WO2011035177A2 - High-throughput screening - Google Patents

Abstract

The invention provides a high throughput screening device having a microwell microarray and a printed microarray of test compound deposits. The layout of the test compound deposits matches the layout of the microwells such that each deposit addresses a single microwell when the two arrays are contacted with each other. The invention further provides methods of screening test compounds for biological activity using the device. The invention also provides methods of fabricating the device.

Description

HIGH THROUGHPUT SCREENING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of the U.S. Provisional Application No. 61/243,283, filed September 17, 2009, the content of which is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant nos. EB009196, EB007249, DE019024, and HL092836 awarded by the National Institutes of Health; and grant no. DMR0847287 awarded by the National Science Foundation. The government has certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to a high throughput screening device, methods of fabricating the device, and using the device for screening test compounds.

BACKGROUND OF THE INVENTION

[0004] High throughput screening (HTS) technologies have been used to successfully identify bioactive compounds, proteins, and small molecules across a broad spectrum of biological fields (Bleicher, et al., (2003) Nat Rev Drug Discov 2(5):369-378); however, screening technologies have not kept pace with the expanding number of potential targets. A vast experimental space has been created at the intersection of the potential targets identified in proteomic and genomic studies, and the chemical space defined by combinatorial chemistries (Geysen, et al., (2003) Nat Rev Drug Discov 2(3):222-230) and natural compounds (Butler M.S. & Buss A.D. (2006) Biochem Pharmacol 71(7):919-929). The disparity between potential targets and the current screening capabilities is confounded by the need to study drug-drug interactions at the early stages of development in order to evaluate potential complications in later-stage trials (Schuster D., Laggner C, & Langer T. (2005) Curr Pharm Des l l(27):3545-3559) Traditionally, HTS is carried-out in dedicated facilities, and access is often limited by high capital costs for automated liquid handling and microscopy. As interest in HTS from varied fields of biological research has increased, a need for cost effective and widely accessible HTS systems has followed. Microscale technologies can help fill this gap in technology by creating simple HTS devices that can be

13133183.4 1 easily fabricated and operated in a scalable manner. Benchtop devices can help decentralize HTS by transferring experimental capabilities from centralized locations to various laboratories or field testing facilities.

[0005] Replicating HTS at the microscale requires isolated reaction chambers for cell- based assays, rapid processing of experiments, high array density to accommodate large chemical libraries, and compatibility with diverse types of chemicals as well as the ability to rapidly test various concentrations and combinations of chemicals. Furthermore, such devices must be simple to use and inexpensive. Additionally, the ability to easily create and screen combinatorial libraries is necessary for generating high quality hits at the benchtop. Combinatorial screening, and the ability to screen for drug-drug interactions can be used as a means of addressing the potential risks associated with absorption, distribution, metabolism, excretion, and toxicology (ADMET) at early stages of drug development (Li A.P. (2001) Drug Discov Today 6(7):357-366 and Kremers P. (2002) Pharmacol Toxicol 91(5):209-217). A number of microscale screening technologies have been developed in attempts to address the technology gap in HTS. For example, live cell microarrays have been developed for small molecule and siRNA screening (Bailey S.N., Sabatini D.M., & Stockwell B.R. (2004) Proc Natl Acad Sci USA 101(46):16144-16149 and Tavana H, et al. (2009) Nat Mater 8(9):736-741), and cell hydrogel microarrays have been developed for screening cytotoxicity to metabolic products (Lee, et al. (2005) Proc Natl Acad Sci USA 102(4):983-987 and Lee, et al. (2008) Proc Natl Acad Sci USA 105(l):59-63) and cell-microenvironment interactions (Lii, et al. (2008) Anal Chem 80(10):3640-3647). These microarray system begin to address many aspects of miniaturized HTS; however, they have not been developed as generalized platforms for combinatorial screening. In some cases, the fabrication of arrayed chemical libraries was complicated. In other cases, open cell-based microarrays were used, therefore miniaturization was limited by diffusion of analytes between arrayed spots.

[0006] Accordingly, there is need for a generalized microarray platform for benchtop high throughput screening and devices for such screening. Especially, there is need for a HTS system that is highly scalable, simple, portable and economical for various types of HTS studies.

SUMMARY OF THE INVENTION

[0007] In one aspect, the invention provides a high throughput screening device, the device comprising: (i) a microwell plate comprising an array of micro wells; and (ii) a coverplate (or coverslip) comprising a microarray of test compound(s), at least one position

13133183.4 2 of the microarray having a deposit comprising a test compound, wherein the layout of at least one of the deposits is aligned with (or corresponds with) at least one of microwells in the microwell plate.

[0008] In another aspect, the invention provides a microarray of test compounds, at least one position of the microarray including a deposit comprising a test compound, wherein the layout of the deposits is aligned to the layout of microwells in a microwell array, and the test compound is encapsulated in a hydrogel and/or the deposit is raised or displaced from the surface of the cover plate such as on a post or a pedestal.

[0009] In yet another aspect, the invention provides a method for screening a test compound for biological activity, the method comprising contacting a microarray with a microwell array, wherein at least one position of the microarray having a deposit comprising test compound and layout of the test compound deposit aligns with the layout of microwells on the microwell array; the microwell array comprises one or more cells in a microwell, and which microwell aligns with the test compound deposit on the microarray when the microarray is contacted with the microwell array.

[0010] In still yet another aspect, the invention provides a method for fabricating a high throughput screening device, the method comprising: (i) generating an array of posts on surface of a first substrate; (ii) generating an array of microwells on surface of a second substrate, wherein the layout of the microwells matches the layout of the posts on the first substrate; and (iii) depositing a test compound on at least one of the posts.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figs. 1A-1H show a microarray system for high throughput screening according to the invention. Fig. 1A is a schematic of the HTS device fabrication and use. (a) micromolding of PEGDA microwell array by photocros slinking; (b) cell seeding in microwell array; (c) a chemical library was deposited on to the tips of arrayed PDMS posts by a microarray printer; (d) cell-seeded microwells and chemical-laden arrayed posts were aligned and pressed together; (e) after chemical exposure, the cell-seeded microwell array were analyzed for toxicity; (f) a single cell-seeded microwell prior to pressing with the chemical-laden arrayed posts; (g) a sealed microwell assay chamber. Figs. IB and 1C are SEM images of arrayed PDMS posts (Fig. IB) and microwells (Fig. 1C) (scale bar = 200 μιη). Fig. ID is a phase contrast micrograph of a selection of seeded MCF-7 breast cancer tumor cells after 24 hours of culture in a sealed microwell system. Fig. IE is high magnification phase contrast micrograph (left) and fluorescent image (right) of microwells

13133183.4 3 treated with calcein AM (green) and ethidium homodimer (red). Fig. IF is a line graph showing the number of cells per microwell as a function of cell seeding density.

[0012] Figs. 2A-2H show characterization and validation of the HTS system. Figs. 2A and 2B are micrographs showing fluorescent images of FITC-dextran and rhodamine B printed on arrayed PDMS posts (Fig. 2A) and in arrayed microwells after sandwiching (Fig. 2B). High magnification image shows a selected 10x10 array of microwells. Fig. 2C is micrograph of PDMS posts printed with chemicals (scale bar = 200 μπι). Fig. 2D is a bar graph showing relative intensity of fluorescence of FITC-dextran and rhodamine B from the selected 10x10 array; Ex/Em: 488/525 and 525/550. Fig. 2E is a bar graph showing cell survival after exposure to PBS in a sealed microwell. Fig. 2F is fluorescent images of selected wells analyzed with Live/Dead viability assay (green/red). Fig. 2G is a scanned fluorescent image of a selected array of microwells exposed to various concentrations of doxorubicin for 24 hours and subsequently stained with calcein AM. Fig. 2H is a line graph showing the dose dependent effect of doxorubicin, for determining IC₅₀ of doxorubicin, in the HTS system of the invention. The lower table compares the IC₅₀ values between microwells and standard 96- well plates.

[0013] Figs. 3A and 3B show the HTS results of a natural compound library performed in the HTS system of the invention. Fig. 3A shows the mean VI of each library compound shown as a color band. Fig. 3B shows the chemical structures, VI, and IC₅₀ of a hit compound (C-BOIO), two non-toxic compounds (C-P011 and C-J005), 0.01% TritonX-100, and 0.1% DMSO in PBS. C-POl 1 and C-J005 are nontoxic to MCF-7 cells at the concentration range <10 μΜ.

[0014] Figs 4A-4E show the Benchtop HTS of drug-drug interactions with the HTS system of the invention. Fig. 4A shows the mean VI of the natural compound library in the presence (right) and absence (left) of 10 μΜ verapamil displayed as color bars. The VI of the library compounds in the absence of verapamil is ordered in descending VI, the VI of each compound in the interaction screen is shown in the adjacent. Fig. 4B shows a scatter plot of the VI of each compound with and without verapamil interactions. Lines indicating the VI of negative (0.1% DMSO) and positive (0.01% TritonX-100) controls are included as visual aids in evaluating the data. Fig. 4C shows the chemical structure of interaction hits, C-L008, C-P013, and C-A005. Fig. 4D is a bar graph showing the VI of hits C-L008, C-P013 and C- A005 in the presence (open bars) and absence (solid bars) of 10μΜ verapamil as measured in the HTS system of the invention. Fig. 4E is a bar graph showing cell survival relative to

13133183.4 4 negative control for ΙΟμΜ of C-L008, C-P013 and C-A005 in the presence and absence of ΙΟμΜ verapamil in 96-well plate.

[0015] Figs. 5A-5D show combination chemical-laden posts. Fig. 5A is a

microphotograph showing the single printing of Rhodamine B (Red fluorescence) on arrayed PDMS posts. Fig. 5B is a microphotograph showing the single printing of FITC-Dextran (Green fluorescence) on PDMS posts. Fig. 5C is a microphotograph showing the multiple printing of both Rhodamine B and FITC-Dextran on the same posts. Fig. 5D is a bar graph showing that there was no significant difference in fluorescence between single printing and multiple printing. The intensity of FITC-Dextran and Rhodamine B were measured by Image J.

[0016] Figs. 6A-6E show the arrayed cell aggregates. Fig. 6A is a microphotograph showing MCF-7 cell aggregates formed within microwell array with bottom made from cell- repellent PEG. Fig. 6B is microphotograph showing that cells in the aggregates remained alive after 1 day culture. Fig 6C is a line graph showing that the diameter of the cell aggregates could be regulated by varying the initial cell seeding density, with a higher seeding density resulting in larger cell aggregates. Fig. 6D is a bar graph showing that during a 6 day culture, the diameter of cell aggregates decreased in the first 2 days, and increased afterwards. Fig. 6E is a microphotograph showing that FITC-dextran printed on the end of arrayed posts was delivered to cell aggregate array (cell aggregates appear as block dots in the fluorescence image).

[0017] Figs. 7A-7D show live/dead controls. Fig. 7A is a fluorescent scanner image (Ex/Em: 532/575+25) of MCF7 breast cancer cells in microwell arrays, stained with Calcein AM (live; green) and TO-PRO-3^®(dead, red), after exposure to DPBS for 6 hours. Fig. 7B is a fluorescent scanner image (Ex/Em: 635/670+20) of MCF7 breast cancer cells in microwell array, stained with Calcein AM (live; green) and TO-PRO-3^® (dead, red), after exposure to 0.01% Triton X-100 for 6 hours. Figs. 7C and 7D are high magnifications images of representative examples of negative (Fig. 7C) and positive (Fig. 7D) controls with live (green) and dead (red) staining.

[0018] Figs. 8A and 8B are bar graph showing the V-index of Trion X-100 (0.1 % V/V) obtained from different positons of one microarray slide as intra-array controls (Fig. 9A) and the same positions of different microarray slides as inter-array controls (Fig. 9B).

[0019] Fig. 9 is a line graph showing the cytotoxicity test of C-BOIO with/without verapamil in 96-well plate (n=2).

[0020] Fig. 10 is a schematic diagram showing the general concept of the HTS device.

13133183.4 5 DETAILED DESCRIPTION OF THE INVENTION

[0021] In one aspect, the invention provides a high throughput screening device, the device comprising: (i) a microwell plate comprising an array of microwells; and (ii) a coverplate (or coverslip) comprising a microarray of test compound(s), at least one position of the microarray having a deposit comprising a test compound, wherein the layout of at least one of the deposits is aligned with (or corresponds with) at least one of microwells in the microwell plate.

[0022] When the coverplate and the microwell plate are aligned and pressed together (sandwiched), each microwell is aligned with a single coverplate microarray deposit. This enables the formation of an array of sealed chambers. Because, the area of the test compound deposit can be less than the opening of a microwell on the microwell array, sealing of the chambers limits and/or inhibits diffusion and cross contamination between arrayed spots. Size of the deposit can be adjusted to compensate for printing tolerance. Thus, higher density arrays can be fabricated as the spacing between assays can be significantly reduced. This provides significant advantages over the currently utilized open microarray systems because in open microarray systems array density is limited by diffusion of analytes from assays to assays. It is to be understood that when the coverplate and the microwell plate are aligned, contacted, or pressed together, it is the compound deposit containing surface of the coverplate that comes in contact with the microwell opening containing surface of the microwell plate.

[0023] The microarray and/or the microwell array can have visual, mechanical, and/or physical guides for aligning the printed microarray with the microwell array so that each test compound deposit aligns with a single microwell on the microwell array. For example, distinct patterns at the corners of the microwell plate and matching features on coverplate can allow easy manual alignment.

[0024] The term "aligns with," as used herein in the context of compound deposit aligning with a microwell, means that when the coverplate and the microwell array are pressed together the deposit projects into and/or is at the opening of a microwell in the microwell array.

Microwell plate

[0025] The microwells of the microwell plate can be of any geometrical shape.

Accordingly, a microwell can be of cylindrical, rectangular, conical, inverse conical,

13133183.4 6 pyramid, or inverse pyramid shape. The microwell can also be a combination of two or more of the above shapes. In some embodiments, the well has a cylindrical shape.

[0026] Similarly, the opening or aperture of the microwell can be of any shape such as circular, elliptical, rectangular, square, or other polygonal shape. In some embodiments, the opening of the microwell is circular, elliptical, rectangular or square.

[0027] Generally, the size of the opening of the microwell can range from about 5 μπι to about 2000μπι. In some embodiments, the microwell aperture is about 5 μπι to about 100 μπι, from about ΙΟΟμπι to about 500 μπι, or from about 500μπι to about ΙΟΟΟμπι. In some embodiments, the microwell aperture is about 400 μπι.

[0028] Similarly, the depth of microwell can also range from about 1 μπι to about 1000 μπι. Accordingly, in some embodiments, the micro wells have a depth of about 1 μπι to about 10 μπι, from about 10 μπι to about 100 μπι, from about ΙΟΟμπι to about 500 μπι, or from about 500μπι to about ΙΟΟΟμπι. In some embodiments, the micro wells have a depth of at least 50 μπι. In some embodiments, the microwells have a depth of about 150 μπι.

[0029] The optimal microwell size can be adjusted depending on the cell type to be employed in the screening protocol/method, for example, the cell size or degree of adhesion to the wells, or the particular processes that will be taking place within the microwells.

[0030] The number of microwells on the microwell plate and the deposits of test compounds on the coverplate depends on the dimensions of the plates and can range from tens to tens of thousands. It is to be understood that the microwells and the deposits can be laid out in any pattern, e.g., rows, columns, circular, triangular, rectangular, square, or any other polygonal shape or geometric pattern.

[0031] In some embodiments, each microwell is at a predefined position on the microwell plate.

[0032] In some embodiments, each deposits is at a predefined position on the coverplate.

[0033] The term "predefined position" relates to any reachable point or area on or within a microwell plate and/or coverplate, which may be selected via suitable means known to the person skilled in the art, e.g. by using appropriate devices or control mechanisms which allow to chose and/or access said reachable points.

[0034] In some embodiments, the microwell plate comprises from 96 to 384 microwells. In some embodiments, the microwell plate comprises at least 500; at least 750; at least 1,000; at least 2,000; at least 5,000; at least 10,000; at least 15,000, at least 20,000; at least 25,000 microwells.

13133183.4 7 [0035] Similarly, in some embodiments, the cover plate comprises from 96 to 384 test compound deposits. In some embodiments, the coverplate comprises at least 500; at least 750; at least 1,000; at least 2,000; at least 5,000; at least 10,000; at least 15,000, at least 20,000; at least 25,000 test compound deposits.

[0036] In some embodiments, the coverplate is a microscope slide having the dimensions 75mm x 25mm.

[0037] In some embodiments, the coverplate is a microscope slide with at least 2,000 test compound deposits.

[0038] In some embodiments, the coverplate is a microscope slide with at least 2,000 test compound deposits arranged in a rectangular pattern.

Cells

[0039] In some embodiments, at least one microwell on the microwell array comprises one or more cells. It is to be understood that number of cells in a microwell will depend on the dimensions of the microwell. Typically, a microwell comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 or more cells. In some embodiments, the microwell comprises at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, or at least 100 or more cells. In some embodiments, a microwell comprises from about 5 to about 100, from about 10 to about 75, or from about 25 to about 55 cells.

[0040] The term "cell" includes any cell such as a mammalian cell, a reptilian cell, an avian cell, a fish cell, an insect cell, a fungal cell, a plant cell, a yeast cell, or a bacterial cell. The term also includes cell lines, e.g., mammalian cell lines such as HeLa cells as well as embryonic cells. Suitable cell lines can be comprised within e.g. the American Type Culture Collection and the German Collection of Microorganisms and Cell Cultures.

[0041] Exemplary insect cell lines include, but are not limited to, Lepidoptera cell lines such as Spodoptera frugiperda cells (e.g. Sf9, Sf21) and Trichoplusia ni cells (e.g. High FiveTM, BTI-Tn-5Bl-4).

[0042] Exemplary fungal cell include, but are not limited to, the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as well as the Oomycota and all mitosporic fungi. Representative groups of Ascomycota include, e.g., Neurospora,

Eupenicillium (or Penicillium), Emericella (ox Aspergillus), Eurotium (ox Aspergillus), and the true yeasts listed above. Examples of Basidiomycota include mushrooms, rusts, and smuts. Representative groups of Chytridiomycota include, e.g., Allomyces, Blastocladiella,

13133183.4 8 Coelomomyces, and aquatic fungi. Representative groups of Oomycota include, e.g., saprolegniomycetous aquatic fungi (water molds) such as Achlya. Examples of mitosporic fungi include Aspergillus, Penicillium, Candida, and Altemaria. Representative groups of Zygomycota include, e.g., Rhizopus and Mucor.

[0043] Fungal cells can be yeast cells. Exemplary yeast cells include, but are not limited to, ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti or Deuteromycota (Blastomycetes). The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is comprised of four sub-families, Schizosaccharomycoideae (e.g., genus Schizosaccharomyces including S. pombe), Nadsonioideae, Lipomyooideae, and Saccharomycoideae (e.g., genera Pichia including P. pastoris, P. guillermondii and P. methanollo ), Kluyveromyces including K. lactis, K. fragilis and Saccharomyces including S. carlsbergensis, S. cerevisiae, S.

diastaticus, S. douglasii, S. kluyveri, S. norbensis or S. oviformis). The basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeasts belonging to the Fungi Imperfecti are divided into two families, Sporobolomycetaceae (e.g., genera Sporobolomyces and Bullera) and Cryptococcaceae (e.g., genus Candida including C. maltose). Other useful yeast host cells are Hansehula

polymorpha, Yarrowia lipolytica, Ustilgo maylis.

[0044] Fungal cells can be filamentous fungal cells including all filamentous forms of the subdivision Eumycota and Oomycota. Filamentous fungi are characterized by a vegetative mycelium composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligatory aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism can be fermentative. In a more preferred embodiment, the filamentous fungal host cell is a cell of a species of, but not limited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, and Trichoderma or a teleomorph or synonym thereof.

[0045] Useful microorganism cells can be unicellular, e.g. a prokaryotes, or non- unicellular, e.g. eukaryotes. Useful unicellular cells are Archeabacteria. Further useful unicellular cells are aerobic bacterial cells such as gram positive bacteria including, but not limited to, the genera Bacillus, Sporolactobacillus, Sporocarcina, Filibacter, Caryophanum, Arthrobacter, Staphylococcus, Planococcus, Micrococcus, Mycobacterium, Nocardia, Rhodococcus; or gram negative bacteria including, but not limited to, the genera Acetobacter,

13133183.4 9 Gluconobacter, Frateuria, Alcaligenes, Achromobacter, Deleya, Amoebobacter, Chromatium, Lamprobacter, Lamprocystis, Thiocapsa, Thiocystis, Thiodictyon, Thiopedia, Thiospirillum, Escherichia, Salmonella, Shigella, Erwinia, Enterobacter, Serratia,

Legionella, Neisseria, Kingella, Eikenella, Simonsiella, Alysiella, Nitrobacter, Nitrospina, Nitrococcus, Nitrospira, Pseudomonas, Xanthomonas, Zoogloea, Fraturia, Rhizobium, Bradyrhizobium, Azorhizobium, Sinorhizobium, Rickettsia, Rochalimaea, Ehrlichia,

Cowdria, Neorickettsia, Treponema, Borrelia, Vibrio, Aeromonas, Plesiomonas,

Photobacterium, Brucella, Bordetella, Flavobacterium, Francisella, Chromobacterium, Janthinobacterium, and lodobacter.

[0046] Suitable plant cells for use in the present invention include dicotyledonous plant cells, examples of which are Arabidopsis Thaliana, tobacco, potato, tomato, and leguminous (e.g. bean, pea, soy, alfalfa) cells. It is, however, contemplated that monocotyledonous plant cells, e.g. monocotyledonous cereal plant cells such as for example rice, rye, barley and wheat, can be equally suitable.

[0047] In some embodiment, the cell is selected from the group consisting of HeLa cells (human); NIH3T3 cells (murine); embryonic stem cells; a cell type such as hematopoietic stem cells, myoblasts, hepatocytes, lymphocytes, and epithelial cells; MCF-7 cells; HEPG2 cells; HL-1 cardiomyocytes; Chinese hamster ovary (CHO) cells, Chinese hamster lung (CHL) cells, baby hamster kidney (BHK) cells, COS cells, THP cell lines, TAg Jurkat cells, hybridoma cells, carcinoma cell lines, and the like. In addition, cells which have been transfected with recombinant genes can also be used in the present invention.

[0048] In some embodiments, a microwell comprises two or more different cell types. As used herein the term "cell type" refers to a cell having a distinct set of morphological, biochemical and/ or functional characteristics that define that cell type (e.g., the ability to internalize a specific compound). The term "cell type" can refer, e.g., to a broad class of cells (e.g., cancer cells, non-cancer cells and nerve cells), a sub-generic class of cells (e.g., prostate cancer cells, HIV-infected cells and breast cancer cells), or a cell line or group of related cell lines (e.g., PC3 and LNCaP).

[0049] The volume of cell culture media in the microwells can range from about lOnL to about lOOnL. In some embodiments, a microwell has from about lOnL to about 75nL of cell culture media. In some embodiments, a microwell has from about 15nL to about 50nL of cell culture media. In one embodiments, a microwell has about 20nL of cell culture media.

[0050] The cells in a microwell can be present as a monolayer, an aggregate, or a suspension of the cells. Furthermore, cells can be encapsulated in a hydrogel and the

13133183.4 10 hydrogel integrated into the microwells. Encapsulating the cells in hydrogel can allow culturing of cells in 3D; thus, mimicking organ and/or tissue organization and structure.

[0051] As used herein, the terms "monolayer" refer to cells that have adhered to a substrate and grow in as a layer that is one cell in thickness.

[0052] As used herein, the term "suspension" refers to cells that survive and proliferate without being attached to a substrate. Suspension cultures are typically produced using hematopoietic cells, transformed cell lines, and cells from malignant tumors.

[0053] Cells can be seeded in the microwells by layering or depositing cell containing media on the arrayed microwells. The amount of media to be layered will depend on the dimensions of the microwell array. Typically, enough media is layered to completely cover the microwell array. In some embodiments, at least 0.5ml, at least 1ml, at least 1.5ml, at least

2ml, or at least 2.5ml of the cell-containing media is layer on the microwell array.

[0054] Skilled artisan can adjust the cell seeding density on the microwell array to adjust the number of cells per microwell. Accordingly, in some embodiments, from about 10⁴ to

7 5 about 10 cells can be layered on the microwell array. In some embodiments, about 10 to about 10⁶ cells can be layered on the microwell array.

[0055] The term "cell culture medium" (also referred to herein as a "culture medium" or "medium") as referred to herein is a medium for culturing cells containing nutrients that maintain cell viability and support proliferation. The cell culture medium may contain any of the following in an appropriate combination: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibiotics, serum or serum replacement, and other components such as peptide growth factors, etc. Cell culture media ordinarily used for particular cell types are known to those skilled in the art.

[0056] In some embodiments, the cell is a differentiated cell. As used herein, by the term "differentiated cell" is meant any primary cell that is not, in its native form, pluripotent as that term is defined herein. The term a "differentiated cell" also encompasses cells that are partially differentiated, such as multipotent cells, or cells that are stable non-pluripotent partially reprogrammed cells. It should be noted that placing many primary cells in culture can lead to some loss of fully differentiated characteristics. Thus, simply culturing such cells are included in the term differentiated cells and does not render these cells non-differentiated cells (e.g. undifferentiated cells) or pluripotent cells. In some embodiments, the term

"differentiated cell" also refers to a cell of a more specialized cell type derived from a cell of a less specialized cell type (e.g., from an undifferentiated cell or a reprogrammed cell) where the cell has undergone a cellular differentiation process.

13133183.4 11 [0057] The term "pluripotent" as used herein refers to a cell with the capacity, under different conditions, to differentiate to cell types characteristic of all three germ cell layers (endoderm, mesoderm and ectoderm). Pluripotent cells are characterized primarily by their ability to differentiate to all three germ layers, using, for example, a nude mouse teratoma formation assay. Pluripotency is also evidenced by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is the demonstration of the capacity to differentiate into cells of each of the three germ layers. In some embodiments, a pluripotent cell is an undifferentiated cell.

[0058] A differentiated cell can be obtained from a stem cell, e.g. an embryonic stem cell or an induced pluripotent stem (iPS) cell. As used here in, the term "induced pluripotent stem cell" or "iPSC" or "iPS cell" refers to a cell derived from a complete reversion or

reprogramming of the differentiation state of a differentiated cell (e.g. a somatic cell).

[0059] The term "reprogramming" as used herein refers to a process that alters or reverses the differentiation state of a differentiated cell (e.g. a somatic cell). Stated another way, reprogramming refers to a process of driving the differentiation of a cell backwards to a more undifferentated or more primitive type of cell. Complete reprogramming involves complete reversal of at least some of the heritable patterns of nucleic acid modification (e.g., methylation), chromatin condensation, epigenetic changes, genomic imprinting, etc., that occur during cellular differentiation as a zygote develops into an adult. Reprogramming is distinct from simply maintaining the existing undifferentiated state of a cell that is already pluripotent or maintaining the existing less than fully differentiated state of a cell that is already a multipotent cell (e.g., a hematopoietic stem cell). Reprogramming is also distinct from promoting the self -renewal or proliferation of cells that are already pluripotent or multipotent, although the compositions and methods of the invention may also be of use for such purposes.

[0060] The term "embryonic stem cell" is used to refer to the pluripotent stem cells of the inner cell mass of the embryonic blastocyst (see US Patent Nos. 5,843,780, 6,200,806, which are incorporated herein by reference). Such cells can similarly be obtained from the inner cell mass of blastocysts derived from somatic cell nuclear transfer (see, for example, US Patent Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein by reference). The distinguishing characteristics of an embryonic stem cell define an embryonic stem cell phenotype. Accordingly, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of an embryonic stem cell such that that cell can be distinguished from other cells. Exemplary distinguishing embryonic stem cell characteristics

13133183.4 12 include, without limitation, gene expression profile, proliferative capacity, differentiation capacity, karyotype, responsiveness to particular culture conditions, and the like.

[0061] As used herein, the term "somatic cell" refers to any cell other than a germ cell, a cell present in or obtained from a pre-implantation embryo, or a cell resulting from

proliferation of such a cell in vitro. Stated another way, a somatic cell refers to any cells forming the body of an organism, as opposed to germline cells. In mammals, germline cells (also known as "gametes") are the spermatozoa and ova which fuse during fertilization to produce a cell called a zygote, from which the entire mammalian embryo develops. Every other cell type in the mammalian body— apart from the sperm and ova, the cells from which they are made (gametocytes) and undifferentiated stem cells— is a somatic cell: internal organs, skin, bones, blood, and connective tissue are all made up of somatic cells. In some embodiments the somatic cell is a "non-embryonic somatic cell", by which is meant a somatic cell that is not present in or obtained from an embryo and does not result from proliferation of such a cell in vitro. In some embodiments the somatic cell is an "adult somatic cell", by which is meant a cell that is present in or obtained from an organism other than an embryo or a fetus or results from proliferation of such a cell in vitro.

Coverplate

[0062] The deposited test compound on the coverplate can be encapsulated in a hydrogel. Alternatively, or in addition, the test compound can be deposited at a position which is raised from or displaced off the surface of the coverplate. The raised position where a test compound is deposited is also referred to as a post herein. The height of the post can be up to the depth of microwells on a complementary microwell array. In some embodiments, the post height is such that on contacting the coverplate with a microwell array, the deposit is in contact with, or partially or fully submerged in the culture media present in the microwell.

[0063] Without limitations, the post height can range from about 5 μπι to about 1000 μπι. Accordingly, in some embodiments, the post height is from about 50 μπι to about 500 μπι, from about 75 μπι to about 250 μπι, and/or from about ΙΟΟμπι to about 200 μπι. In some embodiments, the post height is at least 50 μπι. In some embodiments, the post has an height of about 150 μπι.

[0064] Top surface of the post, at which test compound is deposited, can be of any circular, oval, or polygonal shape. Generally, the post surface matches the shape of the microwell opening on the microwell array. It is to be understood, that a test compound

13133183.4 13 deposit may or may not cover the entire surface of the post. Preferably, area of the test compound deposit is less than the area of the top surface of the post.

[0065] As discussed above, the layout of at least two or more of the posts and/or the test compound deposits aligns the layout of 2 or more microwells of a microwell array. Thus, when the coverplate is placed on, contacted with, combined with or aligned with a matching microwell array, each post and/or test compound deposit aligns with a single microwell on the microwell array. Accordingly, each deposit can be sealed within a single microwell on the microwell microarray.

[0066] Without wishing to be bound by a theory, in some embodiments using the posts on the printed microarray allows better sealing of the microwells; thus, inhibiting diffusion and/or contamination problems associated with open arrays. Additionally, using the posts allows using lower volumes in the microwells because the post can extend into the microwell. Therefore, allowing the test compound deposit to be transferred to the solution in the microwell without cross contamination and/or smearing.

[0067] In one aspect, the invention also provides a printed microarray of test compounds, at least one position of the microarray including a deposit comprising a test compound, wherein the layout of the deposits is aligned with the layout of microwells in a microwell array, and the test compound is encapsulated in a hydrogel and/or the deposit is raised or displaced from the surface of the microarray such as on a post or a pedestal.

[0068] In some embodiments, the printed microarray is a coverplate described herein.

[0069] The printed microarray can be fabricated from any material skilled artisan finds suitable. For example, the printed microarray can be fabricated from materials that are hard, soft, rigid, and/or flexible. A number of materials suitable for use in fabricating the printed microarray are described herein.

Test compounds

[0070] As used herein, the term "test compound" refers to a compounds and/or compositions that are to be screened for their biological activity. Test compounds may include a wide variety of different compounds, including chemical compounds, mixtures of chemical compounds, e.g., polysaccharides, small organic or inorganic molecules, biological macromolecules, e.g., peptides, proteins, peptide analogs, and analogs and derivatives thereof, peptidomimetics, nucleic acids, nucleic acid analogs and derivatives, an extract made from biological materials such as bacteria, plants, fungi, or animal cells, animal tissues,

13133183.4 14 naturally occurring or synthetic compositions, and any combinations thereof. In some embodiments, the test compound is a small molecule.

[0071] As used herein, the term "small molecule" can refer to compounds that are "natural product-like," however, the term "small molecule" is not limited to "natural productlike" compounds. Rather, a small molecule is typically characterized in that it contains several carbon— carbon bonds, and has a molecular weight of less than 5000 Daltons (5 kD), preferably less than 3 kD, still more preferably less than 2 kD, and most preferably less than 1 kD. In some cases it is preferred that a small molecule have a molecular mass equal to or less than 700 Daltons.

[0072] The number of possible test compounds runs into millions. Methods for developing small molecule, polymeric and genome based libraries are described, for example, in Ding, et al. J Am. Chem. Soc. 124: 1594-1596 (2002) and Lynn, et al., J. Am. Chem. Soc. 123: 8155-8156 (2001). Commercially available compound libraries can be obtained from, e.g., ArQule, Pharmacopia, graffinity, Panvera, Vitas-M Lab, Biomol International and Oxford. These libraries can be screened using the screening devices and methods described herein. Chemical compound libraries such as those from NIH Roadmap, Molecular

Libraries Screening Centers Network (MLSCN) can also be used. A comprehensive list of compound libraries can be found at

http://www.broad.harvard.edu/chembio/platform/screening/compound_libraries/index.htm. A chemical library or compound library is a collection of stored chemicals usually used ultimately in high-throughput screening or industrial manufacture. The chemical library can consist in simple terms of a series of stored chemicals. Each chemical has associated information stored in some kind of database with information such as the chemical structure, purity, quantity, and physiochemical characteristics of the compound.

[0073] Generally, the test compound is deposited in a volume of about O.OlnL to about 50nL on the printed microarray. In some embodiments, the test compound is deposited in volume of about O.lnL to about 25nL, about 0.5nL to about lOnL, or about InL to about 5nL on the printed microarray. In some embodiments, test compound is deposited in a volume of about 2nL on the printed microarray.

[0074] The test compound can be deposited at a concentration of from about O.OlnM to about lOOOmM. In some embodiments, the test compound is deposited at a concentration of about O.lnM to about lOOOnM, from about O.lnM to about 500nM, from about InM to about 250nM, and/or about InM to about ΙΟΟηΜ.

13133183.4 15 [0075] The amount of the test compound deposited can range from about 0.01 nanomoles to about 1000 micromoles. In some embodiments, amount of the test compound deposited on a printed microarray position is from about O.Olnanomoles to about 500 micromoles, from about O.lnanomoles to about 1000 nanomoles, from about 0.1 nanomoles to about 500 nanomols, and/or from about 1 nanomoles to about 250 nanomoles.

[0076] Without limitation, the compounds can be tested at any concentration that can exert an effect on the cells relative to a control over an appropriate time period. In some embodiments, compounds are testes at concentration in the range of about O.OOlnM to about lOOOmM, about O.OlnM to about 500μΜ, about Ο.ΟΙμΜ to about 20μΜ, about Ο.ΟΙμΜ to about 10μΜ, about Ο.ΙμΜ to about 5μΜ. It is to be understood that the concentration of the test compound is the final concentration in the microwell and assumes that all of the deposited compound is released into the microwell.

[0077] For combinatorial screening, multiple test compounds can be deposited on the same position of the printed microarray. Such depositing can be accomplished by sequentially depositing the test compounds on the same position, and/or the test compounds can be premixed before depositing on the printed microarray.

[0078] For quality control and/or reproducibility, a test compound can be deposited at more than one position on the printed microarray. When a test compound is deposited at more than one position on the printed microarray, the amount deposited can be the same or different. For example, a test compound can be deposited at different position at different amount to measure a dose response of the compound.

[0079] Compounds to serve as positive controls can also be deposited on the printed microarray. Positive controls can be located at positions throughout the printed microarray, and used as intra-array controls to evaluate depositing consistency on the array. Positions on the printed microarray having no deposited compounds, e.g., deposits of solvents only or no deposit at all, can be used as negative controls.

[0080] In some embodiments, the test compound is encapsulated in a hydrogel. As used herein, the term "hydrogel" refers to polymeric materials which exhibit the ability to swell in water and to retain a significant portion of water within their structure without dissolution. Suitable hydrogels include macromolecular and polymeric materials into which water and small molecules can easily diffuse and include hydrogels prepared through the cross linking, where crosslinking can be either through covalent, ionic or hydrophobic bonds introduced through use of either chemical cross-linking agents or electromagnetic radiation, such as ultraviolet light, of both natural and synthetic hydrophilic polymers, including homo and co-

13133183.4 16 polymers. Hydrogels of interest include those prepared through the crosslinking of:

polyethers, e.g. polyakyleneoxides such as poly (ethylene glycol), poly(ethylene oxide), poly(ethylene oxide)-co-poly(propyleneoxide) block copolymers; polyvinyl alcohol (PVA); poly(lactic acid) (PLA); poly(lactic-co-glycolic acid) (PLGA); poly(vinyl pyrrolidone); polysaccharides, e.g. hyaluronic acid, dextran, chondroitin sulfate, heparin, heparin sulfate or alginate; agarose; proteins, e.g. gelatin, collagen, albumin, ovalbumin or polyamino acids; and the like. Because of their high degree of biocompatibility and resistance to protein adsorption, polyether derived hydrogels are preferred, with poly(ethylene glycol) derived hydrogels being particularly preferred. Furthermore, since, PEG molecules are inert and do not bind with most chemicals, drugs or other proteins can be released from PEG hydrogels based on the molecular diffusion rate as well as the pore diameter of the polymeric network. The behavior photocrosslinkabe PEG is highly dependent upon its molecular weight.

Accordingly, in some instances, low molecular weight PEG diacrylate (PEGDA) is used for fabricating the microwells and high molecular weight PEGDA is used for encapsulating the test compounds on the printed microarray. The low molecular weight PEG hydrogels are often hydrophobic , while higher molecular weight PEG hydrogels are hydrophilic and can be used to release the test compounds in a controllable manner. See, for example, Karp, et al., Lab Chip 7:786-794 (2007).

[0081] In some embodiments, the deposit of the test compound further comprises an analyte capture ligand. As used herein, the term "analyte capture ligand" refers to the binding partner of an analyte. For example, if an analyte capture ligand is an antibody reactive with an antigen in a sample solution, the antigen may be regarded as the "analyte" and the reactive antibody may be referred to as the "analyte capture ligand."

[0082] As used herein, the term "analyte" is a broad term and is used in its ordinary sense and refers, without limitation, to any compound or composition the presence or concentration of which is sought in a sample. For example, an analyte can be an amino acid, peptide, protein, growth factor, saccharide, molecule produced by a cell. In some embodiments, analyte is a molecule produced by a cell in response to a test compound.

[0083] In some embodiments, the analyte capture ligand is selected from the group consisting of antibodies, Fab fragments, scFv, aptamers, nucleic acids, proteins, peptides, other appropriate affinity molecule, and any combinations thereof.

[0084] The test compound and the analyte capture ligand can be mixed together in a deposit on the printed microarray. Alternatively, the test compound and the analyte capture ligand are not mixed in a deposit on the printed microarray. For example, the test compound

13133183.4 17 and the analyte capture ligand can be deposited on separate spots on printed microarray. When the test compound and the analyte capture ligand are deposited on separate spots on printed microarray, the deposits are in close proximity such that both deposits address the same microwell on the microwell array.

[0085] Having a test compound and an analyte capture ligand address the same microwell allows the capture and analysis of a molecule produced by a cell in response to the test compound. Thus, providing a the ability to stimulate cells and subsequently analyze the resulting gene and protein expression. The released molecule can be detected by techniques well known to the skilled artisan, such as ELISA and other antibody-based assays.

Assaying of cellular response

[0086] The devices and methods described herein are useful for screening compounds for their biological activity. As used herein, the term "biological activity" or "bioactivity" refers to the ability of a compound to affect a biological sample. Biological activity can include, without limitation, elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological assay. For example, a biological activity can refer to the ability of a compound to modulate the effect/activity of an enzyme, block a receptor, stimulate a receptor, modulate the expression level of one or more genes, modulate cell proliferation, modulate cell division, modulate cell morphology, or any combination thereof. In some instances, a biological activity can refer to the ability of a compound to produce a toxic effect in a biological sample.

[0087] The biological activity can be determined by assaying a cellular response.

Exemplary cellular responses include, but are not limited to, lysis, apoptosis, growth inhibition, and growth promotion; production, secretion, and surface exposure of a protein or other molecule of interest by the cell; membrane surface molecule activation including receptor activation; transmembrane ion transports; transcriptional regulations; changes in viability of the cell; changes in cell morphology; changes in presence or expression of an internal component of the cell; changes in presence or expression of a nucleic acid produced within the cell; changes in the activity of an enzyme produced within the cell; and changes in the presence or expression of a receptor.

[0088] Assaying of cellular responses can be done in a number of ways. Detection can be by just visual inspection; e.g. cell growth or not, cell morphology, etc. or can be by the use of detector molecules. Detector molecules can be already present in the microwells; e.g. when looking at expression of a gene with a GFP reporter or present in the culture media in the

13133183.4 18 microwell. Alternatively, the detector molecule can be added after the test compound has been allowed to transfer to the cell culture media in the microwell for a sufficient time. Also, the detector molecules can be deposited with the test compound so that the detector molecules are transferred to the cell culture media at the same time as the test compound. The assaying can optionally include a step of washing off excess detector molecule.

[0089] Detector molecules can be selected from the group consisting of nucleic acids including modified analogues thereof, peptides, proteins, and antibodies including antibody fragments, enzyme substrates and specific dyes. Non-limiting suitable examples of specific dyes are well known in the art and include Fluo-3, Fluo-4, calecin AM, ethedium bromide, TO-PRO-3, Alexa Fluor 488 conjugated Annexin V, and Ca-dyes such as e.g. Calcium Green- 1. Other dyes amenable to the present invention include those described available from Molecular Probes (Eugene, Oregon, USA). Dyes such as DAPI and Hoechst can be used for staining cell nuclei to analyze total cell counts.

[0090] Alexa Fluor 488 conjugated Annexin V (Molecular Probes) is a probe used for digital readout of apoptosis in cultures with single cell resolution. This probe binds to membrane phospatidylserine (PS). The propidium iodide (PI) counter stain distinguishes necrotic from apoptotic cells. PI is a high affinity fluorescent nucleic acid stain. It binds to both DNA and RNA. Because PI is highly positively charged, it cannot cross the cell membranes in living cells. However in cells with disintegrated cell membranes, it crosses the membrane to stain the nucleic acids. Therefore, in cells that undergo apoptosis, the PS on the inner surface of the cell membrane will be translocated. As the process of apoptosis progresses, the loss of membrane integrity enables the diffusion of PI into the cell body for binding to the nucleic acids. Therefore, the cells at the apoptotic stage will show first Annexin V positive green ring due to PS translocation and then PI positive red cell nuclei and membrane break-down. On the other hand, for necrotic cells without programmed cell death process, only PI positive red cell nuclei will be seen. Generally, 20 μΐ of PI, diluted with deionized water (1:10) and 10 μΐ of Annexin V are added to per ml of cell culture media. These solution thus allowing real-time monitoring of apoptotis due to their low toxicity.

[0091] Alternatively, label free detection of cellular responses can be envisaged by e.g. calorimetric measurements. This allows the measurement of e.g. metabolic activities in a cell by detection with for example a sensitive IR camera.

[0092] The present Invention also contemplates the monitoring of more than one cellular response, by for example looking at fluorescence at different wavelengths by using e.g. CY3 and CY5 dyes, or by simultaneously employing different methods for detection.

13133183.4 19 [0093] Cells or cellular components can be modified with luminescent indicators for chemical or molecular cellular properties and may be analysed in a living state. Said indicators can be introduced into the cells before or after they are challenged with test compounds and by any one or a combination of a variety of physical methods, such as, but not limited to diffusion across the cell membrane, mechanical perturbation of the cell membrane, or genetic engineering so that they are expressed in cells under prescribed conditions. Live studies permit analysis of the physiological state of the cell as reported by the indicator during its life cycle or when contacted with a test compound such as a drug or other reactive substance.

[0094] In some embodiments, assaying the cellular responses is by luminescence. In some embodiments, the luminescence is fluorescence. Exemplary fluorescent molecule include, but are not limited to, fluorescein isothiocyanate (FITC), rhodamine, malachite green, Oregon green, Texas Red, Congo red, SybrGreen, phycoerythrin, allophycocyanin, 6- carboxyfluorescein (6-FAM), 2',7'-dimethoxy4',5'-dichloro-6-carboxyfluorescein (JOE), 6- carboxy X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein (HEX), 5- carboxyfluorescein (5-FAM), N,N,N',N'tetramethyl-6-carboxyrhodamine (TAMRA), cyanine dyes (e.g. Cy5, Cy3), BODIPY dyes (e.g. BODIPY 630/650, Alexa542, etc), green fluorescent protein (GFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), and the like. Other fluorescent molecules amenable to the present invention include those available, for example, from Molecular Probes.

[0095] Means for detecting signals in general are well known to those of skill in the art. Thus, for example, radiolabels can be detected using photographic film or scintillation counters, fluorescent markers can be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with an enzyme substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and calorimetric labels are detected by simply visualizing the coloured label. Further detection means are for example micro-calorimetry and light microscopy.

[0096] Detection of cellular responses may also be accomplished by multi-step detection practices. Said practices may be, by way of example and not limitation, sandwich assays as are well-known in the art and enzymatic conversions into a detectable product.

[0097] In some embodiments, assaying is performed in real-time.

[0098] In some embodiments, assaying is an end-point assaying.

Screening method

13133183.4 20 [0099] In yet another aspect, the invention provides a method for screening a test compound for biological activity, the method comprising: (i) contacting a microarray with a microwell array; and (ii) assaying a cellular response. At least one position of the printed microarray includes a deposit comprising the test compound. The layout of the deposit aligning with at least one of the microwells in the microwell array when the printed microarray is placed on or contacted with the microwell array.

[00100] In some embodiments, the method comprising the optional step of depositing the test compound on at least one position of a printed microarray.

[00101] In some embodiments, the cell is challenged with a test compound for at least 1 hour, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 24 hours, at least 48 hours, or at least 72 before assaying for cellular response. By challenging a cell with a test compound is meant that the printed microarray is in contact with the microwell array for the stated time.

[00102] In some embodiments, the cell is allowed to culture for at least 1 hour, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 16 hours, at least 24 hours, at least 48 hours, or at least 72 after removal of the printed microarray from the microwell array before assaying for a cellular response. When the cell is allowed to culture for additional time after removal of the printed array from the microwell array, the culture media in the microwell may optionally be changed.

Device fabrication

[00103] In still yet another aspect, the invention provides a method for fabricating a high throughput screening device, the method comprising: (i) generating an array of posts on the surface of a first substrate; (ii) generating an array of microwells on the surface of a second substrate, wherein the layout of the microwells aligns with the layout of the posts on the first substrate; and (iii) depositing a test compound on at least one of the posts.

[00104] A number of materials suitable for use in fabricating the coverplate, printed microarray, and the microwell array have been in described in the art. Exemplary suitable materials include, but are not limited to, poly(ester amide), polystyrene -polyisobutylene- polystyrene block copolymer (SIS), polystyrene, polyisobutylene, polycaprolactone (PCL), poly(L-lactide), poly(D,L-lactide), poly(lactides), polylactic acid (PLA), poly(lactide-co- glycolide), poly(glycolide), polyalkylene, polyfluoroalkylene, polyhydroxyalkanoate, poly(3- hydroxybutyrate), poly(4hydroxybutyrate), poly(3 -hydroxy valerate), poly(3- hydroxybutyrate-co-3-hydroxyvalerate), poly(3-hydroxyhexanoate), poly(4-

13133183.4 21 hyroxyhexanoate), mid-chain polyhydroxyalkanoate, poly (trimethylene carbonate), poly (ortho ester), polyphosphazenes, poly (phosphoester), poly(tyrosine derived arylates), poly(tyrosine derived carbonates), polydimethyloxanone (PDMS), polyvinylidene fluoride (PVDF), polyhexafluoropropylene (HFP), polydimethylsiloxane, poly (vinylidene fluoride- cohexafluoropropylene) (PVDF-HFP), poly (vinylidene fluoride-co-chlorotrifluoroethylene) (PVDF-CTFE), poly(butyl methacrylate), poly(methyl methacrylate), poly(methacrylates), poly(vinyl acetate), poly(ethylene-co-vinyl acetate), poly(ethylene-co-vinyl alcohol), poly(ester urethanes), polyethers, polyethyleneglycols (PEGs), poly(ether-urethanes), poly(carbonate-urethanes), poly(silicone-urethanes), poly(urea-urethanes), and Poly(N- isopropylacrylamide) (pNIPAAM) and any combinations thereof.

[00105] Lithographic or other techniques known to those of skill in the art can be used to pattern practically any material for use as a printed microarray or a microwell microarray. Exemplary additional methods are disclosed in U.S. Pat. No. 6,197,575, content of which is herein incorporated in its entirety.

[00106] In general, the array of posts on the surface of a first substrate can be generated by generating a negative template of arrayed posts on a silicon wafer, and curing a mixture of a silicon elastomer and a curing agent on the patterned silicone master. For example, a silicon substrate may be coated with a photoresist, for example, SU-8 photoresist, available from MicroChem Corp. The resist may be patterned through a mask to produce a negative of the pattern desired for the posts. For example, the resist may be patterned to form an array of islands to produce the posts. The resulting post array can be used to produce a microwell microarray.

[00107] A variety of elastomer precursors and curing agents are commercially available, and one skilled in the art will be able to identify those suitable for use with embodiments of the inventions. Exemplary elastomers include, but are not limited to, silicone elastomers such as PDMS, acrylic elastomers such as VHB 4910, acrylic elastomer as produced by 3M Corporation of St. Paul, Minn., polyurethanes, thermoplastic elastomers, copolymers comprising PVDF, fluoroelastomers, polymers comprising silicone and acrylic moieties, and the like.

[00108] As using herein, the term "curing agent" includes curing agents, crosslinking agents, gelling agents, etc. One exemplary curing agent is s include, but are not limited to, Sylgard 184 (Dow Corning Corporation).

[00109] Generally, the elastomer base solution and the curing agent can be present in any ratio. In some embodiments, the elastomer base solution and the curing agent are in a ratio of

13133183.4 22 about 5:1 to about 50 to:l, from about 5:1 to about 25:1, from about 5:1 to about 20:1, or from about 10:1 to about 15:1.

[00110] The curing reaction can be carried out for any length of time sufficient for curing the particular elastomer chosen. Accordingly, in some embodiments, curing is for at least 30 minutes, at least 1 hours, at least 2 hours, at least 3 hours, at least 4 hours, or at least 6 hours.

[00111] Similarly, the curing reaction can be carried out at a temperature that is optimal for the particular elastomer chosen. Accordingly, in some embodiments, curing is at a temperature of at least 50°C, at least 60°C, at least 70°C, or at least 75°C.

[00112] The array of posts can be used to fabricate the microwell array, which can also be fabricated from any suitable polymeric material. For example, the microwells can be fabricated from a material that repels cells, e.g. a biologically non-fouling material. Non- fouling polymers are well known in the art and include, but are not limited to

polyacrylamides, polysaccharides, phospholipids, poly(ethylene glycol) (PEG), and natural polymers including but not limited to hyaluronic acid and alginate. When the microwells are fabricated from cell repelling material, the microwells can be fabricated on a substrate that provides a cell adhering surface, or the bottom surface of microwells modified, e.g. with ECM molecules such as fibronectin or collagen, to provide a cell adhering surface. Without wishing to be bound by a theory, this results in the formation of nanoliter droplets within the microwells as the liquid is withdrawn form such a microwell. This minimizes water surface tension. See, for example, Suh, et al., Langmuir 20: 6080-6083 (2004), content of which is herein incorporated by reference in its entirety. Cells in these conditions remain viable as long as medium is not evaporated. This can be accomplished by keeping the cell-seeded microwell arrays under saturated humidity conditions. Furthermore, the time in which the nanoliter droplets are exposed to the air is of short duration (e.g., less than 10 minutes, less than 5 minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes, or less than 1 minute), thus minimizing cell death.

[00113] Alternatively, the microwells can be fabricated from a material to which cells can adhere. Suitable substrates on which microwells can be fabricated include, but are not limited to, glass, plastic, polystyrene, polycarbonate, PDMS, nitrocellulose, or a metal. The metal can be one or more of gold, palladium, platinu, silver, steel or alloys or mixtures thereof.

[00114] The microwell microarray can be fabricated by micromolding. For example, a prepolymer can be stamped with a array of posts fabricated above. The prepolymer then polymerized. Without limitations, any polymerization reaction can be employed, such as

13133183.4 23 chemical cross -linking, UV cross-linking etc. Upon removal of the post mold, microwells are formed on the polymer.

[00115] The microwell microarray can also be fabricated by photolithography. For example, a prepolymer solution with the optimal photoinitiator concentration can be placed on a glass surface. To control the height of the microwells, two glass plates can be placed adjacent to the prepolymer solution and photomask placed on top of the prepolymer solution. After UV irradiation, the UV exposed regions will be cross-linked and the uncrosslinked prepolymer can be removed by thorough washing. This produces microwells having a bottom surface of glass, ensuring that cells and proteins can attach to surface while the surrounding walls of the wells can be made of any suitable polymer which can be

biologically non-fouling.

[00116] The inventors have previously reported that PEG microwells arrays can be used to control the size and shape of embryoid bodies (EBs). Karp, et al., Lab Chip 7: 786-794 (2007), content of which is herein incorporated by reference in its entirety. Accordingly, in some embodiments, microwells are fabricated from PEG. In some embodiments, the prepolymer is a mixture of two or more different PEGs. Exemplary PEGs include, but are not limited to PEG258, PEG 400, and PEG500. In some embodiments, prepolymer is polyethylene glycol diacrylate comprising a 1:1 mixture of PEG 258 and PEG 400.

[00117] Methods of printing chemical libraries on microarrays are well known in the art. Accordingly, any such method can be employed for depositing a test compound on at least one of the posts of the microarray.

[00118] The present invention may be defined in any of the following numbered paragraphs:

1. A high throughput screening device, comprising:

(i) a microwell plate comprising an array of microwells, each microwell having a predefined position; and

(ii) a coverplate comprising a microarray of one or more test compounds, at least one position of said microarray having a deposit comprising a test compound, wherein position of at least one of the deposits aligns with the position of at least one of the microwells on the microwell array.

2. The high throughput screening device of paragraph 1, wherein the microwell is of cylindrical, rectangular, conical, inverse conical, pyramid, inverse pyramid shape, or some combination of two or more thereof.

13133183.4 24 The high throughput screening device of any of paragraphs 1-2, wherein the micro well has an aperture of from about 5 μπι to about 1000 μπι.

The high throughput screening device of any of paragraphs 1-3, wherein the microwell has a depth from about 5 μπι to about 1000 μιη.

The high throughput screening device of any of paragraphs 1-4, wherein the microwell array is fabricated from polyethylene glycol (PEG) polyethylene glycol diacrlyate (PEGDA), poly(N-isopropylacrylamide) (pNIPAAM), polyacrylamide (PA A), or polyethylene glycol dimethylacrylate (PEGDMA).

The high throughput screening device of any of paragraphs 1-5, wherein the coverplate is fabricated from polydimethylsiloxane (PDMS) or polyethylene glycol (PEG).

The high throughput screening device of any of paragraphs 1-6, wherein the test compound is encapsulated in a hydrogel and/or the deposit is raised from or offset from the surface of the coverplate.

The high throughput screening device of paragraph 7, wherein the hydrogel is polyethylene glycol (PEG), polyarclyamide (PA A), or alginate.

The high throughput screening device of any of paragraphs 1-8, wherein the test compound is selected from the group consisting of small organic molecules, small inorganic molecules, polysaccharides, peptides, proteins, nucleic acids, an extract made from biological materials such as bacteria, plants, fungi, animal cells, animal tissues, and any combinations thereof.

The high throughput screening device of any of paragraphs 1-9, wherein the test compound is deposited at a concentration in the range of about O.OlnM to about lOOOmM.

The high throughput screening device of any of paragraphs 1-10, wherein the test compound is deposited in a volume in the rage of about O.OlnL to about 50nL.

The high throughput screening device of any of paragraphs 1-11, wherein the deposit of test compound further comprises an analyte capture ligand.

The high throughput screening device of paragraph 12, wherein the analyte capture ligand is selected from the group consisting of antibodies, Fab fragments, scFv, aptamers, nucleic acids, proteins, peptides, other appropriate affinity molecule, and any combinations thereof.

The high throughput screening device of any of paragraphs 12-13, wherein the test compound and the analyte capture ligand are not mixed together. The high throughput screening device of any of paragraphs 1-14, wherein at least one microwell comprises at least one cell.

The high throughput screening device of any of paragraphs 1-15, wherein the cell is attached to surface of the well.

The high throughput screening device of any of paragraphs 1-16, wherein the cell is a mammalian cell, a reptilian cell, an avian cell, a fish cell, a fungal cell, a plant cell, a yeast cell, or a bacterial cell.

The high throughput screening device of any of paragraphs 1-17, wherein the high throughput screening device assays the viability of the cell.

The high throughput screening device of any of paragraphs 1-18, wherein the high throughput screening device assays the presence or expression of an internal component of said cell.

The high throughput screening device of any of paragraphs 1-19, wherein the high throughput screening device assays the presence or expression of a nucleic acid molecule produced within the cell.

The high throughput screening device of any of paragraphs 1-20, wherein the high throughput screening device assays the activity of an enzyme produced within the cell.

The high throughput screening device of any of paragraphs 1-21, wherein the high throughput screening device assays the modulation of a receptor.

Use of a high throughput screening device of any of paragraphs 1-22 for screening a compound for biological activity.

The use of paragraph 23, wherein the screening is a high-throughput screening. The use of any of paragraphs 23-24, wherein the biological activity is elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological assay. The use of any of paragraphs 23-25, wherein the biological activity is selected from the group consisting of modulation of an enzyme activity, inactivation of a receptor, stimulation of a receptor, modulation of the expression level of one or more genes, modulation of cell proliferation, modulation of cell division, modulation of cell morphology, and any combinations thereof.

A microarray of test compounds, at least one position of said microarray comprising a deposit corresponding to a test compound, wherein position of at least one of the deposits aligns with a microwell in a microwell array, and the test compound is encapsulated in a hydrogel and/or the deposit is raised from or offset from the surface of the microarray.

The microarray of paragraph 27, wherein said position is raised from the surface by at least ΙΟμπι.

The microarray of any of paragraphs 27-28, wherein the hydrogel is PEG.

The microarray of any of paragraphs 27-29, wherein the raised position is fabricated from PDMS.

The microarray of any of paragraphs 27-30, wherein the test compound is selected from the group consisting of small organic molecules, small inorganic molecules, polysaccharides, peptides, proteins, nucleic acids, an extract made from biological materials such as bacteria, plants, fungi, animal cells, animal tissues, and any combinations thereof.

The microarray device of any of paragraphs 27-31, wherein the test compound is deposited at a concentration in the range of about O.OlnM to about lOOOmM.

The microarray of any of paragraphs 27-32, wherein the test compound is deposited in a volume in the rage of about O.OlnL to about 50nL.

The microarray of any of paragraphs 27-33, wherein the deposit of test compound further comprises an analyte capture ligand.

The microarray of paragraph 34, wherein the analyte capture ligand is selected from the group consisting of antibodies, Fab fragments, scFv, aptamers, nucleic acids, proteins, peptides, other appropriate affinity molecule, and any combinations thereof. The microarray of any of paragraphs 34-35, wherein the test compound and the analyte capture ligand are not mixed together.

The use of the microarray of any of paragraph 27-36 for screening a compound for biological activity.

The use of paragraph 37, wherein the screening comprises the step of contacting the microarray to a microwell array, and the microwell array comprises one or more cells a cell in the microwell, which microwell is at complementary position to the position of the test compound deposit on the printed microarray.

The use of any of paragraphs 37-38, wherein the screening is a high-throughput screening.

The use of any of paragraphs 37-38, wherein the biological activity is elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological assay. The use of any of paragraphs 38-39, wherein the biological activity is selected from the group consisting of modulation of an enzyme activity, inactivation of a receptor, stimulation of a receptor, modulation of the expression level of one or more genes, modulation of cell proliferation, modulation of cell division, modulation of cell morphology, and any combinations thereof.

The use of any of paragraphs 37-41, wherein the cell is attached to surface of the well. The use of any of paragraphs 34-42, wherein the cell is a mammalian cell, a reptilian cell, an avian cell, a fish cell, a fungal cell, a plant cell, a yeast cell, or a bacterial cell. The use of any of paragraphs 34-43, wherein the screening assays the viability of the cell.

The use of any of paragraphs 34-44, wherein the screening assays the presence or expression of an internal component of the cell.

The use of any of paragraphs 34-45, wherein the screening assays the presence or expression of a nucleic acid molecule produced within the cell.

The use of any of paragraphs 34-46, wherein the screening assays the activity of an enzyme produced within the cell.

The use of any of paragraphs 34-47, wherein the screening assays the modulation of a receptor.

A method for screening a test compound for biological activity, the method comprising:

(i) depositing the test compound on a position of a microarray;

(ii) contacting the microarray with a microwell array, each microwell having a

predefined position, wherein the position of at least one of deposits aligns with the position of at least one of the microwells in the microwell array and the at least one of the microwells comprises one or more cells.

The method of paragraph 49, wherein the method is a high throughput screening method.

The method of any of paragraphs 49-50, wherein the microwell is of cylindrical, rectangular, inverse conical, or inverse pyramid shape, or some combination of two or more thereof.

The method of any of paragraphs 49-51, wherein the microwell has a diameter of from about 5 μπι to about 1000 μπι.

The method of any of paragraphs 49-52, wherein the microwell has a depth from about 5 μπι to about 1000 μπι. 3183.4 28 The method of any of paragraphs 49-53, wherein the microwell array is fabricated from PEGDA and/or PEGDMA.

The method of any of paragraphs 49-54, wherein the microarray is fabricated from PDMS.

The method of any of paragraphs 49-55, wherein the test compound is encapsulated in a hydrogel.

The method of paragraph 56, wherein the hydrogel is PEG.

The method of any of paragraphs 49-57, wherein the position on microarray is raised from the surface of the microarray.

The printed microarray of paragraph 58, wherein said position is raised from the surface by at least 50μπι.

The method of any of paragraphs 49-59, wherein the test compound is selected from the group consisting of small organic molecules, small inorganic molecules, polysaccharides, peptides, proteins, nucleic acids, an extract made from biological materials such as bacteria, plants, fungi, animal cells, animal tissues, and any combinations thereof.

The method of any of paragraphs 49-60, wherein the test compound is deposited at a concentration in the range of about O.OlnM to about lOOOmM.

The method of any of paragraphs 49-61, wherein the test compound is deposited in a volume in the rage of about O.OlnL to about 50nL.

The method of any of paragraphs 49-62, further comprising depositing an analyte capture ligand on the microarray.

The method of paragraph 63, wherein the analyte capture ligand is selected from the group consisting of antibodies, Fab fragments, scFv, aptamers, nucleic acids, proteins, peptides, other appropriate affinity molecule, and any combinations thereof.

The method of any of paragraphs 63-64, wherein the test compound and the analyte capture ligand are at the same position on the microarray.

The method of any of paragraphs 63-65, wherein the test compound and the analyte capture ligand are not mixed together.

The method of any of paragraphs 49-66, wherein the screening is a high-throughput screening.

The method of any of paragraphs 49-67, wherein the biological activity is elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological assay. 69. The method of any of paragraphs 49-68, wherein the biological activity is selected from the group consisting of modulation of an enzyme activity, inactivation of a receptor, stimulation of a receptor, modulation of the expression level of one or more genes, modulation of cell proliferation, modulation of cell division, modulation of cell morphology, and any combinations thereof.

70. The method of any of paragraphs 49-69, wherein the cell is attached to surface of the well.

71. The method of any of paragraphs 49-70, wherein the cell is a mammalian cell, a

reptilian cell, an avian cell, a fish cell, a fungal cell, a plant cell, a yeast cell, or a bacterial cell.

72. The method of any of paragraphs 49-71, wherein the screening assays the viability of the cell.

73. The method of any of paragraphs 49-72, wherein the screening assays the presence or expression of an internal component of said cell.

74. The method of any of paragraphs 49-73, wherein the screening assays the presence or expression of a nucleic acid molecule produced within the cell.

75. The method of any of paragraphs 49-74, wherein the screening assays the activity of an enzyme produced within the cell.

76. The method of any of paragraphs 49-75, wherein the screening assays the modulation of a receptor.

77. A method for fabricating a high throughput screening device, the method comprising:

(i) generating an array of posts on surface of a first substrate, each post having a predefined position;

(ii) generating an array of microwells on surface of a second substrate, wherein position of the microwells aligns with the position of the posts on the first substrate; and

(iii) depositing a test compound on at least one of the posts.

78. The method of paragraph 77, wherein the posts have a height of at least ΙΟμπι.

79. The method of any of paragraphs 77-78, wherein the microwell is of cylindrical, rectangular, inverse conical, or inverse pyramid shape, or some combination of two or more thereof.

80. The method of any of paragraphs 77-79, wherein the microwell has an aperture of from about 5 μπι to about 1000 μπι.

13133183.4 30 81. The method of any of paragraphs 77-80, wherein the microwell has a depth from about 5 μπι to about 1000 μπι.

82. The method of any of paragraphs 77-81, wherein the depositing of the test compound is by a piezo-mciroarrrayer.

83. The method of any of paragraphs 77-82, wherein the test compound is encapsulated in a hydrogel.

84. The method of paragraph 83, wherein the hydrogel is PEG.

85. The method of any of paragraphs 77-84, wherein the test compound is selected from the group consisting of small organic molecules, small inorganic molecules, polysaccharides, peptides, proteins, nucleic acids, an extract made from biological materials such as bacteria, plants, fungi, animal cells, animal tissues, and any combinations thereof.

86. The method of any of paragraphs 77-85, wherein the test compound is deposited at a concentration in the range of about O.OlnM to about lOOOmM.

87. The method of any of paragraphs 77-86, wherein the test compound is deposited in a volume in the rage of about O.OlnL to about 50nL.

88. The method of any of paragraphs 77-87, wherein the deposit of test compound further comprises an analyte capture ligand.

89. The method of paragraph 88, wherein the analyte capture ligand is selected from the group consisting of antibodies, Fab fragments, scFv, aptamers, nucleic acids, proteins, peptides, other appropriate affinity molecule, and any combinations thereof.

90. The method of any of paragraphs 88-89, wherein the test compound and the analyte capture ligand are not mixed together.

91. The method of any of paragraphs 77-90, wherein at least one microwell comprises at least one cell.

92. The method of paragraph 91, wherein the cell is covalently attached to surface of the microwell.

93. The method of any of paragraphs 91-92, wherein the cell is a mammalian cell, a

reptilian cell, an avian cell, a fish cell, a fungal cell, a plant cell, a yeast cell, or a bacterial cell.

94. The method of any of paragraphs 77-93, wherein the high throughput screening

device assays a biological activity of the test compound.

95. The method of paragraph 94, wherein the biological activity is elicitation of a

stimulatory, inhibitory, regulatory, toxic or lethal response in a biological assay.

13133183.4 31 96. The use of any of paragraphs 94-95, wherein the biological activity is selected from the group consisting of modulation of an enzyme activity, inactivation of a receptor, stimulation of a receptor, modulation of the expression level of one or more genes, modulation of cell proliferation, modulation of cell division, modulation of cell morphology, and any combinations thereof.

97. The method of any of paragraphs 77-96, wherein the high throughput screening

device assays the viability of the cell.

98. The method of any of paragraphs 77-97, wherein the high throughput screening

device assays the presence or expression of an internal component of said cell.

99. The method of any of paragraphs 77-98, wherein the high throughput screening

device assays the presence or expression of a nucleic acid molecule produced within the cell.

100. The method of any of paragraphs 77-99, wherein the high throughput screening

device assays the activity of an enzyme produced within the cell.

101. The method of any of paragraphs 77-100, wherein the high throughput screening device assays the modulation of a receptor.

102. The method of any of paragraphs 77-101, wherein the generating the posts comprising the steps of :

(i) generating a negative template of arrayed posts on a silicon wafer; and

(ii) curing a mixture of an elastomer base solution and a curing agent on the

negative template.

103. The method of paragraph 102, wherein the elastomer is PDMS.

104. The method of any of paragraph 102-103, wherein the curing agent is Sylgard 184 (Dow Corning Corporation).

105. The method of any of paragraphs 102-104, wherein the elastomer base solution and the curing agent are in a ratio of about 5:1 to about 50 to:l.

106. The method of any of paragraphs 102-105, wherein said curing is for at least 6 hours.

107. The method of any of paragraphs 102-106, wherein said curing is at a temperature of at least 50°C.

108. The method of any of paragraphs 77-107, wherein the generating the microwell array comprising the steps of:

(i) stamping a prepolymer with the array of posts of paragraph 103; and

(ii) polymerizing the prepolymer.

13133183.4 32 109. The method of paragraph 108, wherein the polymerizing comprises cross-linking the prepolymer.

110. The method of any of paragraphs 108-109, wherein the prepolymer is polyethylene glycol.

111. The method of any of paragraph 108-110, wherein the prepolymer is a mixture of two or more different PEGs.

112. The method of paragraph 111, wherein the two or more different PEGs are selected from the group consisting of PEG258, PEG 400, and PEG575.

113. The method of any of paragraphs 77-112, wherein the microwells are linked to

surface of the second substrate.

114. The method of any of paragraphs 77-113, wherein the second substrate is glass.

115. The method of any of paragraphs 113-114, wherein modified with 3-(trimethoxysilyl) propylmethacrylate (TMSPMA).

116. Use of a high throughput screening device of any of paragraphs 77-116 for screening a compound for biological activity.

Definitions

[00119] Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments of the aspects described herein, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[00120] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[00121] As used herein the term "consisting essentially of refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

13133183.4 33 [00122] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[00123] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about." The term "about" when used in connection with percentages may mean +1%.

[00124] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[00125] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example."

[00126] To the extent not already indicated, it will be understood by those of ordinary skill in the art that any one of the various embodiments herein described and illustrated may be further modified to incorporate features shown in any of the other embodiments disclosed herein.

[00127] The following examples illustrate some embodiments and aspects of the invention. It will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be performed without altering the spirit or scope of the invention, and such modifications and variations are encompassed within the scope of the invention as defined in the claims which follow. The following examples do not in any way limit the invention.

EXAMPLES

MATERIALS AND METHODS

[00128] Fabrication of arrayed posts. Arrayed poly(dimethylsiloxane) (PDMS) posts were fabricated by curing a 10:1 mixture of silicone elastomer base solution and curing agent (Sylgard 184; Dow Corning Corporation) on a silicon negative template. The PDMS elastomer solution was degassed for 15 minutes in a vacuum chamber and cured at 70 °C for 2h before the PDMS molds were peeled from the silicon masters. The generated PDMS replicas had patterns corresponding to the silicon master with protruding columns and were

13133183.4 34 subsequently used for molding of PEG microwells. A negative template of arrayed posts (400 μπι diameter, 150 μπι deep, 600 μπι pitch) was created on a silicon wafer, using standard photolithography techniques (Dusseiller et al., (2005) Biomaterials 26(29):5917- 5925), and the pattern and depth were analyzed with a Dektak surface profiler (Veeco Instruments, Santa Barbara, CA). Photomasks were designed using CleWin Version 2.8 (WieWeb Software, Hengelo, Netherlands) and printed on Mylar™ films at Fineline

Imaging, Inc. (Colorado Springs, CO) with 20,230 dpi resolution.

[00129] Microwell fabrication. Microwells were micromolded from polyethylene glycol diacrylate (1:1 mixture of PEG 258 and PEG 400; Sigma- Aldrich Co., St. Louis, MO) with 1% (w/w) of photoinitiator 2-hydroxy-2-methyl propiophenone (Sigma- Aldrich Co., St. Louis, MO). Arrays of microwells were bonded to 3-(trimethoxysilyl) propylmethacrylate (TMSPMA) (Sigma-Aldrich Co., St. Louis, MO) modified glass slides. A PDMS stamp of arrayed microscale posts was placed on an evenly distributed film of PEG prepolymer solution on 3-(trimethoxysilyl) propylmethacrylate (TMSPMA) modified glass slide and photocrosslinked by UV light (350-500 nm) for 600 s at lOOmW/cm (OmniCure Series 2000, EXFO, Mississauga, Canada).

[00130] Microarray printing. A non-contact piezo-microarrayer (Piezorrayer,

PerkinElimer) was used to deposit 2 nL of reagents on array PDMS posts. All printing was performed at 11 °C and 40% humidity. After printing, the chemical chip was kept in humidified condition until use.

[00131] Chemical library. The chemical library comprised 320 natural compounds with compounds having a greater than 80% purity. Compounds were stored at -80 °C until use. Prior to printing, compounds were diluted to a concentration of 16.7 μΜ in 1% DMSO in phosphate buffered saline (PBS). Compounds B010, J005, L008, P013, A005 and P011 were purchased separately from SPECS Co. Ltd.

[00132] Cell culture. MCF-7 human breast cancer cells (American Type Culture

Collection) were cultured in Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum in a humidified 5% C0₂ incubator (ThermoForma Electron) at 37°C. Microwells were seeded by pipetting lmL of media containing 200,000 cells on to the arrayed microwells. Cells were allowed to settle into the microwells for 30 minutes. Undocked cells were washed with excess media. Cell- seeded microwells were cultured for 12 hours prior to use in the device.

[00133] Scanning electron microscopy. Arrayed posts and microwells were imaged using a FESEM Ultra 55 (Zeiss, Germany) scanning electron microscope. Samples were mounted

13133183.4 35 onto aluminum stages, sputter-coated with Pt/Pd to a thickness of 200 A and analyzed at a working distance of 20 mm.

[00134] Cell viability analysis. To evaluate the cell viability, cells were incubated with 2μΜ calcein AM (Invitrogen) in PBS for 10 min at 37°C. Live cells became fluorescent under blue excitation due to enzymatic conversion of the non-fluorescent calcein to fluorescent calcein AM. Dead cells were fluorescent under green excitation after binding of eithidium homodimer to the DNA of membrane-compromised cells. Fluorescent cells were visualized with appropriate filters under an inverted microscope (Nikon Eclipse TE2000 -U).

[00135] For HTS calcein AM stained micro well arrays were imaged with a GenePix 4100a microarray scanner (Axon Instruments, Union City, CA), and fluorescence images were analyzed with GENEPIX PRO (Axon). To account for variability between independent array experiments a global normalization strategy was implemented. Viability index (VI) was used to evaluate cell viability and calculated as follows:Viability index (VI) = (Χί-μ)/δ (Eqn. 1), where X_;= live cell fluorescence, μ was the average of all the X; for all spots on each slide, and δ was the standard deviation of all the Xj for all microwells on each array.

[00136] In 96 well plate experiments, cytotoxicity was determined by Alarmar blue (Invitrogen), IC₅₀ (concentrations at which 50% inhibition of growth) was calculated by Origin 8.0 software.

Example 1: Device fabrication, operation and characterization.

[00137] A microarray system was fabricated from cell-seeded poly(ethylene glycol) (PEG) microwells and an array of polydimethylsiloxane (PDMS) posts (Fig. 1A). The proof of concept design utilized standard glass slide geometries to generate an array of 2100 posts (400 μπι in diameter, 150 μπι in height; Fig. IB) matching the layout of arrayed microwells (400 μπι in diameter, 150 μπι in height; Fig 1C), as the top and bottom of the device, respectively. When aligned and pressed together (sandwiched), each microwell was addressed by a single PDMS post, thus creating an array of sealed chambers each with a volume of 20 nL. A chemical library was printed on the ends of the arrayed posts by robotic piezo printing. Although, single compounds were printed on individual posts, multiple compounds can be printed on the same spot by repeat printings on the arrayed posts, i.e. in sequential printings, and subsequently delivered to a microwell to enable combinatorial chemical screening (Figs. 5A-5C). Figure 5D shows that there was no significant difference in fluorescence between single and multiple printing of Rhodamine B and FITC-Dextran. Without wishing to be bound by a theory, compound deposited on the end of a post is

13133183.4 36 transferred to the solution contained in the cell-seeded microwell. Thus allowing the microwells and arrayed posts to be used for cell-based screening of small molecule chemical libraries.

[00138] Cells seeded into the microwells adhered to the bottom of the microwells and formed a monolayer (Fig IE). The number of seeded cells per well can be controlled by the initial cell seeding density to enable the formation of fully or partially confluent cell cultures (Fig. IF). In the toxicology assays herein, microwells were seeded with 2xl0⁵ cells per glass slide resulting in 43+7 cells per well. The seeding density was selected so that the density of cells per microwell was similar to that used in a standard 96- well plate assay. See, for example, Yu ST, et al. (2010) Cytotoxicity and reversal of multidrug resistance by tryptanthrin-derived indoloquinazolines. Acta Pharmacologica Sinica 31(2):259-264, content which is herein incorporated by reference. Microwells can also be fabricated from cell- repellant polyethylene glycol (PEG) to create cell aggregates of controlled sizes and shapes (Figs. 6A-6E). The diameter of the cell aggregates can be regulated by varying the initial cell seeding density, with a higher seeding density resulting in larger cell aggregates (Fig. 6C). During a 6 day culture, the diameter of cell aggregates was seen to decrease in the first 2 days, and increase afterwards (Fig. 6D). FITC-dextran printed on the end of arrayed posts was delivered to cell aggregate array as cell aggregates appeared as block dots in the fluorescence image (Fig. 6E). Furthermore, cell-laden hydrogels can be integrated into the microwell arrays to enable culturing of cells in 3D. Thus the device can be used to screen the effects of chemicals on cell monolayers, aggregates and cell-laden hydrogels.

[00139] Accurate yet facile alignment of the cell-seeded microwells and the chemical- laden arrayed posts was possible with the aid of alignment features. Distinct patterns at the corners of the PDMS microwell template produced matching features on both the top and bottom chips for easy manual alignment without magnification. By simply bringing together the two chips, all posts and microwells were sealed simultaneously. Each microwell array was made by molding against the same arrayed PDMS posts, which was used both as template for microwells and for chemical delivery. The matching of arrayed PDMS posts to microwells eased the alignment process, as any imperfections produced during fabrication were identical in both halves of the device. Additionally, both halves of a device were bonded to glass slides to reduce changes in shape and size of the patterned features.

[00140] Fluorescent dyes were used to enable imaging of a working device. Concentration gradients of FITC-labeled dextran and rhodamine B were printed on the arrayed posts (Fig

13133183.4 37 2A). As the arrayed posts and microwells were sandwiched together, the printed solutions (Fig. 2C) were transferred to the solution in the microwells (Fig 2B), without cross contamination and smearing (Fig 2D). Also, no diffusion between neighboring microwells was observed, as none of the microwells was measured to contain both FITC-dextran and rhodamine B.

[00141] Although each sealed microwell contained only 20 nL of culture media, cell viability of MCF7 breast cancer cells, as judged by live/dead (calcein AM/ethidium homodimer) staining, was >90% after 24 hours (Fig. 2E,F). Negative and positive controls of 0.1% DSMO and 0.01% TritonX-100, respectively, resulted in a Z-factor > 0.5 when measuring the mean calcein AM fluorescence from arrayed microwells by fluorescent microarray scanner (Fig. 7). The Z-factor is a statistical measure of the suitability of an assay for HTS that accounts for signal range and variation (Zhang JH, Chung TD, and Oldenburg KR (1999) J Biomol Screen 4(2):67-73) and a Z-factor of 0.5 is the accepted minimum for HTS. Taken together, the positive and negative cell viability controls, and Z-factor analysis demonstrate that the device can be used to evaluate cytoxicity in response to chemical exposure.

[00142] To test the feasibility of the microarray system for high throughput screening of candidate cancer drugs, MCF-7 breast cancer cells in microwells were exposed to chemicals in sealed microwells for 24 hours, and cultured for an additional 24 hours in fresh media. MCF-7 cells were seeded 12 hours prior to the experiments in microwell arrays and cultured in minimal media supplemented with 10% fetal bovine serum. Negative and positive controls of 0.1% DMSO in PBS and 0.01% TritonX-100 in PBS, respectively were included in each chemical screen, and cell viability was determined by measuring calcein AM fluorescence in a microarray fluorescent scanner. It is noteworthy that a fluorescent microscope can also be used for these screening experiments.

[00143] To validate the device, the response of MCF-7 cells to varying doses of doxorubicin, a known chemotherapeutic (Blum RH, & Carter SK (1974) Ann Intern Med 80(2):249-259), was measured and compared to assays in a 96-well plate format.

Concentrations of doxorubicin from 1 nM to 10 μΜ were simultaneously analyzed in a single device. The dose-response cytotoxicity profile of doxorubicin for MCF-7 cells is shown in Fig. 2G and H. Despite the 10⁴-fold miniaturization, the calculated IC₅₀ from the microwell toxicology assays of 12+5.4 nM was in agreement with an IC₅₀ = 17+2.3 nM as determined in 96-well plate format. These results demonstrate that the lower number of cells and tested drug did not reduce the predictive response of the microwell array system.

13133183.4 38 Example 2: Chemical library screening.

[00144] To demonstrate the utility of the HTS device, cytotoxity of a chemical library of 320 natural compounds against breast cancer cells was tested. The library including positive and negative cell viability controls, arrayed in 384-well plates as single compounds (16.7 μΜ in 1% DMSO), was printed on the posts at a volume of 2 nL. As the posts and the microwells were sandwiched, the printed arrays were diluted at a 1:10 ratio inside the 20 nL microwell to generate a final concentration of 1.67μΜ (0.1% DMSO). Each compound from the library was printed on five replicate posts. Positive controls were printed on five replicate posts at three locations throughout the array, and were used as intra-array controls to evaluate printing consistency across arrayed posts. Negative controls of 0.1% DMSO, were printed on 200 posts. As a second negative control, no compounds were printed on the remaining posts.

[00145] Printing the chemical library for five separate devices took approximately 7 hours of printing time using a standard microarrayer. The cytotoxicity assay required 48 hours of culturing time, and data collection required an additional 10-15 minutes per slide. Thus, within a 3-day period (including 2 days of culturing time) 10,500 assays were processed with five separate devices, and up to 2100 assays can be performed with a single device.

[00146] Viability index (VI; Eqn. 1), was used to evaluate cell viability, where low VI indicates high cytotoxicity. The index is a global normalization strategy that account for the variability between independent sandwich arrays, thus allowing for assay comparison between arrays. The mean VI of the screened library is shown as a color intensity map in Fig. 3A, where red represents VI < 0, and green represents VI > 0. The mean VI of the positive viability control was -1.01, and there was no significant difference within the intra- and inter-array control (Figs 8A and 8B). The mean VI of the negative viability control was 1.01. Two hundred and eighty- five compounds (89% of the library) had a VI less than the negative control (0.1%DMSO), and 13 compounds (4%) had a VI less than the positive control (0.01% TritonX-100). Compound B010 (C-BOIO) had the lowest VI, -1.24. Three compounds that span the VI range resultant from the screened library were selected to confirm cytotoxicity in 96-well format (C-P011, C-J005 and C-BOIO). As expected, C-P011 (VI = 0.22) and C-J005 (VI = 1.35) were non-toxic at concentrations less than or equal to 10 mM. In contrast, the IC₅₀ of C-BOIO was determined to be 1.07+0.2 μΜ, indicating its high cytotoxicity.

[00147] C-BOIO, 9-methoxy-camptothecin, is an analogue of camptothecin (CPT), a naturally derived alkoid with anti-tumor efficacy. Camptothecinoids function as DNA

13133183.4 39 topoisomerase inhibitors disrupting normal DNA replication and transcription and leading to cell death. See for example, Hsiang et al., (1985) J Biol Chem 260(27):4873-4878 and Hsiang YH & Liu LF (1988) Cancer Res 48(7):1722-1726. Clinical development of CPT was ceased due to adverse side effects; however, the development of synthetic derivatives has led to the use of CPT analogues for cancer treatment (Pommier Y (2009) Chem Rev 109(7):2894- 2902). Substitutions to carbon-7, -9 and -10 have been shown to increase anti-tumor efficacy, and in some cases reducing toxic side effects (Kehrer et al., (2001) Anti-Cancer Drugs 12(2):89-105). 9-methoxy-CPT has been shown to have cytotoxic effects on MCF-7 and other cell lines (Wu SF, et al. (2008) Molecules 13(6): 1361-1371). The identification of 9- methoxy-CPT as a hit compound demonstrates that the microscale sandwich device is a useful platform for benchtop HTS.

Example 3: High throughput screening of drug-drug interactions.

[00148] To demonstrate the utility of the microarray sandwich system for testing drug- drug interactions, the inventors screened a library of natural compounds while simultaneously delivering a known vasodilator and P-gp inhibitor, verapamil. The response of MCF-7 cells to the chemical library in combination with 10 μΜ verapamil was analyzed (Fig 4).

Comparison of the library screened with and without verapamil is shown in Fig 4A as a color intensity map. The VI of each compound in the absence of verapamil is shown in descending order (Fig 4A, left), and compared to a color bar indicating the VI in the presence of verapamil (Fig 4A, right). The library screens (with and without verapamil) are also compared as a scatter plot in Fig 4B. VI data points that fall along the x-y line in Fig. 4B indicate no interaction between verapamil and the screened compound. For example, in the negative control there was no statistical difference in VI of 0.1% DMSO with or without verapamil indicating that 10 μΜ verapamil has no effect on the VI of MCF-7 cells.

Interaction effects between verapamil and a library compound result in a change in VI, and the greater the interaction effects, the greater the distance from the x-y line. Sixty percent of the library was within one standard deviation (+δ) of the mean distance to x-y line, suggesting insignificant drug-drug interactions with verapamil. This is the case for 9- methoxy-CPT (Fig 4B, yellow; Vim, = -1-23, VI_int = -0.9 ). Other CPT derivatives have been shown to be P-gp substrates including irinotecan (CPT- 11) (Bansal et al., (2009) Eur J Pharm Sci 36(4-5):580-590); however, the presence of verapamil did not potentiate the cytotoxic effects of 9-methoxy-CPT in MCF-7 cells in the library screen or in 96-well plate assays (Figs. 4B and 9).

13133183.4 40 [00149] Hits in the interaction screen were defined as compounds that were >3δ negative of the x-y line and resulted in a VI less than the negative control (data points shown in red, Fig 4B). Three compounds (<1% of the library), C-L008, C-P013 and C-A005 (Figs. 4C and 4D) met the interaction-hit criteria. The cytotoxicity of each hit in the presence and absence of verapamil was verified in 96-well plate assays (Fig. 4E). C-L008 is an analogue of Ovalichalcone, a compound isolated from the seeds of Milletia ovalifolia known to have antibacterial and anti-fungal activities. See, for example, Gupta RK & Krishnamurti M (1977) Phytochemistry 16(2):293-293 and Roy et al., (1986) Phytochemistry 25(4):961-962. C-P013 is an analogue of Amromadendrene, an oil extract of Melaleuca alternifolia with antiinflammatory properties. See, for example, Miyazawa M, Uemura T, & Kameoka H (1995) Phytochemistry 40(3):793-796 and Moreno-Dorado et al., (2003) Tetrahedron 59(39):7743- 7750. C-A005 belongs to triucallane-type triterpenes, a class of compounds that have been widely used as a gastroprotective, hypocholoesterolaemic, and anti-inflammatories. P-gp has broad substrate specificity and it is possible that C-L008, C-P013, and C-A005 are P-gp substrates, thus leading to the increase in cellular concentrations due to the inhibition of efflux by verapamil. See, for example, Robson et al., (1988) Br J Clin Pharmacol 25(3):402- 403 and Hedman et al. (1991) Clin Pharm Ther 49(3):256-262. It is also possible that the cytotoxic effects are potentiated due to other mechanisms for adverse drug-drug interactions.

[00150] A benchtop operated microarray system of the invention was used to identify four natural compounds that have the potential for anti-tumor activities. One compound, 9- methoxy-CPT, is an analogue of CPT, a well-known anti-tumor drug. Derivatives of CPT, including topotecan and irinotecan, have FDA approval for treatment of ovarian, colorectal, and lung cancers (Pommier Y (2009) Chem Rev 109(7):2894-2902). The identification of 9- methoxy-CPT in HTS device of the invention demonstrates the ability to indentify compounds at the benchtop.

[00151] While simple identification of hits is an important step in the drug discovery process, characterizing potential drug-drug interactions of the identified hits is essential to an efficient discovery process. Later-stage failures in drug testing are often attributed to complications that arise due to drug-drug interactions, and early testing of such interactions can help avoid costly failures. As such, the invention provides a HTS system that can be easily adaptable to screening drug-drug interactions and combinatorial libraries.

[00152] Rapid analysis of the experimental screening outcomes was possible with a microarray scanner. Without limitation, the screening results can also be determined It is also with a standard fluorescent microscope with, or without, automated staging. The HTS

13133183.4 41 device of the invention can be characterize as a 'benchtop' as the majority of device components are easily fabricated and operated with equipment that is common in a modern laboratory. For example, device fabrication requires a UV light source capable of generating a minimum of 0.2 mW cm^" . Access to a clean room facility was required for fabrication of a silicon template, although template fabrication is commercially available. Fabrication of the chemical array can be easily accomplished with robotic printing or spotting equipment. Such instruments are common to laboratories or academic research departments in fields that would find use for HTS. A key advantage of this platform is that the chemical library microarrays can be prepared beforehand and stored until use. Further evaluation of the stability of the chemical arrays is required to determine the storage- and shelf-life of the arrays.

[00153] In the microarray sandwich system an array of sealed chambers is created in which isolated cell-based assays are performed. Alignment and sandwiching is simple, and assays are initiated simultaneously. Contaminations between neighboring assays are prevented as each microwell is sealed. Without limitation, using this techniques, higher density arrays can be fabricated as the spacing between assays can be significantly reduced. In open microarray systems array density is limited by diffusion of analytes from assays to assays. Miniaturization of HTS also eliminates the need for large quantities of screening compounds. Here, only 40 fmoles of each library compound was required for a single assay.

Example 4: Control release of chemicals from hydrogel.

[00154] Experiments to test the controlled release and stability of various compounds from PEG hydrogels were also carried out. Briefly, the drugs were encapsulated inside s 0.5μ1 PEGDA hydrogel at a defined concentration (0.1-5% wt). To enable parallel

experimentation each microgel sample was studied in a well of a 96-well plate with a 1ml solution. The release of the compound from the hydrogel was subsequently analyzed using HPLC. The rate of release of the compounds from the hydrogels depended on a variety of factors like the porosity of the hydrogel, the affinity of the comppounds to the hydrogel and the surrounding media. The inventors optimized the porosity of the hydrogel by altering the UV crossliniing time, the concentration and the molecular weight of the PEG polymer. High molecular weight PEGDA macromers (MW 3500-10000) was used to maximize the pore diameter in the gels and to ensure proper diffusion. Furthermore, the release profile before and after drying at various times (1, 3, 10, and 20 days) to demonstrated that the encapsulated chemical arrays can be used long after they have been fabricated.

13133183.4 42 [00155] A chemical library, mixed with hydrogel, was printed on a TMSMA treated PDMS slide. Treatment of PDMS with TMSMA enabled crosslinking of the hydrogel to the substrate (Kang, et al., J. Control Release, 106: 88-98 (2005)). Initially, the 1000-member chemical library was printed in hydrogels (50μπι in diameter) by using the MicroGRID II TAS microarray spotter. The distance between two spots was chosen to be the center-center distance between two microwells, so that when sealed with the microwells, each spot on the glass addresses a single microwell. Once the prepolymerized gels are patterned, they were crosslinked by exposure to UV light. See, for example, Hyan, et al., Langmuir 17:6358-6367 (2001) and Hyan, et al., Adv. Mat. 15: 576-579 (2003). Because the surface of the PDMS slide can be acrylated, the microgels can also be anchored to the substrate. Inventors discovered that the crosslinking dod not significantly change the chemical properties of the materials.

Discussion

[00156] The emergence of combinatorial chemistries and the increased discovery of natural compounds have led to the production of expansive libraries of drug candidates and vast numbers of compounds with potentially interesting biological activities. Despite broad interest in high throughput screening (HTS) across varied fields of biological research, there has not been an increase in accessible HTS technologies.

[00157] The invention provides a simple high through screening device for screening chemical libraries and combinatorial chemical libraries in cell-based assays at the benchtop. An array of sealed chambers is created by covering the microwell array with a coverplate comprising arrayed posted, wherein the layout of the posts aligns with the microwells in the microwell array. The microarray platform delivers chemical compounds to isolated cell cultures by aligning chemical-laden arrayed posts with cell-seeded microwells. In this way, an array of sealed cell-based assays is generated without cross-contamination between neighboring assays. After chemical exposure, cell viability can be analyzed by fluorescence detection of cell viability indicator assays in a standard microarray scanner. By using standard glass slide geometries, the HTS system can be easily integrated into benchtop systems to process more than thousands of individual assays per slide each requiring less than 50 femtomoles of screening compound.

[00158] A device of the invention was used to screen a library of 320 natural compounds for potential anti-tumor agents by determining the cytotoxicity of each compound towards MCF-7 human breast cancer cells. In a second screen, the P-glycoprotein (P-gp) inhibitor,

13133183.4 43 verapamil, was delivered in combination with the library of natural compounds to screen for potentiated cytotoxicity with inhibited P-gp function. P-gp is a membrane -bound ATP- binding cassette (ABC) transport protein, and shows a significant role in drug-drug interactions by acting as an efflux carrier, a known mechanism of multi-drug resistance (Lin, J.H. Drug Deliv. Rev. 55(1): 53-81 (2003)).

[00159] In cytotoxicity screening for potential anti-tumor agents, four hits were identified from a library of 320 natural compounds with the system. Three of the hits exhibited toxicity to MCF7 breast cancer cells via drug-drug interactions with verapamil, a P-glycoprotein (P- gp) inhibitor, and a fourth hit, 9-methoxy-camptothecin, was identified in the absence of verapamil.

[00160] The systems and methods of the invention enable the screening of a wide array of individual or combinatorial libraries in a reproducible and scalable manner. The benchtop cell-based assay can be utilized for rapid and inexpensive chemical screening in the common research lab. The HTS system of the invention is amenable to a broad range of applications, including, but not limited to, combinatorial drug screening, because the system is simple, scalable, robust, and portable.

References

1. Bleicher KH, Bohm HJ, Muller K,Alanine AI (2003) Hit and lead generation: Beyond high-throughput screening. Nat Rev Drug Discov 2(5):369-378.

2. Geysen HM, Schoenen F, Wagner D,Wagner R (2003) Combinatorial compound libraries for drug discovery: An ongoing challenge. Nat Rev Drug Discov 2(3):222- 230.

3. Butler MS,Buss AD (2006) Natural products - The future scaffolds for novel

antibiotics? Biochem Pharmacol 71(7):919-929.

4. Schuster D, Laggner C,Langer T (2005) Why drugs fail— a study on side effects in new chemical entities. Curr Pharm Des l l(27):3545-3559.

5. Li AP (2001) Screening for human ADME/Tox drug properties in drug discovery.

Drug Discov Today 6(7):357-366.

6. Kremers P (2002) In vitro Tests for Predicting Drug-Drug Interactions: The Need for Validated Procedures. Pharmacol Toxicol 91(5):209-217.

13133183.4 44 Bailey SN, Sabatini DM,Stockwell BR (2004) Microarrays of small molecules embedded in biodegradable polymers for use in mammalian cell-based screens. Proc Natl Acad Sci USA 101(46):16144-16149.

Tavana H, et al. (2009) Nanolitre liquid patterning in aqueous environments for spatially defined reagent delivery to mammalian cells. Nat Mater 8(9):736-741.

Lee MY, Park CB, Dordick JS,Clark DS (2005) Metabolizing enzyme toxicology assay chip (MetaChip) for high-throughput microscale toxicity analyses. Proc Natl Acad Sci USA 102(4):983-987.

Lee MY, et al. (2008) Three-dimensional cellular microarray for high-throughput toxicology assays. Proc Natl Acad Sci USA 105(l):59-63.

Lii J, et al. (2008) Real-Time Microfluidic System for Studying Mammalian Cells in 3D Microenvironments. Anal Chem 80(10):3640-3647.

Ma B, Zhang GH, Qin JH,Lin BC (2009) Characterization of drug metabolites and cytotoxicity assay simultaneously using an integrated microfluidic device. Lab Chip 9(2):232-238.

Toh YC, et al. (2009) A microfluidic 3D hepatocyte chip for drug toxicity testing. Lab Chip 9(14):2026-2035.

Gosalia DN,Diamond SL (2003) Printing chemical libraries on microarrays for fluid phase nanoliter reactions. Proc Natl Acad Sci USA 100(15):8721-8726.

Lin JH (2003) Drug-drug interaction mediated by inhibition and induction of P- glycoprotein. Adv Drug Deliv Rev 55(1):53-81.

Yu ST, et al. (2010) Cytotoxicity and reversal of multidrug resistance by tryptanthrin- derived indoloquinazolines. Acta Pharmacologica Sinica 31(2):259-264.

Zhang JH, Chung TD,01denburg KR (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen 4(2):67-73.

Blum RH,Carter SK (1974) Adriamycin. A New Anticancer Drug with Significant Clinical Activity. Ann Intern Med 80(2):249-259.

BLUM RH,CARTER SK (1974) Adriamycin. Annals of Internal Medicine 80(2):249- 259. Hsiang YH, Hertzberg R, Hecht S,Liu LF (1985) Camptothecin induces protein- linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem

260(27):4873-4878.

Hsiang YH,Liu LF (1988) Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res 48(7): 1722-1726. Pommier Y (2009) DNA Topoisomerase I Inhibitors: Chemistry, Biology, and Interfacial Inhibition. Chem Rev 109(7):2894-2902.

Kehrer DFS, Soepenberg O, Loos WJ, Verweij J,Sparreboom A (2001) Modulation of camptothecin analogs in the treatment of cancer: a review. Anti-Cancer Drugs 12(2):89-105.

Wu SF, et al. (2008) Camptothecinoids from the seeds of Taiwanese Nothapodytes foetida. Molecules 13(6): 1361-1371.

Bansal T, Mishra G, Jaggi M, Khar RK,Talegaonkar S (2009) Effect of P- glycoprotein inhibitor, verapamil, on oral bioavailability and pharmacokinetics of irinotecan in rats. Eur J Pharm Sci 36(4-5):580-590.

Gupta RK,Krishnamurti M (1977) A prenylated chalkone from Milletia ovalifolia. Phytochemistry 16(2):293-293.

Roy M, Mitra SR, Bhattacharyya A,Adityachaudhury N (1986) Candidone, a flavanone from Tephrosia Candida. Phytochemistry 25(4):961-962.

Miyazawa M, Uemura T,Kameoka H (1995) Biotransformation of sesquiterpenoids, ( + )-aromadendrene and ( - )-alloaromadendrene by Glomerella cingulata.

Phytochemistry 40(3):793-796.

Moreno-Dorado FJ, Lamers YMAW, Mironov G, Wijnberg JBPA,de Groot A (2003) Chemistry of (+)-aromadendrene. Part 6: Rearrangement reactions of ledene, isoledene and their epoxides. Tetrahedron 59(39):7743-7750.

Robson RA, Fraenkel M, Barratt LJ,Birkett DJ (1988) Cyclosporin-verapamil interaction. Br J Clin Pharmacol 25(3):402-403.

Hedman A, et al. (1991) Digoxin-verapamil interaction: Reduction of biliary but not renal digoxin clearance in humans. Clin Pharm Ther 49(3):256-262.

Dusseiller MR, Schlaepfer D, Koch M, Kroschewski R,Textor M (2005) An inverted microcontact printing method on topographically structured polystyrene chips for arrayed micro-3-D culturing of single cells. Biomaterials 26(29):5917-5925. [00161] All patents and other publications identified in the specification are expressly incorporated herein by reference for all purposes. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

13133183.4 47

Claims

We claim:

1. A high throughput screening device, comprising:

(iii) a microwell plate comprising an array of microwells, each microwell having a predefined position; and

(iv) a coverplate comprising a microarray of one or more test compounds, at least one position of said microarray having a deposit comprising a test compound, wherein position of at least one of the deposits aligns with the position of at least one of the microwells on the microwell array.

2. The high throughput screening device of claim 1, wherein the microwell is of

cylindrical, rectangular, conical, inverse conical, pyramid, inverse pyramid shape, or some combination of two or more thereof.

3. The high throughput screening device of any of claims 1-2, wherein the microwell has an aperture of from about 5 μπι to about 1000 μπι.

4. The high throughput screening device of any of claims 1-3, wherein the microwell has a depth from about 5 μπι to about 1000 μπι.

5. The high throughput screening device of any of claims 1-4, wherein the microwell array is fabricated from polyethylene glycol (PEG) polyethylene glycol diacrlyate (PEGDA), poly(N-isopropylacrylamide) (pNIPAAM), polyacrylamide (PAA), polyethylene glycol dimethylacrylate (PEGDMA).

6. The high throughput screening device of any of claims 1-5, wherein the coverplate is fabricated from polydimethylsiloxane (PDMS) or polyethylene glycol (PEG).

7. The high throughput screening device of any of claims 1-6, wherein the test

compound is encapsulated in a hydrogel and/or the deposit is raised from or offset from the surface of the coverplate.

8. The high throughput screening device of claim 7, wherein the hydrogel is

polyethylene glycol (PEG), polyarclyamide (PAA), or alginate.

9. The high throughput screening device of any of claims 1-8, wherein the test

compound is selected from the group consisting of small organic molecules, small inorganic molecules, polysaccharides, peptides, proteins, nucleic acids, an extract made from biological materials such as bacteria, plants, fungi, animal cells, animal tissues, and any combinations thereof.

13133183.4 48

10. The high throughput screening device of any of claims 1-9, wherein the test compound is deposited at a concentration in the range of about O.OlnM to about lOOOmM.

11. The high throughput screening device of any of claims 1-10, wherein the test

compound is deposited in a volume in the rage of about O.OlnL to about 50nL.

12. The high throughput screening device of any of claims 1-11, wherein the deposit of test compound further comprises an analyte capture ligand.

13. The high throughput screening device of claim 12, wherein the analyte capture ligand is selected from the group consisting of antibodies, Fab fragments, scFv, aptamers, nucleic acids, proteins, peptides, other appropriate affinity molecule, and any combinations thereof.

14. The high throughput screening device of any of claims 12-13, wherein the test

compound and the analyte capture ligand are not mixed together.

15. The high throughput screening device of any of claims 1-14, wherein at least one microwell comprises at least one cell.

16. The high throughput screening device of any of claims 1-15, wherein the cell is attached to surface of the well.

17. The high throughput screening device of any of claims 1-16, wherein the cell is a mammalian cell, a reptilian cell, an avian cell, a fish cell, a fungal cell, a plant cell, a yeast cell, or a bacterial cell.

18. The high throughput screening device of any of claims 1-17, wherein the high

throughput screening device assays the viability of the cell.

19. The high throughput screening device of any of claims 1-18, wherein the high

throughput screening device assays the presence or expression of an internal component of said cell.

20. The high throughput screening device of any of claims 1-19, wherein the high

throughput screening device assays the presence or expression of a nucleic acid molecule produced within the cell.

21. The high throughput screening device of any of claims 1-20, wherein the high

throughput screening device assays the activity of an enzyme produced within the cell.

22. The high throughput screening device of any of claims 1-21, wherein the high

throughput screening device assays the modulation of a receptor.

13133183.4 49

23. Use of a high throughput screening device of any of claims 1-22 for screening a compound for biological activity.

24. The use of claim 23, wherein the screening is a high-throughput screening.

25. The use of any of claims 23-24, wherein the biological activity is elicitation of a

stimulatory, inhibitory, regulatory, toxic or lethal response in a biological assay.

26. The use of any of claims 23-25, wherein the biological activity is selected from the group consisting of modulation of an enzyme activity, inactivation of a receptor, stimulation of a receptor, modulation of the expression level of one or more genes, modulation of cell proliferation, modulation of cell division, modulation of cell morphology, and any combinations thereof.

27. A microarray of test compounds, at least one position of said microarray comprising a deposit corresponding to a test compound, wherein position of at least one of the deposits aligns with a microwell in a microwell array, and the test compound is encapsulated in a hydrogel and/or the deposit is raised from or offset from the surface of the microarray.

28. The microarray of claim 27, wherein said position is raised from the surface by at least ΙΟμπι.

29. The microarray of any of claims 27-28, wherein the hydrogel is PEG.

30. The microarray of any of claims 27-29, wherein the raised position is fabricated from PDMS.

31. The microarray of any of claims 27-30, wherein the test compound is selected from the group consisting of small organic molecules, small inorganic molecules, polysaccharides, peptides, proteins, nucleic acids, an extract made from biological materials such as bacteria, plants, fungi, animal cells, animal tissues, and any combinations thereof.

32. The microarray device of any of claims 27-31, wherein the test compound is

deposited at a concentration in the range of about O.OlnM to about lOOOmM.

33. The microarray of any of claims 27-32, wherein the test compound is deposited in a volume in the rage of about O.OlnL to about 50nL.

34. The microarray of any of claims 27-33, wherein the deposit of test compound further comprises an analyte capture ligand.

35. The microarray of claim 34, wherein the analyte capture ligand is selected from the group consisting of antibodies, Fab fragments, scFv, aptamers, nucleic acids, proteins, peptides, other appropriate affinity molecule, and any combinations thereof.

13133183.4 50 The microarray of any of claims 34-35, wherein the test compound and the analyte capture ligand are not mixed together.

The use of the microarray of any of claim 27-36 for screening a compound for biological activity.

The use of claim 37, wherein the screening comprises the step of contacting the microarray to a microwell array, and the microwell array comprises one or more cells a cell in the microwell, which microwell is at complementary position to the position of the test compound deposit on the printed microarray.

The use of any of claims 37-38, wherein the screening is a high-throughput screening. The use of any of claims 37-38, wherein the biological activity is elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological assay. The use of any of claims 38-39, wherein the biological activity is selected from the group consisting of modulation of an enzyme activity, inactivation of a receptor, stimulation of a receptor, modulation of the expression level of one or more genes, modulation of cell proliferation, modulation of cell division, modulation of cell morphology, and any combinations thereof.

The use of any of claims 37-41, wherein the cell is attached to surface of the well. The use of any of claims 34-42, wherein the cell is a mammalian cell, a reptilian cell, an avian cell, a fish cell, a fungal cell, a plant cell, a yeast cell, or a bacterial cell. The use of any of claims 34-43, wherein the screening assays the viability of the cell. The use of any of claims 34-44, wherein the screening assays the presence or expression of an internal component of the cell.

The use of any of claims 34-45, wherein the screening assays the presence or expression of a nucleic acid molecule produced within the cell.

The use of any of claims 34-46, wherein the screening assays the activity of an enzyme produced within the cell.

The use of any of claims 34-47, wherein the screening assays the modulation of a receptor.

A method for screening a test compound for biological activity, the method comprising:

) depositing the test compound on a position of a microarray;

) contacting the microarray with a microwell array, each microwell having a

predefined position, wherein the position of at least one of deposits aligns with the

13133183.4 51 position of at least one of the microwells in the microwell array and the at least one of the microwells comprises one or more cells.

50. The method of claim 49, wherein the method is a high throughput screening method.

51. The method of any of claims 49-50, wherein the microwell is of cylindrical,

rectangular, inverse conical, or inverse pyramid shape, or some combination of two or more thereof.

52. The method of any of claims 49-51, wherein the microwell has a diameter of from about 5 μπι to about 1000 μπι.

53. The method of any of claims 49-52, wherein the microwell has a depth from about 5 μπι to about 1000 μπι.

54. The method of any of claims 49-53, wherein the microwell array is fabricated from PEGDA and/or PEGDMA.

55. The method of any of claims 49-54, wherein the microarray is fabricated from PDMS.

56. The method of any of claims 49-55, wherein the test compound is encapsulated in a hydrogel.

57. The method of claim 56, wherein the hydrogel is PEG.

58. The method of any of claims 49-57, wherein the position on microarray is raised from the surface of the microarray.

59. The printed microarray of claim 58, wherein said position is raised from the surface by at least 50μπι.

60. The method of any of claims 49-59, wherein the test compound is selected from the group consisting of small organic molecules, small inorganic molecules,

polysaccharides, peptides, proteins, nucleic acids, an extract made from biological materials such as bacteria, plants, fungi, animal cells, animal tissues, and any combinations thereof.

61. The method of any of claims 49-60, wherein the test compound is deposited at a

concentration in the range of about O.OlnM to about lOOOmM.

62. The method of any of claims 49-61, wherein the test compound is deposited in a

volume in the rage of about O.OlnL to about 50nL.

63. The method of any of claims 49-62, further comprising depositing an analyte capture ligand on the microarray.

64. The method of claim 63, wherein the analyte capture ligand is selected from the group consisting of antibodies, Fab fragments, scFv, aptamers, nucleic acids, proteins, peptides, other appropriate affinity molecule, and any combinations thereof.

13133183.4 52

65. The method of any of claims 63-64, wherein the test compound and the analyte capture ligand are at the same position on the microarray.

66. The method of any of claims 63-65, wherein the test compound and the analyte

capture ligand are not mixed together.

67. The method of any of claims 49-66, wherein the screening is a high-throughput

screening.

68. The method of any of claims 49-67, wherein the biological activity is elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological assay.

69. The method of any of claims 49-68, wherein the biological activity is selected from the group consisting of modulation of an enzyme activity, inactivation of a receptor, stimulation of a receptor, modulation of the expression level of one or more genes, modulation of cell proliferation, modulation of cell division, modulation of cell morphology, and any combinations thereof.

70. The method of any of claims 49-69, wherein the cell is attached to surface of the well.

71. The method of any of claims 49-70, wherein the cell is a mammalian cell, a reptilian cell, an avian cell, a fish cell, a fungal cell, a plant cell, a yeast cell, or a bacterial cell.

72. The method of any of claims 49-71, wherein the screening assays the viability of the cell.

73. The method of any of claims 49-72, wherein the screening assays the presence or expression of an internal component of said cell.

74. The method of any of claims 49-73, wherein the screening assays the presence or expression of a nucleic acid molecule produced within the cell.

75. The method of any of claims 49-74, wherein the screening assays the activity of an enzyme produced within the cell.

76. The method of any of claims 49-75, wherein the screening assays the modulation of a receptor.

77. A method for fabricating a high throughput screening device, the method comprising:

(iv) generating an array of posts on surface of a first substrate, each post having a predefined position;

(v) generating an array of microwells on surface of a second substrate, wherein position of the microwells aligns with the position of the posts on the first substrate; and

(vi) depositing a test compound on at least one of the posts.

78. The method of claim 77, wherein the posts have a height of at least ΙΟμπι.

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79. The method of any of claims 77-78, wherein the microwell is of cylindrical, rectangular, inverse conical, or inverse pyramid shape, or some combination of two or more thereof.

80. The method of any of claims 77-79, wherein the microwell has an aperture of from about 5 μπι to about 1000 μπι.

81. The method of any of claims 77-80, wherein the microwell has a depth from about 5 μπι to about 1000 μπι.

82. The method of any of claims 77-81, wherein the depositing of the test compound is by a piezo-mciroarrrayer.

83. The method of any of claims 77-82, wherein the test compound is encapsulated in a hydrogel.

84. The method of claim 83, wherein the hydrogel is PEG.

85. The method of any of claims 77-84, wherein the test compound is selected from the group consisting of small organic molecules, small inorganic molecules,

polysaccharides, peptides, proteins, nucleic acids, an extract made from biological materials such as bacteria, plants, fungi, animal cells, animal tissues, and any combinations thereof.

86. The method of any of claims 77-85, wherein the test compound is deposited at a

concentration in the range of about O.OlnM to about lOOOmM.

87. The method of any of claims 77-86, wherein the test compound is deposited in a

volume in the rage of about O.OlnL to about 50nL.

88. The method of any of claims 77-87, wherein the deposit of test compound further comprises an analyte capture ligand.

89. The method of claim 88, wherein the analyte capture ligand is selected from the group consisting of antibodies, Fab fragments, scFv, aptamers, nucleic acids, proteins, peptides, other appropriate affinity molecule, and any combinations thereof.

90. The method of any of claims 88-89, wherein the test compound and the analyte

capture ligand are not mixed together.

91. The method of any of claims 77-90, wherein at least one microwell comprises at least one cell.

92. The method of claim 91, wherein the cell is attached to surface of the microwell.

93. The method of any of claims 91-92, wherein the cell is a mammalian cell, a reptilian cell, an avian cell, a fish cell, a fungal cell, a plant cell, a yeast cell, or a bacterial cell.

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94. The method of any of claims 77-93, wherein the high throughput screening device assays a biological activity of the test compound.

95. The method of claim 94, wherein the biological activity is elicitation of a stimulatory, inhibitory, regulatory, toxic or lethal response in a biological assay.

96. The use of any of claims 94-95, wherein the biological activity is selected from the group consisting of modulation of an enzyme activity, inactivation of a receptor, stimulation of a receptor, modulation of the expression level of one or more genes, modulation of cell proliferation, modulation of cell division, modulation of cell morphology, and any combinations thereof.

97. The method of any of claims 77-96, wherein the high throughput screening device assays the viability of the cell.

98. The method of any of claims 77-97, wherein the high throughput screening device assays the presence or expression of an internal component of said cell.

99. The method of any of claims 77-98, wherein the high throughput screening device assays the presence or expression of a nucleic acid molecule produced within the cell.

100. The method of any of claims 77-99, wherein the high throughput screening device assays the activity of an enzyme produced within the cell.

101. The method of any of claims 77-100, wherein the high throughput screening device assays the modulation of a receptor.

102. The method of any of claims 77-101, wherein the generating the posts comprising the steps of :

(iii) generating a negative template of arrayed posts on a silicon wafer; and

(iv) curing a mixture of an elastomer base solution and a curing agent on the

negative template.

103. The method of claim 102, wherein the elastomer is PDMS.

104. The method of any of claim 102-103, wherein the curing agent is Sylgard 184 (Dow Corning Corporation).

105. The method of any of claims 102-104, wherein the elastomer base solution and the curing agent are in a ratio of about 5:1 to about 50 to:l.

106. The method of any of claims 102-105, wherein said curing is for at least 6 hours.

107. The method of any of claims 102-106, wherein said curing is at a temperature of at least 50°C.

108. The method of any of claims 77-107, wherein the generating the microwell array comprising the steps of:

13133183.4 55 (iii) stamping a prepolymer with the array of posts of claim 103; and

(iv) polymerizing the prepolymer.

109. The method of claim 108, wherein the polymerizing comprises cross-linking the

prepolymer.

110. The method of any of claims 108-109, wherein the prepolymer is polyethylene glycol.

111. The method of any of claim 108-110, wherein the prepolymer is a mixture of two or more different PEGs.

112. The method of claim 111, wherein the two or more different PEGs are selected from the group consisting of PEG258, PEG 400, and PEG 575.

113. The method of any of claims 77-112, wherein the microwells are linked to surface of the second substrate.

114. The method of any of claims 77-113, wherein the second substrate is glass.

115. The method of any of claims 113-114, wherein modified with 3-(trimethoxysilyl) propylmethacrylate (TMSPMA).

116. Use of a high throughput screening device of any of claims 77-116 for screening a compound for biological activity.

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