MXPA01002960A - Miniaturized cell array methods and apparatus for cell-based screening - Google Patents

Abstract

The present invention devices and methods for maximizing the number of wells that can be imaged at one time while still obtaining adequate pixel resolution in the image. This result has been achieved through the use of fluidic architectures that maximizes well density. The present invention thus provides a miniaturized microplate system with closed fluidic volumes that are internally supplied with fluid exchange, and with wells that are closely spaced to more rapidly detect spatially-resolved features of individual cells.

Description

METHODS AND APPARATUS OF MINIATURIZED CELLULAR ARRANGEMENT FOR CELL-BASED ANALYSIS DESCRIPTION OF THE INVENTION The present invention relates to methods and devices for screening high production and high biological content on cell basis. In the growing field of drug discovery and combination chemistry to generate candidate compounds, it would be very useful to be able to quickly select a large number of substances, by means of a high production screening, for their physiological impact on animals and humans. Before testing the efficacy of a candidate drug "partially qualified in animals, the drug could be selected first for its biological activity and potential toxicity with living cells.The physiological response to the candidate drug could be anticipated after the results of these cell selections. The "lead compounds" have moved rapidly into extensive animal studies that are time consuming and expensive, and the extensive drug testing in animals is becoming less culturally acceptable.The selection of candidate drugs according to their interaction with living cells , prior to animal studies, can reduce the number of animals required in subsequent drug selection processes by eliminating some candidate drugs before going to animal testing, however, manipulation and analysis of drug-cell interactions using current methods does not allow the High production and high biological content, due to the small number of cells and compounds that can be analyzed in a given period of time, the uncomfortable methods required for the supply of the compound, and the large volumes of compounds required for the test. The screening of high production of nucleic acids and polypeptides has been achieved using DNA chip technologies. In typical DNA analysis methods, DNA sequences of 10 to 14 pairs of substrates are unit in defined locations (or places) and up to tens of thousands in number, or on a small glass plate (US Patent No. 5,556,752 , incorporated herein by reference). This creates an array of DNA locations on a given glass plate. The location of a place in the array provides an address for subsequent reference for each DNA location. The DNA sequences are then hybridized with complementary DNA sequences labeled with fluorescent molecules. The signals for each direction on the array are detected when the fluorescent molecules linked to the hybridized nucleic acid sequences bloom in the presence of light. These devices have been used to provide screening for high production of DNA sequences in drug discovery efforts and in the sequential human genome project. Similarly, protein sequences of various amino acid lengths have been joined in discrete places as an array on a glass plate. (US Patent 5,143,854, incorporated herein by reference.) The information provided by an array of nucleic acids or amino acids attached to glass plates is limited according to their underlying "languages." For example, DNA sequences have a language of only 4 nucleic acids and proteins have a language of approximately 20 amino acids In contrast, a living cell, comprising a complex organization of biological components, has a "coarse language with a concomitant multitude of potential interactions with a variety of substances, such such as DNA, RNA, cell surface proteins, intracellular proteins and the like, because a typical selection for drug action is with and within the cells of the body, the cells themselves provide an extremely useful screening tool in the discovery of drug when combined with sensitive detection reagents. It would be more useful to have a screening device of high production, high content to provide high spatial information content at the cellular and subcellular level as well as temporal information about the changes in physiological biochemical and molecular activities.

Microarrays of cells. Methods for making microarrays of a single cell type on a common substrate for other applications have been described. An example of such methods is photolithography, with photochemical protector (Mrksich and Whitesides, Ann. Rev. Biophys., Biomol. Struct. 25: 55-78, 1996), in which a glass plate is uniformly covered with a photoresistor and A photomask is placed on the photoprotective coating to define the desired "arrangement" or pattern. When exposed to light, the photoprotector is removed in the unmasked areas. The entire photolithographically defined surface is uniformly coated with a hydrophobic substance, such as an organosilane, which joins both areas of the exposed glass and the areas covered with the photoresist. The photoprotector is stripped after the glass surface, exposing an array of exposed glass places. The glass plate is then washed with an organosilane having terminal hydrophilic groups or groups capable of reacting chemically such as amino groups. The hydrophilic organosilane is attached to the exposed glass locations with the resulting glass plate having an array of hydrophilic or reactive sites (located in the areas of the original photoresist) through a hydrophobic surface. The arrangement of hydrophilic group sites provides a substrate for non-specific and non-covalent binding of certain cells, including those of neuronal origin. (Kleinfeld et al., J. Neurosci 8: 4098-4120, 1988). In another method based on specific interactions, however non-covalent, stamping is used to produce a gold surface coated with an absorptive alkane thiol protein. (U.S. Patent No. 5,776,748; Singhvi et al., Science 264: 696-698, 1994). The bare golden surface is then coated with alkanethiols terminated in polyethylene glycol which resists protein absorption. After exposure of the entire surface to laminin, a protein that binds cells found in the extracellular matrix, living hepatocytes bind uniformly to, and grow on, the laminin-coated islands (Singhvi et al., 1994). An elaboration involving strong but non-covalent metal chelation has been used to coat gold surfaces with specific protein models (Sigal et al., Anal, Chem. 68: 490-497, 1996). In this case, the golden surface is modeled with alkanethiols terminated with nitriloacetic acid. The discovered gold regions are coated with tri (ethylene glycol) to reduce protein adsorption. After the addition of Ni2 +, the specific adsorption of five proteins marked on histidine is found to be kinetically stable. A more specific single cell type link can be achieved by chemically cross-linking specific molecules, such as proteins, to sites capable of reacting on the patterned substrate. (Aplin and Hughes, Analyt, Biochem 113: 144-148, 1981). Another elaboration of substrate optically modeling creates an arrangement of places capable of reacting. A glass plate is washed with an organosilane that is removed to the glass to coat the glass. The organosilane coating is irradiated with deep UV light through an optical mask that defines a model of an array. The irradiation unfolds the Si-C bond to form a reactive Si radical. The reaction with water causes the Si radicals to form polar silanol groups. The polar silanol groups constitute sites on the array and are further modified to couple other molecules capable of reacting to the sites, as described in US Pat. No. 5,324,591, incorporated herein by reference. For example, a silane containing a biologically functional group such as a free amino moiety can be reacted with the silanol groups. The free amino groups can then be used as covalent binding sites for biomolecules such as proteins, nucleic acids, carbohydrates, and lipids. The unmodeled covalent binding of a lectin, known to interact with the surface of cells on a glass substrate through aminoreactive groups (Aplin & amp; amp;; Hughes, 1981). The optical method to form a simple cell type microarray on a support requires fewer steps and is faster than the photoprotective method, (ie, only two stages), but requires the use of high intensity ultraviolet light from a light source expensive. In all these methods, the result is a microarray of a simple cell type, since the biochemically specific molecules bind to the micro-modeled chemical arrangement uniformly. In the photoprotective method, the cells bind to the arrangement of hydrophilic sites and / or specific molecules linked to the places which, in turn, unite cells. Thus the cells are attached to all the places in the array in the same way. In the optical method, the cells bind to the arrangement of free amino group sites by adhesion. There is little or no difference between the places of free amino groups. Again, the cell adheres to all places in the same way, and thus only a simple type of cell interaction can be studied with these cellular arrangements because each place on the array is essentially the same as another. Such cellular arrangements are inflexible in their usefulness as tools for studying a specific diversity of cells in a single sample or a specific diversity of cellular interactions. Thus, there is a need for arrays of multiple cell types on a common substrate, to increase the number of cell types and specific cellular interactions that can be analyzed simultaneously, as well as methods for producing these microarrays of multiple cell types on a common substrate, to provide a selection of cells of high production and high biological content.

Optical Reading of Cellular Physiology Screening of high production in many thousands of compounds requires the handling and parallel processing of many compounds and reagents test components. High-throughput standard selections use homogeneous mixtures of biological compounds and reagents along with some indicator compound, loaded into arrays of wells in standard 96 or 384 well microplates (Kahl et al., J. Biomol. Ser. 2: 33-40 , 1997). The measured signal from each well, be it fluorescence emission, optical density, or radioactivity, integrates the signal of all the material in the well giving an average total population of all the molecules in the well. This type of test is commonly referred to as a homogeneous test. US Patent No. 5,581,487 discloses an image plate reader that uses a CCD detector (load-coupled optical detector) to image the entire area of a 96-well plate. The image is analyzed to calculate the total fluorescence per well for homogeneous tests. Schroeder and Neagle describe a system that uses low-angle laser scanning illumination and a mask to selectively excite fluorescence within approximately 200 microns of the bottoms of wells in standard 96-well plates to reduce surrounding circumstances when images form the cellular monolayers. (J. Biomol. Ser. 1: 75-80, 1996). This system uses a CCD camera to form the image of the entire area of the bottom of the plate. Although this system measures signals originating from a downhole cell monolayer, the measured signal is averaged over the well area and is therefore still considered a homogeneous measurement, since it is an average response of a population of cells . The image is analyzed to calculate the total fluorescence per well for homogeneous tests on a cellular basis. Proffitt et. to the. (Cytometry 24: 204-213, 1996) describes a semiautomated fluorescence digital imaging system for quantifying relative cell quantities in situ, wherein the cells have been pretreated with fluorescein diacetate (FDA). The system uses a variety of tissue culture plate formats, particularly 96-well microplates. The system consists of an inverted epifluorescence microscope with a motorized stage, video camera, image intensifier, and a microcomputer with a PC-Vision digitizer. Turbo Pascal software controls the stage and scans the plate taking multiple images per well. The software calculates total fluorescence per well, provides daily calibration, and configures for a variety of tissue culture plate formats. Thresholding of digital images and the use of reagents that fluoresce only when absorbed by living cells are used to reduce the fluorescence of surrounding circumstances without removing excess fluorescent reagent. A variety of methods have been developed to image fluorescent cells under a microscope and extract information about the spatial distribution and temporal changes that occur in this cell. A recent article describes many of these methods and their applications (Taylor et al., Am. Scientist 80: 322-335, 1992). These methods have been designed and optimized for the preparation of small numbers of specimens for distribution measurements of spatial and temporal high-resolution imaging, amount of biochemical environment of fluorescent reporter molecules in cells. Useful detection methods are to treat cells with dye and fluorescent reagents, image the cells and design the cells to produce a fluorescent reporter molecule, such as a modified green fluorescent protein (GFP), (Wang et al., In Methods). Cell Biology, New York, Alan R., Liss, 29: 1-12, 1989). The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has a maximum of excitation at 395 nm, a maximum emission at 510 nm, and does not require an exogenous factor. The uses of GFP for the study of gene expression and protein localization are discussed in Chalfie et al., Science 263: 802-805, 1994. Some properties of native GFP are described by Morise et al. (Biochemistry 13: 2656-2662, 1974), and Ward et al (Photochem, Photobiol 31: 611-615, 1980). An article by Rizzuto et al. (Nature 358: 325-327, 1992) discusses the use of native-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 secretion path using native GFP. The expression of GFP in plant cells is discussed by Hu and Cheng (FEBS Letters 369: 331-334, 1995), whereas the expression of GFP in Drosophila embryos is described by Davis et al. (Dev. Biology 170: 726-729, 1995). U.S. Patent No. 5,491,084, incorporated herein by reference, describes the expression of GFP of Aequorea Vitoria in cells as a reporter molecule fused to another protein of interest. GFP mutants have been prepared and used in various biological systems. (Hasselhoff et al., Proc. Nati, Acad. Sci. 94: 2122-2127, 1997; Brejc et al., Proc. Nati 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). The ARRAYSCAN ™ System, as developed by Cellomics, Inc. (U.S. Patent Application Serial No. 08 / 810,983 filed February 27, 1997 and 09 / 031,271 filed February 27, 1998) is an optical system for determining the distribution, environment, or activity of molecules reporter luminescently labeled in cells for the purpose of selecting large numbers of compounds for specific biological activity. The ARRAYSCAN ™ System involves providing cells containing luminescent reporter molecules in an array of locations and scanning numerous cells at each location, converting the optical information into digital data, and using the digital data to determine the distribution, environment or activity of the reporter molecules marked luminescently in the cells. The ARRAYSCAN ™ System includes devices and a computerized method to process, display and store the data, thus increasing the discovery of drugs by providing high cell-based selection, as well as a combination of high production and high cell-base content. a large microplate format. Microfluidics The efficient supply of solutions to a cell array attached to a solid substrate is facilitated by a microfluidic system. Methods and apparatuses for the accurate handling of small liquid samples for ink supply have been described (US Patent No. 5,233,369; US Patent No. 5,486,855; US Patent No. 5,502,467), Biomass Injection (US Patent No. 4,982,739), Storage and reagent supply (U.S. Patent No. 5,031,797), and divided fluidic and microelectronic device arrangements for clinical diagnostics and chemical synthesis (U.S. Patent No. 5,585,069). In addition, methods and apparatus for forming and microchannels have been described in solid substrates that can be used to direct small liquid samples along the surface (US Patent No. 5,571,410; US Patent No. 5,500,071; US Patent No. 4,344,816). For purposes of integrated selection of high production and high cell-based content, particularly for live cell imaging, an optimal microfluidic device would comprise a fluidic architecture with the closest possible well spacing (ie the highest possible well density). , where the fluidic architecture is integrated with the cellular arrangement substrate to allow the supply of efficient fluids to the cells, and eliminate the need to pipe fluids in and out of the wells. Such optimal microfluidic devices would be advantageous for cellular arrangements with interpozo submillimeter distances because it is unmanageable, but impossible to pipe fluids with such a high degree of spatial resolution and precision. In addition, such integrated devices could be used directly for cell-based selection, without the need to remove the cellular substrate from the fluidic architecture to image the cells. An optimal microfluidic device for cell-based selection could additionally comprise a closed chamber to allow environmental control of the cells, and preferably would not directly expose the cells to electrokinetic forces, which may affect the physiology of the cells on the substrate. For example, electrohydrodynamic pumping is less effective with polar solvents (Marc Madou, Fundamentals of Microfabrication, CRC Press, Boca Raton, 1997, p 433). Electro-osmosis is typically accompanied by some degree of electrophoretic separation of charged medium components, such as proteins. US Patent No. 5,603,351 (to patent 351 ') discloses a microfluidic device using a multiple level design consisting of two upper levels with channels and a lower level with reaction wells. However, this device is not designed for use in cell-based selection. The patent 351 patent does not disclose a substrate containing cells or cell binding sites. The microfluidic network described is designed to allow combining two or more reagents in a reaction well, opposite to a microfluidic system of optimal cellular selection that allows to expose live cells cultivated in the bottoms of the wells in a series form to two or more different fluids. The '351 patent describes a device with wells etched into the substrate at a maximum well density of 50 wells / inch2. In addition, the substrate must be separated from the fluidic arrangement for incubation and / or analysis. Finally, the 351st patent directs a system of electrically controlled electrohydrodynamic valves within the wells matrix that are less effective in the aqueous medium used in cell culture, and may also limit the degree of close spacing between the wells in the well. well arrangement. U.S. Patent No. 5,655,560 discloses a clot-free valve system, comprising a fluid distribution system with multiple inlets and multiple outlets incorporating a cross arrangement of microchannels connected vertically at the crossing points by Teflon valves. However, it is clear that it does not disclose a substrate containing a cellular array, nor an integrated fluidic device in combination with the substrate, nor a well density that is optimal for cell-based selection. U.S. Patent 5,900,130 (the '130' patent) describes the active electronic control of the movement of fluids in a capillary-connected structure. This patent does not teach a fluidic architecture that maximizes the area of cellular substrate that can be occupied by cell binding sites. Neither does this patent describe a substrate containing a cellular arrangement, nor an integrated fluidic device in combination with the substrate. In addition, the patent teaches only the control of fluid flow by application of an electric field to the device. U.S. Patent 5,910,287 describes multi-well plastic plates for fluorescence measurements of biological and biochemical samples, including cells, limited to plates with more than 864 wells. This patent does not disclose a microfluidic device with a fluidic architecture integrated with the cellular array substrate. Nor does the patent write a closed chamber to allow environmental control of the cell on the substrate. Thus, none of these prior microfluidic devices provide a fluidic architecture that allows for the closest possible spacing of wells (ie the highest possible well density), where the fluidic architecture is integrated with the cellular array substrate to allow delivery of efficient fluids to the cells, and thus eliminate the need to pipe fluids in and out of the wells. In addition, prior microfluidic devices comprising an array of wells use electrically controlled electrohydrodynamic valves within the well matrix that would be less effective if used with aqueous media for cell culture, and which limits the density of wells. While previous advances in cell array, optical cell physiology reading, and microfluidic technologies provide support technologies that can be applied to the high production and high cell base content section, a need remains in the art for integrated devices and methods that further decrease the amount of time needed-for such selection, as well as for devices and methods that further improve the ability to conduct high-throughput screening and high cell-base content and the ability to flexibly and rapidly change from one to the other . In particular, devices and methods that maximize the density of wells, thereby increasing the number of wells from which images can be formed at a time, and thus greatly increase the production of a selection while maintaining the appropriate resolution of the image , they would be very advantageous. The drug discovery industry already uses 96 and 384 well microplates and is transitioning to the use of 1536 well plates. Nevertheless, further increases in well density using the prior technology are unlikely due to the great difficulty of tubing liquids in and out of wells of very small diameter.

PREVIOUS ART The present invention fills the need in the art for devices and methods that decrease the amount of time needed to drive cell-based selection, and specifically combines the ability to conduct high-throughput screening and high cell-base screening and to change flexibly and quickly from one to the other. The invention provides devices and methods for maximizing the number of wells from which images can be formed at a time while still obtaining adequate pixel resolution in the image. This result has been achieved through the use of fluidic architectures that maximize the density of wells. The present invention thus provides a miniaturized microplate system with closed fluidic volumes that are supplied internally with fluid exchanges, and with wells that are closely spaced to more rapidly detect spatially resulting characteristics of individual cells. In one aspect, the present invention provides a cassette for cellular selection, comprising a substrate having a surface containing a plurality of cell attachment locations; a fluid supply system for supplying reagents to the plurality of cell attachment locations, wherein the fluid delivery system comprises a multiple level chamber that is coupled to the substrate, wherein the multiple level chamber comprises a cross-arrangement of channels inlet and microfluidic outlet channels and a plurality of fluidic locations in fluid connection with the inlet channels and microfluidic outlet channels; and a plurality of wells, wherein an individual well comprises the space defined by the coupling of a cell attachment location and a fluid location, and wherein the wells are present in a density of at least about 20 wells per square centimeter. In another aspect, the present invention provides a cassette for cellular selection comprising a. a substrate having a surface, wherein the surface contains a plurality of cell attachment locations; b. a fluid supply system for supplying reagents to the plurality of cell attachment locations, wherein the fluid delivery system comprises a multiple level chamber that is coupled to the substrate, wherein the multiple level chamber comprises i. a cross-arrangement of input channels and microfluidic output channels, where each well is in fluid connection with one or more input channels and one or more output channels; ii. a plurality of fluidic locations in fluid connection with the inlet channels and microfluidic outlet channels; iii. one or more input interconnects in fluid connection with the microfluidic input channels; iv. one or more output interconnects in fluid connection with the microfluidic output channels; v. at least one source receptacle in fluid connection with one or more output interconnects; and I saw. at least one waste receptacle in fluid connection with one or more output interconnects; and c. a plurality of wells, wherein an individual well comprises the space defined by the coupling at a cellular junction location and a fluid location.

In preferred embodiments, both of the above cassettes further comprise a pump for controlling the flow of fluids within the microfluidic device; a temperature controller of the substrate and / or a controller for regulating the partial pressures of oxygen and carbon dioxide. In another aspect, the present invention provides an improved method for diffusion control in a cassette, wherein the improvement comprises constantly applying a passive restoring force to the valves located within the microfluidic channels of the cassette. In a still further aspect, the present invention provides a method for cellular screening, comprising. a) provide an array of locations that contain multiple cells; b) provide an optical system to obtain images of the arrangement of locations; c) forming serial images of the arrangement sub-arrangements of the locations; and d) acquire data from each of the sub-arrangements in parallel. In a preferred embodiment, arrangement of locations is provided as a cassette, such as those described above. Among other uses, the devices and methods of the present invention are ideal for the selection of high content and / or high cell-based production. The device of the invention is also ideally suited as a cellular support system for a manual diagnostic device (ie: a cell-based test system that forms miniaturized images). The smaller format and sealed content of the current device allows its use in a wrinkled portable system. There is a great economic advantage of using higher density plates. There is an additional economic advantage in faster imaging of sub-arrays when a high-density plate is used; this advantage is not fully realized if the distances between the adjacent wells are not minimized. The present invention provides both of these advantages.

Brief Description of the Figures Figure 1A is a top view of a chemical array of small micro-patterned substrate. Figure 1B is a top view of a chemical array of large micro-patterned substrate. The diagrams in Figure 2 are a method of producing a micro-modeled chemical array on a substrate. A'-Drips of addition of the protecting group (P); B'- Wear with the Hydrophobic Reagent Group (X). Figure 3A is a photograph showing fibroblastic cell growth on a patterned microplate surface, attached to a micro-patterned chemical array and labeled with two fluorescent probes. A-Rodamin-Actin; B-Hoechst-Nucleico. Figure 3B is a photograph showing fibroblastic cell growth in painted models, linked to a chemical or micro-modeling arrangement and marked with two fluorescent probes. A-Sodium Fluorescence attached to a Cell Chip to identify the location of dot patterns of 200 um; B-Fluorescent cell binding of L929 cells to patterned spots (low amplification, 4x); C-Union of fluorescent cell of 3T3 cells to patterned points. Nucleic (anterior), Actin in the same cells (next) (high amplification, 20X); D-Sodium Fluorescence linked to the cell chip to identify the location of dot models. Figure 4 is a diagram of the cassette that is the combination of the microarray of multiple cell types at the bottom and the bottom of the chamber. Figure 5 is a diagram of a chamber having nanofabricated input channels for directing "wells" in the non-uniform array of micro-patterned cells. Figure 6 is a diagram of a camera without channels. Figure 7A is a diagram of the upper part of a chamber with microfilm channels engraved on the substrate. A'-Type of cell. Figure 7B is a side view diagram of a chamber with microfluidic channels engraved on the substrate.

Figure 8A is a diagram of the upper part of a chamber in which the microfluidic channels and wells are formed from a matrix arising from a material stamped on the fluid supply chamber. A'-Type of cell. Figure 8B is a side-view diagram of a chamber in which microfluidic channels and wells are formed from a matrix arising from a material stamped on the fluid supply chamber. Figure 9 is a diagram of a chamber where each well is directed by a channel that originates from one side of the chamber. Figure 10 is a diagram of a chamber in which the wells are directed by channels originating from two sides of the chamber. Figure 11 is a diagram of a chamber in which microfluidic changes are controlled by light, heat or other mechanical means. Figure 12 is a diagram of the luminescence reading instrument. Figure 13 is a diagram of one embodiment of the optical system of the luminescence reading instrument. Figure 14A is a flow chart that provides a complete view of the cell selection method. C-start; D- Establishment start parameters; Self-loading E-Chip; F-Distributed cells and reagents on the chip; G-Macro analysis (Full chip image); H-Any "successful" well ?; l-No, J-Yes; K-Micro analysis (image cells in "successful" wells); L-Database of updated computer scientists; M-Database; N-yes; Ñ-More chips ?; Or not; P-end. Figure 14B is a flow chart of Macro Processing (High Production Mode). B-Field acquired by primary markings (color # 1); C-Background segment wells; D-Wells detected ?; E-Field acquired for other markers (color # 2-4); F-A well location; G-Characteristics of well of measure, H-Criterion gathered for micro analysis ?; I-S¡; J-Pozos designated as a "hit" and the stored location; K-no; L-yes; M - Any unprocessed wells; N-no; Ñ-End. Figure 14C is a Micro Processing flow chart (High Content Mode). A- Background stage in the next field C-Movement to the next hole of success; D-Autofocus of the current field; E-Field acquired by the primary marker (color # 1); F-no; G-EI field contains objects? - H-Objects of background adaptability segments; I-Yes; J-Field acquired by other primary markers (color # 2-4); K-An object located; L-Object of a valid cell ?; M-no; N-Yes; Ñ-no; Or if; P-More cells found in the well ?; Q-Measuring cell characteristics; R-no; S: Made with chip ?; T-no; U-Any objects no. processed? V-s¡; W-yes; W-End Figure 15 is a diagram of the integrated cell selection system. Figure 16 is a photograph of the user interface of the luminescence reading instrument. A-Sample; B- Pigment; C-Camera; D-Parameters; E-selection; F-Analysis; G-Tracking; H-Review; I-Images; J-Data; K-Summary; L- Results; M-Report Figure 17A is a photograph showing lymphoid cells bound non-specifically to an unmodified substrate. A'-control not covered. Figure 17B is a photograph showing lymphoid cells bound non-specifically to a substrate coated with IgM. B'-IgM coating. Figure 17C is a photograph showing lymphoid cells specifically bound to a substrate coated with complete antiserum. C'-Complete coated serum. Figure 18A is a photographic image of the High Production Mode of the luminescence reading instrument that identifies "hits." Figure 18B is a series of photographic images showing the high content mode that identifies the high content of biological information. Figure 19 is a photographic image showing the display of cellular data gathered in the high content mode.

A-Printing; B-Done; C-Cancel; D-Graph 1; E-Graph 2; F-Graph 3; G-Cell # 3; H-Location; I-Excluded; J- Hoechst; K- Cytoplasm; L and M-Not used. Figure 20 represents the optimization of cellular imaging by the simultaneous formation and imaging of a sub-arrangement of cell attachment sites of the complete array. There is a one-to-one correspondence between the arrangement of cell binding sites on the substrate, the arrangement of fluidic locations on the chamber, and the arrangement of wells formed by the union of the two. Figure 21 exemplifies an 8 x 8 array (64 wells, 128 channels) of the microfluidic device in which each fluid location is provided with a separate input and output channel, producing 2n2 channels for an n x n array. Figure 22 represents the microfluidic device of Figure 21 m + n2 valves or pumps are used to control the flow of m liquid or vapor mixtures in an 8 x 8 array. Figure 23 represents a modality of the microfluidic device where the system Fluid supply consists of a non-intercepted cross-arrangement of inlet and outlet channels, and horizontal channels that connect using multiple levels and where the two layers are in the same plane of the well layer. Figure 24 represents a final view and side view of the modality of Figure 23. A-Final view; B-Top view; C-Side view. Figure 25 depicts a modality of the microfluidic device wherein the fluid delivery system consists of a cross-over, non-intercepted arrangement of inlet and outlet channels, and vertical channels that connect using multiple levels, and wherein the two layers are in a plane above the well layer. Figure 26 represents a final view and side view of the embodiment of Figure 25. A-Final view; B-Top view; C-Side view. Figure 27 shows a modality of the microfluidic device wherein the compounds are multiplexed to a simple fluid location by using m positive pressure source reservoirs or pumps, a valveless interconnector, and valve free waste reservoir at atmospheric pressure. Figure 28 shows a modality of the microfluidic device wherein the compounds are multiplexed to a simple fluid location by using m positive pressure source reservoirs or pumps, an interconnector with valves, or a negative pressure waste reservoir or pump with m valves connected to source deposits at atmospheric pressure and deposit of free waste of valves at atmospheric pressure. Figure 29 shows a modality of the microfluidic device in which the arrangement of m positive pressure source reservoirs or pumps is multiplexed to an array of crossed channels n x n of fluidic locations 1 x n output valves and n x 1 interconnected input.

Figure 30 shows a modality of the microfluidic device with m deposits at atmospheric pressure and a waste deposit at negative pressure by means of 1 xn interconnecting inlet and nx 1 outlet valves, where the negative pressure reservoir can be pumped mechanically or thermodynamically mind. Figure 31 shows a modality of a dual pumping system, in which there is only one waste negative pressure tank that must move in coordination with the movement of any particular positive pressure source tank. This negative pressure reservoir can be operated by mechanical or thermodynamic means (for example a capillary action pump). For the capillary action pump, the "movement" of the pump is achieved by means of valves. Figure 32 shows how the pumping and valve scheme of Figure 29 can be extended, using positive pressure source deposits, to work with a cross arrangement of n rows of channels and 2n columns of channels (n channels, common columns plus n channels columns of washing). This requires an interconnector with 2n x 1 output valves. Similarly, the scheme of Figure 30, which employs a negative pressure waste tank, can be extended to work with an array that incorporates wash channels. Figure 33 depicts a modality of the microfluidic device wherein each column channel k has an associated parallel wash channel k *. Figure 34 depicts a modality of the microfluidic device wherein the fluid delivery system consists of a cross-over, non-intercepted arrangement of inlet and outlet channels with vertical connector channels, using multiple levels, wherein the two layers are in a plane above the well layer and each column channel k has an associated parallel wash channel k *. A-Final view; B-Top view; C-Side view. Figure 35 shows, in a case of fluid flow through well (j, k), an example of a method to wash a liquid or vapor segment of row channel j and inside the wash channel k *, where k * is immediately adjacent to the channel k column. Figure 36 depicts a diffusion control method using "normally closed" check valves, comprising a valve element and a valve seat, between each pair of adjacent tracks connecting the channels to the fluidic locations. Figure 37 depicts a modality in which an externally applied magnetic field gradient induces a restoring force on the valve elements and valve seat incorporating a magnetic material. Figure 38 shows a shape of a valve seat with a shape where diffusion out of the ball stops when the externally applied restoring force presses the ball to the left into a round opening in the valve seat. Figure 39 shows a modality allowing a simple magnetic field gradient applied to the complete microfluidic device to induce force on a set of ball valves of the inlet channels and a set of ball valves of the outlet channels. A-Valve open; B-Valve closed; C-Final view; D-Top view; E-Side view. Figure 40 shows one embodiment of the pumping and valve schemes of this invention as they would appear with all pumps and valves on board the cassette. Figure 41 shows one mode, where the pumps are on board and use electrically driven flow, where electric fields are limited to external regions of the well array matrix, and a minimum or no electric field gradient is applied across from any of the wells in which living cells are present.

Detailed Description of the Preferred Modalities All patents, patent applications, and references cited herein are incorporated by reference in their entirety. As used herein, the term "well" is defined as the volume space created by the coupling of a fluid location of the chamber with a cell attachment location on the substrate. The volume space can be created by a depression on the chamber corresponding to the fluidic location, by a depression on the substrate corresponding to the cellular attachment location, by a spacer support that separates the chamber and the substrate (where it does not exist requirement of a depression in the substrate of the chamber), a combination of any of these, any other suitable method to create a volume space between the fluid location and the cell attachment location. As used herein, the term "cell attachment location" refers to a discrete location on the substrate that comprises a plurality of cell binding sites capable of binding cells. The substrate can be derived to create cell binding sites, or the cell binding sites can be naturally present on the substrate. In the plurality of cell attachment sites within an individual cell binding site it may be able to bind only a single cell type, or it may be capable of binding more than one cell type. Different cell binding sites on the same substrate may be identical or different in the type of cells they are capable of binding. The term "fluid location" as used herein refers to a discrete location on the chamber that is a fluid supply site for and / or removal from the cell attachment site. The fluid location may comprise a depression, such as an engraved domain, a raised deposit, or any other type of depression. Alternatively, the fluid location may be flat, such as the term of an input and / or output channel, or the end of the path that is in fluid connection with an input and / or output channel. As used herein, the term "cassette" means the combination of the substrate and the camera. The combination can be modular (more than one piece), with the proviso that a reusable chamber and a disposable or non-modular substrate (single piece), limit any potential interpozo leakage. As used herein, the term "aboard" means integral with the cassette. As used herein, the term "integrated fluidics" means that a microfluidic device comprises a substrate surface for cell attachment and a chamber.

As used herein, the term "chamber" means a fluid delivery system comprising microfluidic channels and fluidic locations, which serves as a specialty substrate cover. As used herein, the term "exchangeable pump" refers to a pump, which includes but is not limited to a syringe pump, a pressure vessel controlled with a valve, or an electrokinetic pump that is used externally (with the optional use of electric shield) with respect to the well arrangement matrix so that no harmful electric field effects are experienced by the cells. As used herein, the term "sub-array" means any contiguous subsection of wells in the complete array of wells, typically in a format corresponding to the shape of an imaging detector array, such as a CCD ( detector device coupled to load). As used herein, the term "cell array matrix" means the space defined by the imaginary parallelepiped (ie, typically a cubic or rectangular box) that would fit around the complete set of wells in the cassette array. As used herein, the term "test components" refers to any component that would be added to a cell selection test, which includes but is not limited to reagents, cells, test compounds, medium, antibodies, and luminescent and reporters. As used herein, the term "luminescent" encompasses any type of light emission, including but not limited to luminescence, fluorescence, and chemiluminescence. In one aspect, the present invention teaches a method for making a microarray of multiple cellular type on a common substrate. As defined herein, a microarray of multiple cell types refers to an array of cells on a substrate that are not distributed in a single uniform coating on the support surface, but rather in a non-uniform manner such that each " "cell binding site" or groups of cell binding sites on the substrate may be unique in their cell-binding selectivity. The method for making a microarray of multiple cell types comprises preparing a micro-patterned chemical array (also referred to herein as a chemically modified array of cell binding sites), chemically modifying the array non-uniformly, and attaching cells to the non-uniform chemical array . In a preferred embodiment, a micro-modeling chemical array comprises a substrate 4 which is treated to produce a hydrophobic surface through which hydrophilic sites or "cell attachment locations" 8 are dispersed at regular intervals (FIG. 1A-1B ). The substrate can be a glass, plastic or silicon wafer, such as a conventional light microscope coverslip, but can also be made from any other suitable material to provide a substrate. As previously described, the term "cell binding sites" is used to describe a specific location on the substrate, and does not require any particular depth The surface of the substrate 4 is preferably approximately 2 cm by 3 cm, but may be larger or smaller In a preferred embodiment, the cell attachment locations 8 of the micro-patterned chemical array contain functional groups capable of reacting such as, but not limited to, amino, hydroxyl, sulfhydryl or carboxyl groups that can bind to cells not specifically or be further chemically modified to bind molecules that specifically bind cells Chemically modified cell binding sites are produced by specific chemical modifications of the cell binding sites on the substrate Cell attachment sites can comprise a variety of different binding molecules cell that allows the union and growth of different cell types at the cell binding sites, or they can allow the binding of only a single cell type. The hydrophobic domains surrounding the cell binding sites on the substrate do not support cell attachment and growth. In one embodiment, a multiple cell-type microarray is made by coating a glass wafer by quimia-absorbance with a layer of a substance having functional groups capable of reacting such as amino groups. In a preferred embodiment, an aminosilane such as 3-aminopropyltrimethoxysilane (APTS) or N- (2-aminoethyl-3-aminopropyl) trimethoxysilane (EDA) is used, but other substances capable of reacting can be used. After this first stage, due to the presence of functional groups capable of reacting, the entire surface of the coated glass wafer is hydrophilic. Secondly, a micro-modeling reaction is carried out where the droplets containing a substance having photo-folding or chemically-removable amino protecting groups are placed in a micromodel of discrete locations on the glass wafer coated with aminosilane. In one embodiment the model comprises a rectangular or square arrangement, but any discrete suitable pattern may be used, such as, but it is not limited to triangular or circular). In one embodiment, the drops vary in volume from 1 nanoliter (ni) to 1000 ni. In a preferred embodiment the drops vary from 250-500 or in volume. . 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 modeling reaction is carried out for 1 to 100 minutes at temperatures ranging from room temperature to 37 ° C, using reagent concentrations between 1 micromole (uM) and 1000 uM. In a preferred embodiment, the reaction is carried out at 37 ° C for 60 minutes using a reagent concentration of 500 μM. The droplets can be placed on the glass wafer coated with aminosilane by means of conventional inkjet technology. (US Patent No. 5,233,369; U.S. Patent No. 5,486,855). Alternatively, an array of pins, defined herein as tapered rods that can transfer between 1 and 1000 and of fluid, is immersed in a bath of the protective amino substance to produce drops of the protective substance at their ends. The pins are then contacted with the glass wafer to transfer the drops to that. In another embodiment, an array of capillary tubes made of glass or plastic, as described in U.S. Patent No. 5,567,294 and 5,527,673, (both incorporated herein by reference), which contains the protective amino substance is contacted with the glass wafer to transfer the droplets to the surface. Thus, the glass wafer is micromodelated with an arrangement of cell attachment sites or locations containing protected amino groups on a hydrophobic surface (Figure 2A-B). Third, a reactive hydrophobic substance with unprotected amino groups is washed on the glass wafer. The hydrophobic substance can be a fatty acid or an alkyl iodide, or any other suitable structure. Certain conditions for such shunting of glass can be found in Prime and Whitesides, Science 252: 1164-1167, 1991, Lopez et al., J. Am. Chem. Soc. 115: 5877-5878, 1993, and Mrksich and Whitesides, Ann. . Rev. Biophys. Biomol. Struct. 25: 55-78, 1996. The fatty acid or alkyl iodide reacts with unprotected amino groups and covalently bonds thereto and the amino groups are 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 an array of cell attachment sites 8 containing protected amino groups on a hydrophobic base (Figure 2C). Fourth, the non-uniform arrangement of cell binding sites occurs by uniformly deprotecting the amino groups in a micro-modeled chemical array produced according to the methods described above. In one embodiment, chemical specificity can be added by chemically crosslinking specific molecules to the cell binding sites. There are a number of well-known homo- or hetero-bifunctional cross-linking reagents such as ethylene glycol bis (succinimidylsuccinate) which will react with the free amino groups at the cell binding sites and crosslink to a specific molecule. Reagents and conditions for crosslinking free amino groups with other biomolecules are well known in the art, as exemplified by the following references: Grabarek and Gergely, Analyt. Biochem 185: 131-135, 1990; McKenzie et al., J. Prot. Chem. 7: 581-592, 1988; Brinkley, Bioconjugate Chem. 3: 12-13; 1992, Fritsch et al., Bioconjugate Chem. 7: 180-186, 1996; and Aplin and Hughes, 1981. In a preferred embodiment, a non-uniform arrangement of cell attachment sites occurs in combination form. The resulting cell binding sites are not uniform (i.e., each cell attachment site or group of cell attachment sites may be unique in their cell attachment selectivity). By the term "combination" is meant that the cell binding sites are treated variably. In one embodiment, the protected amino groups of the modified arrangement of cell attachment sites of step 3 are deprotected and then specific molecules with chemical crosslinking reagents are deposited in a desired pattern. The specific crosslinking agents can be attached to the amino groups and additionally possess a cell binding group. In this step, the type of cell binding group can be varied, from one cell attachment location to another, or from one group of cell attachment sites to another, to create a non-uniform array design. In another embodiment, the amino groups of the chemically modified cell attachment sites of step 3 are uniformly deprotected. A reticular photo activatable is reacted with the deprotected amino groups. An optical mask of a desired pattern is placed on the surface of the cell attachment locations and the exposed cell binding locations are illuminated with a light source. The position and number of cell attachment sites that receive light is controlled by the micromodel of the optical mask. Suitable photoactivatable crosslinkers include arylnitrenes, arylated fluorinated benzophenones and diazopirubatos. The reagents and conditions for optical masking and crosslinking are discussed in Prime and Whitesides, 1991; Sighvi et al., 1994, Sigal et al., 1996 and Mrksich and Whitesides, 1996. The photoactivatable crosslinker is bifunctional because it binds chemically to the amino group on cell attachment sites and, when exposed to light, covalently binds to cell binding molecules, such as antibodies. Reagents and conditions for the photoactivated crosslinking are discussed in Thevenin et al., Eur. J. Biochem. 206: 471-477, 1992 and Goldmacher et al., Bioconjugate Chem. 3: 104-107, 1992. When a photo-reactable crosslinker is used, the glass plate is flooded with cell binding molecules to be linked to the binding sites. cell phone. In one embodiment, cell binding molecules such as antibodies reactive to cell surface antigen, extracellular matrix proteins, (e.g., fibronectin or collagen) or charged polymers (e.g. poly-L-lysine or .poly-L-arginine) they are used in concentrations ranging from about 0.1 to about 1 mM. While the cell binding molecules cover the cell attachment locations, the glass plate is radiated from the underside of the glass plate, at an angle below the critical angle of the glass plate material that results in internal reflection total light. (For discussion of total internal reflection fluorescence microscopy, see Thompson et al., 1993). In one embodiment, the irradiation is carried out at between room temperature and 37 ° C for 0.1 to 10 seconds with light of wavelength between 300 nanometers (nm) at 1000 nm. In a preferred embodiment, the irradiation is conducted at room temperature for one second using light with a wavelength of between about 300 and 400 nm. Optical crosslinking limits the photoactable crosslinking at a short distance within the solution above the cell binding sites, and is described in Baley et al., Nature 366: 44-48, 1993; Farkas et al., Ann. Rev. Physiol. 55: 785-817, 1993; Taylor et al., Soc. Opt. Inst. Eng. 2678: 15-27, 1996; Thompson et al., In Manson, W.T. (ed.), The photo-reactive crosslinker binds cell binding molecules such as antibodies and matrix proteins only at the cell binding sites where the crosslinker was irradiated. For example, a single row of an array of locations of an array of cell attachment locations can be irradiated to produce a single row of cell binding sites with the cell binding molecules attached to the crosslinker. After a wash of the array to remove any unbound cell binding molecule, a second row of cell binding sites can be joined to a second cell binding molecule by subsequent flooding of the glass wafer with the second cell binding molecule while it radiates the second row and optically masks the other rows. The unbound cell binding molecules are removed by washing the array with PBS, or any other suitable regulator. In this form, multiple rows of cell binding sites or groups of cell binding sites can be sequentially illuminated by sequential masking in the presence of a particular cell binding molecule. Alternatively, each cell attachment site can be irradiated one by one using exposure and optical masking. In this form, different cell binding molecules bind to array rows or to individual cell binding sites, creating an uneven array of cells bound to the cell binding sites or any desired pattern. In a further embodiment for producing chemically modified arrays of cell binding sites, a chemically modified array is first produced wherein the amino groups of the cell attachment sites are uniformly protected with photo-blending protective groups. The rows, columns and / or individual cell binding sites are sequentially photo-protected to expose free amino groups by using an optical mask of various models to cover all, except cell binding sites to be unprotected. The exposed cell binding sites (ie, those not covered by the mask) are illuminated, resulting in removal of the protecting groups.The array is flooded with a bifunctional crosslinker which chemically binds to the unprotected amino group and activates the Cellular binding The conditions for the photo-protection of amino groups are discussed Padwa, A., (ed.) "Organic Photochemistry", New York 9: 225-323, 1987, Ten et al., Makromol. Chem. 190: 69- 82, 1989, Pillai, Synthesis 1980: 1-26, 1980, Self and Thompson, Nature Medicine 2: 817-820, 1996 and Senter et al., Photochem, Photobiol 42: 231-237, 1985. Then, the molecules Cell binding sites are flooded within the modified chemical array where they react with the other half of the crosslinker.The array is then washed to remove any unbifunctional bifunctional crosslinkers and cell-binding molecules.Another location of cell attachment or set of cell attachment sites can check out using another optical mask, and the array can then be flooded with a second treatment of a bifunctional crosslinker followed by a different cell binding molecule which binds to this second cell binding site or cell attachment location set of unprotected amino groups. The array is washed to eliminate the second treatment of a bifunctional crosslinker and cell binding molecules. A non-uniform arrangement of cell binding molecules can thus be produced by a repeated sequence of photo-deprotection, chemical cross-linking of specific molecules and washing under a variety of masks. Alternatively, the crosslinking reagents can be delivered to the unprotected cell binding sites together with the cell binding molecules in one step. The concentration gradients of the bound cell binding molecules can be created by controlling the number of exposed unprotected amino groups using an optical mask or by controlling the irradiation dose for the photo-activatable crosslinkers. The chemically modified arrangement of cell binding sites is then used to produce a non-uniform array of cells over the cell binding sites. In one embodiment, the modified chemical array is "seeded" with cells by introducing cells suspended in the array, allowing the cells to attach to the cell binding sites and then washing the wafer to remove weak and unbound bound cells. The cells bind only at the cell binding sites, due to the specific chemical environment at the cell binding sites, in conjunction with the hydrophobic environment that surrounds each of the cell binding sites, allows for selective binding of the cells to the cells. Cell binding locations only. In addition, modification of the cell binding sites with specific cell binding molecules allows selective binding of cells to specific cell binding sites, resulting in an uneven array of cells over the cell attachment sites. In addition, cell surface molecules that bind specifically to cell binding sites may be naturally present or genetically engineered by expressing "cell binding specific location" molecules that have been fused to cellular transmembrane molecules such that the cells interact. with and bind specifically to modified cell binding sites. The creation of an array of cell binding sites with different cell recognition molecules allows a cell binding site, a group of cell binding sites, or the entire array to "recognize", specifically, cell growth and selection from a mixed population of cells. In one embodiment, cells suspended in culture medium at concentrations ranging from about 10 3 to about 10 7 cells per ml are incubated in contact with cell attachment sites for 1 to 120 minutes at temperatures ranging from room temperature to 37 °. C. The unbound cells are washed after the cell binding sites using culture medium or a high density solution to lift the unbound cells from the bound cells. (Channavajjala, et al., J. Cell Sci. 110: 249-256, 1997). In a preferred embodiment, cells suspended in culture medium at concentrations ranging from about 10 5 to about 10 6 cells per ml are incubated in contact with cell attachment sites at 37 ° C for times ranging from about 10 minutes to about 2 minutes. hours. The density of cells bound to the cell binding sites is controlled by the cell density in the cell suspension, the time allowed for cell attachment to the chemically modified cell binding sites and / or the density of the cell binding molecules in the cells. cell binding locations. In one embodiment of the cell-binding procedure, 103-107 cells per ml are incubated at room temperature and 37 ° C for between 1 minute and 120 minutes, with cell binding sites containing between 0.1 and 100 nmol per cm 2 of cellular union. In a preferred embodiment, 105 and 106 cells per ml are incubated for 10 minutes to 2 hours at about 37 ° C, with cell binding sites containing about 10 to 100 nmoles per cm of cell binding molecules. In one embodiment, the cells can be chemically fixed to the cell binding sites as described in Bell et al., J. Histochem. Cytochem 35: 1375-1380, 1987; Poot et al., J. Histochem. Cytochem 44: 1363-1372, 1996; Johnson, J., Elect. Micros Tech. 2: 199138, 1985, and then used for screening at a later time with luminescently labeled molecules such as antibodies, nucleic acid hybridization probes or other ligands. In another embodiment, the cells can be modified with luminescent indicators of cell chemical or molecular properties, seeded on the chemically modified non-uniform array of cell binding sites and analyzed in the live state. Examples of such locators are provided in Giuilano et al., Ana. Rev. Biophys. Biomol. Struct. 24: 405-434, 1995; Harootunian et al., Mol. Biol. Cell 4: 993-1002, 1993; Post et al., .Mol. Biol. Cell 6: 1755-1768, 1995; González and Tsien, Biophys. J. 69: 1272-1280, 199; Swaminathan et al., Biophys. J. 72: 1900-1907, 1997 and Chalfie et al., Science 263: 802-805, 1994. The indicators can be introduced into the cells before or after they are sown on the array by some or a combination of diversity of physical methods, such as, but not limited to, diffusion across the cell membrane (reviewed in Haugland, Hadbook of fluorescent probes and research chemicals, 6th ed Molecular Probes, Inc., Eugene, 1996), mechanical perturbation of the cell membrane (Mceil et al., J. Cell Biology 98: 1556-1564, 1984; Clarke and McNeil, J. Cell Science 102: 533-541, 1992; Clarke et al, BioTechniques 17: 1118-1125, 1994), or engineering genetics so that they are expressed in cells under the prescribed conditions. (Chalfie et al., 1994). In a preferred embodiment, the cells contain luminescent reporter genes, although other types of reporter genes, including those encoding chemiluminescent proteins, are also suitable. Studies in living cells allow the analysis of the physiological state of the cell as reported by luminescence during its life cycle or when it comes into contact with a drug or other reactive substance.

In another aspect of the present invention, a non-uniform cellular array on the cell binding sites are provided, wherein the cells do not bind uniformly to a chemically modified array of cell binding sites on a substrate. The cell array is not uniform due to the underlying non-uniform chemically modified arrangement of cell binding sites providing a variety of cell binding sites of different specificity. Any cell type can be arranged with the proviso that a molecule capable of specifically binding that cell type is present in the chemically modified array of cell binding sites. Preferred cell types for non-uniform cell array over cell attachment sites include lymphocytes, cancer cells, neurons, fungi, bacteria and other prokaryotic and eukaryotic organisms. For example, Figure 3A shows a non-uniform cell array on the cell binding sites containing fibroblast cells grown on a patterned substrate surface and labeled with two fluorescent probes (rhodamine for staining actin and Hoechst for staining nuclei), while the Figure 3B shows a non-uniform cellular array on the cell binding sites containing fibroblastic cell growth (L929 and 3T3 cells) in blunt patterns, labeled with two fluorescent probes and visualized at different magnifications. Examples of cell binding molecules that can be used in the non-uniform cell array on cell binding sites include, but are not limited to antibodies, lectins, and extracellular matrix proteins. Alternatively, genetically engineered cells expressing specific cell surface markers can selectively bind directly to the modified cell binding sites. The non-uniform cellular arrangement on cell attachment sites may comprise fixed or living cells. In a preferred embodiment, the non-uniform cell array on the cell binding sites comprises living cells such as, but not limited to, "tagged" cells with luminescent indicators of chemical or molecular cellular properties. In another aspect of the present invention, there is provided a method for analyzing cells comprising preparing a non-uniform cell array on the cell binding sites wherein the cells contain at least one luminescent reporter molecule, contacting the non-uniform cell array on the cell binding sites with a fluid delivery system to allow reagent delivery to the cells, leading to the selection of high production by acquiring luminescence image from the complete non-uniform cell array at low magnification to detect luminescence signals from all cellular junction locations at the same time to identify those that present a response. This is followed by detection of high content within the corresponding cell binding sites using a set of luminescent reagents with different physiological and spectral properties, examining the selected cell binding sites to obtain luminescence signals from the luminescent reporter molecules in the cells, converting the luminescence signals into digital data and using the digital data to determine the distribution, environment or activity of the luminescent reporter molecules within the cells. Preferred embodiments of I non-uniform cell arrangement are described above on cell binding sites. In a preferred embodiment of the fluid delivery system, a chamber is coupled with the substrate containing the non-uniform cell array. The camera is preferably made of glass, plastic or silicon, but any other material that a camera can provide is adequate. One embodiment of the chamber 12 shown in Figure 4 has an array of labeled domains 13 that couple the cell attachment locations 8 onto the substrate 4. In addition, the input channels 14 are recorded to supply fluid to the recorded domains 13. A series of 16"output" channels for removing excess fluid from the recorded domains 13 can also be connected to the cell attachment locations. The camera 12 and the substrate 10 together constitute a cassette 18. While this embodiment uses recorded domains, any other type of depression 13 formed in the fluidic location 1 may also be used in this embodiment.

Alternatively,. the fluid location may be planar and the cell attachment locations 8 may comprise depressions that couple the fluidic locations. In another alternative, the cell attachment site 8 and the fluid location 1 are flat, and a volume space is created for the well by the use of a spacer support 20 between the substrate 4 and the chamber 12. The chamber 12 is used thus to supply fluid to the arranged cells on the cell binding sites. The fluid may include, but is not limited to, a solution of a particular drug, ligand protein or other substance that binds to expressed portions of cell surface or that are taken up by cells. The fluid for interacting with the arranged cells on the cell binding sites can also include liposomes that encapsulate a drug. In one embodiment, such a liposome is formed of a photochromic material, which releases the drug upon exposure to light, such as photoresponsive synthetic polymers. (Reviewed in Willner and Rabin, Chem. Int. Ed. Engl. 35: 367-385, 1996). The drug can be released from the liposomes in all channels 14 simultaneously, or individual channels, or separate rows of channels can be illuminated to release the drug sequentially. Such controlled release of the drug can be used in kinetic studies and studies of living cells. The control of the fluid supply can be achieved by a combination of microvalves and micropumps that are well known in the capillary action technique. (US Patent Nos. 5,567,294; US Patent No. 5,527,673; US Patent No. 5,585,069, all incorporated herein by reference). Another embodiment of the camera 12 shown in Figure 5 has an array of input channels 14 that couple the recorded domains 13 of the camera which are slightly larger in diameter than the cellular attachment locations 8 on the substrate 4, so that the cell binding sites are immersed within the recorded domains 13 of the chamber 12. The spacers 20 are placed between the chamber 12 and the cells arranged on the cell attachment locations 10 along the sides of the contact. The substrate 4 and the chamber 12 can be sealed together using an elastomer or other sticky coating on the relief region of the chamber. Each recorded domain 13 of the camera 12 can be filled individually or uniformly with a medium that supports the growth and / or health of the arranged cells on the cell binding sites. In a further embodiment (Figure 6), the chamber does not contain input channels, to treat all cells arranged on the cell attachment sites 10 with the same solution. The supply of drugs or other substances is achieved by the use of various modifications of the chamber as follows. A solution of the drug to be tested for interaction with array cells can be loaded from a 96-well microtitre plate into an array of microcapillary tubes. (Figure 7). The arrangement of microcapillary tubes 24 corresponds one by one to the inlet channels 14 with the chamber 12, allowing the solution to flow or be pumped out of the microcapillary tubes 24 into the channels 14. The cassette 18 is inverted so that the locations Cell binding sites are immersed in the embossed domain 13 filled with the fluid (Figure 7B). Once the interaction between the fluid and the cells occurs, the luminescent signals emanating from the arranged cells on the cell binding sites can be measured directly or alternately, the substrate 4 can be lifted out of the chamber for further processing, fixing and marking . Placing the removal of the cell array can be accomplished by means of robotics and / or hydraulic mechanisms. (Schroeder and Neagle, 1996). In a mode of the camera 12 shown in the Figure 7, the channels and domains 13 recorded coupled are recorded inside the chamber chemically (Prime and Whitesides, 1991; López et al., 1993; Mrksich and Whitesides, 1996). The labeled domains 13 are larger in diameter than the cellular binding sites 8. This allows the chamber 12 to be sealed by contact to the substrate 4 leaving room for the cells and a small volume of fluid. The input channels 14 are recorded within each row of the marked domains 13 of the camera 12. Each input channel 14 extends from two opposite ends of the camera 12 and opens at each end. The marked domains 13 of a single row are in fluid communication with the input channels 14 by placing a microcapillary tube 24 containing a solution in contact with the end of the chamber 12. Each row of connected input channels 14 can be filled simultaneously or sequentially During the filling of the input channels 14 by valves and capillary action pumps, each of the channels of the chamber 12 fills and the drug passes to fill each domain 13 recorded in the row of recorded domains 13 connected by channel 14 of entry. In an additional mode of chamber 12, deposits 28 of relief and inlet channels 14 can be placed on the surface of chamber 12 as shown in Figure 8b. In a preferred embodiment, relief tanks 28 and inlet channels 14 can be made of polytetrafluoroethylene or elastomeric material, but can be made of any other sticky material that allows attachment to substrate 4, such as poly (dimethylsiloxane), manufactured by Dow Corning under the trade name SYLGARD 184 ™. The effect is the same as with a camera that has recorded channels and channels and their uses are similar. In another embodiment of the chamber shown in Figure 8A, a first channel 30 extends from one end of the chamber 12 to a first recorded domain 13 or relief deposits 28 and channels. A second channel 32 extends from the opposite end to a second recorded domain adjacent the first recorded domain. The first 30 and second 32 channels are not in fluid communication with each other however they are in the same row of input channels 14 or relief tanks 28. In another embodiment, as shown in Figures 9 and 10, the camera 12 may have an inlet channel 14 extending from each engraved domain 13 or relief reservoir 28 to the end of the chamber. The channels 14 can all originate from one end of the chamber 12 (Figure 9), or from both ends (Figure 10). The input channels 14 can also be divided on both sides of the recorded domains 13 to minimize the space occupied by the input channels 14. Separate fluidic channels allow the operation of kinetic studies where one row at a time or one depression at a time is loaded with the drug. In an additional mode represented in the Figure 11, each recorded domain 13 is in fluid communication with a corresponding input channel 14 having a plug 36 between the end of the channel 14 and the recorded domain 13, which prevents the injected solution from flowing within the recorded domain 13 up to the time wanted. The solutions can be preloaded within the input channels 14 for use at a later time. A plug 36 can likewise be arranged between a terminal recorded domain 13 in a set of recorded domains 13 connected in fluid communication with an input channel 14. Upon releasing the plug 36, the substance flows through and carries all the recorded domains 13 which are in fluid communication with the input channel 14. In one embodiment, plugs 36 are formed of a hydrophobic polymer, such as, but not limited to, proteins, carbohydrates or lipids that have been crosslinked with photodegradable crosslinkers that, upon irradiation, become hydrophilic and pass along with the drug. inside the depression. Alternatively, plug 36 can be formed of a cross-linked polymer, such as proteins, carbohydrates or lipids that have been cross-linked with photo-double crosslinkers that, when irradiated, decompose and pass within the recorded domain 13 together with the solution. The cassette 18, which comprises the substrate 4 and the chamber 12 is inserted into a luminescence reading instrument. The luminescence reading instrument is a mechanical optical device that manages the cassette, controls the environment (for example, the temperature that is important for living cells), controls the supply of solutions to the wells, and analyzes the luminescence emitted from the array of cells, either one well at a time or the complete arrangement simultaneously. In a preferred embodiment (Figure 12), the luminescence reader instrument 44 comprises an integrated circuit inspection station that uses a fluorescence microscope as the reader and micro-robot to manipulate the cassettes. The reader of the present invention can comprise any optical system designed to image a luminescent specimen on a detector. A storage compartment 48 contains the cassettes 18, from where they are removed by a robotic arm 50 which is controlled by the computer 56. The robotic arm 50 inserts the cassette 18 into the luminescence reader instrument 44. The cassette 18 is removed from the luminescence reader instrument 44 by another robotic arm 52, which places the cassette 18 inside a second storage compartment 54. The instrument luminescence reader 44 is a mechanical optical device designed as a modification of the optical base, space is inspection of integrated circuits used to "select" chips "integrated circuits by default. The systems that integrate environmental control, micro-robotics and optical readers are produced by companies such as Cari Zeiss [Jena, GmbH]. In addition to facilitate the robotic handling, the supply of fluids, and the rapid and precise examination, two modes of reading, high content and high production are supported. The reading of high content is essentially the mass that the one made by the reader ArrayScan (North American Application S / N 08/810983). In the high content mode, images are formed at each location on the microarray of multiple cell types at increases of 5-40X or more, recording a sufficient number of fields to achieve the desired statistical resolution of the measurements. In the high production mode, the luminescence reader instrument 44 produces micro array images of multiple cell types at much lower magnifications of 0.2X to 1.0X magnification, providing decreased resolution, but allowing all cell attachment locations on the substrate are registered with a simple image. In one embodiment, a 20mm x 30mm microarray of multiple cell types formed from 0.5X magnification images would fill an array of 1000 X 1500 array of 10um pixels, yielding 20um / pixel resolution, insufficient to define intracellular luminescent distributions but sufficient for record an average response in a single well, and count the numbers of a particular cell subtype in a well. Since the typical integration times are of the order of seconds, the mode of production of reading technology, coupled with loading and automated handling, allows the selection of hundreds of compounds per minute. In one embodiment shown in Figure 13, the luminescence reader instrument comprises a mechanical optical design which is a right or inverted fluorescence microscope 44, which comprises a 64 x, y, z computer controlled stage, a rotating slide 68 computer controlled containing a low magnification objective 70 (eg 0.5 X) and one or more high magnification objectives 72, a 74 lamp white light source with excitation filter wheel 76, a dichroic filter system 78 with filters 80 of emission, and a detector 82 (e.g., charge coupled cooled device) For high production mode, the low magnification objective 70 moves to the location and one or more luminescent images of the entire cell array are recorded. Wells having some selected luminescent responses are further identified and analyzed by means of high content selection, where the slide 68 is rotated to select a higher magnification objective 72 and the 64 x, y, z stage is adjusted to centralize the well "selected" for selection of high cellular and subcellular content, as described in the North American Application S / N 08/810983. In an alternate mode, the luminescence reader instrument 44 may use a laser beam to be swept with a focal or standard lighting mode. The spectral selection is based on multiple laser lines or a group of separate laser diodes, such as those manufactured by Cari Zeiss (Jena, GmbH, Germany) or as discussed in Denk, et al. (Science 248: 73, 1990). Another mode of high throughput screening mode involves the use of a low resolution system consisting of an array (1 x 8, 1 x 12, etc.) of luminescence exciters and luminescence emission detectors that examine subsets of the wells on a non-uniform micro-patterned cell array. In a preferred embodiment, this system consists of cluster optical fibers, but any system that directs luminescence excitation light and collects luminescence emission light from the same well will suffice. By examining the complete microarray of multiple cell types with this system, it produces the total luminescence of each well, of the cells and the solution in which they bathe. This mode allows the collection of luminescence signals from cell-free systems, so-called "homogeneous" tests. Figure 14A shows a method, in the form of a flow chart, for analyzing a microarray of multiple cell types in the high production and high content modes using the luminescence reader instrument, which first uses the high production detection for measure a response from the complete "A" array. (Figure 14B). Any well that responds above a preset threshold is considered a success and the cells in that well are measured by high-content selection. (Figure 14C). The high content mode ("B") may or may not measure the same cell parameter measured during the high production mode ("A"). In another aspect of the invention, a cell selection system is described, wherein the term "selection system" comprises the integration of a luminescence reader instrument, a cassette that can be inserted into the luminescent reader instrument comprising a microarray of multiple cell types wherein the cells contain at least one luminescent reporter molecule and a camera associated with the non-uniform micro-patterned arrangement of cells, a digital detector for receiving data from the luminescence reader instrument, and a computerized means for receiving and processing digital data from the digital detector. Preferred embodiments of the luminescence reader instrument, and the cassette comprise the microarray of multiple cell types and the camera are described in the foregoing. A preferred embodiment of the digital detector is described in the North American Patent Application No. 08/810983, and comprises a high resolution digital camera that acquires luminescence data from the luminescence reader instrument and converts it to digital data. In a preferred embodiment, the computer means comprise a digital cable that transports the digital signals from the digital detector to the computer, a display for the use of interaction and display of the test results, a means for processing test results, and a digital storage means for data storage and archiving, as described in the North American Application S / N 08/810983. In a preferred embodiment, the cell screening system of the present invention comprises the integration of the preferred embodiments of the elements described in the foregoing (Figure 15). The arrangement of the multiple cell types 10 comprises cells bound to chemically modified cell attachment locations 8 on a substrate 4. The chamber 12 serves as a microfluidic delivery system by the addition of compounds to the cells on the substrate 4, and the combination of two comprises the cassette 18. The cassette 18 is placed in a luminescence reader instrument. The digital data is processed as described in the above and in the North American Application S / N 08/810983, incorporated by reference in its entirety. The data may be displayed on a computer screen 86 and made part of a bioinformatics database 90, as described in the North American Application S / N 08/810983. This database 90 allows the storage and retrieval of data obtained through the methods of the invention, and also allows the acquisition and storage of data related to previous experiments with the cell. An example of the computer display screen is shown in Figure 16. Example 1. Coupling of antibodies to microarray of multiple cell types for the binding of specific lymphoid cells 1. The cell line used was a line (A20) lymphoma of mouse B cell that does not express IgM on its surface. A microarray of multiple cell types is prepared by derivation being submerged overnight in 20% sulfuric acid, washed 2-3 times in distilled water in excess, rinsed in 0.1M sodium hydroxide and dried with blotting paper. The microarray of multiple cell types are either used immediately or placed in a glass beaker and converted with parafilm for future use. 2. The microarray of multiple cell types is placed in a 60 mm petri dish, and 3-Aminopropyltrimethoxysilane is layered in the microarray of multiple cell types to ensure full coverage without running over the edges (approximately 0.2 ml during a non-uniform micromolded arrangement 22 x 22 mm of cells, and approximately 0.5 ml during a non-uniform micromolding arrangement 22 x 40 mm of cells). After 4 minutes at room temperature, the microarray of multiple cell types is washed in deionized water and excess water is removed by paper drying. 3. The microarray of multiple cell types is placed in a clean 60 mm petri dish and incubated with 2.5% glutaraldehyde in PBS, approximately 2.5 ml) for 30 minutes at room temperature, followed by three PBS dishes. Excessive PBS is removed by paper drying. 4. The cell nucleus in the microarray of multiple cell types is labeled with a luminescent Hoechst drying during the blocking step. The appropriate number of lymphoid cells (see below) in C-DMEM is transferred to a 15 ml conical tube, and Hoechst drying is added to a final concentration of 10 μg / ml. The cells were incubated for 10-20 minutes at 37 ° C in 5% CO2, and then compressed by centrifugation at 1000 x g for 7 minutes at room temperature. The supernatant containing unbound Hoechst drying is removed and the drying medium (C-DMEM) is added to the resuspension of the cells approximately 1.25-1.5 x 105 in 0.2 ml by 22 x 22 mm of non-uniform micromolded array of cells, and approximately 2.5 x 105 cells in 0.75 ml for the non-uniform micromolished array 22 x 40 mm cells. 5. The microarray of multiple cell types was briefly washed in PBS and transferred to a clean, dry 60 mm petri dish, without touching the sides of the dish. The cells were carefully incubated on top of the microarray of multiple cell types at the density observed in the above. Plates were incubated at 37 ° C in 5% CO2 for 1 hour. Unbound cells were then removed by repeated PBS washes. 6. The antibody solution (Goat Anti-mouse IgG serum or Goat Anti-mouse Total) was stained on a parafilicle (50 μl per non-uniform micromolished array of 22 x 22 mm cells, 100 μl per a micromolished array not uniform 22 x 40 mm cells). The microarray of multiple cell types was inverted in the spots, so that the antiserum covered the entire surface of the microarray treated with multiple cell types without catching air bubbles. The microarray of multiple cell types was incubated with the antibody solution for 1 hour at room temperature. 7. The microarray of multiple cell types was carefully lifted from the parafilm, placed in the clean 60 mm petri dish, and washed three times with PBS. The unreacted sites were then blocked by the addition of 2.5 ml of 10% serum (fetal calf or calf serum in DMEM or Hank's Balanced Salt Solution) for 1 hour at room temperature. 8. Both of the cell lines must bind to the total anti-mouse serum, but only the XI6s must bind to the anti-mouse IgM. The binding of the specific lymphoid cell strains to the chemically modified surface is shown in Figure 17. The mouse lymphoid A20 cell line, surface IgM molecules but unfold IgG molecules, bound much more strongly to the modified surface with the serum goat anti-mouse (Figure 17C) that to the surface modified with the goat anti-mouse IgM (Figure 17B) or an uncoated slider (Figure 17A). Example 2. High Content Screen and Elevated Performance Insulin-dependent stimulation of glucose uptake into cells such as adipocytes and myocytes requires a complex instrumentation of cytoplasmic processes that result in the translocation of GLUT4 glucose transporters from an intracellular compartment in the plasma membrane. A number of molecular events are driven by insulin bound to its receptor, including direct signal transduction events and indirect processes such as the skeletal rearrangements required for the translocation process. Because the actin-cytoskeleton plays an important role in the cytoplasmic organization, the intracellular signaling ions and the molecules that regulate this living gel can also be considered as GLUT4 translocation intermediates. Some two screen levels for insulin mimics are implemented as follows. Cells carrying a stable GLUT4 chimera with a Blue Fluorescent Protein (BFP) are arranged in the microarray of arrays of multiple cell types and then loaded with acetoxymethylester from Fluo-3, a calcium indicator (fluorescent green) . The arrangement of the locations is then simultaneously treated with an array of compounds using the microfluidic delivery system, and a certain sequence of Fluo-3 images of the complete microarray of multiple cell types are analyzed by wells that show a response to calcium in the High-throughput mode, the wells contain compounds that include a response, then are analyzed on a cell per cell basis by evidence of GLUT4 translocation in the plasma membrane (ie the high-content mode) using blue fluorescence detected in the time and space. Figure 18 delineates the sequential images of the total microarray of multiple cell types in the high throughput mode (Figure 18A) and the high content mode (Figure 18B). Figure 19 shows the cell data from the high content mode.

Improved Cassettes and Increased Density In another aspect, the present invention provides devices and methods for maximizing the plated cell area and the number of wells that can be imagined in a sub-array, while still obtaining adequate pixel resolution in the image. This result has been achieved through the use of fluidic architectures that minimize the distance and area between the wells and thus maximize well density. In one embodiment of this aspect there is provided a cell screening cassette comprising a substrate having a surface, wherein the surface contains a plurality of linked cell locations, a fluid supply system for supplying reagents to the plurality of locations cell binding wherein the fluid delivery system comprises a multi-level chamber that joins the substrate, wherein the multiple level chamber comprises i. a cross-arrangement of microfluidic inlet channels and outlet channels, wherein each of the wells is in fluid connection with one or more inlet channels and one or more outflow channels; ii. a plurality of fluidic locations in the fluid connection with the microfluidic inlet channels and outlet channels; iii. one or more multiple inlet tubes in the fluid connection with the microfluidic inlet channels; iv. one or more multiple outlet tubes in the connection of the fluid with the microfluidic outlet channels; v. at least one source receptacle in the fluid connection with one or more multiple inlet tubes; and I saw. at least one wear receptacle in the fluid connection with one or more multiple outlet tubes; and a plurality of wells, wherein an individual well comprises a space defined by the junction of a cell attachment location and a fluid location. In preferred embodiments, the cassettes further comprise a pump for controlling the flow of fluid within the microfluidic device; a substrate temperature controller and / or a controller for regulating oxygen and partial pressures of carbon dioxide within the device. The substrate of the present invention is chemically modified so that the cell selectively adheres to a cell growth substrate and is contained within the small regions, referred to as "cell binding sites," which are sub-millimeters in a few millimeters in size. The combination of the cell attachment location on the substrate and a fluid location on the chamber defines a space referred to as a "well". The substrate may be predominantly flat and the cell attachment locations may be joined with depressions of fluid location in a substrate cover, such that when the two parts are assembled there is some depth in the area of the wells thus formed. Conversely, the fluidic location of the substrate cover may be planar and the substrate may contain depressions at the cellular junction location, so that when the two parts are assembled there is some depth in the belt of the wells thus formed, the fluids and Test compounds are supplied in the wells by means of a chamber, which combines with the substrate to form a cassette. In a preferred embodiment, the chamber acts as the substrate cover and contains fluidic locations in fluid connection with the microfluidic device channels, wherein the fluidic locations comprise depressions that join the cell attachment locations on the substrate, such that the volume from each well is recorded in the chamber. On the surface of the substrate, there are seal regions that lie between and around the cell attachment locations, and corresponding seal regions exist on the underside of the chamber that lies between and around the fluidic locations. When the substrate and chamber are joined, the seal regions or both components join to form a seal that prevents fluid flow through the seal regions between the consequently formed wells. These seal regions of the components can be physically or chemically modified to increase the seal. In a first example, a hydrophobic silane coated with octadecyltrichlorosilane is covered over the seal regions. In a second example, light curable adhesives and cyanoacrylates that are Class IV USP ductile as available from Dymax Corporation are sensitive to use with biomedical devices made of ceramic, glass, plastic, or metal. In a third example, a biocompatible tape material with precision switches joining the wells of the array available from the Avery Dennison Specialty Tape Division. Similarly, biomedical acrylic adhesives can be transferred from tapes to surfaces, such as are available from Tyco Corporation, by applying an adhesive cover exactly in those seal regions. In a fourth example, a light curable silicone elastomer can be used to join and seal between the sealing regions of the substrate and the chamber, such as are available from Master Bond, Inc. The microfluidic cassette of the present invention has advantages. on the prior art of microfluidic devices of cell array in high-content screening systems, including but not limited to: 1) the fastest resemblance of a statically relevant set of cells (based on resemblance of a set of binding locations) cellular (ie "sub-array") simultaneously, see Figure .20) 2) more wells per dish and less physical space occupied per dish (ie, higher well density), 3) more efficient use of the compounds available tests, and 4) more efficient use of cells compared to microplates in which the complete well area is not similar. As the fluid exchange of previous microwell plate, discussed for each well can only be achieved by means of a pipette being inserted in the pzo and either exert or aspirate the fluid in or from the well, respectively. The automated fluid handled in the anterior microwell plates is achieved using robotic pipettes that insert a pipette into each well for each of the transfer steps in a procedure. This pipette is typically also required to move into or from another microplate well for the source or destination of fluid that is exchanged with the microwell plate of interest. Integrated fluidics are advantageous for arrays with internal pit distances in sub-millimeters due to their difficult, if not impossible, handling in 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%. In this manner for a volume (μl) of microliter 100 in the well of a 96-well plate, or acceptable measurement error of 2 μl. For a volume (μl) of 100 nanoliters, a measurement error of 2 must not be required. This level of accuracy is not reliably available with previous automatic pipettes through a spectrum of compositions and viscosities. The minimization of the internal well distances allows the rapid parallel reading of well sub-arrangements. If the integrated fluidics are also bulky (ie: does not allow narrow spreading of wells), when such rapid parallel reading ability is lost. In a preferred embodiment of the present invention, a traversed channel architecture is used, allowing for few channels, valves, and pumps, and therefore further reducing the space taken by the channels in the cassette of the invention. In a more preferred embodiment, the channels are placed at levels higher than the wells, allowing the narrowest possible inner well spreading. In a further preferred embodiment, the use of the porous medium as a drainage "pump" allows for the simple design and manufacture of the system. According to the invention, the channels can be of any size that allows the fluidic architecture in the same defined. In a preferred embodiment, ranges in size from about 0.025 mm to about 0.5 mm in width per square cross section channels, or in diameter per channels of circular cross section. The fluidic architecture of the instant microfluidic cassette allows the control of elements (including, but not limited to pumps, multiple tubes, pressure controlled vessels, and / or valves) to be located outside the cell array matrix, Figure 40 shows a pump mode and dosage schemes of this invention as it will appear with all pumps and valves on the cassette board. All the variants of the pumping and regulation schemes shown in several Figures (and others that will be apparent to those skilled in the art) can be implemented in this way, either with active board devices turned on outside the well array, with off-board reactive devices or with some dash-board devices and some dashboard devices turned off. As a result, the controlled elements can be inside the cassette (board on) or external to the cassette (board off). In any case, they are non-active components within the array of well arrangements that may limit the closed space of the well channels. Because this cassette is particularly designated for the cultivation and analysis of living cells, the fluidic architecture and all its sub-components and functional parts are compatible with, support, and allow the cultivation of living cells. The particular aspects of these sub-components and the functional parts that are designated for live cell culture are identified in the following. In a preferred embodiment, the cassette of the present invention comprises an off-board pump to provide pressure propulsion flow which also controls whose wells are directed by the flow. This design feature ensures that the pumping method, different from many prior art methods, is not ineffective or harmful when used with cell culture medium that is aqueous, polar and contains proteins and salts. For example, electrohydrodynamic pumping is ineffective with polar solvents (Marc Madou, Fundamentals of Microfabrication, CRC Press, Boca Raton, 1997, p 433). Electro-osmosis is typically accompanied by some degree of osmosis typically requiring the use of electric field assist in the range of 100 V / cm at 1000 V / cm, which can affect the physiology of living cells in the device. The control system of the present device does not suffer from the disadvantages of electrically induced methods when using biological fluids. In another embodiment, the pump is on the live board and can use electrically fluidized flow, but the electric fields are limited to external regions in the array of well arrays, and not to electric field gradients that are applicable across any of the wells in which living cells are present. This embodiment is shown in Figure 41, where two electrodes are used to apply electrical potential differences along the segments of fluidic channels corresponding to different source boards (730). Each channel segment with its electrical pair forms an electrokinetic pump (740). Said source deposits (730) can be supplied with means and / or compounds by means of ports (700) carried on the cassette surface. The valves (720) inside the tanks and pumps are closed at any time that the corresponding electric pump is on, to maintain the back pressure and allow the entry of air or fluid, respectively. The voltage control applied to electrode 1 (760) is used to induce electrokinetic flow for fluid flow in the corresponding channel segment. Electrode 2 (780) (closest to the well array) is kept in ground potential as is the wells matrix, to ensure that the minimum or no potential difference and / or the electric field is developed in the vicinity of cells, because electric fields are known to affect cell physiology. Alternatively, the electrokinetic pump can be located downstream of the array to serve as a negative pressure pump. In this mode, the electrophoresis of the medium by the pumping action, if any, should only affect the medium after this has been used by the cells and its mode is extended to the wear deposit. In all modes, the use of off-board or off-board pumps that are external to the well matrix controls the fluid flow that eliminates the need for active valves within the well array, and thus eliminates the problem of the space allocated within the matrix that will prevent the closed spacing of the wells. The control of the fluid path by means of pressure control also allows the optimal use of various diffusion control means which are compatible and effective with the cell culture medium which is aqueous, polar and contains proteins and salts. These means also require minimal space on the device, and thus do not prevent the closed spacing of the wells. U.S. Patent No. 5,603,351 describes a microfluidic device that includes transverse channels, but the channel network is not defined to allow two or more reagents to be combined in a reaction well, but better allows the reagents to be fed into a well in a serial form. The purpose of this cassette is not to expose two or more different fluids to each other, but exposes the living cells cultured in the lower parts of the wells serially to two or more different fluids. The fluidic channels of the cassette of the present invention provide fluid transport and compounds of source receptacle compounds in each of the wells and from each well to one or more wear receptacles, each well can be provided with a separate inlet channel and output (Figure 21), giving channels 2n2 (100, 120) for an array (040) nx n. To control the flow of liquid m or vapor mixtures in an array n x n, valves m + n2 are required (Figure 22). However, for a long arrangement, the number of channels, pumps and valves for this type of architecture becomes unmanageable. A pump that has variable or changeable pressure (for example, a syringe pump) is functionally equivalent to the pressurized reservoir with a valve. Therefore, in the following discussions of pumps, tanks, and valves, it should be understood that these components can be replaced without altering the spirit of the invention.

Multi-level architecture of transverse channel A preferred embodiment of cassette architectures consisting of a non-intersecting, closed array of input (100) and output (120) channels and multiple levels of utilization are described. Specific modalities are given within this class of architectures in Figures 23-26 and in the Figures. 33 and 34. Because architectures in this class require few independent channels per well compared in the class of architectures with separate inlet and outlet channels for each well, these adverse channel architectures also minimize the lateral space (140) that is required between the wells to accommodate the trajectories of the channels. The transverse channel, the multi-level architectures require only 2n channels to direct n2 wells. In a preferred embodiment, the row and column channels by themselves are formed in two distinct layers so that they are not intersected. The row channels lie around the column channels or vice versa. These two layers either in the same plane of the well layer (Figures 23 and 24) or lie in the plane around the valve layer (Figures 25-26 and 33-34). In other modalities, structures with more than two planes are contemplated. In a more preferred embodiment, the channels are placed around the well plane, so that the internal minimum well distance (140) is constrained only by the optical and physical requirements for a certain wall thickness that separates each well. The advantage of placing the channels and levels around the wells is shown by the following examples. Wells that are 0.4 mm x 0.4 mm can be placed over a 0.5 mm pigeonhole with allowance for 0.1 mm well between the wells, producing an area of 5 mm x 5 mm during a 10 x 10 well arrangement. row and column of 0.2 mm wide can easily be formed at higher levels of the device with wide distance between the channels. Conversely, if the 0.2 mm channels with 0.1 mm wall are required within the well plane (and the row and column channel layers are formed in two separate planes within the well plane), the Well increases to 0.8 mm. These yields of an area of 8 mm x 8 mm for a 10 x 10 well arrangement. In this example, the placement of the channels between the wells increases the area required by the arrangement by a factor of 2.6. For different channel architectures with separate inlet and outlet channels for each well, the additional space required between the wells is much greater. Either the channels around or within the level of the wells, horizontal (160) or vertical (180) that connect the channels or pathways are formed from each well (j, k) in their channel j of corresponding row and channel k of column. The manufacture of such a structure of glass, semiconductor, or plastic materials is well known in the art. In such a method, silicon dioxide (SiO2) is lithographically formed within the layers of silicon nitride (Si3N4) using a multistep layer-by-layer process (Turney ahd Craighead, Proc. SPIE 3528: 114-117 ( 1998)). Exposed the structure in an etching to the humid water that eliminates the SiO2 to create the channel network inside the Si3N4 that is not recorded to the water.

Flow control in the transverse channel architecture In a transverse array of rows (A, B, C, etc.) and columns (1, 2, 3, etc.) the fluid flow in and out of well j, k) is produces when, for example, the positive pressure is applied to the row j and the outlet channel valve k is open, where j and k refer to, for example, row D and column 7, respectively. The flow of all the wells of a complete row j occurs when, for example, the positive pressure is applied to the row j and the valves of all the exit channels (1, 2, 3, etc.) are opened. A similar procedure should produce flow in all wells of the column. The pressures discussed in the present are currently different pressures relative to ambient atmospheric pressure, otherwise known as "caliber" pressure. In this way, the positive pressure valve is the amount by which an applied pressure is greater than the atmospheric pressure; The negative pressure valve is the amount by which a pressure is applied at less than atmospheric pressure. These pressures refer to a positive and negative gauge pressure, respectively. The term "fluid path" is used herein to indicate the particular set of wells. { (j, k), (j, n), (m, k), (m, n) ... etc ..}. which is subjected to the flow of fluid by the ease of the flow control devices and the rows j and m, etc. and columns k and n, etc. Note that allowing the flow of two-row and two-column control devices allows a fluid path through four wells. This is defined herein as a simple fluid path involving four wells, rather than four paths involving simple walls.

Reduction in the number of active valves or pumps Several combinations of actively controlled flow control devices (pumps or pressurized tanks with valves) are used to produce flow through a desired well (010) or fluid path. For example, the n compounds can be multiplexed in a single well using positive pressure source reservoirs or pumps (200), a smaller manifold manifold (240) and valve free wear reservoir at atmospheric pressure (300) (Figure 27). This multiple tube (240) of less valve can be placed by a regulated manifold tube [e.g., (260) of Figure 28] to better control of fluid diffusion between sources m. Alternatively, the same functionality can be achieved using the negative pressure wear tank or pump (320) with valves m (260) connected to source tanks m at atmospheric pressure (220) (Fig. 28). The use of source deposits in atmospheric pressure allows convenient bubbling of gases through the source reservoirs to establish and maintain the desired levels of C02 and 02 dissolved in the medium. In another embodiment, a wear deposit filled within a porous medium serves as a capillary action pump (340) and provides negative pressure (Fig. 28). Moisture thermodynamics create a pressure through a liquid-vapor interface defined by a solid-liquid-vapor contact line. This capillary action pressure can be used by tracing a liquid column from any microchannel path in the porous medium of high surface area. A series of such negative pressure vessels can be attached to the arrangement by means of a multiplexed multiplexed tube for flow control, or a single reservoir can be coupled closely to the output channels of the flow control, or a single reservoir it can be coupled closely to the output channels of all the wells simultaneously. A number of porous media are known in the art, including but not limited to a silica gel terminology, porous ceramic materials such as zeolite, a pad of fibers (natural synthetic) and partially hydrophilic hydrated and lyophilized as alginates, alcohol gels polyvinyl, agarose, polyacrylate gels, sugar polyacrylate gels, and polyacrylamide gels. In a preferred embodiment, the porous medium is based on the silica gel technology. A thin membrane or filtration sheet can be used to divide the porous medium of the microchannel array. This method can provide pressure for pumping until the internal surface area of the porous medium is widely wetted by the liquid. By proper selection of the porous medium and the design of this method, suitably the large amounts of the liquid can be pumped to control a selected measurement with the arrangement system of the present invention. In another embodiment, the arrangement of the positive pressure source reservoirs or pumps (200) shown in Figure 27 is multiplexed into a transverse channel arrangement nxn of wells (040) by means of inputs (400) 1 x and multiple tubes of outlet valve (410) nx 1 (Figure 29). Report that for the architectures for the input and output channels for each well (Fig. 22), n + n2 valves or pumps are required in this situation. Again, the smaller manifold manifold 240 can be replaced by a regulated manifold tube [e.g. (260) of FIG. 28] for better fluid diffusion control between sources m. The flow through the well (j, k) is affected when the inlet valve j and the outlet valve k are open. Alternatively, the same functionality can be achieved with deposits m at atmospheric pressure (220) and a negative pressure wear deposit (either 320 or 340) as in Figure 28 by means of multiple inlet pipes (400) 1 xny outlet valve (410) nx 1 (Fig. 30).

Rinsing Channels In a preferred embodiment, a microwell array n x n is increased by a channel k * of additional column or set of column channels. { k *} which allows the passage of a segment of liquid or vapor from a given row j to a wear deposit without going through the wells. { (j, k)} or the column channels. { k *} . These additional column channels. { k *} , refer to rinsing channels (130), because they allow the rinsing of the row channels without requiring flow through the wells. In general, a rinse channel k * (130) can be located adjacent and parallel to a channel of column k (016) but be connected via a channel to row channel j (014) at the point they are transverse [and they are not directly attached to the corresponding well (j, k)]. In one embodiment, each column of channel k has an associated parallel rinse channel k * (Figures 33 and 34). In other embodiments, only one rinse channel provides rinsing to the entire set of row channels. { j} , or there is any combination of rinsing channels. { k *} interposed within the arrangement of column channels. { k} . When there is only one rinsing channel, it may be preferred to have it located adjacent to the last column channel of the array, where k = n. Figure 35 shows, in the case of fluid flow through the well (j, k), an example of a method of rinsing a segment of liquid or vapor from the channel j of row and in the channel k * of rinse, where k * is immediately adjacent to the column channel k. This procedure and architecture allow this segment of fluid to be directed into the wear deposit without passing over the wells. { j, k + 1), (j, k + 2), etc. ..}. that are downstream along the row channel j. This is desirable to limit the diffusion of a given compound from one well (j, k) to other wells downstream. { j, k + 1), (j, k + 2), etc. ..}. where the compound can not be required. Additionally, as described in the following, diffusion can be controlled by introducing steam segments in the micro-channel microwell array.

Figure 32 shows how the pumping and regulated scheme of Figure 29, using positive pressure source reservoirs, can be extended to work with a transverse arrangement of n row channels and 2n column channels (and the ordinary column channels) n plus the rinse column channels n). This requires a multiple outlet valve manifold (420) 2n x 1. In general, for each additional flush channel k * that is added, an additional valve is added to the manifold valve tube connected to the column channels. Similarly, in the note of Figure 30, which employs a pressure wear tank, it can be extended to work with the array incorporating the rinse channels (130).

Dual pumping designs Flow through the trajectory. { j, k), (j, n), (m, k), (m, n) ....}. or more generally through any length of pipe is caused by a continuous pressure gradient that exists along the length of the path or pipe. The control of this pressure gradient by means of pressure applied to the inlets and outlets of the fluid trajectories requires that there is no leakage of flow through the wells of the trajectories. The leakage also requires the proper dosing of the compounds from the wells for drug screening. The seal of the trajectory wells must be suitable for the gauge pressures that are similarly applied. For fluid flow ratios of approximately a few millimeters per minute, path lengths no greater than a few centimeters, and channel transverse section in the order of 100 mm x 100 mm of the Hangen-Poiseuille law predicts that the The required extreme to extreme pressure of? P is less than one atmosphere (1 atm. «14 pounds per square inch). However, considering that various materials and structures known in the art can be implemented by the device of the invention, the possibility of leakage between neighboring wells is minimized by reducing the caliber pressure exerted through the wells of the system. Towards this end, the present device can use positive pressure source reservoirs with a negative pressure wear reservoir. (That is, a "double pumping" system) to essentially cut at half the maximum gauge pressures that must be applied to achieve a given total end-to-end pressure differential. For a "simple pumped" system, that is, a system that is open to atmospheric pressure at one end and that is pumped at the other end (either at a positive or negative gauge pressure, + AP), the difference pressure exerted through the walls of the system is a maximum at the location of the pumped container (i.e., (200) of Figures 27 and 29; or (320, 340) of Figures 28 and 30. The leak is more likely to occur at these maximum gauge pressure points, with positive pressure having to expel fluid out of the path and the negative pressure tends to pull fluid in the trajectory from a neighboring well or channel. (This assumes that neighboring channels and wells are maintained in zero-gauge pressure when they are not subject to flow.) Another method for controlling pressure differentials through the system wells is to apply pressures attached to the neighboring paths while they are not low flow). Intermediate points along the fluid path, the differential pressure across the walls of the system has valves between + AP and zero for positive and negative pumped systems, respectively. The same end-to-end pressure differential AP is exerted through the trajectory by stabilization pressures + AP / 2 in the inlet and outlet tanks, respectively, in this case, the maximum gauge pressures exerted through the wells of the system are + AP / 2 and occurs at the corresponding endpoints of the system. However, the gauge pressure applied within the device is closed to zero if the wells are at approximately midpoints between the inlet and outlet tanks along the length of the path. At the extreme points of the trajectory, the + AP / 2 maximum pressure differences are easily maintained without leakage within the plastic pipe and silica capillaries. The detection of the leak is facilitated because it occurs similarly only at the extreme points of the system. In one embodiment, the two syringes of a dual pumping system are of the same diameter and volume, and because they are attached to the same actuator in the thrust configuration, each syringe moves at the same linear distance and the identical volumes are exchanged. for each increase in movement. As any particular source compound is pushed into the arrangement, an equal volume of wear fluid is pushed. In this way, the syringe arrangement of source m also contains an arrangement of negative pressure wear deposits m. This arrangement of negative pressure wear deposits is multiplexed into the column channels in a smaller manifold tube 1 x m, together as the array of source reservoirs is multiplexed in the row columns. - In another embodiment, the source and waste syringes are of different diameter, and the syringes must be controlled by two separate actuators that drive each syringe in a different linear relationship if it does not produce the same volume changes in each syringe. For example, a small diameter syringe should be used for a compound, while a long diameter syringe should be used as the waste receptacle. If the ratio of diameter is a factor of 2, as the actuator of the small syringe moving a linear unit expels the fluid, the actuator of the long syringe moves only a quarter of the linear unit to the extracted fluid, except that the volumes exchanged They are identical. Figure 31 shows a particular case of this embodiment of a "dual pump" system, in which there is only one waste syringe pump that must be operated in a coordinated manner (for example, by means of computer control) with the actuator of any syringe pump from a particular source. Alternatively, the negative pressure pump can be a capillary pump, also shown in Figure 1. By the capillary action pump, the "activation" of the pump is achieved by means of the valves. In summary, the dual pumping system has the following advantages: 1) reduction in the maximum gauge pressure applied, 2) the gauge pressure applied is smaller near the midpoint of the trajectory, and 3) the leak is already detected and it must similarly occur at the extreme points of the system.

Multiplexing of the highest level The use of multiple multiple port flow tubes between the pumped tanks and the transverse channel arrangement allows the pumps (200) or the tanks (220) in these different modalities, with the liquid mixtures or steam different m for selectively and subsequently producing flow in each well (j, k) of the well arrangement (002) nx n.

More generally, a set of wells define a simple fluid path. { (j, k), (j, n), (m, k), (m, n) ... etc ..}. It can be defined by this system. The flow of only one liquid or vapor mixture can occur through only one liquid or vapor mixture can occur through only one fluid path at one time. The multiplexing of the high level, the simultaneous allowed flow of different liquid or vapor mixtures through different trajectories, is possible with a more complex system of multiple flow tubes. Such a system is easily constructed as a generalization of the present design.

Diffusion control The principle of this mode is that the valve element (520) and valve seal (540) are formed to allow flow in one direction and to suppress diffusion when the flow is stopped. In addition, the forces recovered in all the valves are applied "passively" and constantly. No detailed control system for the check valves is needed, since the control of the fluidic control gradients are the means by which the individual valves are opened. The use of transverse miRNAs with a long number of open paths between, for example, 2n microchannels and n2 wells in some embodiments of the invention are argued by additional methods to control the diffusion of compounds between wells and microchannels. With the different prior art systems, the microfluidic system of the present invention does not need a detailed control system within the device for the control of well-to-well diffusion. Two exemplary means of control diffusion control are described so that they do not require active control of the valves on the board. Both of these interpozzo block diffusion methods by means of fluid flow control from the valves, pumps and multiple tubes are external to the arrangement of the wells, and thus conform the overall design principles of the invention: the optimal representation of sub-arrays of wells in a class of fluidic architectures that require fewer channels and allow for smaller interpolation spacing. The first method uses the insertion of air segments between the different fluid segments. The positive and / or negative pressure methods given in the above modalities are used in the insertion of these air segments. In general, the application of differential pressure in appropriate sequences through a row. { j} particular and columns. { k} or { k *} they can be placed in a number of separate segments in the input channels. { j} between a number of different fluids. In particular, proper sequencing can direct a fluidic segment to another well. { j, k} , and direct another fluidic segment to another well (j.k-1), while also inserting and maintaining an air segment in row j between these two wells. Subsequently, the air segment can be passed out of the system through an unused well or by means of a rinse channel k * (130) (Figure 35). The second diffusion control method uses "normally closed" check valves (500) between each pair of adjacent tracks (via k and via k + 1) (180) which connects the channels to the wells (j, k) and (j) , k + 1) (Figure 36). Similarly, verification valves can be used in architectures that incorporate the rinsing column channels (k *). In other modalities, the check valve can be placed inside the tracks (160, 180) that connect the wells to the microchannels or row channels to rinse the column channels. These valves close unless a pressure gradient is applied through a fluid path of the array. Then, only those valves distributed along the selected fluid path are depressed openings. When the flow stops and the valves are returned to the closed position, the diffusion of valve suppression of the compounds between the wells and the microchannels, or the row channels and the rinsing channels. The recovery forces that closes each valve element (520) in its corresponding valve seal (540) can be provided by any number of methods known in the art, the strength of the recovery force must allow the valves to open in the application of pressure gradients that produce appropriate flow ratios (typically less than 1 cm / min). The methods for the application of recovery forces include mechanical forces by the deformation of metallic, semiconductor, or organic materials; tire pressure; electrostatic force, magnetic force, and the force of gravity. In another embodiment, an externally applied magnetic field gradient (620) induces a recovery force in the valve elements (520) incorporating a magnetic material. This includes but is not limited to the use of paramagnetic beads (600) (for example, provided by Dynal Corp.) as shown in Figure 37. These magnetic beads (600) serve as balls in ball valves that are manufactured within and along a given row and channel column, US Patents Nos. 5,643,738 and 5,681,484 describe a magnetically actuated microbole check valve. However, these patents in particular describe an actively controlled valve that will close the closed ball valve (in demand), and have as their purpose to stop the flow that is imposed by some external pressure. In the present case, the particular advantage of the microfluidic architecture system is that an average recovery force is simultaneously applied to a long assembly of the ball valves through arrangement, and non-active control is required. The movement of the selected balls (at any time if you want to open the valves) along a fluid path is achieved by means of the fluid provocation by itself flows, the ball valve is controlled by the fluid flow , if the fluid flow is controlled by the ball valve. In addition, in this embodiment, the valve seal is particularly designed so that a seal is not obtained in one direction, as opposed to the other direction, where a sufficient average seal that stops diffusion is obtained. In another embodiment, a microbead valve is located between each pair of adjacent tracks (via k and via k + 1) that connect the channels j to the wells (j, k) and (j, k + 1). Each microbole valve (only when a pressure gradient is applied through a path that includes the valve) The design of the valve seal (540) on one side is formed (for example with slots) so that the flow is not stopped when fluid pressure moves the ball in the direction of flow When it does not apply to the pressure gradient (no fluid flows) at the site of a ball valve, a magnetic recovery force presses that subsequent magnetic ball against the other side of the valve seal (540) (where the seal is formed to fix the ball) to suppress the diffusion of the compounds through that portion of the microchannel.This force of recovery is induced by a magnetic field gradient applied to the complete array, so that all the valves are normally closed.

In another embodiment, an externally applied magnetic field gradient (620) induces a restoring force on the valve elements (520) that incorporate a ferromagnetic material. for example, pressure chromium steel balls are available from Glenn Mills Inc., Clifton, NJ in a range of sub-mm to a few mm diameters. Figure 38 shows a form of a formed valve seal exemplifying this main design, specifically, the diffusion along the ball is stopped when the extremely applied recovery force presses the ball in the left in an opening around the seal valve. When the flow is pressed by extremely applied pressure, the ball is forced to the right, but the ball does not stop the flow because the right side of the valve seal is oval in shape and includes a deformation as a groove along a On the other hand, the formation path for the fluid passes through one side of the ball in this open position of the valve. Figure 39 shows a modality that allows a simple magnetic field gradient applied to the complete cassette of the invention to induce force on a ball valve seal of the inlet channels and a ball valve seal of the outer channels. In one embodiment, the direction of the magnetic field gradient is parallel to the plane of the array, but oriented at 45 degrees to both row and column channels. In this way each ball experiences a force component in the direction of the "closed" side of its corresponding valve seal (540). In other embodiments, the magnetic field gradient is applied perpendicular to the plane of the array, and the ball valves operate in the vertical direction within the horizontal microchannels or within the vertical paths. In the latter case (ball valves vertically actuated within the vertical tracks) the track architecture must again allow each ball to be subjected to a component of magnetic force in the direction of the "closed" side of its seal (540) of corresponding valve. An example will be given in a ferromagnetic or paramagnetic valve element (520) of spherical shape, but other forms and other recovery forces may generally be used as that function in the same way.

Dimensions and well characteristics In another aspect, the present invention provides a cassette for cell screening, comprising a substrate containing a surface, wherein the surface contains a plurality of cell attachment locations; a fluid supply system for supplying reagents to a plurality of cell attachment locations wherein the fluid delivery system comprises a multi-level chamber that is bonded to the substrate, wherein the multiple level chamber comprises a cross-linked arrangement of the microfluidic inlet channels and outlet channels and a plurality of fluidic locations in the fluid connection with the microfluidic inlet channels and the outlet channels; and a plurality of wells, wherein an individual well comprises the space defined by the junction of a cell attachment location and a fluid location, and where the wells are present in a density of at least about 20 wells per square centimeter. In a preferred embodiment, the well density is between about 20 wells per square centimeter and about 6400 wells per square centimeter. In a preferred embodiment, the cassette of this aspect of the invention further comprises one or more multiple inlet tubes in the fluid connection with the microfluidic inlet channels in one or more outlet tubes in the fluid connection with the outlet channels microfluidics In another preferred embodiment, the cassette of this aspect of the invention further comprises at least one source receptacle in the fluid connection with one or more multiple inlet tubes and at least one waste receptacle in the fluid connection with one or more multiple output tubes. The various control devices in these modalities is as described in the foregoing. Without limitation they are placed in the well shape geometry in the current invention, as the well shape may differ in different embodiments of the invention. While a rectangular well shape allows for the minimum distance and area between the wells in the array, this shape has the disadvantage of potential differences in cell culture or fluidic properties in the corner regions of the wells, therefore the Well shape that is circular or that is rectangular with rounded corners is used in the preferred embodiment of the invention. The arrangement system of the present invention is designed by the representation of individual cells within the cell field in the drug screen assay system. The screening with the tests involving the measurement of each cell within the cell field, in parallel, is referred to as an Elevated Contour Screening (HCS), due to the detailed information surrounding the intracellular processes that are contained within the image. of single cells. Screening with lower resolution representation or with detectors that integrate a signal over a population of cells refers to High Performance Screening (HTS), because it is faster. In HCS, the cells are typically identified by the representation of the nucleus of cells, which are spherical or ellipsoidal in shape with long axis length that typically average from 5 m to 15 m. The cell array used in the present invention optimizes the HCS ratio using a high-density multiple well format compared to conventional 96-well plates. For HCS, the number of cells that provide a statistically relevant sample, as well as the number of cells that are required per unit area area varies depending on the type of assay and the type of cell being cultured. In this way, the minimum well size required by statistical criteria for each trial is different. However, the material is minimized or optimal the well size is, the ratio in which the wells can be read by adequate image resolution to solve the individual cell nucleus optimized by the present device, which allows several wells that will be represented in a parallel time. This is done more efficiently if there is a minimum wear space between the wells, and the cellular veneered area without the complete image is therefore enlarged. In addition, the present arrangement system optimizes the flexible implementation of HTS and HCS in the same optical-based cell-based screening system. The pixel density required (pixels per miera) is lower for HTS (for example, 0.001 to 0.1 pixels per miera) and high for HCS (for example, 0.1 to 4.0 or more pixels per miera). The present system provides the combined use of HTS and HCS, where a "trigger" identified in HTS is also read in HCS mode for more detailed analysis. In fact, the availability of HCS in the same platform and with the same form, greatly increases the information associated with "shots" identified in the HTS mode. However, the faster possible HCS ratio is advantageous to allow the maximum total screening performance at any time that HCS is used, the minimum pixel density required for HCS in combination with the maximum well density of the array are two critical parameters which define the maximum ratio at which wells can be read in HCS mode. This invention describes how the design of the present array system can extend this HCS relationship, as well as the HTS relationship. In use with 96-well microplates, a conventional microscopic representation system can represent square fields that are 0.1 mm to 10 mm wide centered in each well of diameter of 7 mm. The wells are represented or "read" in series. At each stage position, typically, only one area of the well is read. To read the complete array, (either in HTS modes or HCS modes) the sample stage must move the chip at least 96 times. In this invention, a novel cell screening method is defined, in which many wells are read at a single moment. Because the present arrangement has wells of internal well width and distances that are approximately 10 times smaller than in the 96-well chip (wells that are sub-millimeters in width and total plate dimensions of millimeters differently rather than several centimeters wide), a cell screening system, such as that described in the present invention, can represent many wells simultaneously. That is, the sample stage moves a few times per plate and several wells are read in parallel (Figure 39). This essentially involves the serial representation of "sub-array" (020) of the complete microwell array (040), while acquiring data from the total wells of each sub-array in parallel. For example, the representation of 3 x 3 well sub-arrays requires 9 times less than moments from the sample stage and allows for a faster 9-fold ratio of data acquisition. For a given CCD detector array (080), the amplification of the optical system (060) determines the area of the sub-array (020) that is projected in the detector. For example, an image pixel density of one pixel per micron is achieved by a sub-array of 1000 μm x 1000 μm wells using the 10x optical amplification and a 1000 x 1000 pixel of CCD array of 10 μm x 10 μm elements. In a second example, a sub-array of 2000 μm x 2000 μm wells can be represented on a CCD array of 2000 x 2000 pixels in the same amplification to produce the same pixel density of image. In a third example, an image pixel density of 0.1 pixel per micron is achieved by a 10mm x 10mm sub-array of wells using optical amplification of 1.0x and a CCD array of 1000 x 1000 pixels of 10 μm x 10 μm elements . In a four example, a sub-array 10 mm x 10 mm, the array can be represented on a CCD array of 2000 x 2000 pixels in 2.0x amplification to produce the image pixel density itself. Below the modalities of the particular modalities of well size and recreation according to the present invention that optimizes the representation of sub-arrangement. These well sizes should be long enough to contain a desired number of cells around a desired number of cell binding sites within each location of the array. Due to the considerable range of cell densities desired to be cultured at the cell binding sites (i.e.: very spaced cell cultures or confluent monolayers may be desired), the desired well size may average that which contains only a cell at a smaller cell binding site of about 10 μm in diameter (ie: a well size of 20 to 50 microns), to that which contains either a long cell binding site or an arrangement of several sites of cellular junction (ie: a well size of 1 to 2 mm) comprising a simple location of the array. The previous case leads to high well densities, and the latter to lower well densities. For the high and low well densities, and for the wells containing one or more cell binding sites, the microfluidic architecture class according to the present invention allows the optimum scattering of wells, the minimum wear space between the wells, and the maximum number of wells that can be simultaneously represented in a sub-array and still obtain adequate pixel resolution in the image. The four examples of optical resolution given in the above are used to illustrate how for a preferred range of sub-array sizes, optical amplifications, and pixel resolutions, and based on the advantages of the microfluidic architecture described herein, The present device supports well sizes and spreading that allows for a large increase in the speed at which wells can be represented or read. Additional sub-array sizes, optical amplifications, and pixel resolutions are supported by the methods and devices of the invention. The scope of the invention is not limited by the current state of the art in the number of pixels available, nor in the size of the pixel in the electronic representation systems. 1. For the first example above, a sub-array of 1000 μm x 1000 μm wells is represented in a CCD camera of 1000 x 1000 pixels (with pixel image resolution per miera). Now there are four examples of well sizes and densities that are supported by this invention, (a) First, a 2 x 2 sub-arrangement of 300 μm x 300 μm wells with 200 μm thick walls and well density of 400 wells per cm2. The representation of these wells in groups of four produces a 4-fold speed increase compared to reading the plate in the well at one time. (Second, a 3 x 3 sub-array of 200 μm x 200 μm wells with 100 m wells yields an increased 9 fold in velocity and a well density of 1111 wells per cm2.The number of wells per unit area is a factor greater than 80 that is provided by the commercial microplate of higher current density (the 1536 plate). (c) Third, a 5 x 5 sub-array of 100 μm x 100 μm wells and 100 μm of walls produces an increase in speed of 25 bends and a density of 2500 wells per cm2. (d) Fourth, for a still high density of wells and a greater speed advantage, the well can be 25 μm x 25 μm with 100 μm of walls , producing an 8 x 8 sub-arrangement, a 64-fold speed increase, and a well density of 6400 cells per cm2 2. For a sub-array of 2000 μm x 2000 μm wells represented in a CCD camera 2000 x 2000 pixels (with image resolution of 1 pixel per miera), an additional example of density is considered in tion to the four examples considered in the foregoing, (a) First, a 2 x 2 sub-array of 800 μm x 800 μm wells with walls of 200 μm thickness produces a 4-fold speed increase and a wall density of 100 folds per cm2. The number of wells per unit area is a factor of 5 greater than that provided by the highest density commercial microplate (the 1536 well plate), (b) Second, a 4 x 4 sub-array of 300 μm x 300 μm of wells with walls of 200 μm thickness produce a 16-fold increase in velocity and a well density of 400 wells per cm2. (c) Third, a 6 x 6 sub-array of 200 μm x 200 μm wells with 100 μm walls produces a 36 fold increase in velocity and a density of 900 wells per cm2. (d) Fourth, a 10 x 10 sub-array of 100 μm x 100 μm wells with 100 μm of walls produces a 100 fold increase in velocity and a well density of 2500 wells per cm2. (e) Fifth, a 16 x 16 sub-array of 25 μM x 25 μM wells with 100 μm walls produces a 256 fold speed increase and a well density of 6400 wells per cm2. 3. For a 10 mm x 10 mm sub-array of wells represented in a CCD camera of 1000 x 1000 (the image resolution of 0.1 pixels per meter), the four cases of well size and densities described in the Example 1 above, (a) First, a 20 x 20 sub-array of 300 μm x 300 μm wells with 200 wall thicknesses produces a 400 fold speed increase and a density of 400 wells per cm2. (b) Second, a 30 x 30 sub-array of 200 μm x 200 μm wells with 100 walls produces an increase in speed of 900 bends and a density of wells per cm2. In the above, this case supports a well density 80 times greater than that provided by the current high density commercial microplate, (c) Third, a sub-arrangement 50 x that 50 of 100 μm x 100 μm wells with 100 μm of walls produces a speed increase of 2500 bends and a well density of 2500 wells per cm2. (d) Fourth, an 80 x 80 sub-array of 25 μm x 25 μm wells with 100 μm walls produces a speed increase of 6400 bends at a density of 6400 wells per cm2. For a still high density of walls and a greater speed advantage (2500 bends), the wells can be 100 μm x 100 μm with walls separating the wells being 100 μm wide producing 2500 wells per sub-arrangement, and 2500 wells per cm 2. 4. For a sub-array 10 μm x 10 μm wells represented in the CCD camera of 2000 x 2000 pixels (with image resolution of 0.1 pixel per miera), the cases in previous example 2 of 100, 400, 1111, 2500, and 6400 cm2 wells against the application, but the 10 times greater performance of the speed increase due to the sub-arrangements are 10 times wider on each side. In this way, the increased density for well densities of 100, 400, 1111, 2500, and 6400 wells / cm2 are 400X, 1600X, 3600X, 10,000X, and 25,600X, respectively, in the 10 mm x 10 mm array . The following table compares pit tones by several other devices.

* Estimated speed increase is a comparison of the speed at which the complete array must be represented by compared sub-arrays to represent separately the representation of each well. All of these particular aspects of the instant microfluidic device-the traversed channel, multi-level wellbore density architectures, the fluid flow control in these architectures, the well arrangements in spatially optimized well sub-arrays, and the media Optimal control of the interpozo diffusion of the compounds - are specifically selected and designed to form an integrated system that is compatible with the use of the cell culture medium (an aqueous, polar solution containing biological macromolecules and salts), with the maintenance of desired, physiological levels of dissolved oxygen and carbon dioxide gases in the medium, and with the maintenance of desired, physiological temperature (typically 37 ° C, but other temperatures in the range of approximately 15 ° C to 40 ° C can be desired by particular cell types, particularly, the cell culture medium can be balanced in off-board glasses at desired levels of temperature and dissolved carbon dioxide and oxygen. Then, using the off-board vessels and the valves, the medium is moved through the microchannels and the wells. The seal of these microchannels and wells of the atmosphere allows the partial pressure of gases to be controlled by means of the equilibration of the external container. In a preferred embodiment, the level of carbon dioxide in the medium without the wells can be established and maintained by balancing the average priority of its flow in the device, and / or allowing the exchange of a mixture of carbon dioxide and air with the half as settled inside the well. A number of different environmentally controlled cameras for live cell culture exist. (U.S. Patent Nos. 5,552,321 and 4,974,952; Payne et al., J. Microscopy 147: 329-335 (1987); Boltz et al., Cytometry 17: 128-134 (1994); Moores et al., Proc. Nati. Acad Sci 93: 443-446 (1996), Nature Biotech 14: 3621-362 (1996)). In a further preferred embodiment, the temperature of the entire substrate is controlled by being in contact with, or integrally composed of, a heater and a temperature sensor. An electronic temperature control system regulates the heat to maintain a set temperature set point. Thus, the design of the microfluidic array system described in this invention is already used in a form that supports the live cell culture outside of an external incubator system, Figure 12 shows as this invention provided by the automated reading of the cassettes. The cassettes are maintained under controlled environmental conditions without two storage compartments (48 and 54) before and after reading in the instrument 44 luminescent reader. While read by the luminescence reading instrument, the system maintains the appropriate temperature and composition of gases dissolved in the cell culture medium within the cassette wells. The temperature control inside the wells is provided by hot devices and temperature sensors inside the cassette of the luminescent reader instrument. The control of the composition of dissolved gas within the wells is provided by the equilibration of the medium with a premixed air source and carbon dioxide (commercially available) prior to the medium flow in the cassette wells. Any type of controller for regulating the partial pressures of gas can be used with the current invention. In a preferred embodiment, the system comprises a gas controller comprising a pre-mixed gas source connected to a reservoir or reservoirs containing cell culture medium and / or test compounds. A gas pressure regulator and flow control valve control the flow ratio of this gas mixture and suppression in the contained fluid reservoirs. This equilibrium of the gas mixture with the fluids can be carried out in containers either on board on board or off board of cassette, but in any case outside the wells matrix. The present invention fulfills the need for the technique for devices and methods that decreases the amount of time necessary to drive the high throughput and / or high content cellular based screening. The devices of the invention are also identically suitable as a cellular support system for a portable diagnostic device (ie: a cell-based and miniaturized cell-based assay system). The drug discovery industry already uses 96-well microplates and is transitioning to the use of 384-well plates. The plates with increments to 1536 wells are conceptualized. Thus, there is a major advantage in the efficiency and economy of the use of high density plates such as that of the present invention. The sealed containment of the cells in the cell array of the current invention will provide a strong system that is portable and usable in any orientation. For military and civic toxin testing, the devices of the current invention will provide two main advantages. First, the toxins tested based on a cellular function are advantageously compared to the toxin test based on the molecular structure (for example mass spectrometry or optical spectroscopy), because their ultimate effect tests of the compound in tissue greater than the Related property of the compound whose link to toxicity may be unknown or depend on other conditions. Second, an automated, miniaturized, solid, and portable format for sensor-based cellular function should be advantageous compared to the current state of the art involving long microplates filled by long robotic tubing systems and reading by long microscopic readers. Additionally, other assay capabilities can be integrated into the device of the present invention that is not possible in prior art devices, such as simple plastic microplates. For example, a mass spectrometric or capillary electrophoretic analytical device, or systems for DNA and / or protein analysis, can be integrated into a current device for measuring chemical and structural parameters of the test compounds. Such integration will provide information that is complementary to cell-based, functional parameters, measured by HCS or HTS system. For conventional microplates, such additional assay functions are external to the plate, and therefore require extensive additional equipment for reference of samples from the microplate and for the same analytical equipment. In the miniaturized format with integrated fluidics of the present invention, the sample is integrally pumped to a microanalytical system on the chip or in a second, integrally connected chip. Such microanalytical chips will drive the cost and dramatically increase the speed of cell-based analysis. In this context, the marriage of a micro-scale live cellular analytical system with a microscale chemical analytical system is expected to provide major improvements over existing methods and devices.

Claims (17)

1. CLAIMS 1.
2. A cassette by the cell sieve, characterized in that it comprises: a. a substrate having a surface, wherein the surface contains a plurality of cell attachment locations; b. a fluidic delivery system for delivery test components to a plurality of cell attachment locations, wherein the fluidic delivery system comprises a multiple level chamber that is bonded to the substrate, wherein the multiple level chamber comprises i. a transverse arrangement of microfluidic input channels and output channels; ii. a plurality of fluidic locations in the fluid connection with the microfluidic inlet channels and the outlet channels; and c. a plurality of wells, wherein an individual well comprises a space defined by the junction of a cell attachment location and a fluid location and wherein the wells are present in a density in at least about 20 wells per cm2.
3. The cassette according to claim 1, characterized in that the wells are present in a density of between about 20 wells per square centimeter and about 6400 wells per square centimeter.
4. The cassette according to claim 1, characterized in that the multi-level chamber further comprises: (i) one or more multiple inlet tubes and the fluid connection with the microfluidic inlet channels; and (ii) one or more external multiple tubes in the fluid connection with the microfluidic outlet channels; The cassette according to claim 3, characterized in that it further comprises: (i) at least one source receptacle in the fluid connection with one or more multiple inlet tubes; and (ii) at least one wear receptacle in the fluid connection with one or more multiple outlet tubes.
5. 5. A cassette for cell screening characterized in that it comprises a. a substrate having a surface, wherein the surface contains a plurality of cell attachment locations; b. a fluid supply system for supplying reagents to a plurality of cell attachment locations, wherein the fluid delivery system comprises a multiple level chamber that is joined to the substrate, wherein the multiple level chamber comprises: i. a transverse arrangement of microfluidic inlet channels and outlet channels, wherein each well is in fluid connection with one or more inlet channels or one or more outflow channels; ii. a plurality of fluidic locations in the fluid connection with the microfluidic inlet channels and the outlet channels; and iii. one or more multiple inlet and fluid connection tubes with the microfluidic inlet channels; iv. one or more external multiple tubes in the fluid connection with the external microfluidic channels. v. at least one source receptacle in the fluid connection with one or more multiple inlet tubes; and I saw. at least one wear receptacle in fluid connection with one or more external multiple tubes; and c. a plurality of wells, wherein an individual well comprises a space defined by the union of a cell attachment location and a fluid location.
6. The cassette according to claim 1 or 5 characterized in that it further comprises a pump for controlling the fluid flow within the microfluidic device, wherein the pump is in fluid connection with one or more multiple inlet tubes and one or more tubes multiple output.
7. The cassette according to claim 1 or 5, characterized in that they also comprise a temperature controller.
8. The cassette according to claim 1 or 5, characterized in that it also comprises a controller for regulating gas partial pressure.
9. 9. The cassette according to claim 1 or 5 characterized in that the transverse arrangement of the microfluidic input channels and the output channels is not easy to intersect.
10. 10. The cassette according to claim 1 or 5 characterized in that it also comprises rinsing channels in fluid connection with the outlet channels and the wear tank.
11. The cassette according to claim 1 or 5 characterized in that it further comprises valves between the adjacent wells or within the rinsing channels.
12. The cassette according to claim 11, characterized in that the valves comprise a magnetic element.
13. 13. The cassette according to claim 1 or 5 characterized in that it further comprises an integrated or analytically electrophoretic capillary mass spectrometric device or a system for the analysis of protein and / or DNA.
14. 14. The improved method of cell screening, characterized in that the improvement comprises providing the cassette according to claim 1 or 5 for supplying fluid to a array of locations containing cell attachment sites.
15. 15. The improved method for diffusion control in a cassette, characterized in that the improvement comprises constantly applying a passive recovery force to the valves located without microfluidic channels of the cassette.
16. 16. The method for cell screening, characterized in that it further comprises a) providing an array of locations containing multiple cells; b) providing an optical system to obtain images of the arrangement of locations; c) sub-arrays serially representing the arrangement of locations; and d) acquire data from each of the sub-arrays in parallel.
17. 17. A method for cell screening, characterized in that it comprises a) providing the microfluidic device for cell screening according to claim 1 or 5; b) provide an optical system to obtain array images and locations; c) sub-arrays of serial representation of the arrangement of locations; and d) acquire data from each of the sub-arrays in parallel. SUMMARY The present invention describes methods and apparatus for maximizing the number of wells that can be recorded as an image at one time, while obtaining an adequate pixel resolution therein. This result has been achieved through the use of fluid architectures that maximize well density. The present invention also provides a miniaturized microplate system with close fluid volumes that are supplied internally with the fluid exchange and with closely spaced wells to more quickly detect the specially resolved characteristics of individual cells