JP2019520826A - High Throughput Microbiology Applications High Resolution Systems, Kits, Devices, and Methods Using Combined Media Strategies - Google Patents

Abstract

A method for selecting a medium is provided. The method includes the step of obtaining a microfabricated device including a plurality of microwells, a plurality of different media including a plurality of different media such that each microwell of the plurality of microwells includes a medium and the plurality of microwells includes a plurality of different media. Loading into the microwells, filling at least one cell from the sample into each microwell of the plurality of microwells, culturing the microfabricated device for a predetermined time under predetermined conditions, a plurality of microwells Comparing the contents of the plurality of microwells across and determining at least one of a plurality of different media based on the comparison.

Description

  This application is a continuation-in-part of US application Ser. No. 15 / 135,377 filed Apr. 21, 2016 and priority of US Provisional Patent Application Ser. Claim the priority of which is incorporated herein by reference in its entirety.

  The present invention relates generally to invention innovations in microbiology, microfabrication, chemistry, optics, robotics and information technology. More specifically, the present invention relates to systems, devices, kits and methods for high throughput culture, sorting, isolation, sampling and / or identification of biological entities and / or nutrients.

  The conventional techniques and means for cultivating biological entities from the natural environment and other samples are often slow, laborious, time consuming and expensive. Even with these prior art techniques and tools, often cells and other biological entities will still refuse any attempt to culture and lose the opportunity to obtain information and / or products. Similarly, it is more difficult to select a group of biological entities for specific metabolites, enzymes, proteins, nucleic acids, phenotypes, mutations, metabolic pathways, genes, fitness, performance and / or therapeutic benefit. Need a complicated, effective way. For example, microorganisms live in a very dangerous environment. In order to survive, microbes come from a surprising set of biochemical tools, including novel enzymes, distinctive metabolites, innovative genetic pathways, and manipulation strategies to the environment and adjacent microbes, ie new insights and life-saving antibiotics. We have created powerful solutions that have led to products that extend to fertilizers that improve food production and safety.

  The present invention provides microbiology systems, devices, kits and methods for streamlining culture operations, assisting in high-throughput sorting, and / or creating new ills and products according to embodiments. For example, the device can include a microfabricated device that contains a sample that includes one or more cells. The microfabricated device defines a high density array of one or more cell culture microwells.

  In embodiments, a method of selecting a medium is provided. The method obtains a microfabricated device comprising a plurality of microwells, wherein each microwell of the plurality of microwells comprises a medium and a plurality of microwells comprises a plurality of different media. The microfabricated device is cultured in a predetermined state for a predetermined time, and the contents of the plurality of microwells are compared across the plurality of microwells based on the comparison. Determine at least one of the plurality of different media.

  In embodiments, a method of selecting a medium using a microfabricated device comprising a plurality of microwells is provided, wherein the microfabricated device comprises a plurality of different cells loaded across the plurality of microwells. In this method, at least one cell is loaded from a sample into a plurality of microwells, and the microfabricated device is cultured in a predetermined state for a predetermined time, and the plurality of microwells are traversed across the plurality of microwells. The contents of are compared, and at least one of the plurality of different media is determined based on the comparison.

  In embodiments, a method of selecting a medium using a microfabricated device comprising a plurality of microwells is provided, wherein the microfabricated device comprises at least one cell loaded in each of the plurality of microwells. In this method, a plurality of different media are cultured across a plurality of microwells, and the microfabricated device is cultured under a predetermined condition at a predetermined time, and among the plurality of different media based on the comparison, Determine at least one medium.

  In embodiments, the provided method obtains a microfabricated device comprising a plurality of microwells, wherein each microwell of the plurality of microwells comprises a medium and the plurality of microwells comprises a plurality of different media As such, a plurality of different media are loaded into the plurality of microwells, a biological entity of interest (eg, a cell) is loaded into each microwell of the plurality of microwells, and the plurality of different media based on the comparison. Determine at least one of

  In embodiments of the above-described method, a droplet printer may be used to fill the media in each microwell. Similarly, a droplet printer can be used to load each microwell with at least one cell. Alternatively, each microwell can be filled with at least one cell using a pipette. Comparing the contents of the plurality of microwells also includes evaluating at least one observable property of the contents of the plurality of microwells. In some embodiments, comparing the contents of the plurality of microwells comprises performing digital image analysis of an image of the contents of the plurality of microwells. The loading of the medium can be performed before or after loading the at least one cell. Filling the at least one cell may comprise filling a plurality of cells in each of the microwells.

  A plurality of different media may be loaded into the plurality of microwells, including loading a plurality of different media into different regions of the microwell on the microfabrication apparatus, each of the different regions including one or more microwells Each separate region is the same, and the media contained in the microwells of different regions are different. In some embodiments, comparing the contents of the plurality of microwells comprises comparing statistics of properties of the contents in the microwells of each separate region.

  Multiple microwells can be sealed prior to culturing the microfabricated device. In some embodiments, multiple microwells can be opened to evaporate liquid prior to comparing the contents of multiple microwells.

  In some embodiments, comparing the contents of the plurality of microwells may include evaluating at least one property of the contents at a plurality of time points.

  In some embodiments, the medium loaded in at least one of the plurality of microwells comprises an antibiotic.

  In some embodiments, a kit is provided that includes a microfabricated device that includes a plurality of microwells. Each of the plurality of microwells is configured to include a plurality of different media, and a plurality of different media can be filled into separate regions of the microwell on the microfabricated device. In such a kit, a plurality of different media may be loaded into separate areas of the microwell on the microfabricated device, each separate area constituting a plurality of microwells, the microwell within each separate area The media of are the same, and the microwells of different regions are different.

In some embodiments of the methods or kits described herein, the surface density of the plurality of micro-wells of the microfabricated device is at least 150 per micro well cm ^2, at least 250 microwells per cm ^2, cm ² per at least 400 microwell, at least 500 microwells per cm ^2, at least 750 microwells per cm ^2, at least 1,000 microwells per cm ^2, at least 2,500 microwells per cm ^2, at least 5,000 microwells per cm ^2, cm ² per least 10,000 microwell , at least 50,000 per micro well cm ^2, at least 100,000 microwells per cm ^2, there may be at least 160,000 per micro well 1 cm ^2. In some embodiments of the methods or kits described herein, each microwell of the plurality of microwells of the microfabricated device is about 5 μm to about 500 μm, about 10 μm to about 300 μm, m or about 20 μm to about 20 μm to about It may be at a diameter of 200 μm.

  Other systems, processes and features will be apparent to one of ordinary skill in the art upon review of the following drawings and detailed description. It is intended that all such additional systems, processes and features be included within the description of the present invention, be within the scope of the present invention, and be protected by the accompanying claims.

  The skilled artisan will appreciate that the drawings are primarily for illustrative purposes and are not intended to limit the scope of the inventive subject matter described herein. The drawings are not necessarily to scale, and in some instances the various aspects of the inventive subject matter disclosed herein may be exaggerated or enlarged in the drawings to facilitate an understanding of the different features. In the drawings, the same reference signs denote generally the same features (e.g. functionally similar and / or structurally similar components).

  FIG. 1 is a perspective view illustrating a microfabricated device or chip in accordance with several embodiments.

  2A-2C are top, side and end views, respectively, illustrating the dimensions of a microfabricated device or chip in accordance with several embodiments.

  3A and 3B are an exploded view and a top view, respectively, illustrating a microfabricated device or chip according to several embodiments.

  4A and 4B are illustrations of membranes according to multiple embodiments. FIG. 4C is an image of a thin film surface from which traces formed from contacts with the array of wells in some embodiments can be seen.

  FIG. 5A is an operational step diagram illustrating a method of isolating several cells from a sample according to embodiments. FIG. 5B is an illustration of a method of isolating several cells from a soil sample according to embodiments.

  FIG. 6 is an operational step diagram illustrating a method of isolating and culturing several cells from a sample according to embodiments.

  FIG. 7 is an illustration of a method of isolating and culturing several cells from a complex sample according to embodiments.

  8A-8C are illustrations of sampling with a single pin or multiple pins in accordance with multiple embodiments.

  9A-9D are images showing the selection of one well according to embodiments.

  10A-10D are illustrations of tools for harvesting from one chip in accordance with multiple embodiments.

  FIG. 11 is an image of one well pierced with a thin layer of agar, which graphically illustrates piercing the membrane or sealing layer according to embodiments.

  FIG. 12 is a diagram depicting a cross section of a tip 1200 in accordance with multiple embodiments.

  FIG. 13 is an operation step diagram illustrating a sorting method according to a plurality of embodiments.

  FIG. 14 is a chart illustrating a sorting method in accordance with several embodiments.

  FIG. 15 is a series of images illustrating one screening example in accordance with embodiments.

  16A-16C are images illustrating recovery from one shield in accordance with embodiments.

  FIG. 17A is an exploded view illustrating a sorting chip in accordance with several embodiments. FIG. 17B is a fluorescence image of the chip following sorting according to embodiments. FIG. 17C is an image showing one step of taking a sample from the chip following sorting according to embodiments.

  FIG. 18 is an operation step diagram illustrating a counting method according to a plurality of embodiments.

  FIG. 19 is a chart diagram illustrating a counting method in accordance with several embodiments.

  FIG. 20 is a diagram illustrating an indexing method in accordance with several embodiments.

  21A-21E are summary diagrams depicting a chip with well-specific chemistry according to embodiments.

  22A and 22B are images of different media printed on different microwells of a microfabricated chip in some embodiments.

  FIG. 23A shows microwells on a microfabricated device divided into three regions, each region comprising 3 × 9 microwells and treated with different media. 23B-23D are microscope images of representative microwells from each region shown in FIG. 23A.

FIG. 24 is a plot of digital image analysis results based on average gray values in some embodiments.
Detailed description

  The present invention generally relates to systems, kits, devices and methods for isolation, culture, adaptation, sampling and / or sorting of biological entities and / or nutrients. Disclosed is a microfabricated device (or "chip") for containing a sample comprising at least one biological entity. The term "biological entity" may include microorganisms, cells, cell components, cell products, viruses and others, and the term "species" is used to describe the unit of classification and is manipulated , Taxonomic unit (OTU), genotype, phylotype, phenotype, ecotype, history, behavior or interaction, products, variants, evolutionarily significant species and others.

  The cells can be archaebacteria, bacteria or eukaryotes (eg, fungi). For example, the cells can be microorganisms such as aerobic, anaerobic or conditional aerobic microorganisms. The virus may be a bacteriophage. Other cellular components / products include proteins, amino acids, enzymes, saccharides, adenosine triphosphate (ATP), lipids, nucleic acids (eg, DNA and RNA), nucleosides, nucleotides, cell membranes / walls, flagella, pili, organelles, It may contain metabolites, vitamins, hormones, neurotransmitters, antibodies and others.

  Nutrients may be defined (eg, chemically defined or synthetic media) or undefined (eg, basal or complex media). The nutrient may comprise or be a component of a laboratory formulated and / or commercially produced culture medium (eg, a mixture of two or more chemicals). The nutrient may be a liquid nutrient medium (i.e., a nutrient soup) or one component thereof, such as, for example, marine broth, lysogenic broth (e.g., Luria broth), and the like. The nutrient may be a commercially available agar plate such as liquid medium and / or blood agar medium mixed with agar to form a solid medium or may be one component thereof.

  The nutrients may comprise a selective medium or be one component thereof. For example, selective media can be used for the growth of only certain biological entities or only biological entities having particular properties (eg, antibiotic resistance or specific metabolite synthesis). Nutrients utilize biological properties in the presence of specificity indicators (eg neutral red, phenol red, eosin Y or methylene blue) to make one type of biological entity another type of biological entity Alternatively, it may contain or be a component of a differentiation medium that distinguishes it from other biological entities.

  The nutrient may comprise an extract of the natural environment or a medium derived from the natural environment or may be a component thereof. For example, nutrients can be derived from a natural environment, another environment or multiple environments for a particular type of biological entity. The environment may be one or more biological tissues (eg, connective tissue, muscles, nerves, epithelia, plant epidermis, vascular bundles, soil, etc.), biological fluids or other biological products (eg, amniotic fluid, bile, blood, cerebrospinal fluid). Fluid, earwax, exudates, feces, gastric juice, interstitial fluid, intracellular fluid, lymph fluid, milk, nasal fluid, rumen contents, saliva, sebum, semen, sweat, urine, vaginal secretion, feces etc)) Suspensions, air (eg with various gas contents), supercritical carbon dioxide, soil (eg with minerals, organics, gases, liquids, microorganisms etc.), sediment (eg agriculture, oceans Sediments, etc.), living organic matter (eg, plants, insects, other small organisms and microorganisms), dead organic matter, equine grass (eg, grass, leguminous plants, stored grass, crop residues etc.), minerals, Oil or oil products (eg, animals, plants, petrochemicals May include water (eg, natural fresh water, drinking water, sea water etc.) and / or sewage (eg. Hygiene, commercial, industrial and / or agricultural wastewater, and surface effluents) and others .

The microfabricated device may define a high density array of culture microwells of at least one biological entity. The term "high density" may mean the ability of a system or method to allocate many experiments within a given area. For example, microfabricated device comprising an experimental unit of "high density" is used herein as described further, may incorporate cm ² per about 160,000 or more eye black wells from per cm ² to about 150 micro-wells. Additional examples are shown in Table 1.

  The microfabricated device may incorporate a substrate with a series of functional layers. The series of functional layers defines a first functional layer defining a first array (eg, well) of experimental units, and a next array (eg, microwell) of experimental units within each experimental unit of a preceding functional layer And at least one subsequent functional layer. Each of the experimental units may be configured to contain and culture and / or sort out biological entities and / or nutrients. In particular, the systems, kits, devices and methods described herein may be used for automated and / or high throughput screening of various conditions on high density matrices of cells. For example, the systems, kits, devices and methods described herein examine and compare the effects of one or more different nutrients on microbial growth and / or test for metabolites, enzyme activities, mutations or other cell functions Can be used to

  FIG. 1 is a perspective view illustrating a microfabricated device or chip according to several embodiments. The tip 100 incorporates a substrate shaped in the form of a microscope slide glass with an injection molding mechanism on the upper surface 102. The mechanisms are, in addition to the ejection tags 106, four separate microwell arrays (or microarrays) or the like. The microwells in each of the microarrays are arranged in a grid pattern with a well-free edge around the periphery of the chip 100 and between the microarrays 104.

  FIGS. 2A-2C are top, side and end views, respectively, showing the dimensions of a chip 100 in accordance with several embodiments. In FIG. 2A, the top surface of the chip 100 is approximately 25.5 mm × 75.5 mm. In FIG. 2B, the end face of the chip 100 is approximately 25.5 mm × 0.8 mm. In FIG. 2C, the side of the chip 100 is approximately 75.5 mm × 0.8 mm.

  After loading the sample into the microfabricated device, a membrane may be applied to at least a portion of the device. FIG. 3A is an exploded view of microfabricated device 300 as viewed from the top of FIG. 3B in accordance with embodiments. The device 300 incorporates a chip with a well array 302 containing, for example, soil microorganisms. A membrane 304 is placed on top of the well array 302. A gasket 306 is placed on top of the membrane 304. A polycarbonate cover 308 with a fill hole 310 is placed on top of the gasket 306. Finally, the sealing tape 312 is attached to the cover 308.

  The membrane can cover at least a portion of a microfabricated device incorporating one or more experimental units, wells or microwells. For example, after loading a sample into a microfabricated device, at least one membrane may be affixed to at least one microwell of a microwell high density array. A plurality of films can be attached to portions of the microfabricated device. For example, separate membranes can be attached to a plurality of different subsections of a microwell high density array.

  A membrane is attached, attached, partially attached, affixed and sealed to a microfabricated device to hold at least one biological entity in at least one microwell of a microwell high density array And / or may be partially sealed. For example, the membrane may be reversibly affixed to the microfabricated device by lamination. In order to load at least one biological entity into at least one microwell of the microwell high density array, the membrane is punched, peeled off, desorbed, partially desorbed, removed and / or partially removed It can be done.

  One part of the cell population in at least one experimental unit, well or microwell may be attached to the membrane (eg via adsorption etc). When attached, the cell population in the at least one experimental unit, well or microwell is pulled from the membrane while a portion of the cell solid population in the at least one experimental unit, well or microwell remains attached to the membrane. It can be sampled by peeling it off.

  4A and 4B are illustrations of membranes according to multiple embodiments. FIG. 4A shows a side view of a chip 400 defining a content-filled well array and a membrane 402 sealing the chip 400 over the well array, and thus the surface of the membrane 402 in contact with the chip 400. Has an imprint of each of the wells with the well content sample attached thereto (eg, stuck), as shown in FIG. 4B, when peeled from the chip 400. FIG. 4C is an image of a stamped membrane surface formed by contact with a well array in accordance with embodiments.

  The membrane is impermeable, semi-permeable, selectively permeable, differentially permeable, and / or partially permeable to allow diffusion of at least one nutrient into at least one microwell of the microwell high density array Can be sexual. For example, the membrane may be composed of natural and / or synthetic substances. The membrane may incorporate a hydrogel layer and / or a filter paper. In embodiments, a membrane is selected that has pores of a size small enough to retain at least some or all of the cells in the microwell. For mammalian cells, the size of the pores may be several microns and still retain the cells. However, in embodiments, the size of the holes may be about 0.2 μm or less, such as 0.1 μm. Membrane diameter and pore size are up to the material. For example, a hydrophilic polycarbonate membrane may be utilized, for which the diameter may be in the range of about 10 mm to about 3000 mm and the pore size may be in the range of about 0.01 μm to about 30.0 μm. The impermeable membrane has a pore size approximately equal to zero. In embodiments that use an impermeable membrane, all nutrients must be supplied to the microwell prior to sealing with the membrane. Membranes that are gas permeable but not liquid permeable can receive oxygen to the microwells and carbon dioxide from the microwells. The membrane may have a complex structure which may or may not have a limited pore size. However, their pores may be on the order of nanometers. Other factors in selecting a membrane may include cost, sealing performance and / or sterilization performance and the like.

  The substrate may define a microwell array extended from a first surface to a second surface facing the first surface. The microchannel may have a first opening on the first surface and a second opening on the second surface. The first membrane may be affixed to at least a portion of the first surface such that at least some of the cell populations in the at least one microchannel adhere to the first surface. The second removable membrane may be affixed to at least a portion of the second surface such that at least some of the cell populations in the at least one microchannel adhere to the second surface. The group of cell solids in the at least one microchannel is a cell in the first membrane and / or the cell in the at least one microchannel while at least some of the group of cell solids in the at least one microchannel remain attached to the first membrane The at least some of the solid groups can be sampled by peeling off the second membrane while remaining attached to the second membrane.

  The term "high throughput" may mean the ability of a system or method to perform very large numbers of experiments in parallel or in succession. One example of a "high-throughput" system, as described herein, is one or more biological entities comprising at least one metabolite, enzyme, protein, nucleic acid, phenotype, mutation, metabolic pathway, gene, adaptation It may include an automated apparatus equipped with cell biology techniques to prepare, culture and / or perform multiple chemistries, genetics, pharmaceuticals, optics, and / or image analysis to test for type and ability. According to embodiments, "high throughput" may mean that experiments ranging in size from at least about 96 experiments to at least about 10,000,000 experiments are performed simultaneously or nearly simultaneously.

  The systems, devices, kits and methods disclosed herein can be used for high throughput screening of various conditions against matrices of biological entities (eg cells). The “in-well well” concept refers to any level required or desired by fabricating (eg, microfabricating) a substrate or chip to have multiple levels of functional layers (ie, in-well in-well, etc.) ) Can also be realized. The first functional layer can define an experimental unit (eg, well) array. Each experimental unit provides another functional layer by defining the next experimental unit (eg, microwell) array. This allows high throughput operation because multiple experiments or tests can be performed simultaneously on a single chip.

  For example, in FIGS. 3A and 3B, a gasket 306 is placed on top of the membrane 304, which is applied to the well array 302 of the microfabricated device 300 according to several embodiments. The gasket 306 has only one opening. However, in further embodiments, multiple smaller gaskets with one smaller opening or a single gasket with multiple smaller openings are placed on the top of the device (with or without membranes), This can lead to the step of forming a functional layer, or an array of larger experimental units with the next functional layer, or the next experimental unit array (eg, well 302) located there.

  With multiple levels of functional layers, for example, multiple nutrients or nutrient formulations can be tested simultaneously or nearly simultaneously. The same scheme can be used, for example, to test for specific functions of metabolites or cells or to separate microbes from natural environment-derived nutrients into other nutrients.

  The experimental unit is a predetermined portion of the surface of the microfabricated device. For example, the surface of the chip can be designed to immobilize the cells in a first array of predetermined sites. These predetermined sites may be wells, microwells, microchannels and / or designated immobilization sites. For example, the surface may be microfabricated to define a microwell array. The array can be divided into sections by defining walls or additional walls in the substrate. For example, the surface may be microfabricated to first define a first array of wells, where the inner surface of each well defines a second array of microwells, microchannels or immobilization sites. , Are processed in order. In another example, the surface can be processed to define a microwell array, and another substrate (eg, agar, plastic or other substance) can be defined by the surface and thereby It can be applied to the surface as it divides the microwell. Each well, microwell, microchannel and / or immobilization site may be constructed to accommodate and grow at least one cell, but in use any given well, microwell, microchannel or immobilization site In fact, one or more cells may or may not be accommodated and / or expanded. The type of experimental unit may be interchangeable. For example, the embodiments in this document that specifically describe microwells may also disclose embodiments in which the microwells are at least partially replaced by microchannels, immobilization sites and / or other types of experimental units. Also intended.

  One or more portions of the microfabricated device can be selected, treated, and / or coated with a surface chemistry modifier to have particular surface chemistry. For example, at least a portion of the substrate surface may have a first surface property that reduces the tendency to stick cells and / or a tendency to stick to the cell surface or a second that enhances the tendency to draw cells and stick to the cell surface. Can be designed with surface characteristics of Depending on the type of target cell, the substance and / or the coating may be hydrophobic and / or hydrophilic. At least a portion of the upper surface of the substrate may be treated to have a first surface property that does not repel target cells and / or reduce the tendency of target cells to stick to that surface. On the other hand, at least one part of the inner surface of each experimental unit, well or microwell has a second property that attracts target cells and makes them more prone to occupy experimental units, wells or microwells, It may be processed. One surface of the substrate may have multiple portions with different surface properties.

  The surface chemistry modifier can be applied using chemical vapor deposition, electroporation, plasma treatment and / or electrolytic deposition. The surface chemistry modifier can control surface potential, Rund potential, zeta potential, surface morphology, hydrophobicity and / or hydrophilicity. Surface chemistry modifiers may include silanes, polyelectrolytes, metals, polymers, antibodies and / or plasma and the like. For example, the surface chemistry modifier may be octadecyltrichlorosilane or the like. Surface chemistry modifiers are, for example, dynamic copolymers, such as polyoxyethylene (20) sorbitan monolaurate and / or polyethylene glycol p- (1,1,3,3-tetramethylbutyl) -phenyl ether May be included. The surface chemistry modifier may comprise a static copolymer, such as, for example, poloxamer 407, poly (L-lysine) and / or poly (ethylene glycol) -poly (l-lysine) block copolymer.

  An apparatus for sorting various conditions against a matrix of cells may include a substrate having a surface defining a microwell array. Several sections of the microwell array can be divided into subarrays (eg, by larger wells or walls). The substrate can be made by microfabrication. Each microwell can contain and grow a biological entity (eg, a cell). The resulting matrix of biological entities (eg, cells) can be a high density matrix of biological entities. The first array and / or the second array may be planar, generally planar, and / or multi-planar (eg, on a roll).

  The term "high resolution" may mean the ability of the system or method to distinguish a large number of available experiments. For example, a "high resolution" system or method can select one experimental unit having a diameter of about 1 nm to about 800 μm from a microfabricated device containing a high density experimental unit. The substrate of the microfabricated device or chip can incorporate about 10,000,000 or more microwells. For example, the microwell array may include at least 96, at least 1,000, at least 5,000, at least 10,000, at least 100,000, at least 500,000, at least 1,000,000, at least 5,000,000, or 10,000,000 storage locations.

The surface density of the microwells can be from about 500 microwells per cm ² to about 160,000 microwells or more. The substrate microfabricated device or chip, cm ² per at least 150 microwells cm ² per at least 250 microwells cm ² per at least 400 microwells cm ² per at least 500 microwells cm ² per at least 750 micro-wells, per cm ² of at least 1,000 microwells, per cm ² of at least 2,500 microwells, per cm ² of at least 5,000 microwells, per cm ² of at least 7,500 microwells, per cm ² of at least 10,000 micro-wells, per cm ² of at least 50,000 micro-wells, cm ² per can have a microwell surface density of at least 100,000 microwells or per cm ² of at least 160,000 microwells.

  The size of the microwells can range from nanoscale (eg, a diameter of about 1 to about 100 nanometers) to microscale or more. For example, each microwell may have a diameter of about 1 μm to about 800 μm, a diameter of about 25 μm to about 500 μm, or a diameter of about 30 μm to about 100 μm. The microwell may have a diameter of less than about 1 μm or 1 μm, about 5 μm or less than 5 μm, about 10 μm or less than 10 μm, about 25 μm or less than 25 μm, about 50 μm or less than 50 μm, about 100 μm or less than 100 μm or less than about 200 μm or 200 μm or less , Less than about 300 μm or 300 μm, less than about 400 μm or 400 μm, less than about 500 μm or 500 μm, less than about 600 μm or 600 μm, about 700 μm or less than 700 μm, or less than about 800 μm or 800 μm.

  The microwells can have a depth of about 500 μm to about 5000 μm, a depth of about 1 μm to about 500 μm, or a depth of about 25 μm to about 100 μm. The depth of the microwell may be about 1 μm, about 5 μm, about 10 μm, about 25 μm, about 50 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about It may be 1,000 μm, about 1,500 μm, about 2,000 μm, about 3,000 μm, or about 5,000 μm.

  Each microwell has an opening, and the cross section may have several shapes, for example, circular, hexagonal or square. Each microwell may be accompanied by sidewalls. For microwells with non-circular openings or cross-sections, the diameter of the microwells described herein is shown as the effective diameter of a circle with equivalent area. For example, in the case of a square microwell having a side length of 10 × 10 μm, a circle having an equivalent area (100 square μm) has a diameter of 11.3 μm. The sidewalls may have vertical, beveled or curved cross-sectional shapes. At least one unique regiospecific tag is assigned to at least one microwell of the high density macrowell array to identify species and to specific microwells of the high density array of microwells, as described further below. Can easily be correlated. The at least one specific tag may be assigned and / or arranged at the bottom and / or at least one side of the one microwell. The at least one specific tag is a target specific nucleotide sequence for annealing to a target nucleic acid fragment of at least one biological entity and position specificity for identifying at least one microwell of a high density array of microwells A nucleic acid molecule provided with a nucleotide sequence can be incorporated.

  For example, the substrate of the microfabricated device or chip may have a surface that is approximately 4 inches by 4 inches in size. The surface may define an array of approximately 100 million microwells. The microwell array can be divided by walls into about 100 small compartments and / or the substrate can define an array of about 100 wells, ie, about one million microwells defined in each small compartment or well, totaling about It will be 100 million micro wells. For use cases to test different nutrients, microorganisms from natural environmental samples can be loaded onto the chip such that individual microorganisms or microbial clusters are distributed to the microwells on the chip, where each microwell is It is located at the bottom of a large well. Each larger well can incorporate experimental units so that 100 different nutrients can be tested in parallel or sequentially on the same chip, ie each well covers up to 1 million test cases Can.

  The target cells may be archaebacteria, bacteria or eukaryotes (eg, fungi, plants or animals). For example, target cells may be microorganisms such as aerobic, anaerobic and / or conditional aerobic microorganisms. Different nutrients can be tested in parallel or sequentially using the composition of the target cells, for example, to analyze and compare cell population, proliferation and other effects on cell components and / or cell products. The composition of the target cell can be, for example, one or more viruses (eg, bacteriophage), cell surface (eg, cell membrane, cell wall), metabolites, vitamins, hormones, neurotransmitters, antibodies, amino acids, enzymes, proteins, saccharides, ATP It may be tested for cellular components, products, and / or functions, such as lipids, nucleosides, nucleotides, nucleic acids (eg, DNA or RNA), phenotypes, mutations, metabolic pathways, genes and adaptability.

  The composition of cells may comprise natural environment sample extracts and / or dilutions. The natural environment sample extract and / or dilution may include one or more biological tissue (eg, connective tissue, muscle, nerve, epithelium, plant epidermis, vascular bundles, soil, etc.), biological fluid or other biological product (eg, , Amniotic fluid, bile, blood, cerebrospinal fluid, earlobe, exudates, feces, gastric fluid, interstitial fluid, intracellular fluid, lymph fluid, milk, nasal fluid, rumen contents, saliva, sebum, semen, sweat, urine, vaginal secretion , Sediments, etc.), microbial suspensions, air (eg with various gaseous components), supercritical carbon dioxide, soil (eg with minerals, organics, gases, liquids, microbes etc), eg, agriculture , Marine, etc.), living organic matter (eg, plants, insects, other small organisms and microorganisms), dead organic matter, equine grass (eg, grass, legumes, stored grass, crop residues etc.) , Minerals, (eg, animals, plants, petrochemical ) Oil or oil products, alcohol, buffers, organic solvents, water (eg, natural fresh water, drinking water, seawater etc.) and / or sewage (eg, hygiene, commercial, industrial and / or agricultural wastewater, and surface runoff And others may be included.

  One method may include the step of preparing the composition by combining the cell and a natural environment sample extract and / or dilution prior to application (eg, loading) of the composition containing cells to a microfabricated device . The method may further comprise the step of liquefying the natural environment sample extract and / or dilution. The concentration of cells in the composition may be matched to the target distribution of one cell per experimental unit, well or microwell.

  If the sample contains multiple cells or viruses, those cells in the sample may be lysed after being seeded on the microfabricated device to release the nucleic acid molecules. The cells can be lysed using, for example, chemical treatments such as alkaline exposure, detergents, sonication, proteolytic enzyme K or lysozyme exposure. The cells can also be lysed by heating.

  FIG. 5A is an operational step diagram illustrating one method for isolating cells from one sample according to embodiments. At step 500, a sample is obtained. In step 502, the sample is homogenized and / or dispersed using at least one of physical techniques (eg, mixing and / or sonication) and chemical techniques (eg, chelating agents, detergents and / or enzymes). Turn In step 504, the cells in the homogenized and / or dispersed sample are for example by density centrifugation using Nycodenz® non-particulate medium (available from Progenbiotechnic GmbH, Heidelberg, Germany) It is separated.

  FIG. 5B is a chart illustrating a method of isolating cells from one soil sample according to embodiments. Panel 506 displays the soil sample. Panel 508 displays the samples homogenized and / or dispersed in a test tube. Panel 510 displays samples separated into soluble sediment 512, cells 514, insoluble sediment 516 and Nycodenz® 518 after centrifugation.

  FIG. 6 is a working step diagram illustrating a method of isolating and culturing cells from one sample according to embodiments. At step 600, a sample is obtained. At step 602, at least one cell is extracted from the obtained sample. At step 604, at least one high density microwell array of microfabricated devices or chips is loaded with the at least one extracted cell. Step 604 may provide a cell concentration with the at least one extracted cell, incorporate at least one nutrient / media selection, and / or at least one membrane selection. At step 606, at least a portion of the microwell array is sealed by at least one selected membrane to maintain the cell concentration for those microwells. At step 608, the chip is warmed for culture purposes. Step 608 may incorporate temperature selection, determination of atmosphere (eg, aerobic or anaerobic), and / or determination of incubation time. In step 610, the chip is divided and / or replicated (eg, using a picker), resulting in two parts of cells cultured according to the methods described herein. For example, at least one membrane may peel off a portion of the cultured cells with the attached scissors, or it may be peeled off or pierced to extract the cultured cells as a sample. In optional step 612, a portion of the cultured cells are sacrificed for identification. Step 612 may incorporate PCR, sequencing, and / or various data analysis. At step 614, a strain of interest is identified. Further culturing, testing and / or identification may be performed, for example using the strain of interest and / or the remainder of the cultured cells.

  FIG. 7 is a chart illustrating a method of isolating and culturing complex samples according to embodiments. Panel 700 shows an example of a complex sample, in particular a microflora sample 702 and a soil sample 704. In panel 706, at least one cell has been extracted from the sample using, for example, the protocols illustrated in FIGS. 5A and 5B. In panel 708, at least one extracted cell (and any natural environment extract and dilution) is loaded into a microfabricated device or chip having at least one high density microwell array 710. The tip 710 and the reagent cartridge 712 may be inserted into the incubator 714. The reagents can serve to add fluid to maintain the nutritional needs for growth and / or various selection objectives. Panel 716 shows the product, ie an isolated strain of cultured cells.

  Identification of DNA sequencing, nucleic acid hybridization, mass spectrometry, infrared spectroscopy, DNA amplification, genetic factors or other species identifiers to identify the species or taxonomic lineage of cells or microorganisms growing in one microwell Techniques involving antibody binding and others are required. Many identification methods and processing steps destroy the microbes, thus preventing further cultivation and study of the microbes of interest. Positional conservation and microbial individuals across several experimental units, wells or microwells, to allow both cell and microorganism identification while allowing subsequent culture, research and further breeding of clones of specific objects Further embodiments are designed to take samples from each experimental unit, well or microwell across a single substrate or chip while maintaining group separation.

  As mentioned above, the substrate may allow for sampling and examination of the cell population using a number of additional systems, kits, devices and methods. For example, a collection device can be applied to the first surface of the substrate. The device may incorporate at least one protrusion facing the first surface. The at least one protrusion has a diameter smaller than the opening diameter of each microwell, well or experimental unit. The at least one protrusion is a cell population such that a portion of one population of cells in at least one microwell, well or experimental unit adheres to and / or binds to at least one protrusion. Can be inserted into at least one microwell, well or experimental unit that holds The sample of cell population in at least one microwell, well or experimental unit is attached and / or bound to at least one microwell, well or part of the cell population in experimental unit at least one protrusion It may be recovered by removing the removal device from the first surface of the substrate so as to remain. Each protrusion may be a pin or a plurality of pins or pin assemblies.

  8A-8C are illustrations of single pin or multiple pin harvesting according to embodiments. The tip 800 is provided for inspection by the microscope 802 and collection by the collection controller 804. In FIG. 8A, the harvest control device 804 comprises an arm with only one pin 806. In FIG. 8B, an arm with multiple pins 808 is shown. FIG. 8C is a perspective view of the tip in progress of the harvesting step.

  9A-9D are images that are actually selecting one well according to embodiments. In FIG. 9A, the well is full. In FIG. 9B, the pin has been moved into position. In FIG. 9C, the well is selected. In FIG. 9D, a sample has been removed from the well.

  10A-10D are schematic diagrams illustrating a collection tool from a tip according to embodiments. In FIG. 10A, an instrument with multiple pins is aligned to a chip with multiple wells. In FIG. 10B, the device is lowered so that the pins are immersed in the wells. In FIG. 10C, pins with attached samples are shown, which are transferred to a new chip. On the other hand, in FIG. 10D, the device is turned upside down so that the sample is held by the device itself.

  FIG. 11 is an image of a well pierced with an agar thin layer, illustrating a membrane or sealing layer pierce through according to embodiments.

  On the other hand, when at least one protrusion is immersed in at least one microwell, well or experimental unit, a portion of the cell solid group in the at least one microwell, well or experimental unit is at least one protrusion The amount moved up and around the object, at least some of the movement portion being above the first surface of the substrate and / or the inner surface of at least one microwell, well or experimental unit thereof Do. The method also includes the step of taking the sample of cellular solid mass within the at least one microwell by collecting at least some of the mobile mass portion of the cellular solid mass.

  A similar collection device can be applied to the second surface facing the first surface of the substrate. The device may incorporate at least one protrusion facing the second surface. The at least one protrusion has a diameter approximately equal to or smaller than the diameter of the at least one microwell, well or experimental unit. The at least one protrusion is pushed to the second surface at a location corresponding to at least one microwell, well or experimental unit holding a solid population of cells and / or at least one holding a solid population of cells One microwell, a well or experimental unit is inserted into the at least one microwell, well or part of the cell population in the experimental unit on the first surface of the substrate and / or at least one of the microbes Move up the well, well or the inner surface of the experimental unit. The mobile part of the cell population can then be collected. The cell population is placed on at least one experimental unit, a plug in a well or microwell (e.g. a hydrogel or other soft substance such as agar) and at least one protrusion is pushed to the second surface And when it is at least one inserted into at least one microwell, the plug shall be moved, thereby moving part of the cell population.

  Samples of cell populations from at least one experimental unit, well or microwell may be stored at another location. Prior to further sampling, at least one protrusion may be washed and / or sterilized. At least a portion of the at least one protrusion may be comprised of a material that has been treated and / or coated with a surface chemistry modifier for surface properties that favor cell attachment. The at least one protrusion may be a protrusion array. In applying the device to the first surface of the substrate, the protrusion array can be inserted into the corresponding array of experimental units, wells or microwells. The number of protrusions in the protrusion array may correspond to the number of experimental units in the first array, the number of microwells in one second array of microwells, or the total number of microwells in the substrate.

  Another apparatus for sampling cellular solid groups in a substrate incorporates at least one injection needle and / or nanopipette facing the first surface. The at least one injection needle and / or nanopipette has an outer diameter smaller than the open diameter of each microwell and an inner diameter that can correspond to the target cell diameter. The at least one injection needle and / or nanopipette is inserted into at least one experimental unit, well or microwell, which holds a population of cells. A sample of the at least one experimental unit, well or microwell cell population is collected into the device using pressure to pull the cell population out of the at least one experimental unit, well or microwell.

  Samples of cell populations from the at least one experimental unit, well or microwell may be stored at another location. Prior to further sampling, the at least one injection needle and or nanopipette can be washed and / or sterilized. The at least one injection needle and / or nanopipette can be an injection needle array and / or a nanopipette array. In applying the device to the first surface of the microfabricated substrate, the injection needle and / or nanopipette array can be inserted into a corresponding array of experimental units, wells or microwells. The number of injection needles and / or nanopipettes in the injection needle and / or nanopipette array corresponds to the number of experimental units in the first array, the number of microwells in another array of microwells, or the total number of microwells in the substrate obtain.

  Another method of sampling the cell population in the substrate involves applying focused acoustic energy to at least one experimental unit, well or microwell, holding the cell population in a fluid. . Focused acoustic energy may be applied in a manner effective to eject one small droplet from at least one microwell, such as small droplet ejection by acoustic waves (ADE) (eg, Sackmann et al., "Acoustical Micro- and Nanofluidics: Synthesis, Assembly and Other Applications," Proceedings of the 4th European Conference on Microfluidics (December 2014). The small droplet contains a sample of the cell population in at least one experimental unit, well or microwell. The droplets may be delivered to another container or surface or substrate.

  The substrate at least includes a first piece including at least a portion of the first surface and a second piece including at least a portion of the second surface. The first and second pieces are removably coupled along at least a portion of a plane parallel to the first and second surfaces. The plane divides several experimental units, wells or microwells. The cell population in the at least one experimental unit, well or microwell may, for example, be such that the first portion of the cell population in the at least one experimental unit, well or microwell remains attached to the first piece and at least One experimental unit, a second part of the cell population in a well or microwell, remains attached to the second piece and is sampled by separating the first and second pieces.

  FIG. 12 is a diagram showing one cross section of a chip 1200 according to multiple embodiments. Chip 1200 includes a substrate that defines a content-filled well array 1202. The substrate is composed of a first piece 1206 and a second piece 1208. The first piece 1206 and the second piece 1208 are detachably coupled along a plane parallel to and bisecting the well array 1202. When the first and second pieces 1206 and 1208 are separated, the plurality of wells 1202 and their contents 1204 are divided to provide two copies of the contents 1204 that maintain the isolation and position of the contents 1204 of the chip 1200. Bring.

  Each microwell, experimental unit or microchannel has a partial separation of the microwell, experimental unit or microchannel in a first part and a lower part such that the cell population can grow in both the first part and the lower part Part barriers. Prior to sampling the cell population, the methods described above can include dispersing and / or reducing clumps within the cell population. Dispersion and / or reduction of cell mass within the cell population includes, but is not limited to sonication, shaking, dispensing with small particles, and the like.

  The above method may further comprise the step of identifying at least one cell from a sample of the cell solid population and / or residual cells of the cell population. This may include performing DNA, cDNA and / or RNA amplification, DNA and / or RNA sequencing, nucleic acid molecule hybridization, mass spectroscopy, and / or antibody binding. Alternatively, or additionally, it may also include identifying the experimental unit, well or microwell from which at least one cell was derived. Each experimental unit, well or microwell can be marked with a specific tag incorporating a regiospecific nucleotide sequence. In order to identify the experimental unit, well or microwell, a regiospecific nucleotide sequence is identified in a sequencing and / or amplification reaction, and the regiospecific nucleotide sequence is determined by at least the at least one cell from which it is derived. It can be in correlation with one experimental unit, well or microwell.

  Microfabricated devices as described above may allow for the culture of cells in a sample from a natural environment using additional systems, kits, devices and methods. For example, the sample may be applied to the first surface of the substrate such that at least one of the cells occupies at least one microwell, well or experimental unit. A semipermeable membrane is attached to at least a portion of the first surface (e.g., at least a portion of the inner surface of the experimental unit or well) such that nutrients can diffuse into the at least one microwell, well or experimental unit. Meanwhile, the escape of occupied cells from the at least one microwell, well or experimental unit is prevented and / or reduced. The semipermeable membrane can be, for example, a hydrogel layer. The semipermeable membrane is reversibly or irreversibly attached or affixed to the substrate, for example by lamination. In this way, occupied cells can be cultured in at least one microwell, well or experimental unit provided with at least one nutrient. The cells can be gradually transferred from at least one nutrient to at least one other nutrient or nutrient composition using progressive partial exchange over a period of time, thereby undergoing feeding or remodeling.

  The first nutrient from the natural environment can be used to culture cells occupying at least one first experimental unit, well or microwell, and the second nutrient from the environment is at least It can be used to culture cells occupying one second experimental unit, well or microwell. The above method compares the first nutrient and the second nutrient by comparing at least one first experimental unit, a cell occupying the well or microwell with at least one second experimental unit, a cell occupying the well or microwell It may include the step of analyzing.

For example, one method may include one or more of the steps described below:
Obtaining a chip defining thousands to 10 million microwells in many larger wells or flow cells, where each of the microwells has a diameter of about 1 μm to about 800 μm, a depth of about 1 μm to about 800 μm And the chip has one or more surface chemical agents configured to facilitate transfer of the target microorganism to the microwells;
Applying the natural environment sample or a derivative of the natural environment sample to the chip such that any target microorganism is placed in the microwell;
One or more semi-permeable filters on the chip, hydrogel layers to allow the diffusion of nutrients into the microwell but create a barrier that prevents and / or reduces the escape of microorganisms from the microwell Or installing another barrier;
-Warming the chip with at least one nutrient (eg from natural environment) for culture purpose;
-Gradually changing the nutrient source by progressive partial exchange using at least one other nutrient (e.g. a formulation); and-detecting any growth of the microwell in the microwell.

  The target cells may be archaebacteria, bacteria or eukaryotes. The target virus may be a bacteriophage. If the virus is targeted, the microwells of the chip can also contain host cells on which the virus can grow. Detection of growth of occupied cells or viruses may involve detection of changes in biomass (eg, DNA / RNA / protein / lipid), presence or absence of metabolites, pH, nutrient consumption, and / or gas consumption. Growth detection of occupied cells or viruses can be performed by real-time sequential imaging, microscopy, optical density measurement, fluorescence microscopy, mass spectrometry, electrochemical, amplification (DNA, cDNA and / or RNA), sequencing (DNA and / or Or RNA), nucleic acid molecule hybridization, and / or carrying out antibody binding.

  FIG. 13 is an operational step diagram illustrating a method for performing screening according to multiple embodiments. In step 1300, a sample is obtained. At step 1302, at least one cell is extracted from the obtained sample. At step 1304, at least one high density microwell array of microfabricated devices or chips is loaded with the at least one extracted cell. Step 1304 may comprise providing a cell concentration having the at least one extracted cell, selecting at least one nutrient / media, and / or selecting at least one membrane. At step 1306, at least a portion of the microwell array is sealed with at least one selected membrane to maintain the cell concentration for those microwells. In step 1308, the chip is warmed for culture purposes. Step 1308 may include temperature selection, determination of an atmosphere (eg, aerobic or anaerobic), and / or determination of incubation time. Genetic and / or functional testing may be performed. At step 1310, genetic testing is performed on the chip. At step 1312, the chip will be split and / or replicated (eg, using a picker) to yield two parts of cultured cells according to the methods described herein. For example, at least one membrane may be peeled off, peeled off or punctured such that a portion of the cultured cells remain attached for sampling the cultured cells. In optional step 1314, a portion of the cultured cells is sacrificed for identification. Step 1314 may incorporate PCR, sequencing, and / or various data analysis. At step 1316, a strain of interest is identified. Further culturing, testing and / or identification may be performed, for example using the strain of interest and / or the remainder of the cultured cells. On the other hand, in step 1318, a functional test is performed on the chip. At step 1320, one or more variables are observed and, as at step 1316, the strain of interest is identified.

  FIG. 14 is a chart illustrating a sorting method in accordance with several embodiments. Panel 1400 shows an example of a complex sample, specifically a microbial community-based sample 1402 and a soil sample 1404. In panel 1406, at least one cell has been extracted from the sample using, for example, the protocols shown in FIGS. 5A and 5B. In panel 1408, at least one extracted cell (and any environmental extract and / or dilution) is loaded into a microfabricated device or chip with at least one high density microwell array 1410. The chip 1410 and the reagent cartridge 1412 can be inserted into the incubator 1414. The reagents may be useful for adding fluids to maintain nutritional requirements for growth and / or various sorting purposes. Panel 1416 shows the output, ie, the selection results and the isolated strain of cultured cells.

  FIG. 15 is a series of images illustrating sorting according to embodiments. The images show multiple portions of the chip with a membrane and an acid sensitive layer applied to test for low pH. In the image 1500, more than 1800 50 μm microwells are shown with nine distinct hits 1502. Image 1504 is a magnified screen of enclosure 1504 and image 1506 is a magnified screen of one of the microwells with hit 1502.

  16A-16C are images illustrating retrieval from a shield in accordance with embodiments. In FIG. 16A, at least one well has been drilled using a microscope and a collector having at least one pin. In FIG. 16B, the pins have been removed and cultured in culture. In FIG. 16C, proliferation is apparent.

  FIG. 17A is an exploded view illustrating a sorting chip in accordance with several embodiments. In FIG. 17A, a chip 1700 incorporates a high density array of microwells having, for example, soil microorganisms therein. The film 1702 is attached to the chip 1700. Gasket 1704 is attached to chip 1700 across membrane 1702. Agar 1706 with fluorescent E. coli is attached to chip 1700 through gasket 1704 and membrane 1702. Figures 17B and 17C are images that illustrate sorting according to multiple embodiments. In this example, the shield is for the exclusion zone. FIG. 17B is a fluorescent image of the chip processed like chip 1700 in FIG. 17A and entrained with a shield. FIG. 17C is an image showing the steps of piercing the agar and collecting a sample from this chip.

  In embodiments, after a portion of the sample (or a portion of the portion) has been removed from the device, the position on the device may be correlated with that portion of the sample present at that position. The device may be a microarray or may incorporate a microarray. The microarray has a plurality of locations at which to apply the sample, where after one portion of the sample has been removed from the microarray, each location is marked with a specific tag that can be used to identify the location from which that portion of the sample originated. It is attached.

  The present invention relates to a method of identifying from which position of a microarray a portion of a sample containing at least one nucleic acid molecule is derived after a portion of the sample has been removed from the microarray, which method comprises: Applying one or more portions of the sample to one or more of the plurality of positions on the microarray, wherein each position is (i) a position specific nucleotide sequence and (ii) a first target specific nucleotide (B) annealing the target nucleic acid molecule found in at least one portion of the sample to the marking tag of the position, (c) annealing, wherein the marking is performed with a specific tag having a nucleic acid molecule comprising a sequence; Primer extension, reverse transcription, single-strand ligation reaction or double-strand ligation reaction is performed on the nucleic acid molecule population thus obtained, whereby regiospecific nucleotide sequence is thereby obtained. (E) combining the nucleic acid molecule populations generated in step (c), (d) incorporating into each nucleic acid molecule generated by primer extension, reverse transcription, single strand ligation reaction or double strand ligation reaction, (d) Sequencing of the combined nucleic acid molecule population, thereby obtaining the sequence among one or more regiospecific nucleotide sequences, and (f) at least one position obtained from the combined nucleic acid molecule population Associating that sequence of the specific nucleotide sequence with the position on the microarray that is marked with the tag that includes the position specific nucleotide sequence, whereby the location on the microarray from which a portion of the sample containing at least one nucleic acid molecule was derived Identifying. In embodiments, the sample may comprise at least one cell from which one or more nucleic acid molecules are released after step (a) and before step (b). The sample may comprise at least one cell, and the at least one cell is self-replicating or dividing after step (a) and before step (b). A portion of the portion of the sample can be removed from at least one position prior to step (b), and a portion of the portion of the sample can be removed from the original position of the portion of the sample on the microarray It can be stored in correlated containers. The method of correlating or identifying the position may further comprise the step of amplifying a population of nucleic acid molecules generated in step (c) or combined nucleic acid molecules generated in step (d). The amplification steps are: polymerase chain reaction amplification, multiplex polymerase chain reaction amplification, nested polymerase chain reaction amplification, ligase chain reaction amplification, ligase detection reaction amplification, strand displacement amplification, transcription based amplification, nucleic acid sequence based It may include amplification, rolling circle amplification, or hyperbranched rolling circle amplification. Additional primers can be added during the amplification reaction. For example, both 5 'and 3' primers may be required for PCR reactions. One of the primers used during the amplification reaction may be complementary to the nucleotide sequence in the sample.

  In embodiments, the composition comprising cells and / or viruses is treated with a nucleolytic enzyme before the composition is applied to the microfabricated device so that contaminating nucleic acid molecules are not amplified in subsequent steps. It can.

The sequencing used in the methods and devices of the present invention may be any process that obtains sequence information, including hybridization and the use of sequence specific proteins (eg, enzymes). Sequencing includes Sanger sequencing, sequencing by hybridization, sequencing by ligation, quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer, molecular labeling, TaqMan reporter probes See, eg, digestion, pyrosequencing, fluorescence in situ sequencing (FISSEQ), wobble sequencing, multiplex sequencing, polymerized colony (POLONY) sequencing (see, eg, US Patent Application Publication No. 2012/0270740, which is incorporated herein by reference). See Nanogrid rolling circle (ROLONY) sequencing (eg, US Patent Application Publication No. 2009/0018024, which is incorporated by reference in its entirety into the specification of the present invention), which is hereby incorporated by reference in its entirety. Allele Specificity Oligo Ligated Binding Analysis Sequencing, or Cash Sequencing (NGS) may include sequencing by the platform. Non-limiting examples of NGS platforms, Illumina (R) (San Diego, CA) ^{(e.g., MiSeq TM, NextSeq TM, HiSeq} TM and HiSeqX ^TM), Life Technologies (Carlsbad, CA) (e.g. Ion Torrent ^TM) and Pacific Systems from Biosciences (Menlo Park, Calif.) (Eg PacBio® RS II) are included.

  An organism or species can be identified by comparing the nucleic acid sequences obtained from that organism with various databases containing sequences of numerous organisms. For example, ribosomal RNA sequence data are available from the SIL VA rRNA database project (Max Planck Institute for Marine Microbiology, Bremen, Germany (www.arb-silva.de)), eg Quast et al., "The SIL VA Ribosomal RNA Acids Res. D590-D596 (2013), and Pruesse et al., "SINA: Accurate High-Throughput Multiple Sequence Alignment of Ribosomal RNA Genes," 28 Bioinformatics 1823. No. 18-29 (2012), both papers are hereby incorporated by reference in their entirety into the description of the present invention. Other ribosomal RNA sequence databases include the Ribosomal Database Project (Michigan State University, East Lansing, Michigan (www.rdp.cme.msu.edu): eg, Cole et al., "Ribosomal Database Project: Data and Tools for High Throughput rRNA Analysis 42 Nucl. Acids Res. D633-D642 (2014). This article is incorporated herein by reference in its entirety into the specification of the present invention) and Greengenes (Lawrence Berkeley National Laboratory, Berkeley, California. (www.greengenes.lbl.gov): See, for example, DeSantis et al., "Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB," 72 Appl. Environ. Microbial. 5069-72 (2006). This article contains by reference the entire text of the present invention). The GenBank® gene sequence database contains approximately 260,000 officially described nucleotide sequences of the publicly described species (National Institutes of Health, Bethesda, MD (www. Ncbi. Nlm. Nih. Gov); For example, see Benson et al., "GenBank," 41 Nucl. Acids Res. D36-42 (2013)).

  The verification and identification sequences may include the 16S ribosomal region, the 18S ribosomal region or some other region that provides identifying information. The desired variant may be a species comprising a genotype (eg, a single nucleotide polymorphism (SNP) or other type of variant) or a specific gene sequence (eg, a sequence that specifies the genetic code of an enzyme or protein). Organisms or species may also be identified by matching the sequence to a custom local sequence database. In some cases, at least a predetermined percentage identity (eg, at least 90%) to a known DNA, cDNA or RNA sequence obtained from a species or microorganism from which there is a sequence obtained from a portion of the sample at a certain position. An organism having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity); It can be concluded that the species or organism is found at that position on the microarray.

  The invention further relates to a method of manufacturing a microarray having a plurality of locations for applying a sample, wherein at least one location is marked with a unique tag. The method comprises the steps of: (a) synthesizing a plurality of tags each comprising a nucleic acid molecule comprising (i) a regiospecific nucleotide sequence and (ii) a target specificity nucleotide sequence; Placing the tag in at least one of the positions. In another embodiment, the invention relates to a method of manufacturing a microarray comprising a plurality of locations for applying a sample, at least one location therein being marked with a unique tag, The method comprises the steps of: (a) synthesizing a plurality of tags comprising nucleic acid molecules each comprising a target specific nucleotide sequence and not a regiospecific nucleotide sequence; and (b) among a plurality of positions on the microarray. Placing the tag in at least one position. The target specific sequences may be the same at any position of the microarray. In any of the above embodiments, step (a) may be performed before step (b). The placing step (b) comprises placing the tag at each position by a liquid handling procedure (e.g. pipetting, spotting with solid pins, spotting with hollow pins or deposition with an inkjet device). The at least one tag may comprise the nucleic acid molecule or a portion of the nucleic acid molecule prior to synthesis. Step (a) may be performed simultaneously with step (b). In some embodiments, at least one tag comprises a nucleic acid molecule synthesized at each position by in situ synthesis. The synthesis step (a) may comprise ink jet printing synthesis or photolithographic synthesis.

  Each position of the microarray may be configured to receive a portion of the sample. The position may be tagged or labeled with a nucleic acid molecule (eg, an oligonucleotide) comprising at least one (i) regiospecific nucleotide sequence (eg, a barcode) and (ii) a target specific nucleotide sequence It can. The target specific nucleotide sequence may complement or substantially complement the nucleotide sequence found in the sample. The order of nucleotide sequences from the 5 'end to the 3' end of the nucleic acid molecule may be (1) regiospecific nucleotide sequence, (2) target specificity nucleotide sequence. On the other hand, the order of the nucleotide sequence from the 5 'end to the 3' end of the nucleic acid molecule may be (2) then (1). The nucleic acid molecule may be attached to the microarray at its 5 'end. One or more locations of the device (eg, a microarray) may be untagged or unlabeled.

  The term "complementary" or "generally complementary" refers to an internucleotide or internucleotide such as, for example, between two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid. May mean hybridization, base pairing or duplex formation. Complementary nucleotides are generally A and T / U or C and G. A single stranded nucleotide that is optimally aligned, compared and has a suitable nucleotide insertion or deletion is at least about 80%, usually at least about 90% to 95%, more preferably about at least about 80% of the nucleotides of the other strand. When paired with 98% to 100%, two single stranded RNA or DNA molecules are said to be substantially complementary. On the other hand, substantial complementarity exists when the RNA or DNA strand hybridizes to its complement under selective hybridization conditions. Typically, selective hybridization occurs when there is at least about 65%, at least about 75% or at least about 90% complementarity over a stretch of at least 14 to 25 nucleotides.

  The terms "selectively form a hybrid" or "selective hybrid formation" may mean to detectably and specifically bound. Polynucleotides, oligonucleotides and their fragments selectively hybridize to nucleic acid strands under hybridization conditions and wash conditions that minimize significant amounts of detectable binding to nonspecific nucleic acids. "Highly rigid" or "highly stringent" conditions can be used to achieve selective hybrid formation conditions known in the art and described herein. An example of “highly stringent” or “highly stringent” conditions is a method of culturing one polynucleotide using another polynucleotide, wherein one polynucleotide is 6 × SSPE or SSC, 50% formaldehyde Adhere to a solid surface such as a membrane in a hybridization buffer of 5 × Denhardt's reagent, 0.5% SDS, 100 μg / ml denatured fragmented sperm DNA at 42 ° C. hybridization temperature for 12-16 hours. Followed by two washes at 55 ° C. with 1 × SSC, 0.5% SDS wash buffer.

  The nucleic acid molecule that is part of the positional tag may comprise at least one deoxyribonucleotide or at least one ribonucleotide. The nucleic acid molecule may be single stranded or double stranded. The nucleic acid molecule may be double stranded with single stranded overhangs.

  In embodiments, a positional tag may be used to amplify an annealing nucleic acid molecule. Thus, the positional tag may comprise a nucleic acid sequence further comprising an amplification primer binding site. The amplification primer binding site is at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or alternatively It may be 30 nucleotides in length. The order of the nucleotide sequence from the 5 'end to the 3' end of the nucleic acid molecule may be, for example, (1) amplification primer binding site, (2) regiospecific nucleotide sequence, and (3) target specificity nucleotide sequence .

  In embodiments, the nucleic acid molecule may comprise target-specific nucleotide sequences without including regio-specific nucleotide sequences. In certain embodiments, the nucleic acid molecule may comprise target specific nucleotide sequences without including either regiospecific nucleotide sequences or amplification binding site sequences. In a further embodiment, the nucleic acid molecule may comprise only target specific nucleotide sequences. In still further embodiments, the nucleic acid molecule may be comprised of only target specific nucleotide sequences. Amplification primer binding sites were polymerase chain reaction primers, multiplex polymerase chain reaction primers, nested polymerase chain reaction primers, ligase chain reaction primers, ligase detection reaction primers, strand displacement primers, transcription based primers, nucleic acid sequence based It may be capable of binding to a primer, a rolling circle primer, or a hyperbranched rolling circle primer. Additional primers may be added to the microarray during the amplification reaction. For example, both 5 'and 3' primers may be required for PCR reactions. The target specific nucleotide sequence is amplified at the storage position of the target nucleic acid molecule and can be detected, for example by qPCR, end point PCR and / or a dye to detect the amplified nucleic acid molecule.

It may be desirable to read the sequence of such nucleic acid molecules that anneal to the amplified product based on positional tags or nucleic acid molecules. The positional tag may comprise a nucleic acid sequence further comprising an adapter nucleotide sequence. In certain embodiments, an adapter nucleotide sequence may not be found within the positional tag, but is added to the sample nucleic acid molecule in a secondary PCR reaction or by ligation. The adapter nucleotide sequences, universal adapters or specific sequencing platform (e.g., Illumina (R) or Ion Torrent ^TM) may be adapter. The adapter nucleotide sequence may comprise a sequencing primer binding site. Sequencing primer binding sites include Sanger sequencing, sequencing by hybridization, sequencing by ligation, quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), stepwise ligation and cleavage, fluorescence resonance energy transfer, molecular labeling, TaqMan Reporter probe digestion, pyrosequencing, fluorescence in situ sequencing (FISSEQ), wobble sequencing, multiplex sequencing, polymerized colony (POLONY) sequencing (see eg US Patent No. 2012/0270740), nanogrid rolling circle (see eg US Patent No. 2012/0270740) ROLONY) Sequencing (see, eg, US Patent No. 2009/0018024), Allelic Specificity Oligo Ligation Binding Sequencing, Sequencing on an NGS platform, or primers for any suitable sequencing procedure can be combined. obtain. Non-limiting examples of NGS platforms, Illumina (R) (San Diego, CA) ^{(e.g., MiSeq TM, NextSeq TM, HiSeq} TM and HiSeqX ^TM), Life Technologies (Carlsbad, CA) (e.g. Ion TorrentTM) and Pacific Biosciences Systems from (Menlo Park, Calif.) (Eg PacBio® RS II) are included.

  Regiospecific nucleotide sequences (eg, barcodes) are at least 2, at least 3, at least 4, at least 5, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29, or at least 30 May be the length of

  The target specific nucleotide sequence is at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 75, or at least 100 nucleotides in length.

  At least one position on the microarray can be further marked with a unique molecular identifier tag. Unique molecular identifier tags can be used to quantify growth (eg, growth of microbial colonies or cell replication at that location). The unique molecular identifier may be a disordered nucleotide sequence. Specific molecular identifier usage methods and specific molecular identifier examples have been described in the art, for example reference is made to WO 2013/173394, which is hereby incorporated by reference in its entirety into the specification of the present invention. For example, the unique molecular identifier tag can have the nucleotide sequence NNNANNNCNNNTNNNGNNNANNNNN (SEQ ID NO: 1), where each amplified molecule has a unique (specific) DNA sequence barcode (4 ^ N barcode) Or, several N (the same chaotic mixture of ACGTs) create a large coding space, so as to get 4 ^ 21 to 4 trillion in this example. This sequence can be enumerated without interference from amplification bias or other issues. The fixed bases (A, C, G, T) of SED ID NO: 1 help correct reading of barcodes, eg handling of insertion deletions.

  The present invention combines the use of regiospecific tags for observing and recording the presence or amount of multiple target specific nucleotide sequences (eg, multiplexing) in a sample. At least one position on the microarray is further marked with a second specific tag, for example a nucleic acid molecule comprising (i) an amplification primer binding site, (ii) a position specific nucleotide sequence, and (iii) a target specific nucleotide sequence. It can be attached.

  In embodiments, the nucleic acid molecule may comprise target-specific nucleotide sequences without including regio-specific nucleotide sequences. In certain embodiments, the nucleic acid molecule can comprise target-specific nucleotide sequences, without including either regiospecific nucleotide sequences or amplification binding site sequences. In a further embodiment, the nucleic acid molecule may comprise only target specific nucleotide sequences. In still further embodiments, the nucleic acid molecule may be comprised of only target specific nucleotide sequences. Such target specific nucleotide sequences may be at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 75, or at least 100 nucleotides in length. In certain embodiments, the target specific sequences may be the same at any position on the microarray. Additional target specific nucleotide sequences can be observed and recorded. For example, one or more positions may be marked with at least 10, at least 25, at least 50, at least 75, or at least 100 specific tags, where each tag represents another target in the tag at that position. It contains one target-specific nucleotide sequence which differs from the specific nucleotide sequence.

  Any locus of interest may provide a target specific nucleotide sequence. For example, bacterial 16S ribosomal RNA (rRNA), 18S ribosomal RNA, poly (A) RNA, RNA polymerase gene, DNA polymerase gene, RecA gene, transposase gene sequences, ribosome internal transcription spacer-region (ITS) sequence, enzyme code A portion of any of the gene encoding, control region DNA sequences, binding site DNA sequences, or any of these sequences may serve as target specific nucleotide sequences. The disclosed system, kit, apparatus or method is described in Sundquist et al., "Bacterial Flora-Typing with Targeted, Chip-Based Pyrosequencing," 7: 108 BMC Microbiology (2007) and Wang et al., "Conservative Fragments in Bacterial l6S rRNA Genes and Primer Design for <RTIgt; l6S </ RTI> Ribosomal DNA Amplicons in Metagenomic Studies, "4: 10 PLoS ONE e7401 (2009). One or more of the bacterial 16S rRNA primers described may be used. Each of these documents is incorporated by reference in its entirety into the specification of the present invention.

  The samples used in the disclosed devices and methods can include multiple nucleic acid molecules. The sample may comprise at least one DNA molecule or at least one RNA molecule. The sample may comprise at least one nucleic acid molecule formed by restriction enzyme digestion. The sample may comprise at least one cell (eg, archaebacterial cells, eubacterial cells, fungal cells, plant cells, and / or animal cells). The sample may comprise at least one microorganism. The sample may comprise one or more viruses (eg, bacteriophage) that may require a supply of host cells. One portion of the sample at one position of the microarray may be a single cell or a colony in which a single cell is grown. For example, microorganisms or cells can be individually placed in microwells, and such individual microorganisms or cells can be divided or replicated so that colonies grow inside each microwell in which each microorganism or cell is placed. It can be Thus, one position of the microarray can accommodate a single microbial species or a mixed community of microbial varieties that help each other grow. The sample may comprise any suitable dilution. Nonlimiting examples of samples may include soil, sewage, feces, body cavity contents, biological fluid, living organic matter, dead organic matter, microbial suspension, natural fresh water, drinking water, seawater, wastewater, air Supercritical carbon dioxide, minerals, gases, buffers, alcohols, organic solvents, and / or oils. In embodiments, (a) (i) one regiospecific nucleotide sequence and (ii) one or more target specific nucleotide sequences, or (b) one or more target specific nucleotide sequences (ie, positions) A specific nucleotide sequence is not included) is placed in at least one position of the microarray before one portion of the sample is placed at that position. In other embodiments, (a) (i) a regiospecific nucleotide sequence and (ii) one or more target specific nucleotide sequences, or (b) one or more target specific nucleotide sequences (i.e. regiospecificity) The nucleotide sequence is not included) is placed in at least one position of the microarray after one portion of the sample has been placed in that position. In one example, the sample or a portion of the sample is in one microarray, and the microarray contains at least one of (i) one regiospecific nucleotide sequence and (ii) one or more target specificity nucleotide sequences. The nucleic acid molecule containing can be placed and cultured before being placed. In embodiments, a portion of the portion of the sample may be removed from at least one position of the microarray and stored in a separate container, or alternatively, the nucleic acid molecules may be placed in at least one position of the microarray. The microarray may be divided either before or after being drawn.

The at least one regiospecific tag may comprise a nucleic acid molecule or a portion thereof that has been presynthesized and placed in that position by a liquid handling procedure. For example, the liquid handling procedure may be pipetting, spotting with a solid pin, spotting with a hollow pin or deposition with an inkjet device. The tag can be generated at that position using a number of separately pre-synthesized nucleic acid molecules. At least one tag may comprise a nucleic acid molecule synthesized in situ by in situ synthesis (eg, ink jet printing or photolithography).
(Digital enumeration of species)

  A high density chip device with a surface with high density microwells is described here. The microbes of the microbiota sample are diluted and applied to the device such that the wells contain approximately one microbe per occupied well. The chip may then be warmed for culture purposes so that the microorganisms are replicated in the wells. In addition, a DNA position indexing system is described herein to determine what species species are present in each well. This indexing system provides a number of pre-loaded PCR primers into each well, including addressing barcodes that identify the wells, as well as specific genes within the microbial genome that provide species information or target desired gene sequences. It may involve having a primer sequence directed to an element (eg, 16S). After incubation, microbial DNA is released, their PCR primers amplify the target bacterial DNA region, and amplification products from various wells on the chip can be pooled and further decoded by next-generation sequencing.

  The systems, kits, devices and methodologies described herein can be utilized to provide an absolute count of the number of each microbial species or variant within a sample. Each well may represent a digital event that represents the presence of a single microorganism in the original diluted sample. The position indexing system may allow the user to determine what bacterial species are in the well. One unit of measurement may be "in one well with one bacterial species" and may be independent of the number of bacteria in the well.

  In one example, the mixed sample of microorganisms contains 50% of species 1, 30% of species 2, and 20% of species 3. The sample is diluted and then each occupied well is applied to the chip, with the majority of one microbe. The microorganism is replicated. The replication rate may be different if the species is different. The chip is then processed such that DNA from in-well microorganisms is released and 16S or some other target sequence is amplified. The DNA amplification products from each well can be pooled and sequenced using next generation sequencing. Next generation sequencing data may be analyzed to determine for each well which species are in each occupied well. Many wells may not be occupied at all. The abundance of each species can be determined by dividing the total number of wells occupied by each species by the total number of occupied wells. An absolute abundance determination can be made by multiplying the% abundance of each species from step by the total number of microorganisms in the original sample. The sequencing data can be compared to publicly available sequencing databases to determine what species are in each occupied well. For example, ribosomal RNA sequences can be obtained in the SIL VA rRNA database project described above. Other ribosomal RNA sequence databases include the aforementioned Ribosomal Database Project, Greengenes, and GenBank® gene sequence databases.

  Current methods of estimating microbial species abundance in samples involve the use of traditional techniques such as microscopy, staining, selective media, metabolism / physiology shielding and culture with petri dishes. These methods are often inaccurate due to lack of specificity (microscopy, staining, metabolism / physiology) or lack of ability to reveal all species in the sample (selective medium, culture) That is why many species do not grow well or cause any growth in the traditional approach.

  Current molecular methods to determine the relative abundance of microbial species within a microbial flora sample extract microbial DNA from the sample, perform PCR amplification of 16S or other DNA regions that provide species or other information, and then Next generation sequencing (NGS) is performed on the resulting PCR products. The relative abundance of each species in the original sample is inferred from the relative frequency of species specific DNA sequences in the NGS data. The literature of this type of analysis is abundant, and this method supports the study of many microflora.

  The problems of current methodology are different numbers of 16S genes that may be present in different microorganisms, PCR biases in which sequences from different microbial species are amplified at different rates, and different sequences from different microbial species There is no adjustment to the sequencing bias that can be arranged at a rate. The result is that there is a lot of uncertainty regarding the accuracy of the relative abundance data derived using current methodologies.

  A tabulation of different species can be based on the presence of the species in a single well. This is directly related to the single microorganism from the original sample which splits into its wells during loading. Only PCR / NGS can be used to identify what microbial species are present in each well. The number of sequences identified does not form part of the calculation. Therefore, it is not important whether the method has PCR, NGS or target sequence copy number variation or bias.

  Embodiments can be applied to microbiota research, microbial product discovery and development, medical diagnostics, and any other field where accurate enumeration of microbial species in a sample is desired.

  Thus, embodiments provide an even more accurate measurement of the relative abundance of each species within the bioplex sample, and the relative abundance measurement (to reveal dilutions and / or microorganisms in the original sample. Combined with the measurement of the total number) can provide the ability to convert the absolute abundance or direct aggregation of each species in the original sample. Embodiments may provide new applications of microfabricated high density chips (besides culture and sorting of microorganisms).

  FIG. 18 is an operation step diagram illustrating a tabulation method according to a plurality of embodiments. In step 1800, a sample is obtained. At step 1802, at least one cell is extracted from the acquired sample. At step 1804, at least one high density microwell array of microfabricated devices or chips is loaded with at least one extracted cell. Step 1804 includes providing a cell concentration with the at least one extracted cell, selecting at least one nutrient / media, and / or selecting at least one membrane. At step 1806, at least a portion of the microarray is sealed with at least one selected membrane to maintain cell concentration for the microwells. At step 1808, the chip is warmed for culture purposes. Step 1808 includes selecting a temperature, determining an atmosphere (eg, aerobic or anaerobic), and / or timing the culture. At step 1810, cultured cells may be sacrificed for identification. Step 1810 includes PCR, sequencing, and / or various data analysis. At step 1812, information (eg, microbial community structure) about the sample may be assessed and / or determined.

FIG. 19 is a chart diagram illustrating a tabulation method in accordance with several embodiments. Panel 1900 shows an example of a complex sample, specifically a microflora sample 1902 and a soil sample 1904. In panel 1906, at least one cell has been extracted from the complex sample using, for example, the protocol shown in FIGS. 5A and 5B. In panel 1908, the at least one extracted cell (and any environmental extract and / or dilution) is loaded into a microfabricated device or chip with at least one high density microwell array 1910. A chip 1910 and a reagent cartridge 1912 can be inserted into the incubator 1914. The reagents may be useful for fluid supplementation to maintain the nutrients needed for growth and / or for various sorting purposes. Panel 1916 shows the output, ie the sequence and relative abundance of cultured cells.
(Platform using small droplets)

  Platforms with isolated droplets can be used for separation, culture, and / or sorting in much the same way that chips are used. Small droplets are analogues of microwells that function as nano or picoliter containers. The microdroplet generation method can be utilized to separate microorganisms from complex environmental samples, particularly when combined with a cell-to-chip cell classifier type measurement device. Small droplet addition can be utilized to deliver the microorganisms. Small drop splitting can be used for sequencing or some other degradation test while leaving the biological sample behind. All the preparatory work required for sequencing can be done in the same way in the small droplet mode.

  Embodiments can be utilized to remove microbes from the complex environment into small droplets. For example, a modular system for droplet generation, including cell suspensions, can include one or a few cells. The aqueous droplets may be suspended in the immiscible liquid so as not to approach each other and not touch or stain any surface. Small droplets can be produced in each microwell, for example at 30 Hz, which translates to millions of daily each other.

  A droplet based microfluidic system can encapsulate, manipulate, and / or culture small droplets (eg, about 30 pL). It is noted that cell survival and reproduction are similar to large volume solution control experiments. Small droplets are produced at hundreds of Hz, which means that millions of droplets can be produced in a few hours. A single chip based device can be used to generate small droplets, and those small droplets can be designed to contain single cells.

  Embodiments can be utilized to sort cells in small droplets. Fluorescence sorting after microdroplet culture can be performed on the chip, for example, at a rate of 500 droplets per second. Small droplets can be flowed through one channel at the focal point of an epifluorescent microscope configured for many different measurements. This may be a particularly effective method of sorting for metabolites since the local concentration is quite high due to being trapped in very small droplets.

  Several embodiments may be utilized to classify small droplets. Once cells have been isolated, grown and / or sorted, they can be classified such that a useful sample is removed. Droplets can be classified in a manner similar to commonly used FACS mechanisms.

  Embodiments can be utilized to break up small droplets. Embodiments may require the ability to take a sample, send one part to sequencing (destruction step) and another part that is a viable culture medium to hold the sample. . There are many ways to break up a small droplet, for example by constructing a T-junction with carefully calculated dimensions that will break up as the droplet continues to flow, or electrowetting Not to cause cell lysis), etc., but it is not limited thereto.

  Embodiments may be utilized to coalesce small droplets and / or to add reagents to small droplets. For example, long-term (eg, several weeks) culture of cells requires the ability to add fluid to maintain the nutrients needed for growth. It may also be effective to be able to add reagents for various sorting purposes. Small droplet sorting relies on the ability to combine small droplets containing compound codes with small droplets containing single cells. And then the droplets can be cultured and / or returned to the assay chip to identify compounds through their codes. This may require the ability to accurately combine small droplets as needed.

  Embodiments can be utilized to perform PCR in small droplets. PCR can be used to finally sequence specific gene elements (eg, 16S regions) to identify microorganisms. This can be used to determine what type of microorganism is growing in each well. In small drop systems, this approach is used to determine what microbes are present in each small drop as long as the correct primer sequences are designed to amplify the correct region of the gene. Can be

Embodiments can be utilized to sequence DNA from small droplets (e.g., generated in a PCR step) and / or create a DNA library.
(Position specificity tag for high density chips)

  High density chip devices having a surface with high density microwells may be used. Microorganisms from a microbiota sample can be diluted and applied to the device such that the wells contain approximately one microbe per occupied well. The chip can be warmed for culture purposes so that the microorganisms replicate in the wells and the resulting population of solids represents a single species. A DNA based position indexing system can be used to determine what species are present in each well. The indexing system targets PCR primers pre-loaded into each well containing addressing barcodes that identify the wells, and specificity gene elements (eg, 16S of microbial genes) to provide species information. It may be necessary to have a primer sequence. After incubation, microbial DNA can be released, PCR primers can amplify the target bacterial DNA region, and amplification products from various wells on the chip can be pooled and then decoded by next-generation sequencing.

  The above position indexing system may involve incorporating a different position code for each well on the microchip, in other words reducing the total number of codes needed to code a given number of wells. Each well may incorporate a composite position code. For example, if there are 100 wells in one chip, 100 codes are required if there is one code per well. If two codes were to be decoded from each well, the same chip could be coded with only 20 codes (ie 10 coding for the grid x-axis, 10 coding for the y-axis) .

An example of a PCR strategy incorporating 2 codes per well is shown in Table 2.

An example of a PCR strategy incorporating 3 codes per well is shown in Table 3.

  Three oligo primers are used to make a single PCR product. The advantage of this system of using two oligos to attach a multipart barcode to one end of the molecule is, for example, to reduce the required length of the oligo or to make an extra long barcode , May be included.

  This approach can be generalized to n barcode integrations per reaction. The approach can also include various implementations where there are several barcodes on the same side of the target sequence region. An NGS sequencing adapter can be added to decode the entire sequence for the population of barcoded PCR products by next generation sequencing.

In another implementation, a fixed code can be added to identify the sample number or plate number, and storage of the composite sample / plate in one stroke towards two barcodes as shown in Table 4. May be possible.

In a further implementation, a fixed code can be added to identify the sample number or plate number, and the composite sample / plate can be stored in one stroke towards three barcodes as shown in Table 5. It can be

  Since in all cases the position of the code within the sequence conveys information, the barcodes of code 1, code 2 and code 3 do not necessarily have to be different from one another in the individual wells.

  Making several oligos and printing several chips using a single code coding system is expensive. For example, a 10,000-well chip requires 10,000 repetitions of printing independently of 10,000 single barcodes to place a barcode in a well on the chip. If a two-code system is used, only 200 barcodes may be required in some cases if printing is repeated only 200 times to produce the chips. This implies significant savings in oligo costs, printing time and printing capital investment.

  The use of dual barcoded PCR primers followed by amplification and sequencing analysis to obtain positional data on DNA or RNA containing the randomly divided portion on the microfabricated chip is highly useful. It can have relatively low cost.

FIG. 20 is a diagram illustrating an indexing system in accordance with several embodiments. The microwell 2000 has N rows and M columns, thereby producing N × M unique indices. The position of the microwell in chip 2000 can be considered to have coordinates (N, M). Each column has a common reverse primer sequence (eg, R1, R2, R3, ... RM), and each row has a common forward primer sequence (eg, F1, F2, F3, ... FN) ). For example, a specific tag directed to a particular genetic element within 16S ribosomal ribonucleic acid (rRNA) may comprise forward primer sequence F515 as well as reverse primer sequence R806. Following PCR on chip 2000, the presence of target gene elements can be mapped back to the original unique microwell based on the presence of forward and reverse primer sequences. For example, the presence of F 515 and R 806 directs the user to the microwell with coordinates (515, 806) in chip 2000.
Reduced variability for PCR amplification products across bacteria-containing microwells

  A DNA based position indexing system can be used to determine what species are present in each well. This indexing system targets PCR primers pre-loaded into each well containing addressing barcodes that identify the wells, and specificity gene elements (eg, 16S) within the microbial gene that provide species information. It may be necessary to have the following primer sequences: After incubation, microbial DNA is released, PCR primers amplify the target bacterial DNA region, and amplification products from various wells on the chip can be pooled and decoded by next-generation sequencing.

  Embodiments to limit variations in the amount of PCR product throughout the well may be chipped, such that the amount of PCR primers is limiting in the DNA amplification reaction for most of the expected sample DNA concentrations. A limitation of the amount of PCR primers in the wells can be incorporated during the manufacture of H. Thus, the amount of PCR product produced will be less variable throughout the wells.

  Embodiments to limit variation in the amount of PCR product across the wells are chipped so that production of PCR product is less variation across the wells as compared to the full cycle PCR amplification protocol. It may involve limiting the number of PCR cycles above to less than 3 cycles, alternatively less than 5 cycles, alternatively less than 10 cycles, alternatively less than 15 cycles, alternatively less than 20 cycles, alternatively less than 25 cycles, alternatively less than 30 cycles.

  Embodiments to limit the variation in the amount of PCR product throughout the wells are such that the number of generated PCR amplification products is more related to the amount of nucleotides than the amount of DNA in the original sample, the nucleotides in the reaction mixture It may include limiting the amount. Microwells with large amounts of target DNA deplete nucleotides early in the cycling step, whereas microwells with small amounts of target DNA deplete nucleotides later in the cycling step, but about the same amount Generate amplification product.

  Embodiments to limit variation in the amount of PCR product throughout the wells are available to microorganisms that grow in the wells, such that the cells will self-replicate until the medium is exhausted and replication is stopped. Can include limiting the amount of nutrients.

  Embodiments to limit variation in the amount of PCR product throughout the well place a dye in each well that identifies the PCR product so that the signal becomes more brilliant as more PCR product is generated. Can be included. The dye intensity can be measured and controlled during each PCR cycle so that once the desired signal strength is observed, the sample can be removed from the well.

  Embodiments to limit variations in the amount of PCR product throughout the wells include oligos complementary to barcodes specific to each well to selectively hybridize amplified DNA from each well. It may involve using a mixture of covered hybrid preparation beads. Once the beads are saturated, wash away unbound DNA and release bound DNA from the beads. Next, the amount of DNA from each well is normalized to the saturation limit of the beads.

  Embodiments to limit variations in the amount of PCR product throughout the wells, the fast growing organisms fill the wells rapidly and stop replicating, the slower growing organisms gradually fill the wells, and so on. It may involve warming the chip for culture purposes for extended periods of time to stop growth once the same number of cells are in the wells.

  In embodiments, the use of barcoded primers and Next Generation Sequencing (NGS) within the context of chip format and method dictates which species are growing in which wells on high density microchips Can be used to identify When approximately the same number of bacteria occupy each microwell in the chip, the signal from each well of the NGS data may be almost the same.

  For example, in a typical NGS run that produces 12 million sequence reads, 24 chips, each with 10,000 microwells, will be sequenced in that run, with the same number of bacteria per well, 1 well There will be 50 readings on average.

  However, when different bacteria grow at different rates, the wells will have a small number of bacteria and the wells will have a large number of bacteria. This can distort wells with small numbers of bacteria with the NGS run so that they can not be detected by NGS analysis.

This is why, in a typical NGS implementation that produces 12 million sequence reads, each chip has 10,000 microwells, half of which are 100 times more bacteria than the other half. Suppose that 24 chips contained therein are sequenced in one execution. The probability of detecting bacteria in the slowly growing wells is significantly reduced. in this case,
(10,000 x 24) / 2 x 100 = 12,000,000
(10,000 x 24) / 2 = 120,000

  Infrequent wells correspond to 1% of the total. Thus, of the 12,000,000 readings in one run of NGS, 120,000 are from low frequency wells, ie an average of 1 reading per well.

To minimize the impact of this phenomenon, new methods need to be developed to help homogenize the amount of PCR product across the wells so that all the wells are detected in the NGS run.
(Silicon-based micro-well chip for microbe isolation, growth, sorting and analysis)

  The microfabricated device or chip may be composed at least in part of silicon instead of or in addition to plastic, glass and / or polymer, taking into account the electrical measurement per well. For example, the walls of each well are isolated to create a microcapacitor. In another example, the FETs in each well may be isolated such that the gate surface is exposed to the well contents. Instead of chips purely using silicon, a thin metal layer may be produced on top of existing chips by plating, evaporation and / or arc / flame spraying. This may allow for the addition of more functionality, the use of cheaper and / or cleaner alternative manufacturing methods, and / or smaller and lighter hand-held and / or portable devices.

  Embodiments can be considered for measuring and managing proliferation by electrical measurement. Periodic measurements of impedance can be applied to the measurement of microbial (eg, bacterial) growth. For example, the impedance at both ends of a tube containing E. coli can be determined by the number of cells in that tube in Ur et al., "Impedance Monitoring of Bacterial Activity", 8: 1 J. Med. Microbiology 19-28 (1975). It is being compared. And this article is incorporated by reference in its entirety into the specification of the present invention. To prove that this effect is common, measurements may be performed on other types of bacteria, including Judemonas, Klebsiella and Streptococcus. Wells may be filled with different media to test growth conditions across different formulations.

  Embodiments can allow for sorting by electrical measurement. Electrical measurements can be made per well to allow for sorting. One example was pH. There are a number of different ways to obtain a pH dependent response from the gate of an in-well device including ISFETs, pH meters etc. A well array equipped with an embedded pH sensor can electrically determine which well contains a microorganism that produces acidic or basic metabolites. A simple example is a test for lactic acid production from lactose. The bacteria are diluted and distributed into the wells, grown and then fed with lactose. The wells recording the drop in pH contain a microorganism capable of degrading lactose to lactic acid.

Embodiments may allow for electrical measurement of redox probes. Another way to influence the electrical measurement is to see how the bacteria in the well affect the known redox probes. Basically, a system with a well-defined response can be measured in the presence of bacteria and deviations from the expected behavior can be attributed to the bacterial sample. A typical redox probe is something similar to ferricyanide, [Fe (CN) 6] ^{3-/ 4-} . The reduction of ferricyanide to ferrocyanide is very well characterized, especially as small changes in behavior around the electron transfer from the electrode can be found. This system is "unlabeled" because it can be detected without the need to directly alter the bacteria itself.

  An antibody capable of recognizing a microorganism (eg, E. coli) can be immobilized on an ITO electrode. The electron transfer resistance from the electrode to the ferricyanide containing solution can be measured. E. coli bound to the electrode surface increases the resistance in proportion to the E. coli concentration on the surface. This is an example of a group of measurements that can be used to detect specific types of organisms or metabolites using redox probes.

The work provided with silicon (or at least metal or metal coated plastic) allows more inexpensive chip production (e.g. by taking advantage of existing technology), comprehensive detection capabilities enabling small and / or portable versions, other materials (e.g. , LCR, CV, etc.), integration of newly discovered chip detection modes into existing devices, combination of electrical measurement and sequencing, etc. These advantages benefit all customers using interferometric detection.
(A peelable barrier that protects well specific chemical agents on the chip)

  21A-21E are summary diagrams showing a chip with well-specific chemistry according to embodiments. FIG. 21A shows a microfabricated device or chip having a plurality of microwells. In FIG. 21B, microwell specific chemical agents are assigned to each microwell on the chip. In FIG. 21C, the sealant covers the microwell-specific chemical agent in each microwell of the chip, thereby preventing their interaction with further additives to the wells. There is. In FIG. 21D, the sample is loaded and an experiment is performed on the sample in the macrowell. In FIG. 21E, the trigger (eg, heat) releases the microwell specific chemical agent for interaction with the in-well sample.

  Microwell chips are manufactured, washed and / or their surfaces treated. Specific chemical agents are prepared separately, and then, for example, using a method and / or instrument that allows for the induction of a set of specific chemicals into a particular well (or wells), It is put in the well. Immediately thereafter, sealants may be applied to protect the various chemical agents from the environment and / or from removal, release and / or interference with any limited external triggers.

The microfabricated device or chip may be manufactured in a specific design, for example cleaned and / or surface treated to enhance wetting. The PCR primers can be printed or spotted on specific wells. The chips may be dried and then the wax layer may be deposited by evaporation of the ethanol solution. The optimal concentration is about 1% v / v. The molten wax may be applied directly, or an aqueous or alcoholic solution of the wax may be sprayed. Alternatively, spin coating or evaporation may be used. Various waxes can be used, examples being glyceryl stearate, cetearyl alcohol, 1-hexadecanol, glyceryl stearate, ceteareth-20 (CAS Registry No. 68439-49) with or without polyethylene glycol. -6), and Lotionpro ^TM 165 (Lotioncrafter (registered trademark), Eastsound, Washington, available from) and Polawax ^TM (Croda, Inc., Edison, New Jersey, available) a plurality of commercial products such as from the mentioned Be The underlying chemical agent can be released later, for example by heating to a point at which the wax dissolves. For these compositions, the heating is between 50 ° C and 70 ° C. It is important that the heating temperature be low enough to not damage any chemical components or to boil our aqueous solution.

An important concept is the well specific chemical agent which is partitioned from the tip until the experimenter releases the trigger. This method can be used for barcodes in wells, but can also be applied more broadly to other issues. Various chemical agents useful for encapsulation on chips include, but are not limited to, antibiotics, fluorescers, dyes, PCR primers, solubility enhancers, antibodies, reagents for various metabolic products, etc. is not. Waxes are a good means of sealing things that can be released later by heating, but other substances are used to seal and clear exposure, sonication and / or some other trigger. It is also good. The advantage of this over simple addition of reagents to the chip is the well-to-well control. A similar effect may be achieved by printing chemical agents in the wells after performing a microbial test, but this is a matter of time (the printing step can take a day) and If each well is individually blocked after the microbes are present on the chip, it will introduce the fact that it is not possible to have all the wells acted on in the same amount of time. With the release mechanism, any well can be affected at the same time. In one example, the wax can be deposited onto the chip by solvent casting.
(Isolated microwell for easy and / or relative abundance control)

  High density chip devices may comprise a surface having high density microwells. Microorganisms from microbiota samples, or other cell types, can be diluted and applied to the device such that the wells accommodate approximately one microbe or cell per occupied well. These microwells can be sealed with semipermeable membranes that allow the diffusion of nutrients into the microwells but prevent all or at least some of the microorganisms or cells from migrating out of the microwells.

  Microbial or cell samples can be prepared and then sealed into the chip using an impermeable or gas-only permeable membrane. Because no fluid reservoir is placed on top of the chip or on the membrane, only in-well nutrients at the time of sealing are available to support the growth of microorganisms or cells in the microwell. Two grounds for this feature are: (1) the simplicity of the structure and work flow as a device does not require having a semipermeable membrane or reservoir and there is no need to add nutrients, and The risk of contamination is low, and (2) the relative abundance of premature organisms is reduced by limiting the availability of nutrients. For samples containing multiple early and multiple late-onset organisms, early-onset organisms quickly become source-limited and their growth stops or slows down in their microwells, while late-onset organisms continue to grow. This is to allow the late adult to be shown at a higher relative abundance among the population of microorganisms across the chip as compared to when the nutrient content of the early adult is unlimited. This is important for downstream processing of all sequencing on the chip. It also provides a better detection limit for rare species, as the growth of rare species is not further accelerated by premature growth to the point where the performance of the detection system is limited.

  Current methods attempt to propagate all species regardless of whether they are early or late adult. This has the inevitable result that early-growing species will sweep the community and increase their relative abundance over time. Many types of downstream analysis, such as sequencing or fluorescence sorting, can not analyze all species other than species above a certain relative abundance in a given solid population. If the goal is to preserve diversity and detect rare species, then pre-growth may need to be restricted in some way.

As an example to demonstrate this idea, as a simple illustration of a sample containing two species, one shown in Table 6, one species is doubling every day and another species is doubling every week Consider. Assuming that the late adult species is initially rare with a relative abundance of 5%, it will soon become very rare as both species grow.

(High-density microfabricated array for biobank cells)
Biobanks are designed to open the way for researchers to obtain large numbers of samples from large populations in order to drive certain types of research, such as disease-related biomarker discovery. The state of the art in biobanking provides for tubes or samples to be stored in the form of low density plates, eg 96-well or 384-well plates. This works when the number of samples to be loaded is relatively small and the samples themselves are discrete, isolated groups of solids. Existing approaches to biobanking can be complex and inefficient when storing samples such as microbial samples, where the number of samples may be large and the number of different species or variants within each sample is one There may be hundreds to thousands or even millions of samples per sample. Using existing methods, unless a laborious isolation protocol is performed to separate individual species or variants either prior to or subsequent to the storage step to obtain the desired species or variant. It does not.

  The systems, kits, devices and methodologies described herein may be applied to biobank cells, microorganisms, viruses and other biological entities. A high density chip device comprising a surface with high density microwells may have thousands, hundreds of thousands or millions of microwells per chip. For example, the microbes (or different types of cells or other biological entities such as viruses) from microbiota samples are diluted and applied to the device so that the wells contain approximately one microbe per occupied well Be done. And then, the chip is warmed so that the microorganisms are replicated in the wells and the resulting population is a single species. A nucleic acid based position indexing system may be utilized to determine what species are present in each well. This indexing system may require that each well be preloaded with PCR primers. These PCR primers may contain primer sequences directed to specific genetic elements (e.g. 16S) of microbial genes that provide well-addressed barcodes and species information identifying the wells or target desired gene sequences. . After incubation, microbial DNA is released and PCR primers amplify the target bacterial DNA region. The amplification products from the various wells on the chip can be pooled and subsequently decoded by next-generation sequencing.

  The use of high density microfabricated chips for biobanking requires the use of a large number of species within each sample stored as separate populations without the need to perform a laborious isolation protocol either before or after storage. Or prepare for the variant. The use of a DNA position indexing system or application specific test method allows a more straightforward general approach to the identification of genetic labels or characteristics of the contents of each microwell, such as species information, Give information. Moreover, chip devices provide for a very space efficient method of storing cell populations. For example, a single microscope slide sized chip with 100,000 wells occupies significantly less space than a corresponding conventional storage configuration. The chip format also properly records and manages samples and / or handles a database of subject (eg, patient) information by accommodating many different samples from a single object on one chip May be useful for

  Cells may be placed and / or positioned in the microwells of the device to align the cells in rows using the device. The cells in the microwells can be processed to improve storage effects and then placed in appropriate storage conditions. For example, cells can be treated with agents such as glycerol to ameliorate the effects of freezing. The chips can then be placed under suitable storage conditions, such as a freezer. The cells may be dehydrated, lyophilized and / or vacuum lyophilized and the chip may be placed under appropriate conditions for dry cell protection. Additional structures may be added to the chip to further enhance its usefulness as a storage device. For example, a membrane or other structural component may be placed on top of the chip to seal at least some of the wells prior to storage.

  The chip can be loaded with cells such that each portion of the microwell on the chip is occupied by approximately one cell. The chip is warmed in anticipation of cell replication. The chip is replicated and either the original chip or the replica chip is used to identify cells or species or gene markers present in each well of the chip (e.g. using the position indexing system described above) Be The chip may be processed and / or stored under appropriate conditions. The replication step and / or the identification step may be performed after storage rather than before storage.

  One or more cells from each strain of a set of pre-existing isolated strains may be placed in separate microwells on the chip. The location of the microwell in which each strain or cell type is located is recorded, and the chip can be processed and / or stored under appropriate conditions.

  One type of chip can be created with storage chemistry isolated just below the wax barrier in each well. The isolate can be sealed inside the chip, allowed to grow, and then protected from heat later by heat-induced release of preservatives prior to banking.

  Such devices may be used to store / biobank mixed microbial communities based samples, such as microbial communities based samples from soil, human intestines, seawater, oral cavity, skin etc. The chip can be used to store other types of biological entities such as fungi, archaea, human cells (including germ cells), animal cells and viruses.

  The DNA position labeling system can be used to generate information about the contents of each well across all types of biological entities. The entire chip can be tested for the desired activity using specialized assays such as antibody or substrate analysis. For example, a chip comprising a bank population of T cells can be tested for acquired immune activity.

In one illustrative example, human faecal microbiota samples may be collected from study participants. An individual's fecal microorganism can be biobanked on the chip to maintain a record of the individual's microflora as well as a sample of the microflora for later use as a source of target microorganisms. In another example, a sample of a mixed population of T cells or other acquired immune cells is taken from an individual during a clinical or research study, or as part of a therapeutic workflow (eg, treatment with ex vivo treated cells). In some cases. In yet another example, a soil biome may be stored in conjunction with seed banking.
(High resolution sampling)

  Highly accurate / precise harvesting devices or systems can be designed to perform various harvesting functions from and / or to the microwells on the microfabricated chip described above. The target substrate or tip may be a microscope slide (about 25 mm x 75 mm x 1 mm) with features injection molded on one surface. The microwells can be arranged in a grid pattern with about 4-8 mm wellless edges around the sides of the chip. The size and spacing of the wells can be determined based on the performance of the harvester. The microwells may be squares measuring about 25 μm to about 200 μm along each side, and may be spaced about 25 μm to about 100 μm between the well end and the well end. The microwells may alternatively be circular or hexagonal. The depth of the wells may be about 25 μm to about 100 μm. For example, a 75 mm × 25 mm slide with 7 mm edges, 100 μm square wells with 100 μm spacing between the well ends will have about 16,775 micro wells.

  A highly accurate / precise harvesting system to perform various harvesting functions from and / or to a portion of the membrane corresponding to the microwell on a microfabricated chip as described above Can be designed. The membrane may be a thin sheet already used to encapsulate the proliferating bacteria into the microwells. When peeled from the chip, the membrane can retain not only bacterial samples but also microwell array imprints on its surface after separation from the chip. Thus, the exfoliation membrane can function as a replica of the bacteria grown on the chip.

  The high resolution sampler can receive data input from a user. The input includes at least one pair of chip microwell coordinates, so that the harvester may collect from at least one input pair's coordinates and / or to at least one input pair's coordinates. Mechanical sterilization may be performed between cycles. The high resolution collector may be operable in the anaerobic chamber.

  A high resolution harvester system can receive the chip and align the harvester with the chip, for example using a reference label and / or reference wells. The harvester can harvest, for example, cells grown in microwells on a chip into, for example, a 96-well or 384-well plate containing culture medium. The collector may include a single collection pin or multiple collection pins.

  A high resolution harvester system can receive the membrane and align the harvester to the membrane, for example using a reference well label on the membrane. The harvester may harvest, for example, cells from the membrane into a 96-well or 384-well plate containing, for example, culture medium. The collection pin can have various shapes (e.g. mushroom shaped) and / or a collection surface (e.g. texture) from the membrane. The system may also include one or more mechanisms (eg, floating pins and / or vacuum) to hold and / or flatten the membrane.

  High resolution sampler systems may enable chip replication through chip-to-chip movement. The harvester may receive and align the first chip based on the reference label and / or the reference well. The harvester may also receive and align the second chip based on the reference label and / or the reference well. The harvester may, for example, transfer cells grown in the microwell on the first chip to the microwell on the second chip.

  A high throughput system for automatically harvesting target species of multiple species of at least one biological entity cultured on a microfabricated device may include a port for receiving the microfabricated device. The microfabricated device defines a high density array of microwells. Each microwell of the high density array of microwells is configured to isolate and culture at least one species of at least one biological entity and includes at least one of a plurality of specific tags. Each tag of the plurality of specific tags comprises a nucleic acid molecule, which nucleic acid molecule correlates with a target specific nucleotide sequence for annealing to the at least one biological entity as well as at least one microwell of the high density array of microwells It is intended to include regiospecific nucleotide sequences of interest. The system also incorporates a high resolution harvesting device with at least one protrusion for harvesting at least one biological entity from at least one microwell of a high density array of microwells. The system further incorporates an input device for receiving an indication of the at least one target specific nucleotide sequence and at least one processing unit communicatively coupled to the input device and the high resolution acquisition device. The at least one processor obtains at least one target specific nucleotide sequence from the input device, compares the at least one target specific nucleotide sequence to a plurality of specific tags, and based on the comparison, a high density macro well High resolution to determine at least one microwell containing the target species in the array and to collect at least one biological entity from at least one determined microwell in the high density array of macrowells Control the sampling device.

  A high throughput system is disclosed for automatically harvesting target species among multiple species of at least one biological entity cultured in a microfabricated device. The microfabricated device defines a high density array of microwells, each microwell of the microwell high density array being associated with at least one unique primer of the plurality of unique primers. The system includes a port for receiving the membrane removed from the microfabricated device, the membrane being a high density array of microwells to retain at least one biological entity within the high density array of microwells. As each microwell was sealed, a portion of at least one biological entity corresponding to the high density array of microwells remains attached to the membrane after membrane removal. The system also comprises a high resolution harvesting device comprising at least one protrusion for harvesting at least one biological entity from at least one membrane location corresponding to at least one microwell of the high density array of microwells. An input device for receiving an indication of at least one target specific nucleotide sequence associated with the target species, and at least one processing device communicatively coupled to the input device and the high resolution collection device are incorporated. At least one processor obtains at least one target specific nucleotide sequence representation from the input device, compares the at least one target specific nucleotide sequence to a plurality of specific tags, and has a target species based on the comparison. Determine at least one membrane location corresponding to at least one microwell of the high density array of microwells, and high to extract a portion of the at least one biological entity from the at least one determined membrane location Control the resolution sampling system.

  The above embodiments can be implemented in any of numerous ways. For example, the embodiments may be implemented using hardware, software, or a combination thereof. In a software implementation, the software code may be executed on any suitable processing device or collection of processing devices, whether provided on a single computer or distributed to many computers.

  Moreover, the computer may be embodied in a device that is not generally regarded as a computer but has appropriate processing capabilities, including a personal digital assistant (PDA), a smart phone or any other suitable portable or fixed electronic device.

  Furthermore, a computer may have one or more input and output devices. These devices can be used, inter alia, to provide a user interface. Examples of output devices that may be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other audio generation devices for audible presentation of output. Examples of input devices that can be used for the user interface include a keyboard and a positioning device such as, for example, a mouse, touch pad or digitizing tablet. As another example, a computer may receive input information through speech recognition or other listening schemes.

  Such computers may be interconnected by any suitable form of one or more information communication networks, including a regional information communication network or a wide area information communication network, such as, for example, an enterprise communication network and an intelligent network (IN) or the Internet. obtain. Such an information communication network can be based on any suitable technology, can be operated according to any suitable protocol, and can include a wireless communication network, a wired communication network or an optical fiber communication network.

The various methods or steps outlined herein can be encoded as software executable on one or more processing devices using any one of a variety of operating systems or platforms. In addition, such software may be written using any of a number of suitable programming languages and / or programming or scripting means, and executable machine language code or code executed on a framework or virtual machine or It can also be converted as an intermediate code.
(Combined media strategy)

The systems, kits, devices and methods described above may be used in conjunction with one or more combined Media Strategies, one or more suitable or desired for a particular purpose. Large parallel experiments involving different media on the array of microwells on the microfabricated device disclosed herein may be employed to find the media. For example, combined media strategies
One may determine or select the tremendous potential of different media suitable for rapid growth of a particular microbial species or strain, or suppression of a particular microbial species or strain.

  The medium may be a general growth medium, a different medium, and / or a selective medium, a biological entity having a specific biological entity or a specific property (eg antibiotic resistance or synthesis of a specific metabolite) Growth media, differentiation media, and / or selection media. The medium may comprise nutrients or preparations that promote cell growth, or components that inhibit / delay cell growth, or even kill cells (antibiotics). As used herein, two media loaded in different microwells are considered when their composition is different or they are different in amount or concentration. When completely filled with different media, at least some of the individual microwells of the microfabricated device differ in the media contained therein. The variation of the contents of the medium may be across one or more dimensions of the plurality of microwells to test or discover the formulation or condition for a particular purpose.

  In some embodiments, one or more components of the medium are colorimetric or fluorescent that produce a signal in the microwell when the target analyte (eg, enzyme, metabolite, gene signature) is present. It may comprise a reporter analyte such as a labeling reagent.

  According to some embodiments herein, a method is provided for selecting media from a plurality of different media. The method comprises obtaining a microfabricated device comprising a plurality of microwells, wherein each microwell of the plurality of microwells comprises a medium, and a plurality of microwells comprises a plurality of media. A plurality of microwells are filled, each microwell of the plurality of microwells is filled with at least one cell from the sample, and the microfabricated device is cultured in a predetermined state for a predetermined time, and the plurality of microwells The content of the is compared across, and at least one medium is determined or selected from a plurality of different media based on the comparison result.

  According to some embodiments herein, filling the plurality of microwells with the medium comprises filling a volume of liquid smaller than the volume of the microwells into each microwell and individually A printer capable of controlling and filling with sufficient accuracy or resolution to address the address may be used. For example, the printer may have resolution for individual microwells having a diameter of 100 μm, or 50 μm, or even higher. Such printers have sufficient accuracy to address their microwells and print solutions in thousands of microwells per chip across multiple chips with volumes of 200 pL or less be able to. The volume may be as small as a few tens of picoliters (pL) and allow for smaller components in each microwell and / or more components in each microwell. This allows for the rapid and automatic formation of many different media compositions. Media test libraries may be maintained in the printing process by printing different base solutions in microwells of different concentrations, or may be made prior to printing. The small physical size and inexpensive price of the chip (several thousand plates / compared) make thousands to hundreds of experiments practical. As an example, it may be a droplet printing system that can withdraw liquid from a 96 well plate and then dispense them in as little as 30 pL. The printer may be programmed to take different liquids from the source plate and distribute them in different combinations across the array of microwells.

  The droplet printer described above may be used to load cells or other biological entities (or mixtures of them with cells) into microwells. According to some embodiments herein, methods of loading cells with lower precision may be used. For example, if cells of only one species are the subject of studies to determine their preferred medium among multiple options, or if all of the multiple microwells share part of the same material, A low precision spray system, a tensioner, or a pipette can be used. However, care should be taken to minimize the dislocation of the media already loaded in the microwell when these non-relevant loading methods are used.

  Figures 22A and 22B show matrices of different solutions printed on an array of microwells on a microfabricated device according to some embodiments disclosed herein. FIG. 22A shows printed droplets of media partially filling the microwells. FIG. 22B shows the printed droplet completely filling the microwell.

  According to some embodiments herein, the media may be loaded before, with, or after loading into the microwell. According to some embodiments herein, the filling of cells or other biological entities is performed on a microfabricated device in which the microwells are pre-filled with different media. According to another embodiment of the present invention, filling a plurality of different media may be performed on a microfabricated device in which the microwells are pre-filled with cells to be studied or other biological entities of interest. To be done.

  According to some embodiments herein, the place where the media is first printed on the microwell may be dried. The microfabricated device so treated can be stored. Later, the solution of cells to be tested (e.g. microorganisms or other cells) may be placed on the drying medium in the microwells, for example filled with pipette or printed. The microwells can then be sealed, for example, with a membrane or other material, and then subsequent procedures can be performed, including incubation and observation / comparison.

  According to some embodiments herein, the cell sample solution may be initially placed on the microfabricated device, eg, printed or pipetted, to dry the sample. Multiple media may then be printed on the cells loaded into the microwells. A sealing material (e.g., a membrane) may then be applied to seal the microwell and subsequent procedures including culture and observation / comparison may be performed. In this approach, consistency with the initial filling (eg, the amount of cells to be filled in each microwell, etc.) is important if it is a low precision filling (eg, pipette filling) Doubling cultures can be analyzed meaningfully. In order to improve the results, it is better to first fill the microwell with more cells in order to reduce the overall variability of the microwell.

  According to some embodiments herein, the microwells may be filled with a medium containing multiple nutrients or components. For example, the formulation can be configured in the microwell during the printing process by printing multiple base solutions separately on the microwell through multiple passes of the printer.

  After the media and cells are loaded onto the microfabricated device and prior to culture, the microwells may or may not be sealed. According to some embodiments herein, the microfabricated device is packaged such that the solution can be moved in and out of the microwell while confining the sample. According to some embodiments herein, the microfabricated device can be observed (e.g., using an inverted microscope to monitor growth, etc.) and optically clear materials may be used. According to the first several embodiments desired, the microwells may be sealed with a gas permeable, liquid permeable, or impermeable screen. According to some embodiments herein, polypropylene filled microwells using a laminator at appropriate pressure, temperature, and speed settings to separate microwells while allowing them to be imaged with a microscope Seal with a thin layer of PDMS with a backer.

  Media placed in the microwells across the chip can represent step-by-step or other changes in microwell to microwell content and / or formulation across the chip in one or more dimensions . Unlike looking for slight differences between the contents of two or more petri dishes, the changes in the array of microwells on the chip are the same relative with the same conditions (eg, temperature, humidity, atmosphere, etc.) It occurs over a period of time, allowing more meaningful comparisons between microwells.

  Changes in one or more observable properties (eg, biological, chemical, and / or phenotypic) of the contents of the microwell can be assessed during and / or after culture . The evaluation can be performed one or more times according to a predetermined schedule or time course. For example, such an evaluation may be visual inspection and / or analyte (eg colorimetric change, generation of fluorescent markers etc), real-time sequential imaging, microscopy, optical density, Fluorescence microscopy, microscopy, mass spectrometry, Raman spectroscopy, thermal imaging, electrochemical, amplification (DNA, cDNA, and / or RNA), sequencing (DNA and / or RNA), nucleic acid hybridization (nucleic acid) It may be based on techniques including, but not limited to, hybridization, and / or antibody binding. In some embodiments herein, such an assessment may be based on performing digital image analysis.

  In some embodiments herein, the contents of the microwell image the microwell (as required), based on changes in contents based on optical density, color, fluorescence or any surrogate. It may be assessed by performing digital image analysis of the image.

  In some embodiments herein, the plurality of different media loaded into the plurality of microwells may be loaded into separate regions of the plurality of different media microwells. Each separate area contains the same medium, but microwells in different areas contain different medium. The separate regions can include the same number of microwells, but not necessarily the same number. As an example, four replicas of the small media panel were printed on a microfabricated device. In FIG. 23A, each panel is shown as 2310, 2320, 2330 with 9 × 9 microwells divided into 3 × 9 microwells. All microwells contained Acinetobacter calcoaceticus. The first region 2310 received only PBS (phosphate buffered saline) as a control means. The second region 2320 received R2A, a minimal culture media known to grow Acinetobacter slowly. The third region 2330 received a rich culture media known to rapidly grow Acinetobacter. The microfabricated chips were cultured at room temperature for one day for growth. 23B-23D show microscopic images of the contents of representative microwells from 2310, 2320 and 2330, respectively, which recently demonstrate the effect of different media on the growth rate of cells. As expected, microwells loaded with TSB showed maximal growth of Acinetobacter, whereas microwells loaded with PSB showed no proliferation.

  Beyond simple visual inspection, analysis of the image of the contents of the microwell can provide further information. The digital image of the microwell being examined is via the imaging software to retrieve the attributes of the microwell and the pixels in the microwell, for example, an intermediate gray value (which is a value that represents how dark or light it is) Can be executed and calculated. The analysis result of such an image is shown in FIG. 24 about a microwell. Intermediate gray values of different microwells of the same area, filled in the same medium, can obtain different statistics (such as average) and are calculated as a group of microwells. For example, the median gray values of empty microwells, microwells filled with PBS, microwells filled with R2A, and microwells filled with TSB are 204, 199, 189, and 125, respectively. Intermediate gray values from these images can be converted to optical density measurements by including a standard value known as the known optical density on the microfabricated device to measure with the other microwells.

  In addition to cells (e.g., derived from a microorganism such as recently or a mammal), the above-described strategies and procedures may be applied to other components such as cell components, cell products, viruses, or mixtures, or consortia of different species or entities. It may also be used to find media or growth conditions of biological entities. Some embodiments may be used to optimize chemical reaction conditions, or other conditions where it is desirable to test multiple conditions for a target process or output parameter.

In the case of cells, the medium loaded in the microwell may include one or more nutrients or preparations that can be expected to promote cell growth. In other embodiments, the vehicle may include one or more antibiotics that can be expected to slow or inhibit cell growth. For example, different antibiotics and / or different amounts or concentrations of antibiotics can be placed in different microwells. For example, each microwell may have one of twenty different concentrations of one of thirty different antibiotics at one of twenty different concentrations for six hundred different test conditions, twenty different concentrations of for six thousand different test conditions. Even 300 different antibiotics may be loaded onto a single microfabricated chip. Next, fill the chip so that a small number of microorganisms (eg, material from isolates or pure cultures of microorganisms isolated from clinical material such as blood, urine, or abscess) are located in each well Also good. Chips can also be cultured and growth monitored using the tools and procedures described above. The microbes grow in microwells which are resistant to the antibiotics therein or whose amount of antibiotics therein is insufficient to inhibit the growth of the microorganisms. The microbes will grow slowly or not at all in the microwells where there are inhibitory concentrations of antibiotics to which the microbes are sensitive. Thus, these embodiments allow for the determination of antibiotic susceptibility of microbial isolates, and can also, for example, elicit antibiotic therapy for a subject.
(Conclusion)

  While various embodiments of the present invention have been described and illustrated herein, one of ordinary skill in the art will appreciate that various other implementations of the functionality may be performed to achieve the results described above and / or one or more advantages. Means and structures should be easily imagined, and each such modification and / or modification is considered to be within the scope of the embodiments of the invention described herein. More generally, it is meant by those skilled in the art that all parameters, dimensions, materials and configurations mentioned herein are representative, and actual parameters, dimensions and / or configurations are: It should be readily apparent that the teachings of the present invention depend on the particular application or applications utilized. Those skilled in the art should recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Therefore, the embodiments described above are presented by way of example only, and within the scope of the appended claims and their equivalents, the invention will be different than specifically stated and claimed. It should be understood that embodiments of the can be implemented. Embodiments of the present invention are directed to the individual functions, systems, articles, materials, kits and / or methods described herein. Furthermore, provided that such functions, systems, articles, materials, kits and / or methods do not conflict with one another, a combination of two or more of those functions, systems, articles, materials, kits and / or methods. Are included within the scope of the present invention.

  Moreover, various inventive concepts are embodied in one or more ways, examples of which are provided. The operations performed as a single ring of the method may be ordered in any suitable manner. Thus, embodiments may be configured in which the operations are performed in a different order than that illustrated, including that multiple operations are performed simultaneously, even if shown as sequential operations in the illustrated embodiments. It is good.

  The entire text of each of all the works, patent applications, patents, and other references mentioned herein is incorporated herein by reference.

  All definitions should be understood as controlling dictionary definitions, definitions in documents incorporated as references, and / or the ordinary meaning of the defined terms, as defined and used in this document. It is.

  The indefinite articles "a" and "an" used in the description and claims of the present application are understood to mean "at least one" if "one" or no explicit designation of a number. It should be.

  The phrase "and / or" as used in the specification and claims of this application refers to the elements so linked, ie, in some cases, in a conjugate manner and in other cases in a separate case, It should be understood to mean “one or both” of Numerous elements listed with "and / or" should be construed similarly, ie, one or more of the elements are so linked. In addition to the elements specifically identified by the phrase "and / or", other elements may optionally be present, whether or not they are associated with the identified elements. Thus, as a non-limiting example, when reference to "A and / or B" is used with an unlimited phrase such as "includes", in certain embodiments (elements other than B may be optional Can include only A, but in alternative embodiments (but may optionally include elements other than A), and in yet other embodiments (which may include other elements as options) A and B And so on.

  It is to be understood that "or" as used in the specification and claims of the present application has the same meaning as "and / or" as defined above. For example, when sorting items in a list, "or" or "and / or" also includes anything, ie not only include at least one of the numerous or listed elements but also one or more It shall be interpreted as including, and optionally including items not listed in the additional list. On the contrary, for example, only clearly designated items, such as "only one" or "just one" or "consisting of" used in the claims, are a large number of items or a list of items It will apply to the inclusion of exactly one element in. In general, the term "or" as used in this document may be preceded by an exclusivity term such as "one or the other", "one", "only one" or "just one" It shall only be interpreted as indicating the exclusion of alternatives (ie one or the other, not both) if As used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

  The phrase "at least one" in the context of a list of one or more elements as used in the specification and claims of the present application is selected from any one or more of the list of elements It should be understood to mean at least one and does not necessarily include at least one of all the elements specifically listed in the element list and does not exclude any combination of elements in the element list It should be understood to mean that. This definition also allows elements other than those specifically identified in the list of elements to which the phrase "at least one" relates to to be optionally present, with or without association with those specific elements. I have to. Thus, by way of non-limiting example, “at least one of A and B” (in other words, at least one of A or B, in other words at least one of A and / or B), in one embodiment B is A including at least one, optionally one or more, in the non-existent state (optionally including elements other than B); in another embodiment, A is not present (elements other than A B) optionally containing at least one, optionally one or more, B; in still another embodiment at least one, optionally including one or more, A and at least one, option And B (and optionally including other elements); etc. may be included.

  Also in the claims, as in the above specification, for example, "include", "have etc", "carry", "have", "contain", "accompany", "hold" All transition phrases such as “constituted” should be understood to mean open, ie “including” and “others”. Only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively, as set forth in the Patent Examination Procedure of the United States Patent Office Manual, section 2111.03. .

If the sample contains multiple cells or viruses, those cells in the sample may be lysed after being applied to the microfabricated device to release the nucleic acid molecules. The cells can be lysed using, for example, chemical treatments such as alkaline exposure, detergents, sonication, proteolytic enzyme K or lysozyme exposure. The cells can also be lysed by heating.

As an example to demonstrate this idea, as a simple illustration of a sample containing two species, one shown in Table 6, one species is doubling every day and another species is doubling every week Consider. Assuming that the late adult species is initially rare with a relative abundance of 5%, it will soon become very rare as both species grow.

Assuming that early adult species are limited by physical space for nutrient competition and / or growth, then, as shown in Table 7, the relative abundance of late adults begins to increase after some time has elapsed There is.

(High-density microfabricated array for biobank cells)

  Biobanks are designed to open the way for researchers to obtain large numbers of samples from large populations in order to drive certain types of research, such as disease-related biomarker discovery. The state of the art in biobanking provides for tubes or samples to be stored in the form of low density plates, eg 96-well or 384-well plates. This works when the number of samples to be loaded is relatively small and the samples themselves are discrete, isolated groups of solids. Existing approaches to biobanking can be complex and inefficient when storing samples such as microbial samples, where the number of samples may be large and the number of different species or variants within each sample is one There may be hundreds to thousands or even millions of samples per sample. Using existing methods, unless a laborious isolation protocol is performed to separate individual species or variants either prior to or subsequent to the storage step to obtain the desired species or variant. It does not.

Claims (26)

Obtaining a microfabricated device comprising a plurality of microwells;
Filling a plurality of different media into the plurality of microwells, each of the plurality of microwells comprising a medium, and the plurality of microwells comprising a plurality of different media;
Filling at least one cell from a sample into each of the plurality of microwells;
Culturing the microfabricated device for a predetermined time under predetermined conditions;
Comparing the contents of the plurality of microwells across the plurality of microwells; and
Determining at least one medium from the plurality of different media based on the comparison.   The method of claim 1 including the step of the medium being loaded into each microwell using a droplet printer.   The method according to claim 1, characterized in that the at least one cell is loaded into each microwell using a droplet printer.   The method according to claim 1, comprising the step of filling the at least one cell into each microwell using a pipette.   The method according to claim 1, wherein comparing the contents of the plurality of microwells comprises evaluating at least one observable property of the contents of the plurality of microwells. the method of.   The method of claim 1, wherein comparing the contents of the plurality of microwells comprises performing digital image analysis of an image of the contents of the plurality of microwells. .   The method of claim 1, wherein the step of loading the medium comprises the step performed prior to loading the at least one cell.   The method of claim 1, wherein the step of loading the medium comprises the step performed after loading the at least one cell.   The method of claim 1, wherein the step of filling the at least one cell comprises the step of filling a plurality of cells in each of the microwells.   The step of filling the plurality of microwells with the plurality of different media fills the plurality of different media into different regions of the microwell on the microfabricated device, and the different regions are one or more microwells. In which the microwells within each of the separate regions are the same, and the media included in the different regions of the microwells comprise filling differently. The method described in 1.   The step of comparing the contents of the plurality of microwells comprises the step of comparing statistics of the characteristics of the respective contents of the microwells of the separate regions. 11. The method according to item 10.   The method of claim 1, further comprising sealing the plurality of microwells prior to culturing the microfabricated device.   13. The method of claim 12, further comprising opening the plurality of microwells to evaporate liquid prior to comparing the contents of the plurality of microwells.   The method of claim 1, wherein comparing the contents of the plurality of microwells comprises evaluating at least one property of the contents at a plurality of time points.   The method of claim 1, wherein the medium loaded in at least one of the plurality of microwells comprises a nutrient.   The method of claim 1, wherein the medium loaded in at least one of the plurality of microwells comprises an antibiotic. A method of selecting a medium using a microfabricated device comprising a plurality of microwells, wherein the microfabricated device comprises the plurality of microwells filled transversely with a plurality of different media,
Said method is:
Filling at least one cell from a sample into each of the plurality of microwells;
The microfabricated device is incubated for a predetermined time under predetermined conditions;
Comparing the contents of the plurality of microwells;
Determining at least one of the plurality of different media based on the comparison. A method of selecting a medium using a microfabricated device comprising a plurality of microwells, comprising at least one cell in each of the plurality of microwells,
Said method is:
Filling a plurality of different media across said plurality of microwells;
The microfabricated device is incubated for a predetermined time under predetermined conditions;
Comparing the plurality of cells across the plurality of microwells;
Determining at least one of the plurality of different media based on the comparison. Obtaining a microfabricated device comprising a plurality of microwells;
Filling a plurality of different media into the plurality of microwells, each of the plurality of microwells comprising a medium, and the plurality of microwells comprising a plurality of different media;
Loading a biological entity of interest into each microwell of the plurality of microwells;
Comparing the contents of the plurality of microwells across the plurality of microwells;
Determining at least one medium from the plurality of different media.   20. The method of claim 19, comprising at least one cell of the biological entity. Said plurality of at least 150 micro-well surface density per cm ² of microfabricated device, at least 250 microwells per cm ^2, at least 400 microwells per cm ^2, at least 500 microwells per cm ^2, at least 750 microwells per cm ^2, at least 1,000 micro wells per cm ^2, at least 2,500 micro wells per cm ^2, at least 5,000 micro wells per cm ^2, at least 7,500 micro wells per cm ^2, at least 10,000 micro-wells per cm ^2, cm ² per at least 50,000 micro-well, cm ² at least 100,000 microwells per or a method described in any one of claims 1 to 20 is at least 160,000 microwells per cm ^2.   22. A microwell of each of the plurality of microwells of the microfabricated device, wherein the diameter is about 5 μm to about 500 μm, about 10 μm to about 300 μm, or about 20 μm to about 200 μm. The method described in either. A microfabricated device constituted by a plurality of microwells; and
A kit, wherein a plurality of different media are filled in the plurality of microwells, and each of the plurality of microwells is constituted of the plurality of different media.   The plurality of different media are filled in microwells of separate areas on the microfabricated device, each of the separate areas being constituted by at least one or more microwells, each of the separate areas being The kit according to claim 23, characterized in that the microwells in the range are the same and the media contained in different regions of the microwells are loaded differently. Said plurality of at least 150 micro-well surface density per cm ² of microfabricated device, at least 250 microwells per cm ^2, at least 400 microwells per cm ^2, at least 500 microwells per cm ^2, at least 750 microwells per cm ^2, at least 1,000 micro wells per cm ^2, at least 2,500 micro wells per cm ^2, at least 5,000 micro wells per cm ^2, at least 7,500 micro wells per cm ^2, at least 10,000 micro-wells per cm ^2, cm ² per at least 50,000 micro-well, cm ² per at least 100,000 microwells, or kits according to any one of claims 23 or 24 is at least 160,000 microwells per cm ^2.   26. A microwell of each of the plurality of microwells of the microfabricated device, wherein the diameter is about 5 μm to about 500 μm, about 10 μm to about 300 μm, or about 20 μm to about 200 μm. The kit described in either.

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