JP2002525600A - Miniaturized cell array method and device for cell-based screening - Google Patents

Abstract

SUMMARY The present invention relates to an apparatus and method for maximizing the number of wells that can be imaged at one time, while still having adequate pixel resolution in the image. This result was achieved through the use of a fluid construct to maximize well density. Thus, the present invention comprises a fluid exchange and a closed fluid volume in which closely spaced wells are provided to more quickly detect the spatially resolved features of individual cells. To provide a miniaturized microplate system.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD [0001] The present invention relates to methods and apparatus for cell-based high throughput and high biological content screening.

BACKGROUND OF THE INVENTION This application is incorporated herein by reference in its entirety, September 22, 1998.
It is a continuation-in-part of U.S. Provisional Patent Application Ser.

[0003] In the expanding competition of drug discovery and combinatorial chemistry to create candidate compounds, a very large number of substances are rapidly screened via high-throughput screening for their physiological impact on animals and humans. What you can do will be very useful. Before testing the effect of a "partially qualified" drug candidate on an animal, the drug may first be screened on living cells for its biological activity and potential toxicity. The biological response to the drug candidate can be predicted from the results of these cell screens.

[0004] Traditionally, "lead compounds" have been rapidly transferred to a wide range of animal experiments that are time consuming and costly. In addition, extensive drug testing in animals has become more differentially unacceptable. Screening drug candidates according to their interaction with living cells prior to animal testing is necessary in subsequent drug screening processes by removing some drug candidates before going to animal testing. The number of animals can be reduced. However, manipulation and analysis of drug-cell interactions using current methods requires only a small number of cells and compounds that can be analyzed at a given time, the cumbersome methods required for compound delivery, and the compounds required for testing very large volumes. Therefore, it does not allow both high throughput and high biological content screening.

[0005] High throughput screening of nucleic acids and polypeptides has been achieved using DNA chip technology. In a typical DNA analysis method, 10 to 14
Base paired DNA sequences are deposited on small glass plates at defined locations (or spots) of up to tens of thousands (see US Pat.
556, 752). This creates an array of DNA spots on a given glass plate. The location of the spots in the array provides an address for later reference for each spot of DNA. The DNA sequence is a complementary DN labeled with a fluorescent molecule.
Hybridizes with the A sequence. Signals from each address on the array are detected when fluorescent molecules attached to the hybridizing nucleic acid sequence fluoresce in the presence of light. These devices have been used to provide high-throughput screening of DNA sequences in drug discovery efforts and human genome sequencing projects.
Similarly, protein sequences of various amino acid lengths have been attached to spots that are distinguished as arrays on glass plates (US Pat. No. 5,14,14, incorporated by reference).
3,854).

[0006] The information provided by arrays of either nucleic acids or amino acids bound to glass plates is limited according to their underlying "language." For example, D
The NA sequence has a language of only four nucleic acids, and the protein has a language of about 20 amino acids. In contrast, living cells that contain a complex organization of biological components contain DNA, RN
A. It has an enormous "language" with comparable magnitudes of potential interactions with various substances such as cell surface proteins, intramolecular proteins and the like. Since the typical target for drug action is somatic and intracellular, the cells themselves provide an extremely useful screening tool in drug discovery when combined with sensitive detection reagents. Thus, having a high-throughput, high-content screening device to provide high-content spatial information at the cellular and subcellular levels and temporal information about changes in physiological, biochemical and molecular activities Would be most useful.

Microarrays of Cells Various methods have been proposed for making single-cell type microarrays on conventional substrates for other applications. One example of such a method is photochemical resist-photolithography (Mrksich and Whitesides, Ann. Rev. Biophys. Biomol. Struct. 25: 55-78, 1996), wherein:
The glass plate is uniformly coated with photoresist and a photomask is placed over the photoresist to define the desired "array" or pattern. Upon exposure to light, the photoresist in the unmasked areas is removed. The entire surface defined by photolithography is uniformly coated with a hydrophobic material, such as an organosilane, that binds to both the exposed glass and the photoresist-coated areas. Then, the glass plate is washed with an organosilane having a chemically reactive group such as a terminal hydrophilic group or an amino group. The hydrophilic organosilane binds to the exposed glass and the resulting glass plate crosses the hydrophobic surface (
It has an array of hydrophilic or container spots (located in the area of the original photoresist). An array of spots of hydrophilic groups provides a substrate for the non-specific and non-covalent attachment of certain cells, including those of neuronal origin.

In another method based on specific but non-covalent interactions, stamping is used to generate protein-adsorbing alkanethiol-coated gold surfaces (US Pat. No. 5,776,748). No .; Singhvi et al., Science 264:
696-698, 1994). The bare gold surface is then coated with a polyethylene glycol-terminated alkanethiol that resists protein adsorption. After whole surface exposure to laminin (a cell-binding protein found in the intracellular matrix), live hepatocytes uniformly bind to and proliferate on laminin-coated islets (Singhvi et al., 1994).
). Techniques involving strong but non-covalent metal chelation have been used to coat gold surfaces with specific protein patterns (Sigal et al., Anal. Chem. 68: 490-).
497, 1996). In this case, the gold surface is patterned with alkanethiol terminated with nitroacetic acid. Bare areas of gold are coated with tri (ethylene glycol) to reduce protein adsorption. After the addition of Ni ²⁺ , the specific adsorption of the five histidine-tagged proteins was found to be dynamically stable.

[0009] More specific single cell type binding can be achieved by chemically cross-linking specific molecules, such as proteins, to reactive sites on the patterned substrate (
Aplin and Hughes, Analyt. Biochem. 113: 144-148, 1981). Another technique of substrate patterning produces an array of reactive spots, if desired. The glass plate is washed with an organosilane that chemisorbs the glass and coats the glass.
The organosilane coating is illuminated by deep ultraviolet light through an optical mask that defines the pattern of the array. Irradiation breaks Si-C bonds to form reactive Si radicals. Reaction with water causes the Si radicals to form polar silanol groups. The polar silanol groups constitute spots on the array and are further modified to spot other reactive molecules as disclosed in US Pat. No. 5,324,591 which is incorporated herein by reference. Allow coupling. For example, silanes containing biological functional groups such as free amino groups can react with silanol groups.
Free amino groups can be used as sites of covalent attachment for biomolecules such as proteins, nucleic acids, carbohydrates and lipids. Non-patterned covalent attachment of lectins known to interact with the surface of cells to glass substrates via reactive amino groups has been demonstrated (Aplin and Hughes, 1981). Optical methods for forming single-cell type microarrays on a support require fewer steps and are faster than photoresist methods (ie, only two steps), but high intensity ultraviolet light from expensive light sources. Requires the use of

In all of these methods, the result is a single cell type microarray.
This is because biochemically specific molecules bind uniformly to micropatterned chemical arrays. In the photoresist method, cells bind to a spot of free amino groups by adhesion and / or an array of specific molecules that attach to spots that in turn bind to the cell. Cells bind to all spots on the array in a similar manner. In the optical method, cells bind to an array of spots of free amino groups. There is little or no difference between free amino group spots. Again, the cells adhere to all spots in a similar manner, and thus only a single type of cell interaction can be studied on these cells. This is because each spot on the array is substantially identical to one another. Such a cell array is inflexible in its utility as a tool for studying particular cells or particular cell interactions in a single sample. Thus, comprising an array of multiple cell types on a common substrate, and a high throughput and high biological content screening of cells to increase the number of cell types and specific cell interactions that can be analyzed simultaneously There is a need for a method for producing these microarrays of multiple cell types on a common substrate.

Optical Readings of Cell Biology Performing high-throughput screening on thousands of compounds requires parallel handling and processing of many compounds and assay compound reagents. Standard high-throughput screening uses a homogeneous mixture of compound and biological reagents with several indicator compounds loaded into an array of wells in a standard microplate with 96 or 384 wells (Kahl J. Bi
omol.Scr. 2: 33-40, 1997). The signal measured from each well (either fluorescent emission, optical density, or radioactivity) integrates the signal from all materials in the well and gives the overall population average of all molecules in the well. This type of assay is commonly referred to as a homogeneous assay.

US Pat. No. 5,581,487 describes an imaging plate reader that uses a CCD detector (charge coupled photodetector) to image the entire area of a 96 well plate. The image is analyzed to calculate the total fluorescence per well for a homogeneous assay.

[0013] Schoeder and Neagle selectively excite fluorescence within approximately 200 microns at the bottom of a well in a standard 96-well plate when imaging a cell monolayer. A low-angle laser scanning irradiation and mask to reduce blemishes are described (J. Biomol. Scr. 1: 75-80, 1996). This system uses a CCD camera to image the entire area at the bottom of the plate. This system measures the signal from the cell monolayer at the bottom of the well, but the measured signal is averaged over the area of the well and is therefore still considered a uniform measurement. Because it is the average response of a population of cells. Analyzing the image,
Calculate total fluorescence per well for a cell-based homogeneous assay.

[0014] Proffitt et al. (Cytometry 24: 204-213, 1996) describe semi-automated fluorescence digital imaging with a system for quantifying relative cell numbers in situ, where cells are fluorescein Pre-treated with acetate (FDA). The system utilizes various tissue culture plates, especially 96-well microplates. The system consisted of an inverted fluorescence inverted microscope equipped with a motorized stage, a video camera, an image intensifier, and a microcomputer with a PC-vision digitizer. Turbo Pascal software controls the stage, scans the plate and takes multiple images per well. The software calculates total fluorescence per well, provides daily calibration, and configures various tissue culture plate formats. The threshold setting of digital images and the use of reagents that fluoresce only when taken up by living cells are used to reduce background fluorescence without removing excess fluorescent reagents.

[0015] Various methods have been developed to image fluorescent cells under a microscope and extract accurate information about the spatial distribution and temporal changes occurring in these cells. Recent papers describe many of these methods and their applications (Taylor (
Taylor) et al., Am. Scientist 80: 322-335, 1992). These methods have been designed and optimized for the preparation of a small number of analytes with high spatial and temporal resolution of the distribution, quantity and biochemical environment of the fluorescent reporter molecule in the cells.

Treatment of cells with dyes and fluorescent reagents, imaging of cells, and generation of cells to generate fluorescent reporter molecules such as modified green fluorescent protein (GFP) are useful methods (Wang et al.). , In Methods in Cell Biology, New York, Alan R. Liss, 29: 1-12, 1989). The jellyfish (Aequorea victoria) green fluorescent protein (GFP) has an excitation maximum of 395 nm and an emission maximum of 510 nm and does not require any extrinsic factors. The use of GFP for studying gene expression and protein localization is discussed in Chalfie et al., Science 263: 802-805, 1994. Some properties of wild-type GFP are described by Morise et al. (Biochemi
stry 13: 2656-2662, 1974) and Ward et al. (Photochem. Photobiol. 31: 6).
11-615, 1980). Rizzulo et al. (Nature)
358: 325-327, 1992) discusses the use of wild-type GFP as a tool for visualizing subcellular organelles in cells. Kaether and Gerdes (FEBS Letters 369: 267-271, 1995) report the visualization of protein transport along the secretory pathway using wild-type GFP. G in plant cells
Expression of FP was determined by Hu and Cheng (FEBS Letters 369: 331-334,19).
95), while GFP expression in Drosophila embryos is
vis) et al. (Dev. Biology 170: 767-729, 1995). No. 5,491,084, incorporated herein by reference, discloses the expression of GFP from Aequorea victoria in cells as a reporter molecule fused to another protein of interest. Mutants of GFP have been prepared and used in several biological systems (Hasselhoff et al., Proc. Natl. Acad. Sci. 94: 2122-2127, 1997; Bre.
Jc et al., Proc. Natl. Acad. Sci. 94: 2306-2311, 1997; Cheng et al., Nature Biotech, 14: 606.
-609, 1996; Heim and Tsien, Curr. Biol. 6: 178-192, 1996; Ehrig et al., FEBS Letters.
367: 163-166, 1995).

An array scan (ARR) developed by Cellmics, Inc.
AYSCAN ^™ ) system (U.S. patent application Ser.
No. 810,983 and U.S. Patent Application No. 09, filed February 27, 1998.
No./031,271) is an optical system for measuring the distribution, environment, or activity of a fluorescently labeled reporter molecule in a cell for the purpose of screening a very large number of compounds for specific biological activities. The (ARRAYSCAN ^™ ) system has a cell array containing fluorescent reporter molecules in a position array, scans a large number of cells at each position, converts optical information into digital data, and uses the digital data to label fluorescent cells in the cells. Measuring the distribution, environment or activity of the reporter molecule. (ARRAYSCAN ^TM
2.) The system processes, displays, and stores data, thus providing devices and computerized methods for increasing drug discovery by providing high content cell-based screening, and large microplates. Includes a combined high-throughput and high-content cell-based screen in format.

Microfluidic The efficient delivery of a solution to an array of cells attached to a cell substrate is facilitated by a microfluidic system. Precise handling of small liquid samples for ink delivery (US Pat. No. 5,233,369; US Pat. No. 5,486,855; US Pat. No. 5,502,467), biological sample aspiration (US Pat. No. 4,982,739), reagent storage and delivery (US Pat. No. 5,031,797), and distributed microelectronic and fluidic device arrays for clinical diagnostics and chemical synthesis (US Pat. No. 5,031,797). No. 585,069). In addition, a method and apparatus for the formation of microchannels in a solid substrate that can be used to detect small liquid samples along a surface has been described (US Pat. No. 5,571,410; US Pat. Patent No. 5,500,071; US Patent No. 4,34
4,816).

In particular, for the purpose of integrated high-throughput and high-content cell-based screening for live cell imaging, optimal microfluidic devices enable the closest possible well spacing (ie, highest possible well density). Fluid construct. Here, the fluid construct is integrated with the cell array substrate to enable efficient fluid delivery to the cells, eliminating the need for pipetting fluid into and out of the wells. Such an optimal microfluidic device would be advantageous for cell arrays at well internal distances below millimeter. This is because it is practical, if not impossible, to pipette fluids with such high spatial resolution and accuracy. Further, such integrated devices can be used directly in cell-based screening without the need to remove the cell substrate from the fluid construct to image the cells.

Optimal microfluidic devices for cell-based screening further include a closed chamber that allows for environmental control of the cells, preferably with electrodynamic forces that can affect the physiology of the cells on the substrate. Will not expose cells directly. For example, electrohydrodynamic pumping is less efficient with polar solvents (Marc Madou, Fundamentals of M
icrofabrication, CRC Press, Boca Raton, 1997, p. 433). Electro-osmotic pressure typically involves some electrophoretic separation of charged components such as proteins.

US Pat. No. 5,603,351 (“the '351 patent”) describes a microfluidic device that uses a multi-level design consisting of an upper level with two channels and a bottom level with reaction wells. However, this device was not designed for use in cell-based screening. "351 patent"
Does not disclose substrates containing cells or cell-engaging sites. The disclosed microfluidic network allows two or more reagents to react, as opposed to an optimal cell screening microfluidic system that allows live cells cultured on a well to be exposed in a lineage format to two or more different fluids. It is designed to allow it to be combined in a well. The '351 patent discloses a device having wells etched into a substrate at an optimum well density of 50 wells / inch 2. Further, the substrate must be detached from the fluid array for incubation and / or analysis. Finally, the '351 patent discloses a system of electronically controlled electrohydrodynamic values in a matrix of wells that is less efficient in aqueous media used in cell culture, and also in arrays of cells. The degree of close spacing between wells can be limited.

US Pat. No. 5,655,560 discloses a fluid distribution system with multiple inputs and multiple outputs that captures an orthogonal array of microchannels that are vertically coupled at orthogonal points by Teflon® valves. Disclose a valveless clogging system including However, this patent does not disclose a substrate containing a cell array, does not disclose an integrated fluidic device associated with the substrate, and does not disclose a well density that is optimal for cell-based screening.

US Pat. No. 5,900,130 (the “130” patent) describes the active and electrical control of fluid movement in an interconnected capillary structure. This patent does not teach a fluid construct that maximizes the area of the cell substrate that can be occupied by cell binding sites. Nor does this patent disclose a substrate containing the cell array, nor does it disclose an integrated fluidic device associated with the substrate. Further, the patent only teaches the control of fluid flow by applying an electric field to the device.

US Pat. No. 5,910,287 describes a multi-well plastic plate for fluorescence measurement of biological and biochemical samples, including cells, limited to plates with more than 864 wells I do. This patent does not describe a microfluidic device with a fluid construct integrated with a cell array substrate. Also, the patent does not disclose a closed chamber that allows environmental control of cells on the substrate.

Thus, in each of these prior microfluidic devices, the fluid construct is integrated with the cell array substrate to allow for efficient fluid delivery to the cells, and to provide the fluid into and out of the wells. It does not provide a fluid construct that allows for the closest possible well spacing (ie, highest possible well density) that eliminates the need for petting.
In addition, prior microfluidic devices, including arrays of wells, would be less efficient if used in aqueous media for cell culture and would have electronically controlled electronics within a matrix of wells that would limit well density. Use hydrodynamic values.

Although the foregoing advances in cell arrays, optical cell physiology readings, and microfluidic technology provide support technologies that can be applied to improved high-throughput and high-content cell-based screens, furthermore, Apparatus and methods for reducing the amount of time required for efficient screening, as well as for improving the ability to perform high-throughput and high-content cell-based screening and the ability to switch to each other flexibly and quickly There still exists a demand. In particular, an apparatus and method that maximizes well density, thereby increasing the number of wells that can be imaged at one time, thus greatly increasing screening throughput while maintaining adequate image resolution Would be very advantageous.

The drug discovery industry has already used 96- and 384-well microplates, which are shifting towards the use of 1536-well plates. However, further increases in well density using the prior art are unlikely due to the great confusion of pipetting liquid into and out of very small diameter wells.

DISCLOSURE OF THE INVENTION The present invention reduces the amount of time required to perform cell-based screening, and the ability to perform high-throughput and high-content cell-based screening and to switch flexibly and rapidly with each other. Satisfies the need in the art for devices and methods that specifically combine capabilities. The present invention provides an apparatus and method for maximizing the number of wells that can be imaged at one time, while still obtaining adequate pixel resolution in the image. This result was achieved through the use of a fluid construct that maximized well density. The present invention thus provides a miniaturized fluid exchange and closed fluid volume with well-spaced closely spaced wells to more quickly detect the spatially resolved features of individual cells. The present invention provides an optimized microplate system.

[0029] In one aspect, the invention includes a substrate having a surface containing a plurality of cell binding sites, a fluid delivery system for delivering a reagent to the plurality of cell binding sites, wherein the fluid delivery system comprises a substrate. Wherein the multi-level chamber includes an orthogonal array of microfluidic input and output channels and a plurality of fluid locations in fluid communication with the microfluidic input and output channels; and Multiple wells, where
A cell characterized in that each well comprises a space defined by the joining of one cell binding location and one fluid location, wherein the wells are present at a density of at least about 20 wells per cm ² A screening cassette is provided.

In another aspect, a. A substrate having a surface containing a plurality of cell binding sites, b. A fluid delivery system for delivering a reagent to a plurality of cell binding locations, wherein the fluid delivery system includes a multi-level chamber that interfaces with a substrate, the multi-level chamber comprising: i. An orthogonal array of microfluidic input and output channels, wherein each well is in fluid communication with one or more input channels and one or more output channels; ii. A plurality of fluid locations in fluid communication with the microfluidic input and output channels; iii. One or more input manifolds in fluid communication with the microfluidic input channel; iv. One or more output manifolds in fluid communication with the microfluidic output channels; At least one source container in fluid communication with one or more input manifolds; vi. Including at least one waste container in fluid communication with one or more output manifolds; c. A cassette for cell screening is provided, wherein the plurality of wells, wherein each well includes a space defined by the joining of one cell binding location and one fluid location.

In a preferred embodiment, both of the cassettes further comprise a pump for controlling the fluid flow in the microfluidic device, a temperature controller for the substrate and / or a controller for regulating the partial pressure of oxygen and carbon dioxide. including.

In another aspect, the present invention provides an improved method for diffusion control in a cassette, wherein the improvement is a passive restoration for a valve located in a microfluidic channel of the cassette. Including constant application of force.

In yet a further aspect, the present invention provides a method comprising: a) providing a position array containing a plurality of cells; b) providing an image of the position array with optics; c) sequentially sub-arraying the position array. And d) obtaining data from each of the sub-arrays in parallel.

In a preferred embodiment, the location array is provided as a cassette, such as those disclosed above.

[0035] Among other uses, the devices and methods of the present invention are ideal for high content and / or high throughput cell-based screening. Also, the device of the present invention is ideally suited as a cell support system for hand-held diagnostic devices (ie, a miniaturized imaging cell-based assay system). The smaller format and sealed containment of the device of the present invention allows its use in rigid portable systems. There are significant economic advantages from using higher density plates. In addition, there is an economic advantage in faster imaging of the sub-array when using high density plates, which cannot be fully realized if the distance between adjacent wells is not minimized. The present invention provides both of these advantages.

[0036] All patents, patent applications, and references cited herein are hereby incorporated by reference in their entirety.

As used herein, the term “well” is defined as a volumetric space created by joining one fluid location of a chamber with one cell binding location on a substrate. The volume space is separated by a recess on the chamber corresponding to the fluid location, by a recess on the substrate corresponding to the cell binding location, a spacer support separating the chamber and the substrate (where the recess in the substrate of the chamber is located). There is no requirement), any combination of these, or any other method for creating a volumetric space between a fluid location and a cell binding location.

As used herein, the term “cell binding site” refers to a distinct location on a substrate that contains multiple cell binding sites that can bind to cells. The substrate can be derivatized to create a cell binding site, or the cell binding site can be naturally present on the substrate. Multiple cell binding sites within an individual cell binding location can bind only to a single cell type, or can bind to more than one cell type. Different cell binding sites on the same substrate may be of the same or different types of cells that can be bound.

As used herein, the term “fluid location” refers to a distinct location on a chamber that is the site of delivery and / or removal of fluid to a cell binding location. The fluid location may include a recess, such as an etched domain, a raised container, or any other type of recess. Alternatively, the fluid location may be flat, such as at the end of an input and / or output channel, or at the end of a passage in fluid communication with the input and / or output channel.

As used herein, the term “cassette” refers to a combination of a substrate and a chamber. The combination can be any module (more than one piece), with reusable chambers and disposable substrates, or limit leakage in any potential wells rather than modules (single piece) I do.

As used herein, the term “on board” means integrated with the cassette.

As used herein, the term “integrated fluid” means that the microfluidic device includes both a substrate surface and a chamber for cell binding.

As used herein, the term “chamber” means a fluid delivery system that includes microfluidic channels and fluid locations, which acts as a specialized substrate cover.

As used herein, the term “switchable pump” includes, but is not limited to, a syringe pump, a pressure-controlled vessel with a valve, or a deleterious electric field effect not experienced by cells. As such, referring to pumps, including conductivity pumps used externally (with the desired use of electrical shielding) with respect to the matrix of the array of wells.

As used herein, the term “subarray” typically refers to a CCD (
Means any adjacent subsection of a well in the entire array of wells in a format corresponding to the shape of the imaging detector array (e.g., charge coupled device detector).

As used herein, the term “matrix of a cell array” refers to the space defined by a virtual parallelepiped that will fit around the entire set of wells in an array of cassettes. means. As used herein, the term "assay component" refers to a component added to a cell screening assay, including, but not limited to, reagents, cells, test compounds, media, antibodies, fluorescence and reporters. Say any component that might be.

As used herein, the term “fluorescence” includes, but is not limited to,
Also includes any type of luminescence, including cold light, luminescence and chemiluminescence.

In one aspect, the invention teaches a method of making a microarray of multiple cell types on a common substrate. As defined herein, a microarray of multiple cell types is not distributed over a uniform single coating on the surface of the support, but rather on each "cell binding site" or cell binding site on the substrate. Refers to an array of cells on a substrate that is in a heterogeneous format such that the group can be very rare in its cell binding selectivity.

A method for making microarrays of multiple cell types involves preparing a micropatterned array (also referred to herein as a chemically modified array of cell binding sites) and heterogeneously chemically modifying the array. And then attaching the cells to a heterogeneous chemical array.

In a preferred embodiment, the micropatterned array comprises a substrate 4 that has been treated to produce a hydrophobic surface across which hydrophilic spots or “cell binding sites” 8 are regularly spaced. (FIGS. 1A-1B). The substrate can be a glass, plastic or silicon wafer, such as a conventional light microscope cover glass, but can be made from any other suitable material that provides the substrate. As described above, the term "cell binding site" is used to describe a specific spot on the substrate and does not require any particular depth. The surface of the substrate 4 is preferably about 2 cm × 3
cm, but can be larger or smaller. In a preferred embodiment, the cell binding locations 8 of the micro-patterned array are not limited, but can be non-specifically bound to cells or further chemically bound to molecules that specifically bind to cells. Contains reactive functional groups such as aminohydroxyl, sulfhydryl or carboxyl groups that can be modified.

A chemically modified cell binding site is created by specific chemical modification of a cell binding site on a substrate. The cell binding site can include a variety of different cell binding molecules that can allow different types of cells to attach and proliferate at the cell binding site, or allow only a single cell type to attach. The hydrophobic domain surrounding the cell binding site on the substrate does not support cell attachment and proliferation.

In one embodiment, a micro-array of a plurality of cell types is created by coating glass with a layer of a material having a reactive functional group such as an amino group via chemisorption. In a preferred embodiment, an aminocyan such as 3-aminopropyltrimethoxysilane (APTS) or N- (2-aminoethyl-3-aminopropyl) trimethoxysilane (EDA) is used, but other reactable materials are used. Can also be used. Following this first step, the entire surface of the coated glass wafer is hydrophilic due to the presence of reactive functional groups.

Second, a micropatterning reaction is performed and droplets containing a photocleavable or chemically removable amino-protecting substance are deposited at distinct locations on the aminosilane-coated glass wafer. Put in a fine pattern. In one embodiment, the pattern includes a rectangular or square array, but any suitable distinguishing pattern (such as, but not limited to, a triangle or a circle) may be used. In one embodiment, the droplets range in volume from 1 nanoliter (nl) to 1000 nl. In a preferred embodiment, the droplet has a volume of 2
It is in the range of 50-500 nl. Suitable photochemically removable amino protecting substances include, but are not limited to, 4-bromomethyl-3-nitrobenzene, 1- (
4,5-dimethoxy-2-nitrophenyl) -ethyl (DMNPE) and butyloxycarbonyl. In one embodiment, the patterning reaction uses a reagent concentration of between 1 micromolar (μM) and 1000 μM, from ambient to 37 ° C.
C. for 1 to 100 minutes at a temperature in the range of .degree. In a preferred embodiment,
The reaction is performed at 37 ° C. for 60 minutes using a reagent concentration of 500 μM.

The droplets can be placed on aminosilane-coated glass wafers via conventional inkjet technology (US Pat. No. 5,233,369; US Pat. No. 5,486,855). Alternatively, an array of paper-coated rods and pins as defined herein capable of transferring between 1 nl and 1000 nl of fluid may be immersed in a bath of aminoprotective material and the ends of the protective material solution Generates a drop. The pins are brought into contact with the glass wafer to transfer droplets thereto. In another embodiment, US Pat. No. 5,5 containing an amino protecting substance (incorporated by reference).
An array of capillaries made of glass or plastic as described in 67,294 and 5,527,673 are contacted with a glass wafer to move droplets to the surface. Thus, a glass wafer is micropatterned with an array of spots or cell binding sites containing amino groups protected on a hydrophobic surface (FIG. 2A-
B).

Third, the hydrophobic material that is reactive with the unprotected amino groups is washed on the glass wafer. The hydrophobic material can be a fatty acid or an alkyl iodide, or any other suitable structure. Certain conditions for such derivatization of glass are Prime and Whitesides, Science 252: 11.
64-1167, 1991, Lopez et al., Journal of the American Chemical Society 115: 5877-5878, 1993, and Murksich and Whitesides, Ann. Rev. .Bi
omol. Struct. 25: 55-78, 1996. The fatty acid or alkyl iodide reacts with and is covalently bonded to an unprotected amino group, and the amino group is now hydrophobic due to the fatty acid or alkyl iodide group. The resulting modified array of cell binding sites 9 comprises a glass wafer 4 with a cell binding site array 8 containing protected amino groups on a hydrophobic background (FIG. 2C).

Fourth, a heterogeneous array of cell binding sites is created by uniformly deprotecting amino groups in a micropatterned array generated according to the method described above. In one embodiment, chemical specificity can be added by chemically crosslinking a specific molecule to a cell binding site. A number of well-known homo- or hetero-bifunctional crosslinkers, such as ethylene glycol bis (succinimidyl succinate), which will react with free amino groups at the cell binding site and crosslink to specific molecules, is there. Reagents and conditions for cross-linking free amino groups with other biomolecules are described in the following references: Grabarek and Gergel
y), Analyt. Biochem 185: 131-135, 1990: McKenzie et al., J. Prot. Chem. 7: 581-592,19.
88; Brinkley, Bioconjugate Chem. 3: 12-13; 1992, Fritsch et al., Bioco.
njugate Chem. 7: 180-186, 1996; and Aplin and Hughe
s), well-known in the art as exemplified by 1981.

In a preferred embodiment, the heterogeneous array of cell binding locations is generated in a combinatorial format. The resulting cell binding sites are heterogeneous (ie, each cell binding site or group of cell binding sites can be extremely rare in its cell binding selectivity). The term combinatorial means that cell binding sites are variably processed.

In one embodiment, the protected amino groups of the modified array of cell binding positions in step 3 are deprotected, and then the specific molecule is deposited in a desired pattern with a chemical cross-linking agent. The specific cross-linking agent can bind to an amino group and can carry a cell-binding group. In this step, the type of cell binding group can be varied from one cell binding site to another, or from one group of cell binding sites to another, resulting in a heterogeneous design in the array. .

In another embodiment, the amino group at the chemically modified cell binding position of step 3 is heterogeneously deprotected. The photoactivated crosslinker is reacted with the deprotected amino group.
An optical mask having a desired pattern is placed on the surface of the cell binding position, and the exposed cell binding position is irradiated with a light source. The location and number of cell binding sites that receive light are controlled by the fine pattern of the optical mask. Suitable photoactivated crosslinkers include arylnitrenes, fluorinated arylazi compounds, benzophenone and diazopyruvate. Reagents and conditions for optical masking and cross-linking are Prime
And Whitesides, 1991; Sighvi et al., 1994, Sigal et al., 1996, and Murksich and Whitesizes (Wh).
itesides), 1996. Photoactivated crosslinkers are bifunctional in that they chemically bind to amino groups at cell binding sites and, when exposed to light, covalently bind to cell binding molecules such as antibodies. Reagents and conditions for photoactivated cross-linking are described in Thevenin et al., Eur. J. Biochem. 206: 471-477, 1992 and Goldmacher et al., Bioconjugate Chem. 3: 104-107, 1992. Is being discussed.

If a photoactivated crosslinker is used, the glass plate is filled with cell binding molecules and bound to cell binding sites. In one embodiment, a cell binding molecule such as a surface antigen-reactive antibody, an extracellular matrix protein (eg, fibronectin or collagen) or a charged polymer (eg, poly-L-lysine or poly-L-arginine) is reduced to about 0. Use at concentrations ranging from 0.1 to about 1 mM. While the cell-binding molecules cover the cell-binding site, the glass plate is illuminated from the lower surface of the glass plate at an angle less than the critical angle of the material of the glass plate, resulting in total reflection of light. (For a discussion of total internal reflection fluorescence microscopy, see Thompson
Et al., 1993). In one embodiment, the irradiation is 300 nanometers (
nm) to 1000 nm for between 0.1 and 10 seconds at ambient temperature and 37 ° C. In a preferred embodiment, the irradiation is about 300 and 400 n
m for 1 second at room temperature using light of a wavelength between m. Optical crosslinking limits the photoactivated crosslinking to a short distance into the solution above the cell binding site, which is described in Bailey et al., Nature 366: 44-48, 1993; Farkas et al., Ann. .Rev.Physiol
.55: 785-817, 1993; Taylor et al., Soc. Opt. Instr. Eng. 2678: 15-27, 1996.
Thompson et al. , in Mason, WT, “Fluorescent and Luminesce
nt Probes for Biological Activity ”San Diego: Academic Press 405-
419, 1993.

The photoactivated cross-linking agent binds to cell-binding molecules such as antibodies and matrix proteins only at the cell-binding sites irradiated by the cross-linking agent. For example, a single row of a cell binding location array can be illuminated to produce a single row of cell binding locations with a cell binding molecule bound to a crosslinker. Following washing of the array to remove any unbound cell-bound molecules, the glass wafer is subsequently illuminated with the second cell-bound molecules while illuminating the second row and optionally masking other rows. Filling allows the second row of cell binding locations to bind to a second cell binding molecule. Unbound cell-bound molecules are removed by washing the array with PBS or any other suitable buffer. In this manner, a plurality of locations of a cell binding location or group of cell binding locations can be sequentially illuminated by sequentially masking in the presence of a particular cell binding molecule. Alternatively, each cell binding location can be illuminated by using pinpoint exposure and optical masking. In this way, different cell binding molecules are bound to rows or individual cell binding locations of the array, resulting in a heterogeneous array of cells bound to any desired pattern of cell binding locations.

In a further embodiment for producing a chemically modified array of cell binding sites, a chemically modified array is first generated, wherein the amino groups at the cell binding sites are homogenized with photocleavable protecting groups. Protected. Rows, columns and / or individual cell binding sites are sequentially photodeprotected to expose free amino groups by using various patterns of optical masks to cover except for the cell binding sites to be deprotected. Illuminate the exposed cell binding sites (ie, those not covered by the mask, thereby removing the protecting groups; chemically binding to the deprotected amino groups and activating the cell binding sites) The array is filled with a bifunctional cross-linking agent.The conditions for photodeprotection of amino groups are described in Padwa, A. (eds.) “Organic Photochemist.
ry ", New York 9: 225-323, 1987, Ten et al., Makromol. Chem. 190: 69-
82,1989, Pillai, Synthesis 1980: 1-26,1980, Self and Thompson (Thom
pson), Nature Medicine 2: 817-820, 1996, and Center (Senter) et al., Photo
chem. Photobiol. 42: 231-237,1985. Next, the cell binding molecules are loaded onto the modified chemical array, where they react with the other half of the crosslinker. The array is then washed to remove any unbound bifunctional crosslinker and cell binding molecules. Another cell binding site or set of cell binding sites can be deprotected using another optical mask, and the array can be bifunctionally cross-linked, followed by this second cell binding site of deprotected amino groups. Alternatively, it can be filled with a second treatment of distinct cell binding molecules that bind to the set of cell binding sites. The array is washed to remove the bifunctional crosslinker and the second treatment of cell binding molecules. In this way, a heterogeneous array of cell-bound molecules can be generated by repeated sequences of photodeprotection, chemical cross-linking of specific molecules, and washing under various masks. Alternatively, the cross-linking agent can be delivered to the deprotected cell binding site along with the cell binding molecule in one step.
The concentration gradient of attached cell-binding molecules can be created by controlling the number of exposed deprotected amino groups using an optical mask or by controlling the amount of irradiation for photoactivated crosslinkers. Can be.

[0063] The chemically modified array of cell binding sites is used to generate a heterogeneous array of cells on the cell binding sites. In one embodiment, introducing the suspected cells onto the array "spreads" the cells onto the modified chemical array, allowing the cells to bind to the cell binding sites, and then rinsing the wafer and Remove bound and weakly bound cells. Cells are allowed to bind only at cell binding sites. This is because, in combination with the hydrophobic environment surrounding each of the cell binding sites, the specific chemical environment at the cell binding site allows for selective binding of cells only to the cell binding sites. Furthermore, modification of a cell binding site with a specific cell binding molecule allows for selective binding of cells to the specific cell binding site, resulting in a heterogeneous array of cells at the cell binding site. In addition, cell surface molecules that specifically bind to the cell binding site are either naturally occurring or interact with the cell binding site where the cell has been modified,
It can be engineered by "cell binding site-specific" molecules fused to cell transmembrane molecules to specifically bind to it. The creation of an array of cell binding sites with different cell recognition molecules involves one cell binding site, a group of cell binding sites, or the entire array specifically "recognizing" cells from a mixed population of cells.
, Allowing it to grow and screen.

In one embodiment, cells suspended in culture at a concentration in the range of about 10 ^{3 to} about 10 ⁷ cells per liter are incubated with the cells at a temperature ranging from ambient to 37 ° C. for 1 to 120 minutes. And incubate. Unbound cells are then rinsed from the cell binding sites using medium or high density solution to lift unbound cells from the bound cells (Channavajjala et al., J. Cell.
Sci. 110: 249-256.1997). In a preferred embodiment, cells suspended in medium at a concentration ranging from about 10 ^{5 to} about 10 ⁶ cells per ml are contacted with the cell binding site at 37 ° C. for a time ranging from about 10 minutes to about 2 hours. And incubate.

The density of cells attached to a cell binding site is determined by the cell density in the cell suspension, the time allowed for cell binding to the chemically modified cell binding site, and / or the density of cell binding molecules at the cell binding site. Is controlled by In one embodiment of the cell attachment procedure, 10 ³ to 10 ⁷ cells per ml are incubated between 1 minute and 120 minutes between ambient temperature and 37 ° C., and the cell binding site is 0.1% per cm ² of cell binding molecule. And between 100 nanomolar. In a preferred embodiment, 1
Incubate 105 and 106 cells per ml at about 37 ° C. for 10 minutes to 2 hours, and the cell binding sites contain about 10 to 100 nanomoles per cm ² of cell binding molecule.

In one embodiment, the cells are derived from Bell et al., J. Histochem. Cytochem 3
5: 1375-1380, 1987; Poot et al., J. Histochem. Cytochem 44: 1363-1372,199.
6; chemically attached to the cell binding site as described by Johnson, J. Elect. Micros. Tech. 2: 129-138, 1985, and used as an antibody, nucleic acid hybridization probe or other Used in screening at a later time with fluorescently labeled molecules such as ligands.

In another embodiment, cells are modified with a fluorescent indicator of the chemical or molecular properties of the cells, seeded on a chemically modified array of heterogeneous cell binding sites, and then analyzed live. be able to. Examples of such indicators are:
Giuilano et al., Ann. Rev. Biophys. Biomol. Struct. 24: 405-434, 1995; Harootunian et al., Mol. Biol. Cell 4: 993-1002, 1993; Post et al., Mol. Bi.
ol. Cell 6: 1755-1768, 1995; Gonazalez and Tsien, Bi.
ophys. J. 69: 1272-1280, 1995; Swaminathan et al., Biophys. J. 72:19.
00-1907, 1997 and Chalfie et al., Science 263: 8.
02-805, 1994. Indicators may be, but are not limited to, as they are expressed under the conditions described (Chalfie et al., 1994).
Diffusion across cell membranes, mechanical perturbation of cell membranes (reviewed in Haugland, Handbook of Fluorescent Probes and Exploratory Chemicals, Sixth Edition, Molecular Probes, Inc., Eugene, 1996). (McNeil et al., J. Cell Biology 98: 1556-1564, 1984; Clarke and McNeil (
McNeil), J. Cell Science 102: 533-541,1992; Clarke
) Et al., BioTechniques 17: 1118-1125, 1994), or by any one or a combination of various physical methods, such as genetic engineering, before or after inoculation on the array. In a preferred embodiment, the cells contain a fluorescent reporter gene, but other types of reporter genes are also suitable, including those encoding chemiluminescent proteins. Living studies of living cells allow the analysis of the physiological state of cells as reported by fluorescence during the cell cycle or when contacted with drugs or other reactive substances.

In another aspect of the present invention, there is provided a heterogeneous cell array of cell binding locations, wherein cells are bound to a chemically modified array of cell binding locations on a substrate. Cell arrays are heterogeneous because the heterogeneous chemically modified arrays underlying cell binding sites provide a variety of cell binding sites of different specificities. Although any cell type can be aligned, it is assumed that molecules capable of specifically binding to that cell type are present in a chemically modified array of cell binding sites. Preferred cell types for heterogeneous cell arrays at cell binding sites include lymphocytes, cancer cells, neurons, fungi, bacteria and other prokaryotes and eukaryotes. For example, FIG.
Is at a cell binding site containing fibroblasts growing on a surface patterned substrate and labeled with two fluorescent probes (rhodamine to stain actin and Hoechst to stain nuclei) FIG. 3B shows a heterogeneous cell array, while FIG. 3B shows a heterogeneous cell binding site containing fibroblast proliferation (L929 and 3T3 cells) labeled with two fluorescent probes and visualized at different magnifications. 3 shows a cell array.

Examples of cell binding molecules that can be used in heterogeneous cell arrays at cell binding locations include, but are not limited to, antibodies, lectins, and extracellular matrix proteins. Alternatively, genetically engineered cells expressing a specific cell surface marker can be selectively attached directly to the modified cell binding site. Heterogeneous cell arrays at cell binding sites can include either fixed or live cells. In a preferred embodiment, the heterogeneous cell array at the cell binding site comprises living cells, such as, but not limited to, cells "labeled" with a fluorescent indicator of the chemical or molecular properties of the cells.

In another aspect of the invention, a heterogeneous cell array at a cell binding site is prepared,
Here, the cells contain at least one fluorescent reporter molecule, contacting the heterogeneous cell array at the cell binding site with a fluid delivery system to enable reagent delivery to the cells, and providing a low magnification total heterogeneous cell array. A method for analyzing cells characterized in that high-throughput screening is performed by obtaining fluorescent images of the cells, a fluorescent signal is detected at once from all cell-binding positions, and those exhibiting a response are identified. You. This is followed by high content detection within the responding cell binding sites using a set of fluorescent reagents with different physiological and spectral properties, and scanning the selected cell binding sites to detect the fluorescent reporter molecule in the cell. From a fluorescent signal, converting the fluorescent signal into digital data, and using the digital data to measure the distribution, environment or activity of the fluorescent reporter molecule in the cell.

[0071] Preferred embodiments of the heterogeneous cell array at the cell binding site are disclosed above. In a preferred embodiment of the fluid delivery system, the chamber is bonded to a substrate containing the heterogeneous cell array. The chamber is preferably made from glass, plastic or silicon, but any other material that can provide a chamber is suitable. One embodiment of the chamber 12 shown in FIG. 4 has an array of etched domains 13 that match cell binding locations 8 on the substrate 4. In addition, an input channel 14 is etched to supply fluid to the etched domain 13. A series of "output" channels 16 for removing excess fluid from the etched domains 13 can also be coupled to the cell binding locations. The chamber 12 and the substrate 10 are brought together to form a cassette 18.
Is configured. Although this embodiment utilizes etched domains, any other type of recess 13 formed at fluid location 1 can be utilized in this embodiment. Alternatively, the fluid location may be flat and cell binding location 8 may include a recess matching fluid location 1. In another alternative, the cell binding site 8 and the fluid location 1 are both flat and the volume space for the well is created by the use of a spacer support 20 between the substrate 4 and the chamber 12.

Thus, the chamber 12 is used for the delivery of a fluid to the cells aligned at the cell binding location 10. Such fluids include, but are not limited to, solutions of certain drugs, proteins, ligands or other substances that bind to or are taken up by cell surface expression sites. The fluid that interacts with the cells aligned at the cell binding site 10 can also include liposomes that encapsulate the drug. In one embodiment, such liposomes are formed from photochromic materials, which release drugs upon exposure to light, such as photoreactive synthetic polymers (Willner and Rubin). ), Edited by Chem. Int., Engl. 35: 367-385, 1996.
Has been reviewed). The drug can be released simultaneously from the liposomes in all chambers 14, or the individual channels or another row of channels can be irradiated to release the drug sequentially. Controlled release of such drugs can be used in genetic and live cell experiments. Control of fluid delivery can be achieved by a combination of micro-valves and micro-pumps well known in the capillary action art (US Pat. No. 5,567,2, incorporated herein by reference).
No. 94; US Pat. No. 5,527,673; US Pat. No. 5,585,069).
.

Another embodiment of the chamber 12 shown in FIG. 5 has a smaller diameter than the cell binding locations 8 on the substrate 4 so that the cell binding sites are immersed in the etched domains 13 of the chamber 12. Large chamber etched domain 13
Has an array of input channels 14 that match A spacer support 20 is placed between the cells aligned with the chamber 12 and the cell binding location 10 along the sides of the contact. The substrate 4 and the chamber 12 can be sealed together using an elastomer or other adhesive coating in the raised area of the chamber. Each etched domain 13 of the chamber 12 can individually or uniformly fill a medium that supports the growth and / or health of cells aligned at the cell binding location 10. In a further embodiment (FIG. 6), the chamber does not contain an input channel to treat all cells aligned at cell binding location 10 with the same solution.

Delivery of drugs or other substances is achieved through the use of various modifications of the chamber, as follows. Solutions of the drug to be tested for interaction with the cells of the array can be loaded from a 96-well microtiter plate into an array of micro-capillaries 24 (FIG. 7). The array of micro-capillaries 24 is
1: 1 to allow the solution to flow from the micro-capillary tube 24 to the chamber 14 or to be pumped. The cassette 18 is inverted so that the cell binding sites 8 sink into the fluid-filled etched domains 13 (FIG. 7B). Once the interaction between the fluid and the cells has taken place, the signal emitted from the cells aligned at the cell binding site 10 can be measured directly or, alternatively, the substrate 4 can be post-treated, fixed and labeled. Can be lifted away from the chamber. Placement and removal of the array of cells can be accomplished via robots and / or hydraulics (Schröder (Schr.
oeder) and Neagle, 1996).

In one embodiment of the chamber 12 shown in FIG. 7, channels and matching etched 13 are chemically etched into the chamber (
Prime and Whitesides, 1991; Lopez
Et al., 1993; Murksich and Whitesides, 1996.
). The etched domain 13 is larger in diameter than the cell binding position 8. This allows the chamber 12 to be contact sealed to the substrate 4, leaving space for cells and a small volume of fluid. Input channels 14 are etched into each row of etched domains 13 of chamber 12. Each input channel 14 extends from two opposite edges of the chamber 12 and opens at each edge. A single row of etched domains 13 contains a micro-
The capillary 20 is in fluid communication with the input channel 14 by making contact with the edge of the chamber 12. Each row of the combined input channels 14 can be filled simultaneously or sequentially. During filling of the input channels 14 by valves and pumps or by capillary action, each of the channels of the chamber 12 is filled, and the drug passes through each etch in the row of the etched domains 13 bound by the input channels 14. Satisfies the domain 13 that has been set.

In a further embodiment of the chamber 12, an elevated container 28 and the input channel 14 can be placed on the surface of the chamber 12, as shown in FIG. In a preferred embodiment, the taller container 28 and input channel 14 can be made from polytetrafluoroethylene or an elastomeric material, but they are made of poly (D) manufactured by Dow Corning under the trade name SYLGARD 184 ^TM. It can be made from any other tacky material that allows it to adhere to the substrate 4, such as dimethylsiloxane). The effect is identical to the chamber with the etched channels,
Channels and their use are similar.

In another embodiment of the chamber shown in FIG. 8A, the first channel 30
Extends from one edge of the chamber 12 to the first etched domain 13 or the higher vessel 28 and channel. A second channel 32 extends from the opposite edge to a second etched domain adjacent to the first etched domain. The first 30 and second 32 channels are not in fluid communication with each other, but are in the same row of the input channel 14 or higher vessel 28.

In another embodiment, shown in FIGS. 9 and 10, the chamber 12 can have an input channel 14 that extends from each etched domain 13 or higher vessel 28 to the edge of the chamber. The channel 14 can originate from one edge of the chamber 12 (FIG. 9), or both edges (FIG. 10). Also, the input channel 14 can be divided on both sides of the etched domain 13 to minimize the space occupied by the input channel 14. Separate fluid channels allow the performance of dynamic experiments where one row at a time or one recess at a time is filled with drug.

In a further embodiment depicted in FIG. 11, each etched domain has a plug 36 between the end of the channel 14 and the etched domain 13.
In fluid communication with the corresponding input channel 14, which prevents the injected solution from flowing into the etched domain 13 until the desired time. The solution can be pre-loaded into the input channel 14 for use at a later time. Similarly, a plug 36 can be disposed between the terminal etch domains 13 in the set of connected etch domains 13 in fluid communication with the input channel 14. Upon release of the plug 36, the substance is placed in the input channel 1
Flow through and fill all the etched domains in fluid communication with 4.

In one embodiment, the plug 36 is hydrophilic upon irradiation and is cross-linked with a photocleavable cross-linking agent that passes with the drug into the recess, including but not limited to proteins, carbohydrates, and the like. Or formed from hydrophobic polymers such as lipids. Alternatively, the plug 36 may be degraded when irradiated and crosslinked with a photocleavable crosslinker that passes along the solution to the etching domain 13, a crosslinked polymer such as a protein, carbohydrate or lipid. Can be formed from

The cassette 18 including the substrate 4 and the chamber 12 is inserted into a fluorescence reader. Fluorescent reader equipment handles the cassettes, controls the environment (eg, the temperature that is important for living cells), controls the delivery of solutions to wells, and from the array of cells (one well at a time or the entire array at the same time). An optical-mechanical device that analyzes the emitted fluorescence. In a preferred embodiment (FIG. 12), the fluorescence reader equipment 44 includes an integrated circuit monitoring station using a reader to operate the cassette and a fluorescence microscope as a microrobot. The reader of the present invention can include any optical system that is truncated to image a fluorescent analyte with a detector. The storage compartment 48 holds the cassettes 18, which are then retrieved by a robot arm 50 controlled by a computer 56. The robot arm 50 inserts the cassette 18 into the fluorescence reader equipment 44. Cassette 18
Is removed from the fluorescence reader device 44 by another robot arm 52 that places the cassette in a second storage compartment 54.

The fluorescence reader equipment 44 is an optical-mechanical device designed as a modification of an optical-based integrated circuit monitoring station used to “screen” the integrated circuit “chip” for defects. is there. Carl Zeiss [Jena, Gm
bH]. In addition to facilitating robot handling, fluid delivery, and fast and accurate scanning, two reading modes, high content and high throughput are supported. The high content readout is substantially similar to that performed by an ArrayScan reader (US application Ser. No. 08 / 810,983).
. In the high content mode, each location on the micro-array of multiple cell types is imaged at a magnification of 5-40X or higher to record a sufficient number of fields to achieve the desired statistical resolution of the measurement.

In the high-throughput mode, the fluorescent reader equipment 44 has a size of 0.2 × to 1.
Image micro-arrays of multiple cell types at much lower magnifications of 0x magnification, providing reduced resolution, but allowing all cell binding locations on the substrate to be recorded in a single image . In one embodiment, a 20 mm × 30 mm micro-array of multiple cell types imaged at 0.5 × magnification is a 1000 × 10 × 10 μm pixel.
Sufficient to fill 1500 arrays and define the intracellular fluorescence distribution, but not enough to record the average response in a single well and to count the number of specific cell subtypes in a well A resolution of 20 μm / pixel. Because typical incorporation times are on the order of seconds, the high throughput mode of readout technology coupled with automatic loading and handling allows screening of hundreds of compounds per minute.

In one embodiment, shown in FIG. 13, the fluorescence reader equipment includes a computer-controlled x, y, z-stage 64, a low magnification objective lens 70 (eg, 0
. A computer controlled rotating objective stage 68 holding 5 ×) and one or more high magnification objectives 72, a white light source lamp 74 with an excitation filter wheel 76, a dichroic filter system 78 with an emission filter 80, Upright or inverted fluorescence microscope 4 including a detector 82 (eg, a cooled charge-coupled device)
4 including an optical-mechanical design. In the high throughput mode, the low-magnification objective lens 70 is moved to a predetermined position, and one or more fluorescent images of the whole cell array are recorded. Wells that exhibited some selected fluorescent response were identified and further analyzed via high-content screening, where the objective mount 68, as described in US application Ser. No. 08 / 810,983, was used. Rotated and high magnification objective lens 72
And the x, y, z-stage 64 is adjusted to center on the "selected" well for both cell and subcellular high content screening.

In another embodiment, the fluorescence reader equipment 44 can utilize a laser beam scanned in either a confocal or standard illumination mode. Multiple laser lines or separate lasers manufactured by Carl Zeiss (Jena, GmbH, Germany) or discussed in Denk et al. (Science 248: 73, 1990). Based on a group of diodes.

Another embodiment of the high-throughput screening mode consists of an array of fluorescent exciters and detectors (1 × 8, 1 × 12, etc.) scanning a subset of wells in a heterogeneous micropatterned array of cells. Including the use of low resolution systems. In a preferred embodiment, the system consists of bundled optical fibers, but any system that directs fluorescence excitation light and collects fluorescence emission from the same well would be sufficient. Scanning the entire micro-array of multiple cell types with this system yields, from each well, total fluorescence from both the cells and the solution to which they are attached. This embodiment allows for the collection of a fluorescent signal from a cell-free system (a so-called "homogeneous" assay).

FIG. 14A shows the analysis of multiple cell-type micro-arrays in both high throughput and high content modes using a fluorescent reader instrument that first measures response from the entire array “A” using high throughput detection. 3 shows a method in the form of a flowchart for performing the method. (FIG. 14B). Any well that responds above a preset threshold is considered a hit and the cells in that well are measured via high content screening. (FIG. 14C). The high content mode ("B") may or may not measure the same cell parameters measured during the high throughput mode ("A").

In another embodiment of the present invention, a cell screening system is disclosed, wherein the term “screening system” is equipped with a fluorescent reader, a plurality of cell types, wherein the cells have at least one fluorescent reporter molecule. Containing) micro-
A cassette that can be inserted into a fluorescence reader instrument containing a chamber associated with a heterogeneous micropatterned array of cells and cells, a digital detector for receiving data from the fluorescence reader instrument, and acquiring digital data from the digital detector And the integration of computer means for processing.

A preferred embodiment of a cassette comprising a fluorescence reader and a micro-array and chamber of multiple cell types has been disclosed above. A preferred embodiment of a digital detector is disclosed in U.S. Ser. No. 08 / 810,983, and includes a high-resolution digital camera that acquires fluorescence data from a fluorescence reader and converts it to digital data. In a preferred embodiment, the computer means comprises a digital cable for transporting digital signals from a digital detector to a computer, a display for user interaction and display of assay results, as described in US application Ser. No. 08 / 810,983. Includes means for processing assay results, and digital storage media for data storage and retrieval.

In a preferred embodiment, the cell screening system of the present invention comprises an integration of the previously disclosed preferred embodiment of the elementi (FIG. 15). The array of cell types 10 includes cells attached to chemically modified cell binding sites 8 on substrate 4. The chamber 12 serves as a microfluidic delivery system for the addition of the compound to the cells 10 on the substrate 4 and the two combinations include a cassette 18. Cassette 18 is placed in fluorescence reader equipment 44. The digital data is processed as described in U.S. application Ser. No. 08 / 810,983, which is incorporated herein by reference and which is hereby incorporated by reference in its entirety. The data can be displayed on a computer screen 86 and form part of a bioinformatics database, as described in US application Ser. No. 08 / 810,983. This database 90 allows for the storage and retrieval of data obtained by the method of the present invention, as well as the acquisition and storage of data relating to previous experiments on cells. An example of a computer display screen is shown in FIG.

Example 1 Coupling Antibodies to a Micro-Array of Multiple Cell Types for Attachment of Specific Lymphoid Cells The cell line used was a mouse B-cell lymphoma line that does not express IgM on its surface (
A20). Micro-arrays of multiple cell types were soaked in 20% sulfuric acid overnight, washed 2-3 times in excess distilled water, washed in 0.1 M sodium hydroxide, blotted and dried Prepared for derivatization. Micro-arrays of multiple cell types were used immediately or placed in clean glass beakers and coated with paraffin for future use.

[0092] 2. Multiple cell type micro-arrays were placed in 60 mm Petri dishes and 3-aminopropyltrimethoxysilane was overlaid on multiple cell type micro-arrays to ensure complete coverage without running on edges. (22 × 22 mm of cells
Approximately 0.2 ml per heterogeneous micro-patterned array, and 22 x 40 m of cells
m for heterogeneous micro-patterned arrays). After 4 minutes at room temperature,
Micro-arrays of multiple cell types were washed in deionized water and excess water was removed by blotting.

[0093] 3. Micro-arrays of multiple cell types were placed in clean 60 mm Petri dishes and incubated with glutaraldehyde (2.5% in PBS, 2.5 ml) for 30 minutes at room temperature, followed by three PBS washes. Excess PBS was removed by blotting.

[0094] 4. During the blocking step of cell nuclei in a micro-array of multiple cell types,
Removed by fluorescent Hoechst dye. Transfer the appropriate number of lymphoid cells (see below) in C-DMEM to a 15 ml conical tube and add 10 μg / ml Hoechst dye.
To a final concentration of. Cells were incubated for 10-20 minutes at 37 ° C. in 5% CO ₂ and then pelleted at 1000 × g for 7 minutes at room temperature. The supernatant containing unbound Hoechst dye was removed and fresh medium (C-DMEM) was added to suspend the cells as follows: 0.2 μm per 22 × 22 mm heterogeneous micropatterned array of cells. Approximately 1.25-1.5 × 10 ⁵ cells in 2 ml, and 22 of cells
Approximately 2.5x in 0.75ml for x40mm heterogeneous micro-patterned array
10 ⁵ cells.

[0095] 5. Micro-arrays of multiple cell types were washed briefly in PBS and transferred to a clean, dry 60 mm Petri dish without touching the sides of the dish. Cells were carefully pipetted on top of a micro-array of multiple cell types at the density described above. The dishes were incubated for 1 hour at 37 ° C. in 5% CO ₂ . Unbound cells were then removed by repeated PBS washes.

[0096] 6. Antibody solution (goat anti-mouse IgM or goat anti-mouse whole serum) was spotted on parafilm (50 μl per 22 × 22 mm heterogeneous micropatterned array of cells, per 22 × 40 mm heterogeneous micropatterned array of cells). 100
μl). The micro-arrays of the multiple cell types were inverted on the spots such that the antisera covered the entire surface of the treated micro-arrays of the multiple cell types without trapping air bubbles. Micro-arrays of multiple cell types were incubated with the antibody solution for 1 hour at room temperature.

[0097] 7. Micro-arrays of multiple cell types were carefully shifted from parafilm, placed in clean 60 mm Petri dishes, and washed three times with PBS. Unreacted sites were then blocked by the addition of 2.5 ml of 10% serum (calf or fetal calf serum in DMEM or Hank's balanced salt solution) for 1 hour at room temperature.

[0098] 8. Both cell lines should bind anti-mouse whole serum and only X16 should bind anti-mouse IgM. Binding of specific lymphoid cell lines to chemically modified surfaces is shown in FIG. A mouse lymphoid A20 cell line that lacks surface IgM molecules but presents IgG molecules has a goat anti-mouse IgM modified surface (FIG. 1).
7B) or bound to whole goat anti-mouse serum modified surface (FIG. 17C) more than uncoated slides (FIG. 17A).

Example 2 High Content and High Throughput Screening Insulin-dependent stimulation of glucose uptake into cells such as adipocytes and muscle cells results in the transfer of GLUT4 glucose transport from the intracellular compartment to the plasma membrane Requires complex orchestration of the resulting cytoplasmic processes. Numerous molecular events are triggered by insulin binding to the receptor, including direct signal transduction events and indirect processes such as cytoskeletal reorganization required for the metastatic process. Since the actin-cytoskeleton plays an important role in cytoplasmic organization, the intracellular signaling ions and molecules that regulate this living gel can also be considered intermediates in GLUT4 translocation.

A two-level screen for insulin coagulation is considered as follows.
Cells carrying a stable chimera of GLUT4 along with blue fluorescent protein (BFP) were placed on a micro-array of multiple cell type arrays and loaded with the acetoxymethyl ester form of Fluo3, a calcium indicator (green fluorescence). The location array was then treated with an array of compounds using a microfluidic delivery system, and short sequences of Fluo-3 images of all micro-arrays of multiple cell types were analyzed for wells exhibiting a calcium response. Wells containing the compound that elicited the response are then analyzed on a cell-by-cell basis (ie, high content basis) as evidence of GLUT4 transfer to the plasma membrane using blue fluorescence detected in time and space. .

FIG. 18 shows the high throughput mode (FIG. 18A) and the high content mode (FIG. 18A).
FIG. 4B shows a sequential image of the entire micro-array of multiple cell types in B). FIG.
Figure 4 shows cell data from high content mode.

Improved Cassette and Increased Well Density In another form, the present invention provides a method for obtaining a suitable pixel resolution in an image.
Devices and methods are provided for maximizing cell plating area and the number of wells that can be imaged in a sub-array. This result was achieved through the use of a fluid construct that maximized the distance and area between wells, and thus maximized well density.

In one embodiment of this embodiment, a substrate having a surface, wherein the surface contains a plurality of cell binding sites; a fluid delivery system for delivering a reagent to the plurality of cell binding sites, wherein Wherein the fluid delivery system includes a multi-level chamber bonded to the substrate;
Wherein the multi-level chamber comprises: i. An orthogonal array of microfluidic input and output channels, wherein each well is in fluid communication with one or more input channels and one or more output channels; ii. A plurality of fluid locations in fluid communication with the microfluidic input and output channels; iii. One or more input manifolds in fluid communication with the microfluidic input channel; iv. One or more output manifolds in fluid communication with the microfluidic output channels; At least one source container in fluid communication with one or more input manifolds; vi. A plurality of wells, wherein each well includes a space defined by the junction of one cell binding location and one fluid location, including at least one waste container in fluid communication with one or more output manifolds. A cassette for cell screening is provided.

In a preferred embodiment, the cassette further comprises a pump for controlling fluid flow in the microfluidic device; a substrate temperature controller and / or a controller for adjusting the partial pressure of oxygen and carbon dioxide in the device. including.

The substrate of the present invention may be chemically modified such that the cells selectively adhere to the cell growth substrate and are contained within a small area, called the “cell binding site,” that is sub-millimeter to several millimeter in size. Qualified. The combination of the cell binding location on the substrate and the fluid location on the chamber defines a space called a "well". The substrate is overwhelmingly flat and the cell binding locations match the fluid location recesses on the substrate cover so that when the two parts are assembled there is some depth in the area of the well thus formed can do. Conversely, the substrate cover fluid location may be flat and the substrate may have recesses in the cell binding locations so that there is some depth in the area of the well thus formed when the two parts are assembled. Can be contained. Fluid and test compound are supplied to the wells through a chamber that combines with the substrate to form a cassette. In a preferred embodiment, the chamber acts as a substrate cover and contains fluid locations in fluid communication with the microfluidic channels of the device, wherein the fluid locations are located on the substrate such that the volume of each well is etched into the chamber. Includes a recess that matches the cell binding position of

On the surface of the substrate, there is a sealing area between and around the cell binding locations and a corresponding sealing area on the bottom surface of the chamber between and around the fluid locations. When the substrate and chamber are joined, the sealing areas of both components associate to form a seal, which impedes the flow of fluid across the sealing area between the wells formed thereby. These sealing regions of both components can be physically and / or chemically modified to enhance sealing. First
In an embodiment, the sealing region is coated with a hydrophobic silane coating of octadecyltrichlorosilane. In a second embodiment, Dyma
USP class IV-compliant photocurable adhesives and cyanoacrylates, such as those available from x Corporation), can be used in biochemical devices made from ceramics, glass, plastic, or metal. In a third embodiment, a biocompatible tape material having a precision cutout that matches the wells of the array is provided by Avery Dennison Specialty Tape Division (Avery).
Dennision Specialty Tape Division). Similarly, a biomedical acrylic adhesive, such as that available from Tyco Corporation, can be transferred from the tape to the surface to accurately apply the adhesive to these sealing areas. In a fourth embodiment, a photocurable silicone elastomer, such as that available from Master Bond, Inc., can be used in bonding and sealing between the sealing area of the substrate and the chamber. .

The microfluidic cassettes of the present invention include, but are not limited to: 1) (based on simultaneous imaging of a set of cell binding sites (ie, a “subarray”; see FIG. 20). 2) more wells per plate and more physical space occupied per plate (ie, higher well density), 3) more effective use of valuable test compounds, and 4 ) More efficient use of cells compared to microplates where all wells are not imaged; including advantages over prior art cell array microfluidic devices used in high content screening systems.

As discussed above, the preceding microwell plate fluid exchange for each well can only be achieved by pipettes inserted into the wells and ejecting or aspirating fluid into or out of the wells, respectively. Automatic fluid handling in a preceding microwell plate is achieved by using a robotic pipettor that inserts a pipette into each well for each fluid transfer step in the procedure. The pipette is typically required to be transferred to or into another microplate well for the source or destination of the fluid to be exchanged for the microwell plate of interest.

[0109] Integrated fluidics is advantageous for arrays with millimeter lower well internal distance. This is because, if not possible, it is not practical to pipette fluids with an acceptable degree of spatial resolution and accuracy. An acceptable level of accuracy requires that the measurement error should not exceed 2%. Thus, a 100 microliter (μl) volume in the wells of a 96-well plate would require a measurement error of 2 μl. For a 100 nanoliter (nl) volume, a 2nl measurement error would be required. This level of accuracy is not reliably available with a pre-automated pipettor across the spectrum and viscosity of the composition.

Minimizing the well-to-well distance allows for fast parallel reading of a sub-array of wells. If integrated fluidics is too bulky (ie, does not allow for close spacing of wells), such fast parallel reading capability is lost. In a preferred embodiment of the present invention, an orthogonal channel construction is utilized, allowing fewer channels, valves and pumps, thereby further reducing the space taken by the channels on the cassette of the present invention. In the most preferred embodiment, the channel is located at a level above that of the wells, allowing for the closest possible inter-well spacing. In a further preferred embodiment, the use of a porous medium as a drain "pump" allows for a simpler design and manufacture of the system. According to the present invention, the channels may be of any size that allows for the defined fluid constructs herein. In a preferred embodiment, they range in size for a square section channel, or a diameter for a round section channel, between about 0.025 mm to about 0.5 mm.

The fluid constructs of the microfluidic cassettes of the present invention include, but are not limited to,
Pumps, manifolds, controlled pressure vessels, and / or valves) can be located outside the matrix of the cell array. FIG. 40 shows one embodiment of the pumping and valving scheme of the present invention where all pumps and valves appear to be contained in the cassette. All variations of the pumping and valving schemes shown in the various drawings (and as would be apparent to one skilled in the art) can be implemented in a built-in active device outside the matrix of wells, in an external active device, or in some This is done in an internal device and some in an external device in this way. As a result, the control element can be inside the cassette (built-in) or outside the cassette (external). In either case, there is no active ingredient in the matrix of the array of wells that can limit the close spacing of channels and wells. Since this cassette was specifically designed for the cultivation and analysis of living cells, the fluid construct and all of its sub-components and functional parts are compatible with the support and allow the culturing of living cells. The specific morphology of the sub-components and functional parts designed in living cell culture will be confirmed later.

In a preferred embodiment, the cassette of the present invention includes an external pump to provide a pressure-driven flow that also controls which wells are addressed by the flow. This design feature differs from many prior art methods in that the pumping method
Ensures that it is not effective or harmful when used in a cell culture medium that is aqueous, polar and contains proteins and salts. For example, electrohydrodynamic pumping is not effective with polar solvents (Marc Madou)
, Fundamntals of Microfabrication, CRC Press, Boca raton, 1997, p. 433).
Electroosmotic pressure typically involves some electrophoretic separation of charged media components, such as proteins. Also, electroosmotic pressure typically requires the use of electric field strengths in the range of 100 V / cm to 1000 V / cm, which can affect the physiology of living cells in the device. The fluid control system of the present invention does not suffer from the electrically induced method when using biological fluids.

In another embodiment, the pump is self-contained and may utilize electro-driven flow, but the electric field is restricted to a matrix of an array of wells and an electric field gradient across any of the wells where cells are present. Does not apply. This embodiment is shown in FIG. 41, where two electrodes are used to apply an electric field potential difference along a segment of a fluid channel corresponding to a different self-contained source container (730). Each channel segment with that electrode pair forms an electrokinetic pump (740). These source containers (
730) via the filling port (700) at the cascade surface
Or a compound can be supplied. Valve between container and pump (720
) Is always closed when the corresponding electrokinetic pump is off and always open when the corresponding electrokinetic pump is on to maintain the back pressure and to allow air and fluid intake, respectively. Make it possible. With the control of the voltage applied to electrode 1 (760),
Inducing the electrokinetic flow of the fluid in the corresponding channel segment. Electrode 2 (780), which is closest to the matrix of wells, is maintained at a ground potential, as in the matrix of wells, with minimal or no potential difference and / or electric field occurring near the cells. This is because electric fields are known to affect cell physiology. Alternatively, the electrokinetic pump can be located downstream from the array and act as a negative pressure pump. In this embodiment, electrophoresis of the medium, if any, by pumping will only affect the medium after it has been used by cells and is on its way to a waste container.

In all embodiments, the use of an internal or external pump external to the matrix of wells to control fluid flow obviates the need for an active valve within the matrix of wells. In addition, it eliminates the problem of allocating space in the matrix that would prevent close spacing of wells.

Control of the fluid pathway by pressure control allows the use of any of a variety of diffusion means that are aqueous, polar, compatible with cell media containing proteins and salts, and are effective. Also, these measures require minimal space on the device and do not obstruct the closed space of the well.

Although US Pat. No. 5,603,351 includes orthogonal channels, the channel network is not defined to allow two or more reagents to be combined in a reaction well, but rather the reagents are serialized. Be supplied to the well. The purpose of the cassette of the invention is not to expose two or more different fluids to each other, but to expose live cells cultured at the bottom of the well in series to two or more different fluids.

The fluid channels of the cassette of the present invention provide for the transport of fluids and compounds from a source container to each well and from each well to two or more waste containers. Each well can be provided with separate input and output channels (FIG. 21)
, 2 × ² arrays (100, 120) per n × n array (040). Controlling the flow of m fluids or vapor mixtures into an n × n array requires m + n ² valves or pumps (FIG. 22). However, for large arrays, the number of channels, pumps and valves for this type of construction becomes impractical. Pumps with variable or switchable pressure (eg, syringe pumps) are functionally equivalent to pressurized vessels with valves. Thus, in the following discussion of pumps, vessels and valves, it should be understood that these components can be substituted without changing the spirit of the invention.

Orthogonal-Channel Multi-Level Construct A preferred embodiment of a multi-level cassette construction consisting of an orthogonal non-crossing array of input (100) and output (120) channels is disclosed. Specific embodiments within this class of constructs are shown in FIGS.
3 and 34.

[0119] These orthogonal-channel constructs have the advantage of requiring fewer independent channels per well, as the constructs in this case require fewer independent channels per well compared to a class of constructs with separate input and output channels for each well. Lateral space required between wells to accommodate pathways for (140)
Is minimized.

[0120] Orthogonal - 2n for channel multi-level construct address the wells ^{n 2}
It just needs a channel. In a preferred embodiment, the row and column channels themselves are formed in two distinct layers so that they do not intersect.
The row channel is above the column channel or vice versa. These two layers are either in the same plane of the well layer (FIGS. 23 and 24) or in the plane above the well layer (FIGS. 25-26 and 33-34). In other embodiments, structures having more than two surfaces are contemplated.

In the most preferred embodiment, the channels are above the plane of the wells such that the minimum inter-well distance (140) is only constrained by optical and physical requirements for the thickness of certain walls separating each well. To be placed. The advantage of placing the channel at a level above the well is illustrated by the following example. 0.4mm × 0.4mm
Wells can be placed on a 0.5 mm pitch grid with tolerance for 0.1 mm walls between wells, 5 mm x 5 m per 10 x 10 well array
This produces an m region. Row and column channels 0.2 mm wide can be easily formed at the upper level of the device with sufficient distance between the channels. In contrast, if 0.2 mm channels with 0.1 mm walls are required in the plane of the well (and the row and column channel layers are formed on two separate planes in the plane of the well), The pitch increases to 0.8 mm. This yields an 8 mm x 8 mm area per 10 x 10 well array. In this embodiment, the displacement of the channels between the wells increases the area required for the array by a factor of 2.6. With different constructs having separate input and output channels for each well, the additional space required between wells is quite large.

For channels above or within the level of a well, horizontal (160) or vertical (180) coupling channels or passages from each well (j, k) to its corresponding row channel j and column channel k. It is formed. Glass, semiconductor,
Or the manufacture of such structures from plastic materials is well known in the art. In one such method, a silicon dioxide (
SiO ₂ ) is formed by lithography in a silicon nitride (Si ₃ N ₄ ) layer (
Turner and Craighead, Proc. SPIE 3528: 114-
117 (1998)). Exposure of the structure to a wet etch removes the SiO ₂ and creates a channel network in the unetched Si ₃ N ₄ .

Control of Flow in Orthogonal-Channel Constructs In an orthogonal array of rows (A, B, C, etc.) and columns (1, 2, 3, etc.), fluids to and from wells (j, k) The flow is, for example, when a positive pressure is applied to row j and the valve of output channel k is opened (where j and k
Occur, for example, in row D and column 7, respectively). Flow to all wells in all rows j occurs, for example, when positive pressure is applied to row j and the valves (1, 2, 3, etc.) of all output channels are opened. A similar approach will result in flow to all wells in a row.

The pressures discussed here are, in reality, the normal pressure of the atmosphere, otherwise “gauge”
Pressure difference from what is known as pressure. Thus, a positive pressure valve is an amount by which the applied pressure is greater than normal pressure;
The amount by which the applied pressure is below normal pressure. These pressures are referred to as positive and negative gauge pressure, respectively.

As used herein, the term fluid path refers to rows j and m etc. and columns k and n
A specific set of wells subjected to the flow of fluid by enabling a flow control device such as (j, k), (j, n), (m, k), (m, n)... Is shown. Note that enabling a two row and two column flow controller results in a fluid path through four wells. This is defined here as a single fluid pathway involving four wells, rather than four pathways involving a single well.

Reducing the Number of Active Valves or Pumps Various combinations of actively controlled fluid control devices (pumps or pressurized vessels with valves) can be used to pass through the desired well (010) or fluid path. Cause the flow to occur. For example, m compounds may include m positive pressure source vessels or pumps (200).
, Valveless manifold (240) and pressure free valve-free waste container (30
0) can be multiplexed into a single well (FIG. 27). This valveless manifold (240) is a manifold with a valve [for example, FIG.
8 (260)] to better control the diffusion of fluid between the sources of m. Alternatively, the same function can be achieved by using a pressureless waste vessel or pump (320) with an m valve (260) connected to an m pressure source vessel (220) at normal pressure (Figure 2). 28). Use of the source container of atmospheric pressure, as can the convenient valving gas through the source container, establish and maintain a desired level of dissolved CO ₂ and O ₂ in the medium.

In another embodiment, the waste container filled with the porous medium is a capillary action pump (
340) and provides no pressure (FIG. 28). The thermodynamics of wetting creates pressure across a liquid-gas interface bounded by solid-liquid-gas contact lines. This pressure of capillary action can be used to draw a row of liquid from any microchannel channel into a high surface area porous medium. A series of such non-pressure vessels can be connected to the array via a manifold with multiplexed valves for flow control, or a single vessel can be closely connected to the output channels of all wells simultaneously. Can be coupled. Without limitation, silica gel technology, porous ceramic materials such as zeolites, pads of hydrophilic (synthetic or natural) fibers, and alginates, poly (vinyl) alcohol gels, agarose, sugar polyacrylate gels and polyacrylamides Numerous porous media are known in the art, including partially hydrated or lyophilized hydrogels such as gels. In a preferred embodiment, the porous medium is based on Sirigel technology. A membrane or filter sheet can be used to separate the porous medium from the microchannel array. This method can provide pumping pressure until the interior region of the porous medium is wetted by the fluid.
With the proper choice of porous medium and the design of the method, a suitably large volume of liquid can be pumped to make selected measurements with the array system of the present invention.

In another embodiment, the positive pressure source container or pump (20) shown in FIG.
0) array with lxn inputs (400) and nxl outputs (41)
0) Multiplexed to n × n orthogonal channels (040) of well by valve manifold (FIG. 29). For constructs with separate input and output channels for each well, to m + n ² of the valve or pump is specified to be necessary in this situation. Again, the valveless manifold (240) can be replaced with a valved manifold [eg, (260) in FIG. 28] to better control fluid diffusion between sources of m. Flow through well (j, K) occurs when input valve j and output valve k are open. Alternatively, an lxn input (400) and an nxl output valve (41
0) The manifold allows the same function to be achieved with a container (220) at m atmospheric pressure and a non-pressure disposal container (either 320 or 340) as in FIG. 28 (FIG. 30).

Wash Channel In a preferred embodiment, well {(j, k)} or column channel {k}
N × n microwell array with an additional column channel k ^* or set of column channels {k ^* } to allow liquid or gaseous segments to pass from a given row j to the waste container without proceeding through Is increased. These additional row channels {k ^* } are called wash channels (130). Because they allow the washing of the row channels without the need for flow through the wells.

In general, the wash channels k ^* (130) can be located adjacent and parallel to the column channels k (016), but at a point where they are orthogonal to the channel j (014). [Corresponding wells (
j, k). In one embodiment, each row channel k has an associated parallel wash channel k ^* (FIGS. 33 and 34). In another embodiment, only one provides cleaning for the entire set of row channels {j}.
There may be one wash channel, or there may be any combination of wash channels {k ^* } present in the array of row channels {k}. If there is only one wash channel, it will preferably be located adjacent to the last column channel of the array (where k = n).

FIG. 35 shows the liquid or gas from row j to the wash channel k ^* (where k ^* is immediately adjacent to the column channel k) for fluid flow through well (j, k). 3 illustrates an embodiment of a method for rinsing a segment of a. The approach and construct are such that the fluid segment is downstream along the row channel j, the well ｛(j, k
+1), (j, k + 2), etc., without passing through. This is desirable to limit the diffusion of a given compound from one well (j, k) to other downstream wells {(j, k + 1), (j, k + 2), etc.} (where the compound is Will not be required). In addition, diffusion can be controlled by introducing gas segments into the microchannel microarray, as described below.

FIG. 32 illustrates how the pumping and valving scheme of FIG. 29 using a positive pressure source vessel with an orthogonal array of n row channels and 2n column channels (n normal column channels + n wash column channels). Show if you can expand to work. This requires a 2n × 1 output valve manifold (420). Generally, for each additional wash channel k ^* added, one additional valve is added to the valve manifold coupled to the row channel. Similarly, the arrangement of FIG. 30 using non-pressure waste containers can be expanded to work with arrays that integrate wash channels (130).

Dual-Pumped Design Any of the fluid paths x {(j, k), (j, n), (m, k), (m, n). Flow through a length of tube is caused by a continuous pressure gradient that exists along the length of the path or tube. Control of this pressure gradient by the pressure applied to the inputs and outputs of the fluid path requires that there be no leakage of flow across the walls of the path. Also, leaks jeopardize the proper dose of compound to wells for drug screening. The sealing of the path walls must be suitable for the gauge pressure to be applied. At flow velocities of roughly a few millimeters per minute, path lengths of a few centimeters or less, and channel cross-sections on the order of 100 mm × 100 mm, the Hagenpoise law states that the required end-to-end pressure difference ΔP is one atmosphere (1 Atmospheric pressure to less than 14 pounds per square inch). Nevertheless, considering that various materials and structures known in the art can be implemented in the device of the present invention, by reducing the leak between adjacent wells, the gauge pressure acting across the walls of the system, Be minimized. To this end, the apparatus of the present invention is applied to achieve a given full end-to-end pressure differential using a positive pressure source vessel (ie, a "double pumped" system) with a non-pressure waste vessel. The maximum gauge pressure that must be achieved can be substantially reduced by half.

“In a single-pumped system, that is, a system that is open to barometric pressure at one end and pumped at the other end (to either positive or negative gauge pressure ± ΔP), across the system wall The acting pressure differential is the position of the pumped container, ie (200) in FIGS. 27 and 29; or (320, 340) in FIGS. Leaks are most likely to occur at these points of maximum gauge pressure, with positive pressure tending to remove fluid out of the path and non-pressure tending to draw fluid from adjacent wells or channels into the path (which is Assuming that channels and wells are held at zero gauge pressure when not subjected to flow.Another way to control the pressure differential across the walls of the system is to use adjacent paths when they are not under flow. Applying matching pressure). At the mid-point along the flow path, the pressure difference across the system wall has a value between ± ΔP and zero for positively and negatively pumped systems, respectively.

By establishing a pressure ± ΔP / 2 at the input and output vessels, respectively, the same end-to-end pressure difference ΔP works across the path. In this case, the minimum gauge pressure acting across the system wall is ± ΔP / 2 and occurs at the corresponding end point of the system. Furthermore, if the well is approximately at the midpoint between the input and output vessels along the length of the path, the gauge pressure in the adapted device will be close to zero. At the end of the path, the maximum pressure difference ± ΔP / 2 is more easily retained without leakage in the plastic tube or silica capillary. Leak detection is facilitated because it seems to occur only at the end of the system.

In one embodiment, each syringe moves the same linear distance because the two syringes of the dual pumping system are of the same diameter and volume and are connected to the same actuator in a push-pull configuration. The same capacity is exchanged for each increment of movement. Any particular source compound is pushed into the array and an equal volume of waste fluid is withdrawn. Thus, an array of m source syringes contains an array of m pressureless waste containers. This array of unpressured waste containers is multiplexed into column channels by an lxm valveless manifold, just as the array of source containers is multiplexed into a matrix.

In another embodiment, the source and waste syringes are of different diameters, and the syringe must be controlled by two separate drivers driving each syringe at different linear speeds, but with the same volume change in each syringe Is generated. For example, a smaller diameter syringe can be used per compound, while a larger diameter syringe can be used as a waste container. If the ratio of the diameters is a factor of two, as the driver of the small syringe moves one linear unit to remove the fluid, the driver of the large syringe moves only one quarter of the linear unit to collect the fluid However, the exchanged capacity is the same. FIG. 31 shows “Dual-
One particular case of this embodiment of the "pumped" system is shown, wherein one waste that must be operated in concert (eg, by computer control) in the operation of any particular source syringe pump. There is only a syringe pump. Alternatively, the non-pressure pump can be a capillary action pump, also shown in FIG. In a capillary action pump, "actuation" of the pump is achieved by a valve.

In summary, the dual-pumped system has the following advantages: 1) reduced maximum applied gauge pressure, 2) applied gauge pressure is small near the midpoint of the path, and 3) leaks are easily detected and the system Most likely to occur at the end of
Having.

Higher Levels of Multiplexing The use of a multi-port flow manifold between a pump vessel and an orthogonal channel array allows the use of m different pumps (200) in these various embodiments with m different fluid or gas mixtures. ) Or container (220) can selectively and sequentially generate flow in each well (j, k) of the n × n well array (002). More generally, a single fluid path ｛(j, k), (j, n), (
The set of wells that define m, k), (m, n), etc. can be defined by this system. The flow of only one liquid or gas mixture can occur through only one fluid path at any one time. Higher levels of multiplexing that allow simultaneous flow of different liquid or gas mixtures through different paths are possible with more complex systems of flow manifolds. Such a system is easily constructed as a generalization of the design of the present invention.

Controlling the Diffusion The principle of this embodiment is that the valve element (520) and the valve seat (540) allow one-way flow and suppress diffusion when flow is stopped. It is to be made into such a shape.

Furthermore, the restoring force on all valves is applied “passively” and constantly.
No detailed control system for the check valve is required. This is because the control of the fluid pressure gradient is the means by which the individual valves are opened.

[0142] For example, the use of orthogonal microchannels with many open path between the micro channel and n ² wells 2n, in some embodiments of the present invention, among the wells and microchannels It can be increased by additional methods for controlling the diffusion of the compound. Unlike prior art systems, the microfluidic system of the present invention does not require a detailed control system in the apparatus for controlling inter-well diffusion. Two exemplary means for controlling inter-well diffusion without requiring active control of the built-in valve are disclosed. Both of these methods are external to the array of wells and thus block interwell diffusion by controlling fluid flow from valves, pumps and manifolds that are compatible with the overall design principles of the present invention. Optimal imaging of sub-arrays of wells in a class of fluid constructs that requires fewer channels and allows for smaller interwell spacing.

The first method uses the insertion of air segments between different fluid segments
. Using the positive pressure and / or negative pressure methods given in the previous embodiment, these
Insert air segment. In general, a particular row {j} and column {k} or {k ^* The application of different pressures in a suitable arrangement across the ｛
Many separate air segments in j｝ can be placed between many different fluids
You. In particular, also insert the air segment in row j between these two wells
While maintaining proper sequencing, one fluid segment can be stored in one well (j, k).
And directing another fluid segment directly to another well (j, k-1).
Can be. Subsequently, either through the unused wells or through the cleaning channel
Le k^*Passing the air segment out of the system via (130)
(Fig. 35).

A second method of diffusion control is to add channel j to wells (j, k) and (j, k +
1) between each pair of adjacent passages (passage k and passage k + 1) (180) that joins
A "normally closed" check valve (500) is used. Similarly, a check valve can be used in a construct that incorporates a wash column channel {k ^* }. In other embodiments, a check valve can be placed in the passage (160, 180) connecting the well to the microchannel or the row channel to the wash column channel. These valves are closed unless a pressure gradient is applied across the fluid path of the array. Then, only the valves distributed along the selected fluid path are pushed open. When flow is stopped and the valve returns to the closed position, the valve inhibits compound diffusion between the well and microchannels or between the row and wash channels. The restoring force for closing the valve element (520) in its corresponding valve seat (540) can be provided by any number of methods known in the art. The strength of the restoring force must be able to open the valve upon application of a pressure gradient that produces a suitable flow rate (typically less than 1 cm / min). Methods for application of restoring forces include deformation of metals, semiconductors or organic materials; pneumatics; electrostatic forces, magnetic forces, and mechanical forces due to gravity.

In another embodiment, an externally applied magnetic field gradient (620) induces a restoring force on the valve element (520) incorporating magnetic material. This includes, but is not limited to, the use of paramagnetic beads (600) shown in FIG. 37 (eg, supplied by Dynal Corp.). These paramagnetic beads (600) serve as balls in microball valves manufactured in and along each row and column channel. U.S. Patent Nos. 5,643,738 and 5,681,484 describe magnetically actuated microball check valves. However, these patents specifically describe actively controlled valves that allow the ball valve to be closed "on demand" and have as its purpose to stop the flow imposed by some external pressure. In the case of the present invention, a particular advantage to the microfluidic construction system is that a gentle restoring force is applied simultaneously to a large ensemble of ball valves over the entire array and no active control is required. Movement of the selected ball along the fluid path (whenever it is desired to open the valve) is achieved by means of flowing the fluid itself. The ball valve is controlled by the flow of the fluid, not the flow of the fluid by the ball valve. Further, in this embodiment, the valve seat is specifically designed so that a seal is not obtained in one direction, unlike the other direction, if a mild seal is obtained that is sufficient to stop diffusion. .

In one embodiment, one microball valve is located between each pair of adjacent passages (passage k and passage k + 1) connecting channel j to wells (j, k) and (j, k + 1). . Each microball valve opens only when a pressure gradient is applied across the path containing the valve. The design of the valve seat (540) on one side is shaped (eg, in a groove) so that the flow is not stopped when fluid pressure moves the ball in the direction of flow. If no pressure gradient is applied (and no fluid is flowing) at the location of a particular ball valve, the magnetic restoring force will force the paramagnetic ball against the other side of the valve seat (540), where The valve seat is shaped to fit the ball), which inhibits the diffusion of the compound across that portion of the microchannel. This restoring force is induced by the magnetic field gradient applied to the entire array so that all valves close normally.

In another embodiment, an externally applied magnetic field gradient (620) induces a restoring force on the valve element (520) incorporating the ferromagnetic material. For example,
Precision chrome steel balls are available from Glenn Mills Inc., Clifton, NJ, ranging in diameter from millimeters down to several millimeters.

FIG. 38 shows an embodiment of a valve seat shaped to illustrate this design principle. In particular, if the restoring force applied from the outside pushes the ball to the left and into the round opening in the valve seat, diffusion through the ball is stopped. When the flow is activated by an externally applied pressure, the ball is pushed to the right, but the ball does not present the flow. Because the right side of the valve seat is oval in shape, including a groove-like deformation along one side,
This is because it creates a path for the fluid to avoid the ball in this open position of the valve.

FIG. 39 shows that a single magnetic field gradient applied to all cassettes of the present invention can induce forces on both the input channel ball valve set and the output channel ball valve set. 1 shows an embodiment.

In one embodiment, the direction of the magnetic field gradient is parallel to the plane of the array, but at a 45 degree orientation for both row and column channels. Thus, each ball experiences a force component in the direction of the "closed" side of its corresponding valve seat (540). In other embodiments, the magnetic field gradient is applied perpendicular to the plane of the array and the ball valve operates vertically in a horizontal microchannel or in a vertical passage. In the latter case (a vertically actuated ball valve in a vertical passage), the passage construction again causes each ball to be subjected to a magnetic component in the direction of the "closed" side of its corresponding valve seat (540). Must be able to

Although an example of a spherically shaped ferromagnetic or paramagnetic valve element (520) has been given, other shapes and other restoring forces that function similarly can generally be used.

Well Dimensions and Features In another form, the invention relates to a surface, wherein the surface contains a plurality of cell binding sites; a fluid delivery system for delivering reagents to the plurality of cell binding sites, wherein In addition, the fluid delivery system includes a multi-level chamber in contact with a substrate, wherein the multi-level chamber includes a microfluidic input channel and an output channel and a plurality of fluids fluidly coupled to the microfluidic input and output channels. A plurality of wells, where each well includes a space defined by the junction of one cell binding location and one fluid location, wherein the wells have at least about 20 per cm ^2. A cell substrate comprising a substrate having a density of wells. To provide a cassette for cleaning. In a preferred embodiment, the well density is between about 20 wells and 1 cm ² per about 6400 wells per 1 cm ^2.

In a preferred embodiment, the cassette of this aspect of the invention further comprises one or more input manifolds fluidly coupled to the microfluidic input channels and one or more output manifolds fluidly coupled to the microfluidic output channels. . In another preferred embodiment, the cassette of this aspect of the invention further comprises at least one source container in fluid communication with one or more input manifolds and at least one source fluid in fluid connection with one or more output manifolds.
Includes one waste container. Various control devices in these embodiments are as described above.

No limitation is imposed on the geometry of the well shapes in the present invention, as the well shapes may differ in different embodiments of the present invention. Although a rectangular well shape allows for minimum distance and area between wells in the array, this shape has the disadvantage of potential differences in cell culture or fluid properties in the corner regions of the well. Therefore,
A circular, rectangular well shape with rounded corners is used in a preferred embodiment of the present invention.

The array system of the present invention is designed for imaging individual cells within the field of view of a cell in a drug screening assay system. Screening in an imaging assay that involves the parallel measurement of each individual cell within the field of view of the cell is referred to as high content screening (HCS). This is because detailed information about intracellular processes is contained within the image of a single cell. Lower resolution imaging or screening with a detector than integrating the signal over a population of cells is referred to as high throughput screening (HTS) because it is faster. In HCS, cells are typically 5 μm to 15 μm
It is identified by imaging nuclei of cells that are spherical or ellipsoidal in shape with a major axis length in the range of m. The cell array used in the present invention optimizes the rate of HCS by using a higher density multi-well format as compared to a normal 96-well plate.

In the HCS, the number of cells providing a statistically relevant sample as well as the number of cells required per unit surface area will vary depending on the type of assay and the type of cells to be cultured. Thus, the minimum well size required by the statistical criteria for each assay will vary. Nevertheless, whatever the minimum or optimal well size, the speed at which the wells can be read at a suitable image resolution and resolve individual cell nuclei is optimized by the device of the present invention. This allows several wells to be imaged in parallel at one time. This is most efficiently done if there is minimal extra space between the wells and the cell plating area within the entire image is therefore maximized.

In addition, the array system of the present invention optimizes the flexible implementation of both HTS and HCS for the same optical imaging cell-based screening system. The required pixel density (pixels per micron) is lower for HTS (eg, 0.001 to 0.1 pixels per micron) and higher for HCS (eg, 0.001 to 0.1 pixels per micron).
For example, 0.1 to 4.0 or more pixels per micron). The system of the present invention provides for a seamless combination of HTS and HCS, and the "hits" identified in HTS are also read in HCS mode for more detailed analysis.
In fact, the availability of HCS on the same platform and on the same sample is
It greatly increases the information associated with the "hit" identified in the TS mode. Nevertheless, the fastest possible rate of HCS is advantageous to allow for maximum total screening throughput whenever HCS is used. The minimum pixel density required by the HCS in combination with the maximum well density of the array is two very important parameters that define the maximum speed at which a well can read in HCS mode. The present invention describes how the design of the array system of the present invention can maximize this HCS rate as well as the HTS rate.

For use in a 96-well microplate, a typical microscope imaging system can image a square field of 0.1 mm to 10 mm width at the center of each 7 mm diameter well. Wells are imaged or "read" sequentially
. At each location on the stage, typically only one area of the well is read. To read the entire array (either in HTS or HCS mode), the sample stage must move the microplate at least 96 times.

In the present invention, we define a novel cell screening method in which many wells are read at once. The arrays of the present invention have well widths and inter-well distances roughly 10 times smaller than in 96-well microplates (widths are millimeters below, and overall plate dimensions are several millimeters rather than centimeters wide). ), Cell screening systems such as those disclosed in the present invention can image many wells simultaneously. In this way, the sample stage is moved less times per plate and some wells are read in parallel (FIG. 39).

This substantially involves the sequential imaging of “subarrays” (020) of the entire microwell array (040), while acquiring data from all wells of each subarray in parallel. For example, imaging a 3 × 3 well sub-array requires nine times less movement of the sample stage and allows roughly nine times faster speed of data acquisition.

For a given CCD detector array (080), the magnification of the optical system (060) determines the area of the sub-array (020) projected on the detector. For example, the image pixel density of one pixel per micron is 10 × optical magnification and 10 μm × 1
This is achieved for a 1000 μm × 1000 μm sub-array of wells by using a 1000 × 1000 pixel CCD array of 0 μm elements. In a second example, a 2000 μm × 2000 μm sub-array of wells was
The same image pixel density can be obtained by forming an image on a 00 × 2000 pixel CCD array. In a third embodiment, an image pixel density of 0.1 pixels per micron is achieved per 10 mm × 10 mm sub-array of wells by using a 1000 × 1000 pixel CCD array of 1.0 × optical magnification and 10 μm × 10 μm elements. Is done. In a fourth embodiment, a 10 mm × 10 mm sub-array of the array can be imaged at 2000 × magnification onto a 2000 × 2000 pixel CCD array to obtain the same image pixel density.

In the following, we give examples of special embodiments of well size and spacing according to the present invention to optimize the imaging of sub-arrays. These well sizes must be large enough to contain the desired number of cells bound to the desired number of cell binding sites within each location of the array. The possible range of desired cell densities to be cultured at the cell binding site (ie, for very widely spaced cells,
Or a dense monolayer culture would be desirable), the desired well size would be one containing only one cell at a very small cell binding site of about 10 μm in diameter (
That is, a well containing a large cell binding site or an array of multiple cell binding sites including a single location on the array (ie, a well size of 1-2 mm). Can be The former can lead to higher well densities, and the latter can lead to lower well densities.

At both high and low well densities, and for wells containing one or more cell binding sites, a class of microfluidic constructs according to the present invention provides optimal spacing of wells, minimal extra space between wells, And a maximum number of wells that can be imaged simultaneously in the sub-array and still obtain adequate pixel resolution in the image.

We now demonstrate how, within the preferred ranges of subarray size, optical magnification, and pixel resolution, and based on the advantages of the microfluidic constructs described herein, The four examples of optical resolution listed above are used to illustrate whether to support well size and spacing settings that allow a large increase in the speed at which wells can be imaged or read. Additional subarray sizes, optical magnifications, and pixel resolutions are supported by the methods and apparatus of the present invention. The scope of the present invention is not limited by the current state of the art in the number of available pixels, nor is it limited by the pixel size in electronic imaging systems. In the first embodiment described above, a 1000 μm × 1000 μm sub-array of wells has a 1000 × 1000 pixel CC (with an image resolution of one pixel per micron).
An image is formed on the D camera. We now provide four examples of well sizes and densities supported by the present invention. (A) First, 300 μm × 300 with 200 μm-thick walls and a well density of 400 wells per cm ²
2 × 2 subarray of μm wells. Imaging these wells in group 4
This results in a 4-fold speed increase compared to reading the plate one well at a time. (B) Second, 3 × of 200 μm × 200 μm well with 100 μm wall
The three sub-arrays produce a 9-fold increase in speed and well density of 1111 wells per cm ² . The number of wells per unit area is a factor 80 times greater than that provided by the current highest density commercial microplates (1536 well plates). (C) Third, a 100 μm × 100 μm well and 1
A 5 × 5 subarray with 00 μm walls results in a 25-fold increase in speed and a density of 2500 wells per cm ² . (D) Fourth, with the advantage of even higher well density and greater speed, the wells have 25 μm × 100 μm walls.
It can be 25 μm, resulting in an 8 × 8 subarray, a 64-fold speed increase, and a well density of 6400 cells per cm ² .

[0165] 2. 2000 x 2000 pixels CC (at an image resolution of 1 pixel per micron)
For a 2000 μm × 2000 μm subarray of wells imaged by a D camera,
One additional example of well density is conceivable other than the four examples discussed above. (A) First, a 2 × 2 subarray of 800 μm × 800 μm wells with 200 μm-thick walls produces a 4 × speed increase and a well density of 1000 wells per cm ² . The number of wells per unit area is a factor five times greater than that provided by current highest density commercial microplates (1536 well plates). (B) Second, 200 μm-300 μm × thickness ×
A 4 × 4 subarray of 300 μm wells results in a 16-fold speed increase and a well density of 400 wells per cm ² . (C) Third, a 6 × 6 subarray of 200 μm × 200 μm wells with 100 μm walls results in a 36-fold speed increase and a well density of 900 wells per cm ² . (D) Fourth, 100
A 10 × 10 subarray of 100 μm × 100 μm wells with μm walls has 100
This results in a fold speed increase and a well density of 2500 wells per cm ² .
(E) Fifth, a 25 μm × 25 μm 16 × 16 subarray with 100 μm walls results in a 256-fold speed increase and a well density of 6400 wells per cm ² .

[0166] 3. 1000 x 1000 pixels (at an image resolution of 0.1 pixels per micron)
For a 10 mm x 10 mm subarray of wells imaged by a CCD camera,
The authors again reported that the four fields of well size and density described in Example 1 above were used.
Describe the case. (A) First, 300 μm × 300 μ with a 200 μm-thick wall
A 20 × 20 sub-array of m-wells provides a 400-fold speed increase and 1 cm²Ah
Yields a density of 400 wells. (B) Second, 200 μm having a 100 μm wall
A 30 × 30 subarray of mx200 μm wells can increase speed 900 times and
1cm²Per well yields 1111 wells. As mentioned above, this
If not, than provided by the current highest density commercial microplate
Also support 80 times greater well density. (C) Third, 10 having a 100 μm wall
A 50 × 50 well subarray of 0 μm × 100 μm wells is 2500 times faster
And increase 1cm²A well density of 2500 per well results. (D) Fourth
The 80 × 80 subarray of 25 μm × 25 μm wells with 100 μm walls
, 6400 times speed increase and 1cm²Yields a density of 6400 wells per
You. Still the advantages of higher well density and greater (2500 times) speed
For the wells, the wells are 100 μm × 100 μm wide with walls separating the wells.
100 μm, 2500 wells per subarray, and 1 cm ² This yields 2500 wells.

[0167] 4. (10 mm x 10 mm subarray of wells imaged on a 2000 x 2000 pixel CCD camera with an image resolution of 0.1 pixels per micron
The cases from Example 2 above of 0, 400, 1111, 2500 and 6400 well cm ² apply again, but the sub-arrays are 10 times wider on each side, resulting in a 100 times greater speed increase. Thus, 100, 400, 1111, 2500
And the speed increase for a well density of 6400 wells / cm ² is 10 m
400 ×, 1600 ×, 3600 ×, 10 ×
000x and 25600x.

The table below compares the well pitch for various other devices.

[0169]

[Table 1] * Estimated speed increase is a comparison of the speed at which the entire array can be imaged by the sub-array compared to imaging by imaging each well separately.

All of these specific forms of the microfluidic devices of the present invention-orthogonal channels, multi-level constructs, high well densities, fluid flow control to these constructs, spatially optimized sub-arrays of wells Any means of well placement and control of compound inter-well diffusion may include the use of cell culture media (a polar aqueous solution containing biological macromolecules and salts) and dissolved oxygen and carbon dioxide in the media. Maintaining physiological levels of carbon gas, and the desired biological temperature (typically 37 ° C., but other temperatures in the approximate range of 15 ° C. to 40 ° C.) may be desirable for certain cell types. ) Is specifically selected and designed to form an integrated system compatible with the maintenance of

In particular, the cell culture medium can be equilibrated in a storage vessel to the desired level of temperature and dissolved carbon dioxide and oxygen. In this way, the culture medium is transferred to the well through the microchannel using the external storage container and the valve. Sealing these microchannels and wells from the atmosphere allows the partial pressure of the gas to be controlled by equilibration of the outer vessel. In a preferred embodiment, the level of carbon dioxide in the medium in the well is determined by equilibrating the medium prior to its flow into the device and / or as the mixture of carbon dioxide and oxygen enters the well. It can be established and maintained by replacing the medium. There are a number of different environmentally controlled chambers for living cell culture (US Pat. Nos. 5,552,321 and 4,974,9).
No. 52; Payne et al., J. Microscopy 147: 329-335 (1987); Boltz et al., Cytom.
etry 17: 128-134 (1994); Moores et al., Proc. Natl. Acad. Sci. 93: 443-44.
6 (1996); Nature Biotech. 14: 3621-362 (1996)).

[0172] In a further preferred embodiment, the temperature of the entire substrate is controlled by contact with a heater or by integrating a heater and contacting a temperature sensor. Electronic temperature control systems adjust the heater to achieve a selected temperature set point. Thus, the design of the microfluidic array system described in the present invention is easily used in a way to support live cell culture without an external incubator system. FIG. 12 shows how the present invention provides for automatic reading of cassettes. The cassette is kept under controlled environmental conditions in two storage compartments (48 and 54) before and after reading in the fluorescence reader equipment 44. As read by the fluorescent reader equipment, the system maintains the proper temperature and composition of the dissolved gas in the cell culture medium in the wells of the cassette. Temperature control in the wells is provided by heating the instrument and temperature sensors in a cassette equipped with a fluorescent reader. Control of the dissolved gas composition in the wells is provided by equilibration of the media with a source of premixed (commercially available) air and carbon dioxide prior to flowing the media into the wells of the cassette. . Any type of controller for adjusting the gas partial pressure can be used in the present invention. In a preferred embodiment, the system includes a gas controller that includes a premixed gas source coupled to a container or containers containing the cell culture medium and / or test compound. Gas pressure regulators and flow control valves control the rate of flow of the gas mixture and its pressure in the connected fluid container. This equilibration of the gaseous mixture with the fluid can be carried out in a container built in or external to the cassette, but in any case outside the matrix of wells.

The present invention satisfies the need in the art for devices and methods that reduce the amount of time required to perform high throughput and / or high content cell-based screens. Also, the device of the present invention is ideally suited as a cell support system for hand-held clinical devices (ie, a miniaturized imaging cell-based assay system). The drug discovery industry is already using 96-well microplates and is shifting towards the use of 384-well plates. 1
Plates with up to 536 wells are conceivable. Thus, there are significant advantages in both throughput and economy from the use of higher density plates, such as those of the present invention.

The sealed containment of cells in the cell arrays of the present invention provides a rigorous system that is portable and can be used in any orientation. In military and civil toxin tests, the device of the present invention will provide two major advantages. First, a toxin test based on cell function is advantageous compared to a toxin test based on molecular structure (eg, mass spectrometry or optical spectroscopy.
Because the link to toxicity is unknown or tests the ultimate effect of the compound on tissue, rather than the relevant properties of the compound, which may depend on other conditions. Second, automated, miniaturized, rugged and portable formats for cell function-based sensors are compatible with the current state of the art, including large microplates filled by large robotic pipetting systems and read by large microscope readers. It would be advantageous in comparison.

In addition, other assay capabilities can be incorporated into the devices of the present invention that were not possible with prior art devices such as simple plastic microplates. For example,
Mass spectrometry or capillary electrophoresis analyzers, or systems for DNA and / or protein analysis, can be incorporated into the devices of the invention to measure chemical and structural parameters of test compounds. Such uptake will provide information that supplements the cell-based functional parameters measured by the HCS or HTS system. In conventional microplates, such additional assay functions are external to the plate, thus requiring a wide range of additional instruments for transferring samples from the microplate and the analytical instrument itself. In the miniaturized format with integrated fluidics of the present invention, the sample is pumped integrally to the microanalytical system on a chip or on a second chip that is integrally connected. Such a micro-analysis chip would reduce cost and dramatically increase the speed of cell-based analysis. In this sense, the integration of microscale living cell analysis systems with microscale chemical analysis systems is expected to provide significant improvements over existing methods and equipment.

[Brief description of the drawings]

FIG. 1A is a plan view of a small substrate micropatterned chemical array.

FIG. 1B is a plan view of a large substrate micropatterned chemical array.

FIG. 2 illustrates a method of producing a finely patterned chemical array on a substrate.

FIG. 3A is a photograph showing fibroblast growth on a surface patterned chip, attached to a micropatterned chemical array and labeled with two fluorescent probes.

FIG. 3B is a photograph showing a fibroblast proliferation in a spot pattern, attached to a micropatterned chemical array and labeled with two fluorescent probes.

FIG. 4 is a diagram of a cassette that is a combination of a microarray at the top and chamber bottom of multiple cell types.

FIG. 5 is a diagram of a chamber with nanofabrication input channels for addressing “wells” in a heterogeneous micropatterned array of cells.

FIG. 6 is a diagram of a chamber without a channel.

FIG. 7A is an overhead view of a chamber with microfluidic channels etched on a substrate.

FIG. 7B is a side view of a chamber having microfluidic channels etched on a substrate.

FIG. 8A is an overhead view of a chamber formed from a raised matrix of material with microfluidic channels and wells engraved on the fluid delivery chamber.

FIG. 8B is a side view of a chamber in which microfluidic channels and wells are formed from a raised matrix of material engraved on a fluid delivery chamber.

FIG. 9 is an illustration of a chamber where each well is addressed by a channel from one side of the chamber.

FIG. 10 is an illustration of a chamber in which wells are addressed by channels from two sides of the chamber.

FIG. 11 is an illustration of a chamber in which the microfluidic switch is controlled by light, heat, or other mechanical means.

FIG. 12 is a diagram of a device equipped with a fluorescent reader.

FIG. 13 is a diagram of one embodiment of a fluorescence reader equipped optical system.

FIG. 14A is a flowchart comprising an overview of a cell screening method.

FIG. 14B is a flowchart of a macro (high throughput mode) process.

FIG. 14C is a micro (high content mode) processing flowchart.

FIG. 15 is a diagram of an integrated cell screening system.

FIG. 16 is a photograph of a user interface equipped with a fluorescent reader.

FIG. 17A is a photograph showing lymphoid cells non-specifically attached to an unmodified substrate.

FIG. 17B is a photograph showing lymphoid cells non-specifically attached to a substrate coated with IgM.

FIG. 17C is a photograph showing lymphoid cells specifically bound to a substrate coated with total antiserum serum.

FIG. 18A is a photographic image from a high throughput mode equipped with a fluorescent reader to identify “hits”.

FIG. 18B is a series of photographic images showing the high content mode identifying high content biological information.

FIG. 19 is a photographic image showing a display of cell data collected from the high content mode.

FIG. 20 shows optimization of cell imaging by simultaneous imaging of subarrays of cell binding locations on all arrays. Between the cell binding location array on the substrate, the fluid location array on the chamber, and the array of wells formed by joining the two:
There is one dependency.

FIG. 21 shows an 8 × 8 array of microfluidic devices (64 wells, 128 channels) with separate input and output channels at each fluid location, resulting in 2n2 channels for an n × n array. For example.

FIG. 22 utilizes m + n ² valves or pumps to control the flow of m liquid or gas mixtures into an 8 × 8 array.

FIG. 23 shows that the fluid delivery system consists of an orthogonal non-intersecting array of input and output channels, and a horizontal coupling channel utilizing multiple levels, wherein:
The two layers represent an embodiment of the microfluidic device on the same side of the well layer.

FIG. 24 shows an end view and a side view of the embodiment of FIG. 23.

FIG. 25 shows that the fluid delivery system consists of an orthogonal non-intersecting array of input and output channels, and a vertically coupled channel utilizing multiple levels, wherein:
Figure 3 shows an embodiment of a microfluidic device with two layers on the upper surface of the well layer.

FIG. 26 shows an end view and a side view of the embodiment of FIG. 25.

FIG. 27 illustrates an embodiment of a microfluidic device in which compounds are multiplexed into a single fluid location by using a m positive pressure source vessel or pump, a valveless manifold, and a pneumatic valve-free waste vessel. .

FIG. 28 illustrates a negative pressure waste container or pump or a barometric valve free waste container with an m valve coupled to an m positive pressure source container or pump, a valved manifold, or an atmospheric pressure m source container. Figure 4 illustrates an embodiment of a microfluidic device, where used to multiplex compounds into a single fluidic location.

FIG. 29 shows an array of m positive pressure source vessels or pumps with 1 × n inputs and n
Figure 3 shows an embodiment of a micro device multiplexed by a xl output valve manifold into an nxn orthogonal channel array of fluid locations.

FIG. 30 shows an embodiment of a microfluidic device with a m pressure vessel and a negative pressure waste vessel with l × n input and n × 1 output valve manifolds, wherein the negative pressure vessel is It can be pumped mechanically or thermodynamically.

FIG. 31 illustrates an embodiment of a dual pumped system where there is only one waste negative pressure container that must be operated in conjunction with the operation of any particular positive pressure source container. The vacuum vessel can function by either mechanical or thermodynamic (eg, a capillary action pump) means. For capillary action pumps, the pump "
"Activation" is achieved by a valve.

FIG. 32 illustrates how the pumping and valving scheme of FIG. 29 using a positive pressure source vessel, with an orthogonal array of n row channels and 2n column channels (n normal column channels + n wash column channels). Indicates that it can be expanded to work. This requires a 2n × 1 output valve manifold. Similarly, the arrangement of FIG. 30 using negative pressure waste containers can be expanded to work with arrays that incorporate wash channels.

FIG. 33 shows an embodiment of a microfluidic device having a parallel wash channel k ^{* with} which each row channel k associates.

FIG. 34 shows that the fluid delivery system consists of an orthogonal non-intersecting array of input and output channels with vertical coupling channels utilizing multiple levels, where the two layers are above the well layer FIG. 3 shows an embodiment of a microfluidic device having a parallel wash channel k ^* with each row channel k associated therewith.

FIG. 35 shows that in the case of fluid flow through well (j, k), from row channel j to wash channel k ^* (where k ^* is immediately adjacent to column channel k). 1 illustrates an embodiment of a method for rinsing a liquid or gas segment.

FIG. 36 illustrates a method of diffusion control using a “normally closed” check valve including a valve element and a valve seat between each pair of adjacent passages coupling a channel to a fluid location.

FIG. 37 illustrates an embodiment in which an externally applied magnetic field gradient induces a restoring force on the valve element and valve seat that take up the magnetic material.

FIG. 38 shows an embodiment of a shaping valve seat in which diffusion through the ball is stopped only when an externally applied restoring force pushes the ball to the left and into a round opening in the valve seat.

FIG. 39 shows an implementation that allows a single magnetic field gradient applied to a whole microfluidic device to induce forces on both the input channel ball valve set and the output channel ball valve set. The form is shown.

FIG. 40 illustrates one embodiment of the pumping and valving scheme of the present invention, such that all pumps and valves built into the cassette are present.

FIG. 41 utilizes a flow where the pump is embedded and driven by electricity, where:
FIG. 7 illustrates an embodiment where the electric field is confined to regions that are external to the matrix of the array of wells and there is minimal or no electric field gradient applied across any of the wells where live cells are present. (Explanation of Symbols) 001 Fluid position 002 Fluid position array 003 optionally having a recess 003 Subarray 004 Substrate 008 Cell binding position 009 Chemical modification of cell binding position 010 Cells aligned on cell binding position 012 Chamber 012 Recess formed in fluid position 014 Input channel 016 Output channel 018 Cassette 020 Spacer support 024 Micro-capillary tube 028 Higher vessel 030 First channel 032 Second channel 036 Plug 040 Fluid position array 044 Fluorescent reader instrument 048 First storage section 050 First robot arm 052 Second robot arm 054 Second storage section 056 Computer 060 Optics 064 Computer controlled xyz stay 068 Computer-controlled rotary objective stage 070 Low-magnification objective lens 072 High-magnification objective lens 07 White light source lamp 076 Excitation filter wheel 078 Two-color filter system 080 Emission filter 082 Detector (eg, cooled CCD) 086 Computer screen 090 Database 140 well Horizontal direction 160 Horizontal coupling channel 180 Vertical coupling channel 200 Switchable positive pressure source container or pump 220 Normal pressure source container 240 m × l valveless manifold 300 Waste container 320 Switchable negative pressure waste container or pump 340 Thermodynamics Waste pump 400 l × n valve manifold 410 n × l valve manifold 420 2n × l valve manifold 500 check valve 5 0 valve element 540 valve seat 560 field (B) gradient 580 magnetic bowl check filling of resilience 600 beads 620 field 700 port for valve 710 channels 720 valve 730 container 740 Dodengaku pump 760 electrode 1 780 electrode 2

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 21/03 G01N 33/50 Z 27/447 37/00 103 33/50 27/26 301A 37/00 103 301Z C12N 5/00 E (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL) , SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Go, Albert H. United States 15116 Glenshaw Louise Drive, Pennsylvania 2014 (72) Inventor Taylor, D. Lansing United States 15215 Pittsburgh, PA Notre Dame Place 910 F-term (reference) 2G045 AA40 CB01 DA12 DA13 DA36 FA11 FB03 FB12 GC15 HA14 2G057 AA04 AB03 AC01 BA01 BA03 BB01 EA06 4B029 AA07 BB11 CC03 Q08 Q04 Q03 Q08 Q04 Q03 Q08 Q04 Q03 Q08 Q04 Q04 QR82 QS36 QS39 QX02 4B065 AA94X BA21 CA46 CA60

Claims (17)

[Claims] 1. A method comprising: a. A substrate having a surface containing a plurality of cell binding sites, b. A fluid delivery system for delivering an assay component to a plurality of cell binding locations, wherein the fluid delivery system includes a multi-level chamber in contact with a substrate, wherein the multi-level chamber comprises: i. Orthogonal array of microfluidic input and output channels ii. A plurality of fluid locations in fluid communication with the microfluidic input and output channels; c. A plurality of wells, wherein each well includes a space defined by the joining of one cell binding location and one fluid location, said wells being present at a density of at least about 20 wells per cm ^2. Cassette for cell screening. Wherein said well, cassette according to claim 1, characterized in that present in a density of between about 20 wells per 1 cm ² and 1 cm ² per 6400 wells. 3. The multi-level chamber further comprises: (i) one or more input manifolds in fluid communication with the microfluidic input channel; and (ii) one or more output manifolds in fluid communication with the microfluidic output chamber. The cassette according to claim 1, comprising: 4. The method of claim 1, further comprising: (i) at least one source vessel fluidly coupled to one or more input manifolds; and (ii) at least one waste vessel fluidly coupled to one or more output manifolds. Item 4. The cassette according to Item 3. 5. A method according to claim 1, wherein: a. A substrate having a surface containing a plurality of cell binding sites; b. A fluid delivery system for delivering a reagent to a plurality of cell binding locations, wherein said fluid delivery system includes a multi-level chamber that interfaces with a substrate, wherein the multi-level chamber comprises: i. An orthogonal array of microfluidic input and output channels, wherein each well is in fluid communication with one or more input channels and one or more output channels; ii. A plurality of fluid locations in fluid communication with the microfluidic input and output channels iii. One or more input manifolds in fluid communication with the microfluidic input channel iv. One or more output manifolds fluidly coupled to the microfluidic output channels v. At least one source vessel in fluid communication with one or more input manifolds vi. Including at least one waste container in fluid communication with one or more output manifolds; c. A cassette for cell screening, wherein each well comprises: a plurality of wells each including a space defined by one cell binding site and one fluid site junction. 6. The apparatus of claim 1, further comprising a pump for controlling fluid flow in the microfluidic device, wherein the pump is in fluid communication with one or more input manifolds and one or more output manifolds. Or the cassette according to 5. 7. The apparatus according to claim 1, further comprising a temperature controller.
Or the cassette according to 5. 8. The cassette according to claim 1, further comprising a controller for adjusting a gas partial pressure. 9. The cassette according to claim 1, wherein orthogonal arrays of microfluidic input channels and output channels do not intersect. 10. The cassette according to claim 1, further comprising a washing channel fluidly connected to the output channel and the waste container. 11. The cassette according to claim 1, further comprising a valve between adjacent wells or in the washing channel. 12. The valve according to claim 1, wherein the valve includes a magnetic element.
2. The cassette according to 1. 13. The cassette according to claim 1, further comprising an integrated nodal analysis or a capillary electrophoresis analyzer, or a system for protein and / or DNA analysis. 14. The improvement comprises a cassette according to claim 1 or 5,
An improved method of screening cells comprising providing a fluid to a positional array containing cell binding sites. 15. An improved method for controlling diffusion in a cassette, wherein the improvement applies constant passive restoring force to a valve located in a microfluidic channel of the cassette. 16. A position array containing a plurality of cells, b) an image of the position array is acquired with optics, c) a sub-array of the position array is imaged sequentially, d) a sub-array. A method for cell screening, comprising obtaining data from each of the above in parallel. 17. A microfluidic device for cell screening according to claim 1 or 5; b) an image of a position array is acquired with an optical system; c) a subarray of the position array is sequenced. And d) obtaining data from each of the sub-arrays in parallel.