarily due to the insufficient level of regulation of interference patterns at pressure nodes. The inability to accurately and reproducibly control interference patterns of pressure nodes severely limits the capability of standing SAW-based intercellular studies. As such, there is a need for new methods of manipulating cells in either unicellular or multicellular assemblies to improve the understanding of interactions between cells. [0010] An acoustic control apparatus, acoustic manipulation process, and an acoustic control apparatus fabrication process that show one or more improvements in comparison to the prior art would be desirable in the art.

BRIEF DESCRIPTION OF THE INVENTION

[0011] In an embodiment, an acoustic control apparatus includes a first interdigital transducer arrangement positioned to direct a first acoustic wave, a second interdigital transducer arrangement positioned to direct a second acoustic wave in a non-parallel direction relative to the first acoustic wave, and a manipulation region at least partially defined by an interference pattern at least partially formed by interaction between the first acoustic wave and the second acoustic wave.

[0012] In another embodiment, an acoustic control process includes directing a first surface acoustic wave generated from a first transducer arrangement, directing a second surface acoustic wave generated from a second transducer arrangement in a non-parallel direction relative to the first surface acoustic wave, and at least partially defining a manipulation region by an interference pattern formed by the first surface acoustic wave and the second acoustic wave.

[0013] In another embodiment, an acoustic control apparatus fabrication process includes depositing metal on a photoresist-patterned substrate to produce a metal deposit, performing a lift-off process on the metal deposit to produce one or more interdigital transducer arrangements, aligning one or more microfluidic channels with the one or more interdigital transducer arrangements to produce an assembly, and maintaining the assembly at a temperature range of between 0°C and 90°C to produce the acoustic control apparatus.

[0014] Other features and advantages of the present invention will be apparent from the following more detailed description, taken in conjunction with the accompanying drawings which illustrate, by way of example, the principles of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1 is a perspective view of an embodiment of an acoustic control apparatus with a first interference pattern, according to the disclosure.

[0016] FIG. 2 is a perspective view of an embodiment of an acoustic control apparatus with a second interference pattern, according to the disclosure.

[0017] FIG. 3 is an interference pattern having a net-like array of two-dimensional pressure nodes, according to an embodiment of the disclosure.

[0018] FIG. 4 is an interference pattern having a dot-array like pattern of pressure nodes, according to an embodiment of the disclosure.

[0019] FIG. 5 is a graphical depiction of cell-cell distance over time in response to an input signal, according to the disclosure.

[0020] FIG. 6 shows two HEK cells with different intercellular distances of 20 micrometers, 15 micrometers, 5 micrometers, and 0 micrometers, controlled according to an embodiment of the disclosure.

[0021] FIG. 7 shows cells in direct contact, with fluorescence dye transferred to the neighboring cell after one hour, indicating the formation of functional gap junctions, according to the disclosure.

[0022] FIG. 8 shows separated cells, with no transfer of fluorescence dye after one hour, controlled according to an embodiment of the disclosure.

[0023] FIG. 9 shows an acoustic potential distribution with a defined acoustic potential forming an elongate ovular shape, controlled according to an embodiment of the disclosure.

[0024] FIG. 10 shows an acoustic potential distribution with a defined acoustic potential forming an elongate perpendicular ovular shape relative to the elongate ovular shape of FIG. 9, controlled according to an embodiment of the disclosure. [0025] FIG. 11 shows an acoustic potential distribution with a defined acoustic potential forming a generally circular shape, controlled according to an embodiment of the disclosure.

[0026] FIG. 12 shows an acoustic potential distribution with a defined acoustic potential forming a generally circular shape compressed relative to FIG. 11, controlled according to an embodiment of the disclosure.

[0027] FIG. 13 shows a two-cell cell chain controlled by an acoustic potential distribution, according to an embodiment of the disclosure.

[0028] FIG. 14 shows a three-cell cell chain controlled by an acoustic potential distribution, according to an embodiment of the disclosure.

[0029] FIG. 15 shows a four-cell cell chain controlled by an acoustic potential distribution, according to an embodiment of the disclosure.

[0030] FIG. 16 shows a five-cell cell chain controlled by an acoustic potential distribution, according to an embodiment of the disclosure.

[0031] FIG. 17 shows a single-layer cluster of two rows of cells controlled by an acoustic potential distribution, according to an embodiment of the disclosure.

[0032] FIG. 18 shows a single-layer generally triangular cluster of cells controlled by an acoustic potential distribution, according to an embodiment of the disclosure.

[0033] FIG. 19 shows a single-layer generally circular cluster of cells controlled by an acoustic potential distribution, according to an embodiment of the disclosure.

[0034] FIG. 20 shows a single-layer cluster of cells controlled by an acoustic potential distribution, according to an embodiment of the disclosure.

[0035] FIG. 21 shows a schematic axial view of an acoustic control apparatus, according to the disclosure.

[0036] FIG. 22 shows bright-field image and time-lapse fluorescence images of two cells trapped within an acoustic pressure node, with the left cell being preloaded with Calcein-AM dye results in dye transfer between the two cells over time, controlled according to an embodiment of the disclosure.

[0037] FIG. 23 shows bright-field image and time-lapse fluorescence images of a three-cell system trapped by an acoustic pressure node, with the left cell being preloaded with Calcein-AM dye results in transfer of fluorescent molecules throughout the cell assembly, controlled according to an embodiment of the disclosure.

[0038] FIG. 24 shows bright-field image and time-lapse fluorescence images of a linear cell assembly trapped by a linear acoustic pressure node, results in dye molecules being transferred sequentially because of the defined linear assembly, controlled according to an embodiment of the disclosure.

[0039] FIG. 25 shows bright-field image and time-lapse fluorescence images of a two- dimensional, multiple-cell system trapped by a spherically shaped acoustic pressure node, allowing dye transfer to occur with all neighboring cells simultaneously, controlled according to an embodiment of the disclosure.

[0040] FIG. 26 schematically depicts an acoustic control apparatus with linear assemblies of cells formed under the control of a tunable acoustic well, whereby upon removal of the acoustic field, the cells drop to the surface and attach, according to the disclosure.

[0041] FIG. 27 shows in-suspension and attachment of HEK 293T cells over time controlled by acoustic potential, according to an embodiment of the disclosure.

[0042] FIG. 28 shows in-suspension and attachment of HMVEC cells over time controlled by acoustic potential, according to an embodiment of the disclosure.

[0043] FIG. 29 shows dye transfer of HMVEC cells over time controlled by acoustic potential, according to an embodiment of the disclosure.

[0044] FIG. 30 shows in-suspension and attachment of HeLa S3 cells over time controlled by acoustic potential, according to an embodiment of the disclosure. [0045] FIG. 31 shows dye transfer of HeLa S3 cells over time controlled by acoustic potential, according to an embodiment of the disclosure.

[0046] Wherever possible, the same reference numbers will be used throughout the drawings to represent the same parts.

DETAILED DESCRIPTION OF THE INVENTION

[0047] Provided are an acoustic control apparatus, acoustic control process, and an acoustic control apparatus fabrication process. Embodiments of the present disclosure, for example, in comparison to concepts failing to include one or more of the features disclosed herein, permit improved ability to understand the mechanisms and processes of cell-cell interactions (for example, in physiological and pathological processes, such as embryogenesis, differentiation, cancer metastasis, immunological interactions, and diabetes), permit precise spatial and temporal control of cells, permit creation of defined cellular assemblies, permit control and/or separation without modification to a cell's active state, permit control and/or separation without removing or affecting nutrients and/or osmolality, permit control and/or separation without restricting space available for the cells to grow, permit control and/or separation without labeling and/or lasers, permit control and/or separation with higher precision (for example, controlling intercellular distance at micron-scale) and/or higher throughput (for example, forming arrays of cell assemblies with tunable geometric configurations), permit simultaneous control and/or separation of multiple cells/particles, permit control and/or separation cell assemblies in suspension (for example, cells within cell-matrices), permit regulation of interference patterns at pressure nodes, permit improved separation of particles or immiscible fluids from fluids, permit improvements to scientific techniques (for example, relating to tissue engineering, developmental biology, and/or cancer therapy), provide other advantages and distinctions, or provide a combination thereof.

[0048] Referring to FIG. 1, an acoustic control apparatus 101 includes a first interdigital transducer arrangement 103 positioned to direct a first acoustic wave 105 forming a first acoustic wave pattern and a second interdigital transducer arrangement 107 positioned to direct a second acoustic wave 109 forming a second acoustic wave pattern, for example, surface acoustic waves (SAWs). The first acoustic wave 105 (and the first acoustic wave pattern) and the second acoustic wave pattern 109 (and the second acoustic wave pattern) at least partially define a manipulation region 1 11, for example, by forming an interference pattern 1 13 based upon being positioned in a non-parallel arrangement.

[0049] The first interdigital transducer arrangement 103 and the second interdigital transducer arrangement 107 are arranged in any suitable configuration capable of producing the interference pattern 1 13 based upon the non-parallel arrangement. Suitable non-parallel arrangements include the first interdigital transducer arrangement 103 and the second interdigital transducer arrangement 107 being relatively positioned between 45 degrees and orthogonal (90 degrees), orthogonal or substantially orthogonal, 75 degrees, 60 degrees, 45 degrees, 30 degrees, 15 degrees, 10 degrees, 5 degrees, or any suitable combination, sub-combination, range, or subrange therein.

[0050] Although only the first interdigital transducer arrangement 103 and the second interdigital transducer arrangement 107 are shown, suitable configurations include fewer or more interdigital transducer arrangements. For example, in one embodiment, the acoustic control apparatus 101 includes or consists of more than two interdigital transducer arrangements, such as, three interdigital transducer arrangements, four interdigital transducer arrangements, five interdigital transducer arrangements, or more than five interdigital transducer arrangements. Alternatively, in one embodiment, the acoustic control apparatus 101 consists of two or three interdigital transducer arrangements.

[0051] The interference pattern 1 13 controls or manipulates particles (for example, micrometer- sized and/or nanometer-sized particles), fluids (for example, immiscible fluids), or any other material responsive to acoustic energy. For example, based upon a first input frequency and/or phase, the first acoustic wave 105 (and the first acoustic wave pattern) is generated and, based upon a second input frequency and/or phase, the second acoustic wave 109 (and the first acoustic wave pattern) is generated. In one embodiment, the input frequencies are generated by radiofrequency (RF) signals. In one embodiment, the first input frequency and/or phase differs from the second input frequency and/or phase, but the resonance frequency range is shared. Sites within the interference pattern 1 13 are known as acoustic wells. In one embodiment, as many as 1,600 acoustic wells are defined by the interference pattern 113. [0052] In one embodiment, the interference pattern 113 is able to be finely tuned, thereby permitting manipulation of the geometry of particles, such as, cell assemblies, in response to a change in the first input frequency (and/or phase) and/or the second input frequency (and/or phase). For example, in one embodiment, the acoustic control apparatus 101 is capable of creating isolated half-wavelength sized square pressure nodes. In response to input frequencies, cells or other particles are capable of being manipulated and/or controlled, for example, by acoustic radiation force and/or drag force driven by acoustic streaming. By modulating the two input frequencies, the acoustic control apparatus 101 is capable of precisely activating and tuning the position and dimension of the particles within the interference pattern 113. As a result, a cluster of cells or particles within the interference pattern 1 13 is capable of being precisely manipulated based upon quantity, orientation, and/or configuration. In a further embodiment, suspended cell assemblies are capable of being allowed to settle to the surface at any time to attach and optionally spread.

[0053] In one embodiment, the interference pattern 1 13 is finely tuned by adjusting any suitable property of the SAWs forming the interference pattern 1 13, including, but not limited to, the power, amplitude, angular frequency, phase constant, wavelength, frequency, waveform, space coordinate, time coordinate, wavenumber, period, of combination thereof, of one of or dependently or independently both of the SAWs forming the interference pattern 1 13. The fine tuning of the interference pattern 1 13 may include, but is not limited to, increasing fringe spacing, decreasing fringe spacing, increasing depth in a three-dimensional configuration, decreasing depth in a three-dimensional configuration, and combinations thereof.

[0054] In one embodiment, the geometric configuration of particles, manipulated by the fine tuning of the interference patter 1 13 is any suitable one-dimensional, two-dimensional or three dimensional geometry, simple geometry, or complex geometry, including, but not limited to a point, line segment, curve, triangle, quadrilateral, square, rectangle, rhombus, parallelogram, trapezoid, kite, pentagon, hexagon, octagon, nonagon, decagon, circle, oval, ellipse, parabola, hyperbola, sphere, spheroid, polygon, tetrahedron, pentahedron, hexahedron, heptahedron, octahedron, enneahedron, decahedron, dodecahedron, cone, symbol, written character, letter, numeral, alphanumeric, glyph, pictogram, syllabogram, logogram, or combination thereof. [0055] In one embodiment, the interference pattern 113 includes a pressure gradient capable of directing cells into the middle of the manipulation region. In one embodiment, as shown in FIG. 3, the interference pattern 1 13 is a net-like array of two-dimensional pressure nodes. In one embodiment, as shown in FIG. 4, the interference pattern 113 is a dot-array like pattern of pressure nodes. The interference pattern 1 13 is contained within a chamber having an acoustic aperture that is larger than the dimensions of the chamber. For example, in one embodiment, the interference pattern 113 is within a square chamber having dimensions of 6 millimeters by 6 millimeters and the acoustic aperture is designed to permit SAWs of 9 millimeters in both directions.

[0056] The interdigital transducer arrangements include any suitable material(s) capable of generating acoustic waves to define the interference pattern 1 13. In one embodiment, electrodes 117, for example, 30 to 50 pairs (such as, 40 pairs) are arranged on any suitable substrate 1 19, for example, with spacing gaps of between 50 micrometers and 300 micrometers, between 60 micrometers and 200 micrometers, between 60 micrometers and 100 micrometers, between 70 micrometers and 80 micrometers, or any suitable combination, sub-combination, range, or subrange therein.

[0057] Suitable substrates 119 include a lithium niobate substrate, a lithium tantalate substrate, lead zirconium titanate substrate, a polymer such as polyvinylidene fluoride (PVDF) or another fluoropolymer, quartz, another material, or a combination thereof. Additionally or alternatively, the substrate 1 19 is capable of being a 128° Y-cut piezoelectric substrate, a piezoelectric substrate with a 45° angle to the X-direction, or a combination thereof.

[0058] In one embodiment, the first interdigital transducer arrangement 103 and/or the second interdigital transducer arrangement 107 include(s) a metal deposit on a photoresist-patterned substrate, for example, a titanium layer and a gold layer. Suitable thicknesses of the titanium layer include, but are not limited to, less than 10 nanometers, less than 7 nanometers, between 1 nanometer and 10 nanometers, between 3 nanometers and 7 nanometers, between 4 nanometers and 6 nanometers, 5 nanometers, or any suitable combination, sub-combination, range, or subrange therein. Suitable thicknesses of the gold layer include, but are not limited to, less than 200 nanometers, less than 120 nanometers, between 20 nanometers and 300 nanometers, between 50 nanometers and 150 nanometers, between 80 nanometers and 120 nanometers, 100 nanometers, or any suitable combination, sub-combination, range, or sub-range therein.

[0059] Referring again to FIGS. 1-2, in one embodiment, one or more microfluidic channels 1 15 extend through the manipulation region 1 1 1. The microfluidic channel(s) 1 15 includes a polydimethylsiloxane material, a collagen coating along the flow path, a fibronectin coating along the flow path, or a combination thereof. Alternatively, in one embodiment, the acoustic control apparatus 101 is devoid of the microchannel(s) 1 15 and operates as a static well for analytical purposes.

[0060] The acoustic control apparatus 101 is fabricated by any suitable fabrication process. One suitable process includes depositing metal on a photoresist-patterned substrate to produce a metal deposit, performing a lift-off process on the metal deposit to produce one or more interdigital transducer arrangements, aligning one or more of the microfluidic channels 1 15 with the first interdigital transducer arrangement 103 and the second interdigital transducer arrangement 107 as an assembly, and maintaining the assembly at a temperature range of between 0°C and 90°C (for example, between 10°C and 90°C, between 20°C and 90°C, between 30°C and 90°C, between 10°C and 70°C, between 20°C and 70°C, between 30°C and 70°C, between 30°C and 40°C, between 35°C and 40°C, or any suitable combination, sub- combination, range, or subrange therein) and/or for a duration of at least 1 second (for example, at least 12 hours, at least 24 hours, between 1 second and 4 weeks, between 1 day and 3 weeks, between 1 day and 3 days, between 1 hour and 2 days, between 12 hours and 2 days, or any suitable combination, subcombination, range, or sub-range therein) to produce the acoustic control apparatus 101.

[0061] The microfluidic channel(s) 1 15 are capable of being produced using standard lithography and PDMS mold replication methods. In a further embodiment, the collagen coating and/or the fibronectin coating is/are applied, for example, with 1 mg/mL collagen type 1 or fibronectin overnight in a 37°C cell culture incubator, prior to rinsing with phosphate buffered saline (PBS buffer) before each use of the acoustic control apparatus 101.

[0062] The acoustic control apparatus 101 is capable of being used for separating and/or controlling the position of particles and/or fluids. In one embodiment, the acoustic control apparatus 101 is used in a biocompatible manner, such as, by modulating cell-cell interactions or interactions between cells and other objects (such as other cells, solid substrates, matrices, viruses, bacteria, or fluorescent beads), without the interference of cell-surface interactions. Other illustrative examples of a particle include solid or hollow particles, and unicellular or multicellular organisms. More specific examples include cells, platelets, micelles, liposomes, illustratively phospholipid vesicles or other vesicles, bacteria, viruses, fluorescent beads, fungal spores, pollen, dust, cell fragments illustratively a nucleus or a mitochondria, and non-biological particles. In one embodiment, the acoustic control apparatus 101 is used to explore gap junction based intercellular communication and/or functional intercellular communication, for example, by visualizing dye coupling between cells.

[0063] Communication between cells is generally achieved through soluble factors (for example, paracrine, autocrine, and/or exosomes) or through contact such as gap junctions. The control of intercellular distance for regulating these processes, as communication through soluble factors is highly dependent upon the distance between cells, and gap junction based communication occurs when cells are in contact with one another. Considering this, study of these processes is dependent upon the precise spatial control of cell assemblies. In one embodiment, the acoustic control apparatus 101 is able to control both cell-cell distance and cell arrangement and employs the formation of isolated pressure nodes with tunable pressure gradients.

[0064] In one embodiment, the acoustic control apparatus 101 enables the label-free study of the correlation between cell-cell adhesion and cell-matrix adhesion (for example, the cross talk between cadherin and integrin).

EXAMPLES

[0065] For experimental setup, the SAW microfluidic device is placed inside of a customized stage cell culture chamber (INUBTFP-WSKM-GM2000A, Prior Scientific). A solution of cells is injected into the device using a syringe pump (KDS210, KD Scientific). Two independently controllable AC signals generated by a function generator (3102C, AFG) and amplified by two amplifiers (25A100A, Amplifier Research) are connected to two pairs of interdigital transducer arrangements to generate two sets of orthogonal propagated standing SAWs. The power of the applied SAW is maintained at a range from 10 to 40 mW (working area of 5.8 cm ). [0066] When necessary, cell culture conditions inside the SAW microfluidic device are maintained by a custom environmental chamber. Humidified air with 5% C0₂ is continuously flowing into the incubation chamber throughout the processes, and humidity saturation is maintained by evaporation of deionized water inside the chamber. The temperature is maintained at 37 °C with an integrated temperature control system. The cells are kept inside the sealed incubation chamber at a stable 37°C and 5% C0₂ condition.

[0067] Data acquisition is performed by microscopy. The incubator chamber with the SAW microfluidic devices is mounted onto an inverted microscope (Nikon TE2000U). Images (phase contrast and fluorescence) are acquired with a 10X objective and a charge-coupled device (CCD) camera (CoolSNAP HQ2, Photometries, Tucson, AZ) connected to a computer. To remove the double image effect from the lithium niobate substrate, a polarizer is placed on the top. All of the Images are analyzed with the Image J software package (U.S. National Institutes of Health) to characterize the intercellular distance.

[0068] Once the array of pressure nodes is established as described above, two cells are located within the range of four pressure antinodes (150 μιη x 150 μιη area), both of them are pushed to move towards the same point (pressure node). Thus, by controlling the movement towards the pressure node, the distance between two cells is controlled.

[0069] To achieve precise control over intercellular distance, the movement of cells is stopped immediately upon removal of the acoustic field. This is first tested with 10 μηι polystyrene beads. To attain a greater degree of control, particles are first aligned to a line with random distance by only turning on one pair of IDTs, thus creating standing waves aligned in one direction only. Once particles are stable, a modulated RF signal is applied in the orthogonal direction by activating the orthogonal pair of IDTs in order to push cells towards one another. The modulated signal is set to a pulse signal with a 500-millisecond duration and a 2-second interval. The whole process is then recorded and analyzed to study the movement process. When plotted, the pattern of movement shows a clear step-like shape that matches the period of the modulated input signal. The results indicate that the movement of particles is fully controlled by the input signals. This demonstrates tuning of the distance between two objects with sub-micron resolution. [0070] This technique is further demonstrated by controlling the intercellular distance of HEK 293T cells. Two distinct states, direct contact and non-contact with a small distance, in the context of forming cell-cell junctions for intercellular communication studies are analyzed. Gap junction based intercellular communication (GJIC) involves direct contact between cells, while communication that relies upon soluble factors is highly dependent upon the distance between the cells sending and receiving the signals. The generation of these two states is important in order to isolate effects from the two types of intercellular communication. FIG. 5 demonstrates that the distance between cells is capable of being controlled using the manner described above, with blue and red color indicating low and high acoustic potential, respectively, and arrows showing the direction of radiation force. Two HEK 293T cells are able to be positioned at any distance smaller than their initial distance. FIG. 6 shows two HEK cells with different intercellular distances of 20, 15, 5, and 0 μιη, respectively.

[0071] Functional gap junction communication is then probed using the gap junction permeable fluorescent dye, Calcein-AM. Cells stained with Calcein-Am are mixed with unstained cells at a ratio of 1 : 1. Although not intending to be bound by theory, the efficiencies of forming two cell pairs are believed to be dependent on the loading concentration. In this experiment, the concentration is approximately 5x10⁵ cells/mL. Cells are loaded into the microfluidic channel using a syringe and the SAW field is used to position cells either to be in direct contact or separated by a small distance. Once cells are moved to the desired positions, the SAW field is removed and cells are maintained in cell culture medium at 37°C and 5% C0₂ environment. FIG. 7 illustrates that when the cells are in direct contact, fluorescence dye can be transferred to the neighboring cell after one hour, indicating the formation of functional gap junctions. As shown in FIG. 8, when cells are separated, even by a very small distance (for example, 3 μιη), no transfer of fluorescence is observed after the same period of time.

[0072] In addition to enabling the control of intercellular distance, the well-defined acoustic pressure nodes created by the acoustic control apparatus 101 are also found to be suitable for manipulating a group of cells in order to form different geometric configurations. When a group of cells is present within the confines of the interference pattern 1 13, they are manipulated in concert and assembled into defined patterns using the acoustic radiation force. The interference pattern 1 13 is determined to be highly tunable in terms of size and shape as indicated in both simulation and experimental results of FIGS. 9-12, which show acoustic potential distribution with different acoustic amplitudes corresponding with different geometries defined by an acoustic potential boundary 901.

[0073] When different input powers and frequencies (10 mW and 13.45 MHz, 30 mW and 13.35 MHz, respectively) are applied to the two orthogonal interdigital transducer arrangements, a rectangular shaped acoustic well is generated as shown in FIG. 9. Using this shaped acoustic well, cell chains with a cell number of two (FIG. 13), three (FIG. 14), four (FIG. 15), and five (FIG. 16) are formed by controlling the concentration of HeLa cells loaded into the device.

[0074] The direction of the rectangle interference pattern 1 13 is re-oriented by 90°, as shown in FIG. 10, by switching the input powers of the interdigital transducer arrangements (30 mW and 13.45 MHz, 10 mW and 13.35 MHz, respectively). Similarly, the same amplitude (20 mW) is applied in both directions, forming a square-shaped or circular-shaped interference pattern (see FIGS. 1 1 and 12), thereby assembling single-layer clusters as shown in FIGS. 17-20, illustrating capabilities of reorienting, for example, to produce spherically and/or single layer cell assemblies.

[0075] The size of the interference pattern 1 13 is decreased by increasing the input power (30 mW) as shown in FIG. 12, forming three-dimensional cell spheres as a result. Cells present in the acoustic field tend to be held at a fixed distance above the substrate by the acoustic radiation force and acoustic streaming induced hydrodynamic force. As a result, the acoustic control apparatus 101 is found to be capable of maintaining cell assemblies in suspension.

[0076] In addition to the ability to manipulate and pattern cells, the acoustic control apparatus 101 is used to initiate and investigate functional GJIC. HEK 293T cells are known to form gap junctions when they are in contact and can exhibit vivid dye coupling properties. To examine whether these cells can form functional gap junction channels in suspension state, Calcein-AM stained HEK 293T cells and unstained cells are combined at a ratio of 1 :2 to 1 :4 and then loaded into the acoustic control apparatus 101. After applying the SAW field, the cells are patterned into linear arrays and maintained in culture medium for the entire duration of the experimental period with the acoustic field as depicted in FIG. 21. [0077] After 30 minutes of initial incubation, bright field and fluorescent images of suspended cell assemblies are recorded every 5 minutes. Vivid dye coupling from donor cells to receiver cells is observed in all the linear arrays regardless of cell number. The larger the cell numbers, the longer it took to observe evident dye coupling at the terminal cells, as is shown in FIGS. 22- 24. For example, as shown in FIG. 22, bright-field image and time-lapse fluorescence images of two cells trapped within an acoustic pressure node, with the left cell being preloaded with Calcein-AM dye result in dye transfer between the two cells over time. As shown in FIG. 23, bright-field image and time-lapse fluorescence images of a three-cell system trapped by an acoustic pressure node, with the left cell being preloaded with Calcein-AM dye result in transfer of fluorescent molecules throughout the cell assembly. As shown in FIG. 24, bright-field image and time-lapse fluorescence images of a linear cell assembly trapped by a linear acoustic pressure node, result in dye molecules being transferred sequentially because of the defined linear assembly.

[0078] If the cells are patterned in a linear array, their communication (as observed by the transfer of dye) occurs linearly. If cells are patterned in a cluster, their communication format is changed as well. As shown in FIG. 25, after tuning the acoustic well to assemble cells into a cluster, multiple cells receive the signal simultaneously from the donor cell, as shown by bright- field image and time-lapse fluorescence images of a two-dimensional, multiple-cell system trapped by a spherically shaped acoustic pressure node, allowing dye transfer to occur with all neighboring cells simultaneously. As a control, gap junction inhibitor 18, a-glycyrrhetinic acid, is used to exclude the possibility of dye leakage. Collectively, these data demonstrate that HEK 293T cells are capable of forming functional gap junction channels in suspension, without the need for adhesion to a substrate. The acoustic control apparatus 101, therefore, provides a simple and rapid way to examine the formation and function of gap junction mediated intercellular communication in suspended cultures. Arranging cells with defined number and connection also simplifies the model for quantitative characterization of GJIC (for example, gap junction permeability). Moreover, the acoustic control apparatus 101 is capable of controlled patterning of cells, enabling the study of intercellular communication within groups of cells with varied architectures (for example, linear vs. sphere). [0079] In another experiment, a linear pattern of HEK 293T cells is created using a method corresponding to FIG. 26, which schematically depicts the acoustic control apparatus 101 with linear assemblies of cells formed under the control of a tunable acoustic well, whereby upon removal of the acoustic field, the cells drop to the surface and attach. When the SAW is present, the linear cell assembly is maintained in suspension for 1 hour due to the combination of acoustic radiation force and acoustic streaming induced hydrodynamic force. During this period, only cell-cell adhesion occurs, despite the presence of a receptive surface coated with collagen to facilitate cell attachment (see FIG. 27). After 1 hour, the SAW is removed, which allows the cells to drop to the surface and attach to form a cell-matrix interaction. After 40 minutes, HEK 293T cells show proper adhesion and expansion morphology on the surface.

[0080] After assembling cells in suspension, the geometric configuration of cell assemblies is translated to the surface as cells became adherent. To exploit the capability of this method for the study of intercellular communication under adherent conditions, the distinct gap junction based dye coupling properties of hTERT-HMVEC (Human microvascular endothelial cells, CRL- 4205) and HeLa S3 (CCL-2.2) cell lines is investigated. Endothelial cells are known to express gap junction proteins (e.g., Cx43) and allow small molecules to pass through the gap junction channels.

[0081] Calcein-AM stained HMVEC cells are mixed with unstained HMVEC cells at a ratio of 1 :4 and loaded into the microchannel. A one-dimensional standing SAW field is formed as described in the previous section and these HMVEC cells are patterned into linear assemblies. Once the linear pattern is stable, the SAW field is removed to allow cells to settle down and attach to the surface. FIG. 28 shows that HMVEC cells start to spread 15 minutes after settling down. After 25 minutes, most of the cells attach and spread on the surface while the geometric configuration is still maintained. After all the cells become adherent to the substrate, time-lapse fluorescence images are taken to monitor the transfer of fluorescence molecules from stained (donor) cells to unstained (receiver) cells shown in FIG. 29. As soon as 20 minutes after beginning measurements, trace amounts of fluorescence increase are detected in the receiver cells. At 60 minutes, the fluorescence intensity in receiver cells is much more evident, indicating that significant dye transfer occurs between donor and receiver cells. [0082] To further confirm this increase of fluorescence intensity is a result of gap junction based dye transfer and not due to dye leakage, a similar experiment using the HeLa S3 cell line is performed. This HeLa cell line is reported to be incapable of expressing connexins and lacks the ability to exhibit dye transfer. As shown in FIG. 30, HeLa S3 cells show similar attachment and spread dynamics after patterning in suspension. However, dye coupling between adherent HeLa S3 cells is not observed even 2 hours after they became completely adherent, as shown in FIG. 31.

[0083] The following publications are incorporated herein by reference in their entirety: Hyafil, F., Babinet, C. & Jacob, F. Cell-cell interactions in early embryogenesis: a molecular approach to the role of calcium. Cell 26, 447-54 (1981); Streuli, C.H., Bailey, N. & Bissell, M.J. Control of mammary epithelial differentiation: basement membrane induces tissue-specific gene expression in the absence of cell-cell interaction and morphological polarity. The Journal of cell biology 115, 1383-95 (1991); Stetler-stevenson, W.G., Aznavoorian, S. & Liotta, A. Tumor cell interactions with the extracellular matrix during invasion and metastasis. Annual review of cell biology 9, 541-573 (1993); Manz, B.N. & Groves, J.T. Spatial organization and signal transduction at intercellular junctions. Nature reviews. Molecular cell biology 11 , 342-52 (2010); Ablasser, A. et al. Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP. Nature 503, 530-4 (2013); Kaizuka, Y., Douglass, A.D., Varma, R., Dustin, M.L. & Vale, R.D. Mechanisms for segregating T cell receptor and adhesion molecules during immunological synapse formation in Jurkat T cells. Proceedings of the National Academy of Sciences of the United States of America 104, 20296-301 (2007); Brownlee, M. Biology of diabetic complications. Nature 414, 813-820 (2001); El-Ali, J., Sorger, P.K. & Jensen, K.F. Cells on chips. Nature 442, 403-11 (2006); Guo, F. et al. Probing cell-cell communication with microfluidic devices. Lab on a chip 13, 3152-62 (2013); Zhang, H. & Liu, K.-K. Optical tweezers for single cells. Journal of the Royal Society, Interface / the Royal Society 5, 671-90 (2008); Voldman, J. Electrical forces for microscale cell manipulation. Annual review of biomedical engineering 8, 425-54 (2006); Pan, Y., Du, X., Zhao, F. & Xu, B. Magnetic nanoparticles for the manipulation of proteins and cells. Chemical Society reviews 41, 2912-42 (2012); Di Carlo, D., Wu, L.Y. & Lee, L.P. Dynamic single cell culture array. Lab on a chip 6, 1445-9 (2006); Suri, S. et al. Micro fluidic-based patterning of embryonic stem cells for in vitro development studies. Lab on a chip 13, 4617-24 (2013); Skelley, A., Kirak, O., Suh, H., Jaenisch, R. & Voldman, J. Microfluidic control of cell pairing and fusion. Nature methods 6, 147-52 (2009); Thery, M. Micropatterning as a tool to decipher cell morphogenesis and functions. Journal of cell science 123, 4201-13 (2010); Yun, H., Kim, K. & Lee, W.G. Cell manipulation in microfluidics. Biofabri cation 5, 022001 (2013); Laurell, T., Petersson, F. & Nilsson, A. Chip integrated strategies for acoustic separation and manipulation of cells and particles. Chemical Society reviews 36, 492-506 (2007); Bazou, D. et al. Gap junctional intercellular communication and cytoskeletal organization in chondrocytes in suspension in an ultrasound trap. 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[0084] While the invention has been described with reference to one or more embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed as the best mode contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. In addition, all numerical values identified in the detailed description shall be interpreted as though the precise and approximate values are both expressly identified.

Claims

Claims What is claimed is:

1. An acoustic control apparatus, comprising:

a first interdigital transducer arrangement positioned to direct a first acoustic wave;

a second interdigital transducer arrangement positioned to direct a second acoustic wave in a non-parallel direction relative to the first acoustic wave; and

a manipulation region at least partially defined by an interference pattern at least partially formed by interaction between the first acoustic wave and the second acoustic wave.

2. The acoustic control apparatus of claim 1, wherein the manipulation region includes at least two cells arranged for monitoring of cell-cell interactions.

3. The acoustic control apparatus of claim 1, comprising one or more microfluidic channels extending through the manipulation region.

4. The acoustic control apparatus of claim 1, wherein the interference pattern includes a pressure gradient capable of directing cells into the middle of the manipulation region.

5. The acoustic control apparatus of claim 1, wherein the first interdigital transducer arrangement is positioned orthogonal to the second interdigital transducer arrangement.

6. The acoustic control apparatus of claim 1, wherein the first interdigital transducer arrangement has a first input frequency or phase and the second interdigital transducer arrangement has a second input frequency or phase, the first input frequency or phase differing from the second input frequency or phase.

7. The acoustic control apparatus of claim 1, wherein the interference pattern is a net-like array of two-dimensional pressure nodes or a dot-array like pattern of pressure nodes.

8. The acoustic control apparatus of claim 1, wherein the first interdigital transducer arrangement and the second interdigital transducer arrangement are on a lithium niobate piezoelectric substrate.

9. The acoustic control apparatus of claim 1, wherein the first interdigital transducer arrangement and the second interdigital transducer arrangement are on a 128° cut piezoelectric substrate.

10. The acoustic control apparatus of claim 1, wherein the first interdigital transducer arrangement and the second interdigital transducer arrangement are on a piezoelectric substrate with a 45° angle to the X-direction.

11. The acoustic control apparatus of claim 1 , wherein one or both of the first interdigital transducer arrangement and the second interdigital transducer arrangement include a metal deposit on a photoresist-patterned substrate.

12. The acoustic control apparatus of claim 11, wherein the metal deposit includes titanium and gold.

13. The acoustic control apparatus of claim 12, wherein the titanium has a thickness of between 1 nanometers and 10 nanometers.

14. The acoustic control apparatus of claim 12, wherein the gold has a thickness of between 20 nanometers and 300 nanometers.

15. The acoustic control apparatus of claim 1 , wherein at least one of the one or more microfluidic channels includes a polydimethylsiloxane material.

16. The acoustic control apparatus of claim 1, wherein at least one of the one or more microfluidic channels includes a collagen coating.

17. The acoustic control apparatus of claim 1 , wherein at least one of the one or more microfluidic channels includes a fibronectin coating.

18. The acoustic control apparatus of claim 1, wherein one or both of the first interdigital transducer arrangement and the second interdigital transducer arrangement include electrodes with spacing gaps of between 50 micrometers and 300 micrometers.

19. An acoustic control process, comprising:

directing a first surface acoustic wave generated from a first transducer arrangement; directing a second surface acoustic wave generated from a second transducer arrangement in a non-parallel direction relative to the first surface acoustic wave; at least partially defining a manipulation region by an interference pattern formed by the first surface acoustic wave and the second surface acoustic wave.

An acoustic control apparatus fabrication process, comprising:

depositing metal on a photoresist-patterned substrate to produce a metal deposit; performing a lift-off process on the metal deposit to produce one or more interdigital transducer arrangements;

aligning one or more microfluidic channels with the one or more interdigital transducer arrangements to produce an assembly; and

maintaining the assembly at a temperature range of between 0°C and 90°C to produce the acoustic control apparatus.