AU2002249884A1 - Organ system microarrays - Google Patents

Description

ORGAN SYSTEM MICROARRAYS

Related Applications

[001] This application claims priority to, and the benefit of U.S.S.N.

09/710,697 filed November 10, 2000, the disclosure of which is incorporated by reference herein in its entirety.

Technical Field

[002] The invention relates to organ cultures arrayed at a plurality of locations on a substrate. Each location comprises a scaffold for supporting the growth and/or proliferation of cells and at least one cell type. These organ system microarrays are useful for a wide range of applications.

Background of the Invention

[003] Most standard methods for drug screening rely on evaluating interactions between a drug and a bioeffector molecule involved in a particular pathology. Typically, isolated bioeffector molecules are arrayed on a substrate and exposed to a variety of test compounds, or, alternatively, a variety of test compounds are arrayed on a substrate and exposed to a bioeffector molecule. Test compounds which have a desired effect on a bioeffector molecule's activity (e.g., its ability to bind to another molecule or to mediate a reaction) are then identified as lead compounds for further testing in vivo. However, the disparity between the physiological relevance of the screening system used and the in vivo environment in which these compounds are required to function reduces the ability of such assays to predict the effectiveness of a lead compound in vivo. As a consequence, a large amount of time and money is expended by drug manufacturers in following "leads" that ultimately have no clinical utility. [004] Drug screening using simple cell systems has been described and provides a means for more closely replicating the in vivo conditions in which a drug must work. However, these systems are limited in that cells typically develop as monolayers in culture or in suspension (i.e., do not maintain the normal relationships found in vivo) and after a period of time become undifferentiated, losing normal function.

[005] Functional three-dimensional organ systems have been produced from in oculums isolated from organ explants of predominantly normal epithelial cells and predominantly normal differentiated mesenchymal cells. The primary utility of such organ systems has been to provide a source of tissue for tissue transplantation and/or tissue reconstruction. The idea of using microarrays to provide high throughput screening systems has been exploited in the field of genomics, where purified oligonucleotides arrayed at a plurality of locations on a substrate have been used to screen for gene expression, perform sequence analysis, and to identify compounds which bind to DNA molecules of interest. Likewise, arrays of isolated polypeptides have been used to determine protein expression profiles, and to analyze interactions between polypeptides and test compounds such as drugs. However, because these arrays use isolated biomolecules, there is a gap between the information obtainable from a given array and the in vivo biological system the array is supposed to represent. Accordingly, there is a need in the art for systems that better mimic functioning organs for use in drug screening, therapeutic design, genomic evaluation, and other applications.

Summary of the Invention

[006] The present invention provides arrays which comprise miniature organ systems, or organoids, arrayed at a plurality of locations on a substrate. The organ systems of the invention are able to maintain biological functions of the organs from which they are derived. Thus, the present invention provides a platform for predicting in vivo responses to a variety of external stimuli.

[007] According to the invention, an organoid microarray comprises a plurality of miniaturized organ systems at different locations on one or more substrates. In one embodiment, miniature organ systems (organoids) of the invention are arrayed on a substrate at a plurality of locations. For example, the substrate may be a microtiter plate and the locations may be wells of the microtiter plate. In another embodiment, each location is on a different substrate, for example a different cell culture plate. The locations may be analyzed simultaneously, sequentially, or a combination of the above. [008] Each location of the invention preferably comprises a scaffold for supporting the growth and proliferation of cells comprising at least one organ-specific cell type. Preferably, the scaffold comprises at least one component of an extracellular matrix (either naturally or synthetically derived). In a preferred embodiment of the invention, each location on the substrate contains at least two different cell types. At each location, the resulting miniature organ system mimics at least one aspect of a functional organ system. In a preferred embodiment of the invention, the organ system at each location mimics substantially all of the physiological functions of an organ. [009] Organ system microarrays are useful to predict responses to therapeutic intervention among members of a diverse patient population, and to correlate those responses with genotypic and phenotypic characteristics of the population. Accordingly, microarrays of the invention are useful to establish individualized treatment programs for patients, as well as to establish genotype- specific treatment regimens. Because organ systems of the mvention are miniaturized, the invention allows multiple potential treatments to be simultaneously screened. Alternatively, treatments are simultaneously screened on organoids prepared from samples obtained from genetically diverse members of a population in order to identify optimum treatments. Organ system microarrays are also generated to represent individual(s) having a particular genetic background (e.g., individual(s) having a congenital disease) to identify test compounds effective against a particular genetic-based disease.

[010] Organ system microarrays of the invention are also useful to categorize disease tissue based upon its responsiveness to treatment. For example, organoids comprising tumor cells are arrayed according to the underlying mutation giving rise to the tumor, and a variety of drug candidates are applied to determine which ones work best against specific tumor variants. Organoids are also useful to identify drugs that function optimally at different stages of tumor progression. In another embodiment, organoid systems are arrayed which represent a plurality of different types of cancerous tissue. In this embodiment, the microarray is used to identify drugs capable of targeting many different types of cancers. Locations representing normal organ tissue may also be provided on the microarray to identify compounds which have toxic effects restricted to cancer cells. [Oil] Organ system microarrays are also used in methods to determine the system- wide effects of a particular test compound. For example, organoids are provided which represent a plurality of different organ systems from a single individual or from a representative population of individuals. In one embodiment, organoids are generated from individual(s) having a particular genetic background (e.g., individual(s) with congenital diseases) to identify compounds effective in treating individuals with certain inherited diseases. [012] In a different aspect of the invention, organ system microarrays are provided in which organ systems representing different developmental stages of an organ or organ system are arrayed at different locations on a substrate. Alternatively, the microarray can include locations comprising organ systems from individuals having a specific type of congenital disease, as well as control organ systems at varying developmental stages. These types of organ system microarrays are used to compare developmental expression profiles in individuals having different genetic backgrounds.

[013] Organ system microarrays of the invention also serve as repositories for organ systems for future expansion and implantation into an animal, preferably a human. Organ systems for use in the invention may be genetically modified to provide cells having a normal or improved function. In still another embodiment, organoids are used to determine gene and/or protein expression profiles of organs prior to and after therapeutic intervention in order to predict the effect of a test compound. Test compounds are identified which produce a substantially similar expression profile in an organ system affected by the same disease to identify lead compounds which achieve a desired therapeutic result (e.g., amelioration of symptoms). In a preferred embodiment, the desired therapeutic result is one that mimics results obtained using a known therapeutic treatment. [014] The invention also provides a platform for identifying, confirming, and evaluating disease markers, such as genetic polymorphisms. L this embodiment, a genetic polymorphism(s) is correlated with the disease state of the tissue(s) in which it occurs. Organ system microarrays of the invention are also useful to identify other characteristics of tissue that can serve as markers for disease. Such characteristics include proteins, such as cell surface markers, cell morphology, cell-cell interactions, and others. The invention overcomes the limitations of nucleic acid arrays and protein arrays known in the art by providing proteins and nucleic acids, as well as all the other biological molecules associated with organs, at each location on the microarray, allowing for multiplex analysis of all of these molecules in an environment that more closely mimics an in vivo environment. [015] In a highly preferred embodiment, an organoid mimics substantially all of the functions of an organ in its natural environment. For example, a preferred kidney organoid comprises different types of cells that are representative of substantially all the cell types of a kidney. This organoid replicates in vivo functions of a kidney, and includes cell-cell interactions that occur in a kidney.

Detailed Description of the Invention

I. The Microarrays

[016] The present invention provides miniature organ systems arrayed at a plurality of locations on one or more substrates. Each location preferably comprises a scaffold, and most preferably a polymeric scaffold, that supports the growth and proliferation of cells and at least one cell type. In one aspect of the invention, the cells grow in and on scaffolds, forming three-dimensional structures which retain the functional properties of the organs from which the cells were derived. In a preferred embodiment, the locations comprise at least two different cell types, and preferably three to four different cell types, each of which is characteristic of the organ system represented by the microorganoid. In a more preferred embodiment, a location comprises at least five cell types. In a most preferred embodiment, a location comprises substantially all the specific cell types of an organ so as to mimic the natural cellular environment of each cell type.

A. Substrates

[017] A substrate may be any solid support that permits cells to be exposed to liquid culture media and suitable supplies of oxygen without substantial contamination. In one embodiment of the invention, the substrate is a microtiter plate and individual locations of the microarrays are formed within the wells of the microtiter plates. In another embodiment of the invention, the substrate is a microtiter plate comprising a filter at its base to permit the diffusion of nutrients and oxygen within the individual wells of the plate. In this embodiment, all the cells of the microarray are exposed to a uniform culture medium, although individual locations are separated from each other by the well walls of the plate, hi other embodiments of the invention, the substrate is a cell culture plate, a flask, a test tube, or other suitable carrier.

[018] In one embodiment of the invention, the substrate is treated so that cell attachment at sites other than a location for organoid growth is inhibited. In this embodiment, the substrate is treated with a denatured protein such as heat-inactivated albumin at sites other than organoid locations. As a consequence, cell contamination of the substrate is prevented outside of locations for organoid growth.

B. Scaffold Polymers

[019] Scaffolds used in the invention preferably are formed of either natural or synthetic polymers. Examples of natural polymers which may be used include, but are not limited to, proteins, such as albumin, collagen, synthetic polyamino acids, prolamines, and polysaccharides, such as alginate, hyaluronic acids, chitosans, and other naturally occurring biodegradable sugar polymers. Scaffold material may be homogeneous, heterogeneous (comprising different types of polymers or natural materials), or derived from a source of organ tissue (wherein scaffold material is associated with cells and does not need to be highly purified). [020] Synthetic polymers that are useful include bioerodible polymers such as poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide)

(PLGA), poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates and degradable polyurethanes. PLA, PGA and PLA/PGA copolymers are particularly useful for forming biodegradable matrices. PGA is the homopolymer of glycolic acid (hydroxyacetic acid). PLA polymers are usually prepared from the cyclic esters of lactic acids. Both L(+) and D(-) forms of lactic acid can be used to prepare the PLA polymers, as well as the optically inactive DL-lactic acid mixture of D(-) and L(+) lactic acids. Methods of preparing polylactides are well known (See, e.g., U.S. Patents, U.S. Patent No. 1,995,970 to Dorough; U.S. Patent No. 2,703,316 to Schneider; U.S. Patent No. 2,758,987 to Salzberg; U.S. Patent No. 2,951,828 to Zeile; U.S. Patent No. 2,676,945 to Higgins; and U.S. Patent No. 2,683,136; 3,531,561 to Trehu, all incorporated by reference herein). [021] Non-degradable materials may also be used to form the scaffold. Non- degradable materials include, but are not limited to, polyacrylates, ethylene-vinyl acetate polymers and other acyl substituted cellulose acetates and derivatives thereof, non-erodible polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), chlorosulphonated polyolifms, polyethylene oxide, polyvinyl alcohol, Teflon^®, and nylon. In one embodiment of the invention, pieces of polyvinyl alcohol sponges (e.g., Ivalon,™ from Unipoint Industries), or alkylated or acylated derivatives thereof, are used as scaffold materials. Methods for making non-erodible polymers are described, for example, in U.S. Patent No. 2,609,347 to Wilson; U.S. Patent No 2,653,917 to Hammon, U.S. Patent No 2,659,935 to Hammon, U.S. Patent No 2,664,366 to Wilson, U.S. Patent No 2,664,367 to Wilson, and U.S. Patent No 2,846,407 to Wilson, the teachings of which are incorporated by reference herein.

C. Scaffold Microarchitecture

[022] In an embodiment of the invention in which a scaffold is used, the three-dimensional organization of the cell type(s) at each location of the microarray is generally regulated by the microstructure of the scaffold. Specific pore sizes/density and structure of the scaffold may by controlled to regulate the pattern of cell adhesion, organization, and even function of cells. In one embodiment, the scaffold is formed of polymers having sufficient interstitial spacing to allow for free diffusion of nutrients and gases, e.g., with the range of about 100-300 microns. The three- dimensional geometry of the scaffold may further be controlled by including various synthetic materials, such as string, suture material, or sponge material within the scaffold. [023] The shape of the scaffold may be designed to maximize cell growth at each location or for future implantation of the scaffold and cells into the body of a mammal. In one embodiment of the invention, the scaffold is a flat surface, while in another embodiment of the invention, the scaffold has a tubular shape. In still another embodiment of the invention, the scaffold is a disc. Other scaffold architectures are also possible and encompassed within the scope of the invention. In embodiments of the invention in which the scaffold is a crosslinked polymer, shaping of the scaffold may be done by selectively controlling crosslinking while the polymer is cast or molded. Polymers may be cast by solvent casting to obtain a branched fiber structure. In this embodiment, a solution of polymer in an appropriate solvent, such as methylene chloride, is cast as a branching pattern relief structure. After solvent evaporation, a thin film is obtained and can be compression molded (e.g., at 30,000 psi) into an appropriate pattern.

[024] In another embodiment, molten polymers are drawn into filaments and a mesh is formed by compressing fibers together. Meshes can be solitary or entwined with other fibers. The use of branching fibers allows for an increase in surface area, maximizing the number of cells at a particular location (e.g., increasing the density of the microarray). Polymer fibers for use in the invention also include commercially available materials such as Polyglactin, an absorbable synthetic suture material which is a 90:10 copolymer of glycolide and lactide and is manufactured as NICRYL™ braided absorbable suture (Ethicon Co., Somerville, Ν.J.). See, Craig et al.: "A Biological Comparison Of Polyglactin 910 And Poly gly colic Acid Synthetic Absorbable Sutures." Vol. 141, Surg., p. 1010 (1975), the entirety of which is incorporated by reference herein. Polyglycolide fibers can be used as supplied by the manufacturer.

[025] When fibers are used to form the polymeric scaffold, cells typically attach to the fibers in multiple layers and retain their normal configurations and interrelationships. In one embodiment of the invention, the scaffold may be pre- seeded with stromal cells such as fibroblast cells, which are allowed to deposit extracellular matrix materials on the fibers and thus "prime" the fibers for the addition of more cells (See, e.g., U.S. Patent Nos. 6,022,743; 5,962,325; and 5,902,741 each of which is incorporated by reference herein).

[026] In another embodiment, cell adhesion molecules are deposited directly on the scaffold. Examples of such cell adhesion molecules include, but are not limited to, fibronectin, laminin, chondronectin, epinectin, uromorulin, and the like. In one embodiment, polymers containing attachment peptides, such as the attachment peptide RGD (Arg-Gly-Asp) are synthesized and incorporated into the matrix. Additional basement membrane components or analogues may be provided, including collagens, agar, agarose, gelatin, glycosaminoglycans, and the like. Commercially available ECM-like matrix materials such as Matrigel^® may also be used. In one embodiment, factors including nutrients, growth factors, inducers of differentiation or de-differentiation, or drugs, are incorporated into the membrane. [027] In another embodiment, a scaffold may be formed of different materials to optimize attachment of various types of cells at specific locations. Mixtures of polymers, both degradable and non-degradable, natural and synthetic, are used in another embodiment. In still a further embodiment of the invention, the scaffold material is a heterogeneous material derived from the organ tissue used to form a particular location on the array. In this embodiment, the architecture of the scaffold is substantially as it is found in vivo and can include attached cells.

[028] Scaffolds for use in the present invention are characterized with respect to mechanical or biological properties by a number of assays routinely used in the art. For example, tensile strength may be determined using an Instron tester while polymer molecular weight may be assayed by gel permeation chromatography (GPC). Glass transition temperature may be determined by differential scanning calorimetry (DSC) and bond structure by infrared (IR) spectroscopy. In vitro cell attachment and viability can be assessed using microscopy, histology, and/or by quantitative assessment using radioisotopes (e.g., tritiated thymidine) or vital stains (e.g., tetrazolium dye, trypan blue).

D. Source of Cells

[029] In one embodiment, cells are obtained by biopsy from an organ of a patient or patient population. As defined herein, an organ comprises an extracellular matrix component and at least one cell type. Typically, an organ comprises a plurality of cells types, each cell type having a defined relationship with at least one other cell type (e.g., a spatial relationship or an inductive relationship in which contact with a first cell type changes the gene expression patterns of a second cell type, or a functional relationship in which each cell type has a different function within the system). In one embodiment, the organ sample is selected from the group consisting of skin, neural tissue, muscle, bone, cartilage, liver, spleen, kidney, bladder, ureter, adrenal glands, pancreas, urothelial cells, mammary gland tissue, ductus deferens tissue, testes, trachea, arteries, thyroid glands, parathyroid glands, cardiac tissue, lung tissue or other respiratory tissue, gastrointestinal tissue, and other mesenchymal, endothelial, or epithelial tissues. [030] In one embodiment according to this aspect of the invention, cells are dissociated from the organ sample, which may be a biopsy core, and transferred to a cell culture buffer (e.g., Phosphate Buffered Saline, Hanks Buffered Saline). Cells can be dissociated by teasing apart with forceps or tweezers, or by using more force (e.g., through the use of homogenizers, French presses, grinders, blenders, sieves, insonators, and the like). Cells can further be dissociated using standard techniques such as digestion with collagenase, typsin, chymotrypsin, elastase, hyaluronidase, DNAse, pronase, dispase, or other enzyme solutions (e.g. other nuclease or protease solutions). After washing the cells several times, cells are concentrated (e.g., by centrifugation) or further purified (e.g., by adherence, flow sorting, differential centrifugation in ficoll hypaque, clonal selection followed by expansion, or filtration) and resuspended in a small volume of serum-containing media (e.g., especially fetal bovine serum or calf serum) or serum-free media, and transferred at a predetermined concentration to location(s) in the microarray. Cells may be transferred immediately, or expanded first in culture, hi a preferred embodiment, 10,000-100,000 cells per square cm are seeded at a given location. Dead or dying cells may be removed from a location by replacing the growth medium with fresh medium. [031] In another embodiment according to this aspect of the invention, cells are obtained from established cell culture lines and mixed. Cells used in the present invention may be at various stages of differentiation, and include embryonic cells, terminally differentiated cells, and/or various stages in between. [032] In one embodiment of the invention, the cells are unmodified after isolation from a biological source such as the body of a mammal. However, in other embodiments of the invention, cells are genetically or phenotypically altered to provide some additional function or to provide a normal function that the cell was previously lacking.

[033] Methods of altering cells encompassed within the scope of the invention include introducing genetic material [e.g., DNA (including vectors, genes, portions of genes, portions of regulatory sequences, origins or replication), RNA, antisense molecules, triple helix forming molecules, ribozymes, modified nucleic acids (including PNA molecules), and aptamers)] which may be provided as naked nucleic acids or within a carrier (e.g., within liposomes, or encapsulated by viral coat proteins). Genetic material may also be changed through mutagenesis (i.e., site- directed or random) through the introduction of point mutations, deletions, or insertions into the genome. In one embodiment of the invention, a reporter gene is introduced into specific "control cells" at each location. In this embodiment of the invention, a gene which produces an easily assayable phenotype, e.g., fluorescence, may be provided downstream from a cell-specific/organ-specific control element, such that induction of the reporter gene indicates that the cell is functioning properly within the miniature organ system at a particular location.

[034] Phenotypic modification includes inducing the expression of proteins or cell antigens by changing culture conditions (e.g., providing growth factors, such as cytokines, interferons, or by providing other bioactive compounds, including drugs. However, phenotypic modifications may also be caused by the genetic modifications discussed above.

[035] Finally, cells may be seeded on an extracellular matrix designed to produce tissues or organs having a defined function (e.g., bladder, kidney, or other organ function) in order to produce an organ system for use in the invention.

E. Forming the Microarrays

[036] In one embodiment, scaffolds comprising cells are formed directly at individual locations by seeding cells on previously microarrayed scaffolds. Alternatively, scaffolds are seeded with cells in larger culture dishes and then pieces of scaffold with cells attached are transferred to individual locations on the substrate. Cells may also be seeded simultaneously at locations with scaffolds or extracellular matrices. For example, cells are seeded on scaffolds or matrices derived from the same organ that is the source of the cells. [037] In one embodiment according to this aspect of the invention, polymer fibers used to generate the scaffolds are placed into a culture medium comprising cell type(s) from a selected organ system and incubated until the appropriate number of cells attach. Viability of cells on the scaffold and cell density are assayed using standard techniques known in the art (e.g., microscopically, testing representative samples with vital cell stains or measuring the incorporation of radioactive labels into cells). The scaffold with cells attached is then cut into pieces and each piece is placed at a location on the microarray. In one embodiment of the invention, the microarray comprises 1 to 40 locations with 10,000 to 10,000,000 cells at each location. Preferably, the microarray comprises at least 1-3 locations. [038] In a preferred embodiment, cells are kept oxygenated using bioreactors which may be incorporated into the culture plates. A microarray according to the invention may be kept indefinitely. In one embodiment, the microarray is frozen, and may be stored frozen and thawed for future use. In another embodiment, the microarray is maintained by changing the culture medium every 1-3 days. In a preferred embodiment, the culture medium comprises tissue specific nutrients (e.g. cytokines).

[039] Cell types in locations within the microarray may be derived from a single individual or from a plurality of individuals, hi one embodiment of the invention, the microarray comprises a plurality of locations such that the entire microarray provides the genetic diversity representative of a population. In another embodiments of the invention, the microarray reflects the genetic diversity of a defined population of individuals (e.g. Askhenazi Jews, individuals with sickle cell anemia, a pedigree). In further embodiments, the locations represent organ systems from a variety of organs from individuals with a congenital disease. In still further embodiments, each location represents a different type of tumor or cancer cell or different grades of the same cancer. A single organ system at different stages of development may also be arrayed at different locations in a single microarray. [040] In order to determine that each location comprises cell types representative of a desired organ system and that the cells have the same function and interrelations that they do in the corresponding organ from which they derive, an aliquot of the cell type is obtained to test for the expression of organ-specific markers. A variety of assays may be used, as are well known in the art, including assays to determine the presence of specific RNA molecules (e.g., Northerns, dot blots, RT- PCR, RNAse protection studies, and the like) or of antigens (e.g., immunoassays, Western blots). Secretion of organ-specific products or the production of organ- specific metabolites may also be determined by obtaining an aliquot of cell culture media from desired locations and assaying for these products or metabolites. Cell morphology is another indicia of the proper function of an organ system. The organ systems at each location mimic at least one aspect of that organ's function. In a preferred embodiment, organ systems mimic substantially all physiological functions of their in vivo counterparts (e.g., genotype, expression of biomolecules, cell-cell interactions, secretion products, metabolites, reproductive and differentiation capacities, and other characteristics).

II. Methods of Using Organ System Microarrays

[041] Organ system microarrays according to the present invention may be used in both screening assays and expression studies, providing powerful tools to examine drug interactions in diverse genetic systems. A. Population Microarrays

[042] In one aspect of the invention, a microarray comprises a plurality of locations representative of a population of individuals. These organ system microarrays reflect the sensitivities of a plurality of patients to both diseases and drugs so that when a lead compound is selected for use in a pre-clinical or clinical trial, it is more likely to be effective and safe in an "average patient" having a particular genetic background. In a preferred embodiment, samples are obtained from three individuals, and each sample is preferably represented at 1-3 locations on a microarray. In one embodiment, organs from different individuals are pooled at a given location to generate a representative organ at that location.

[043] In one embodiment according to this aspect of the invention, the population represented by the microarray is a population of individuals having a particular congenital disease. Examples of such diseases include, but are not limited to, Ataxia-telangiectasia, hypothyroidism, congenital heart disease, Osteogenesis imperfecta, Canavan disease, Castleman disease, Charcot-Marie-Tooth disease, congenital enzyme deficiencies, Kostmann's disease, sickle cell disease, Tay-Sachs disease, von Recklinghausen's disease, Cystic fibrosis, Huntington disease, muscular dystrophy, hemophilia, Zellweger syndrome, and the like. [044] In another embodiment according to this aspect of the invention, the population represented by the microarray is a population of individuals particularly susceptible to developing a particular disease (e.g., Ashkenazi Jews, susceptible to Tay-Sachs disease, African Americans, susceptible to sickle cell anemia, Native Americans, susceptible to SCID). In a further embodiment, microarrays are generated which represent organ systems from individuals within a pedigree, sharing a heritable defect (e.g., familial breast cancer, familial Alzheimer's). In still a further embodiment of the invention, the microarray comprises locations representing organ systems from a plurality of individuals suspected of having apolygenic disease (e.g., bipolar disorder, and schizophrenia), hi still another embodiment, the microarray comprises locations of organ systems of individuals exposed to a particular environment (e.g., a geographic area associated with a high risk of cancer).

[045] Organ system microarrays provide a platform for determining a genetic profile associated with a disease state. As defined herein, a "genetic profile" is a genetic marker or series of genetic markers (e.g., polymorphisms) which correlate with a disease state in which they occur. Because organ system microarrays according to the invention provide the ability to correlate genotype with phenotype at a microlevel, identifying these polymorphisms provides useful markers for disease. The invention is not limited to examining nucleic acid differences in such systems, but can be used to identify multiple different types of tissue characteristics as markers. Such characteristics include proteins, cell surface markers, cell morphology, cell-cell interactions and others. In some embodiments, combinations of characteristics may be used to provide a disease profile.

[046] In one embodiment, the organ system microarrays are used for both expression profiling (e.g., assaying expression profiles of RNA or proteins of the organ systems) and for assaying the efficacy of test or lead compounds. As defined herein "expression profiles" refer to data relating to the expression of at least one gene product (i.e., RNA and/or protein), and preferably multiple gene products. Where a pattern of gene expression is diagnostic of an organ system state, a "signature profile" is obtained. Comparing an expression profile to a signature profile allows a determination to be made concerning a test organ system's state. When a test organ system's expression profile is substantially similar to a signature profile (i.e., is statistically 95% likely to be the same), the test organ's system is confirmed as having that particular state. [047] In one embodiment of the invention, the effect of test compounds on the expression profiles of an organ system exposed to a disease is evaluated. In this embodiment, the expression profile of organ system(s) from a patient with a disease is determined, as well as the expression profile of the same organ system(s) from a healthy individual, to derive signature profiles associated with both a disease state and a disease-free state. [048] In a further embodiment of the invention, the expression profile of an organ system derived from a patient having the disease and being treated with a drug known to be effective is determined. A signature profile is determined for this organ system when a particular therapeutic endpoint is reached (e.g., when the patient no longer shows symptoms) to obtain an expression profile for a treated state. Test compounds are then assayed for their effects on test organ systems derived from patients having the disease, and lead compounds are identified which are able to generate expression profiles in these organ systems substantially similar to either a healthy state or a treated state. B. Tumor Microarrays

[049] In one aspect of the invention, organ system microarray provides a plurality of locations, with subsets of locations representing different grades of a single type of tumor. In one embodiment according to this aspect of the invention, each subset represents a population of individuals. In a different embodiment, the microarrays may be designed to represent a genetic background particularly susceptible to a certain type of cancer. The microarrays are used for both expression profiling and for testing compounds such as anti-neoplastic agents. In another embodiment, a correlation between the expression profile of a grade of tumor which is untreated and one wliich is treated with a compound known to be effective in vivo is determined and applied to the identification of new lead compounds likely to be effective in vivo.

[050] In a further aspect of the invention, microarrays also include locations representing normal organ tissue. In this embodiment, the microarrays may be used to identify test compounds which specifically target cancer cells and have mimmal toxic effects on non-cancer cells.

[051] In another aspect of the invention, organ systems which represent a plurality of different types of cancerous organs are arrayed. In this embodiment, the microarray is used to identify drugs capable of targeting many different types of cancers. In another embodiment accordmg to this aspect of the invention, the microarray is used to identify expression profiles of RNA and/or proteins to identify universal markers or groups of markers unique to cancer cells. Organ systems may also be derived from tumors affecting a single tissue, but caused by different genetic mutations (e.g., breast tumors obtained from patients with BRAC1, BRAC2, HERNEU mutations, and others).

C. Multiple Organ System Microarrays

[052] In another aspect of the invention, organ system microarrays are provided in which each location represents a different kind of organ system, i.e., providing a microarray that reflects the body of an individual or a population of individuals. In this embodiment, the "body microarray" may be used to determine system-wide effects of a particular test compound. As disclosed above, body microarrays may be obtained from populations of individuals representing diverse genetic backgrounds or genetic backgrounds of interest (e.g., from individuals having congenital diseases).

D. Developmental Organ System Microarrays

[053] In a different aspect of the invention, the organ system microarrays comprise locations, each location representing a different developmental stage in the development of a specific organ. In a further embodiment according to this aspect of the invention, the microarray comprises a plurality of subsets of locations, each subset comprising developmental stages corresponding to a different organ (e.g., one subset would comprise locations including different stages of heart development, while another subset would comprise locations at different stages of liver development). In a further embodiment according to this aspect of the invention, a microarray is provided comprising control organ systems at varying developmental stages and organ systems from individuals having a specific type of congenital disease. Such microarrays are useful in expression profiling applications and to determine the effects of test compounds at specific stages of an organ's development. In one embodiment of the invention, the microarrays are used to identify teratogenic effects of compounds prior to testing in vivo.

E. Tissue Banking

[054] In one aspect of the invention, the microarray is used as a bank of transplantable organ cells which are stored for future implantation, hi this embodiment, the scaffold is provided in a form designed to be suitable for implantation in vivo into the body of a mammal. The scaffold is designed to have a sufficient surface area and exposure to nutrients such that growth and differentiation of cells can occur and such that blood vessels will ingrow into the scaffold in vivo. [055] When using the microarrays as "organ banks," the scaffold polymers are selected to meet the mechanical and biochemical parameters necessary to provide adequate support for the cells that will assimilate into the host's body and become part of the host's body (i.e., connected to the host's vasculature). Since the time required for successful implantation of an organ is greatly reduced if cells are transplanted into a prevasularized matrix, in one embodiment of the invention, the locations are used to generate small blood vessels suitable for creating larger vascular networks in vivo. [056] In one embodiment according to this aspect of the invention, the microarrays provide a bank of genetically modified organ systems. In this embodiment, cells are isolated from an organ source as described above and genetically modified by means routinely used in the art (transfection, electroporation, ballistic methods, viral infection, mutagenesis), introducing new genetic material [e.g., DNA (including vectors, genes, portions of genes, portions of regulatory sequences, origins of replication), RNA, antisense molecules, triple helix forming molecules, ribozymes, modified nucleic acids, (including PNA molecules) and aptamers], or by modifying endogenous genetic material (e.g., by mutagenesis). Genetic material within a cell may be introduced or modified prior to or after seeding the cell on the scaffold at a location. Microarrays according to this aspect of the invention provide a means of testing the biological impact of a genetic modification on an organ system prior to testing in vivo, hi a further embodiment according to this aspect of the invention, locations on the microarrays comprise at least two cell types and at least one cell type is genetically modified (either before or after placement on the scaffold). The expression profile of the at least two cell types is determined and if a desired expression profile is obtained, genetically modified cells from a location are implanted into the body of a mammal.

III. Preparing Organ System Specific Locations

A. Bladder Organ Systems

[057] In one aspect of the invention, at least one location comprises an organ system representative of human bladder tissue. In this embodiment according to this aspect of the invention, specimens are obtained and processed within one hour after surgical removal of a biopsy sample (e.g., a transmural bladder biopsy) from a patient or from an organ banking source. The sample is placed in transport media (e.g., PBS, commercial media) until processing. In this embodiment, individual tissue components of the bladder (e.g., smooth muscle cells, urothelial cells) are isolated for separate culture and reconstitution at bladder organ system location(s) on the microarray. Alternatively, the individual tissue components may themselves be deposited at locations on the microarray to generate smooth muscle cell locations and urothelial cell locations. i. Bladder Smooth Muscle Cell Cultures

[058] In one embodiment, the bladder specimen is placed under a dissecting microscope and urothelial and muscle layers are separated. Muscle layers are cut into 2-3 mm muscle segments (e.g., with iris scissors) and are spaced evenly onto 100 mm cell culture plates. The plates are left uncovered inside a cell culture hood and the segments are allowed to dry and adhere to the plate (e.g., approximately 10 minutes). Fifteen ml of Dulbecco's Modified Eagle's medium (DMEM) (HyClone Laboratories, hie, Logan, Utah) is added and the plates are covered and left undisturbed for five days. [059] Media is changed on or about the sixth day and non-adherent tissue fragments are removed. When small islands of smooth muscle cells form, as determined by inspection under a dissecting microscope, tissue fragments are removed and the media is changed. When the islands have expanded to produce a sufficient number of cells (e.g., at least 10,000 cells), the cells are trypsinized, washed, centrifuged, and resuspended in Dulbecco's Modified Eagle's medium

(DMEM) (HyClone Laboratories, Inc., Logan, Utah) with 10% fetal calf serum. Ten ml of cells at approximately 10,000 to 100,000 cells/ml are plated onto 10 cm plates. [060] Cells are fed by removing supernatant and adding new culture medium every three days, or as needed, depending on the cell density. Cells are passaged when they are 80-90% confluent by removing the medium from a plate, adding 10 ml PBS/EDTA (0.5 M), and incubating for 4 minutes to separate the cells. Separation of cells is confirmed using a phase contrast microscope by examining the separation of cell junctions. When 80-90% of the cells are separated, 5 ml of medium is added and the cells are aspirated into a 15 ml test tube and centrifuged at 1000 rpm for 5 minutes.

[061] After removing the supernatant, cells are resuspended in 5 ml of medium. Cells are assayed for viability by exposing a 100 μl aliquot of the cell suspension to trypan blue stain and counting the number of blue cells (e.g., dead cells) and total cells on a hemocytometer to determine percent viability. Approximately 1 ml volumes of 10,000 to 100,000 cells/ml are aliquoted onto 100 mm culture plates and medium is added to a total volume of 10 ml. Cells are incubated until needed in a 37°C incubator with 5% CO₂. ii Urothelial Cell Culture

[062] In one embodiment, a bladder specimen is obtained to provide a source of urothelial cells. To minimize cellular injury, the specimen is ideally sharply excised rather than cut with an electrocautery. The serosal surface is marked with a suture to ensure there is no ambiguity as to which side represents the urothelial surface.

[063] The specimen is transported in a 15 ml tube in ice cold culture medium

(e.g., Keratinocyte-SFM from GIBCO BRL (Cat. No. 17005), with Bovine Pituitary Extract (Cat. No. 13028, 25 mg/500 ml medium) and Recombinant Epidermal Growth Factor (Cat. No. 13029, 2.5 μg/500 ml medium) as supplements). The specimen is handled using sterile technique as is well known in the art of tissue culture. The specimen may be stored with refrigeration at 4°C for several hours; however, in one embodiment, the specimen is processed in a laminar flow cell culture hood as soon as possible, using sterile instruments. [064] To wipe blood from the specimen, the specimen is placed in a first 10 cm cell culture dish with ten ml of 4°C medium and is gently agitated by back and forth motion of the dish. The specimen is transferred to a second dish containing the same amount of medium where the process is repeated, and is finally transferred to a third dish, comprising 3.5 ml of medium. The urothelial surface is then scraped gently with a No. 10 scalpel blade without cutting into the specimen. Urothelial cells are observable as tiny opaque material dispersing into the medium. [065] In another embodiment of the invention, cells are isolated from a patient by introducing a catheter into the bladder to fill the bladder with an enzyme solution (e.g., a mild collagenase solution from about 0.05 to about 0.40 percent collagenase). Following irrigation of the bladder and collection of the rinses, urothelial cells are collected into medium to form a urothelial cell/medium suspension.

[066] The urothelial cell/medium suspension obtained by either biopsy or bladder irrigation is aspirated and six wells of a 24-well cell culture plate are seeded with approximately 0.5 ml of the cell suspension in each well. Another 0.5 to 1 ml of medium is added to each well to give a total of 1 to 1.5 ml per well and the cells are incubated in a 37°C incubator with 5% CO₂. On the following day (Day 1 post harvesting), medium in each well is aspirated and fresh medium is applied. The cells which are removed in the process of aspiration are centrifuged at 1000 rpm for 4 minutes and resuspended in 3 to 4.5 ml of fresh medium (warmed to 37°C) to seed an additional 3 wells in the same 24-well plate.

[067] The medium is replaced with fresh warm (37°C) medium every 2 to 3 days thereafter, until the cells are 80 to 90% confluent (e.g., about 7 to 10 days from the time of harvesting). The cells are passaged whenever they reach up to 80 to 90% confluence by removing the medium and adding PBS/EDTA solution followed by Trypsin/EDTA as above. When 80-90% of the cells appear separated from each other under a microscope, 700 μl of soy bean trypsin inhibitor is added in an additional 5 ml of Keratinocyte medium. Cells are centrifuged and resuspended in 5 ml of medium and assayed for viability using trypan blue. Each 80 to 90% confluent 10 cm dish can be passaged into three or four 10 cm dishes containing approximately 10 ml of medium. Cells are incubated until sufficient cell quantities are available for seeding onto locations comprising scaffolds (e.g., in microtiter plates) or until needed.

iii. Polymer Preparation

[068] In one embodiment of the invention, bladder organ system locations comprise biodegradable polymer scaffolds. In this embodiment, a synthetic polymer polyglycolic matrix is coated with a liquefied copolymer (poly-DL-lactide-co- glycolide 50:50, 80 mg/ml methylene chloride) in order to achieve adequate mechanical characteristics (e.g. as determined by tensiomiter, pressure studies, and elasticity compliance) and a desired shape. After sterilization with ethylene oxide, the scaffold is seeded with the cultured bladder muscle cells and/or urothelial cells. [069] In another embodiment according to this aspect of the invention, the polymer scaffold is made of naturally derived acellular collagen. A tissue source of collagen is obtained by surgical removal from a desired source, placed in a flask with sterile distilled water, and stirred by magnetic stirring at moderate speed for 24 - 48 hours at 4°C to lyse cell membranes and remove cellular debris. [070] Cells are then treated with Triton X 100 (0.5%), to remove nuclear components, and ammonium Hydroxide (0.05%), to lyse cell membranes and cytoplasmic proteins and placed in fresh distilled water for 72 more hours in a stirring flask at 4°C, with a change of water, and stirring for an additional 24 - 48 hours. A small piece of tissue is obtained and analyzed for histology to confirm the presence of any cell remnants. Typically, a small amount of tissue mass is decellularized at this time. Dense tissue may also require additional treatments with Triton X 100 and Ammonium Hydroxide. The process of washing in distilled water is repeated, until substantially all that remains is the decellularized collagen polymer which is used for the scaffold. After a final wash with distilled water, the polymer is rinsed with 1 x PBS overnight, and then packed and sterilized in cold gas (e.g., ethylene oxide) for 72 hours, to be stored until use (e.g., frozen, packaged). When the polymer scaffold is ready to use it is equilibrated in medium overnight prior to seeding. If the scaffold is used for direct applications (e.g., as scaffolding for tissue regeneration), it is equilibrated in sterile saline or PBS for 20 minutes prior to use.

[071] In a further embodiment of the invention, the microarray is used as a source of bladder cell tissue to generate an artificial organ for implantation into the body of a mammal in vivo. In this embodiment, a 10 x 10 cm synthetic polymer matrix is configured into a bladder-shaped mold using biodegradable sutures, coated with liquefied copolymer, and seeded with cells obtained from locations on the microarray. The mold may be implanted at this time or stored until used.

iv. Generating the Microarray

[072] In one embodiment, approximately 32 confluent 25 cm plates of each cell type, muscle and urothelium, is processed for seeding on scaffolds of the microarray. The polymer scaffold (e.g., biodegradable polymer or acellular collagen) is arrayed at a plurality of locations on the substrate (e.g., 1-3 wells of a microtiter dish). Alternatively, a single scaffold matrix is first seeded in a culture dish and cut into sections for depositing at different locations on a substrate. In one embodiment, the polymer scaffold is shaped to provide an exterior surface and a luminal surface at each location. The exterior surface of the polymers is seeded with resuspended smooth muscle cells and the cell-seeded polymers are incubated in DMEM, with changes of medium at 12 hour intervals to ensure a sufficient supply of nutrients. After 48 hours of incubation, the urothelial cells are processed in a similar fashion and are seeded onto a luminal surface of the polymer. The microarrays are maintained at 37°C in the presence of 5% CO₂.

[073] Attachment of cells to the scaffold may be determined microscopically. [074] To assay for the integrity and function of the organ sample, in one embodiment of the invention, cell aliquots are obtained from location(s) and lysed on a solid support (e.g., a nylon membrane) to assay the binding of the sample to antibodies which detect markers unique to urothelial cells and muscle cells (e.g., actin). It should be readily apparent to those of skill in the art that a number of different types of specific protein assays may be employed including, but not limited to asymmetric unit membranes. In some embodiments of the invention, RNA expression is monitored (e.g., by dot blotting) to determining the expression of organ- specific RNAs. In one embodiment of the invention, RT-PCR assays may be performed on a few cells, or even one cell, to determine the presence or absence of organ-specific RNA . Implantation into athymic mice may also be used to test the in vivo function of individual organ systems.

B. Kidney Organ Systems

[075] As above, in preparing kidney organ systems according to one embodiment of the invention, tissue components are isolated individually for later reconstitution of an organ system on a scaffold. In this aspect of the invention, isolated renal cells and endothelial cells may be used to form individual locations, or may be combined to generate kidney organ systems on locations on the microarray.

i. Renal Cell Isolation

[076] In one embodiment of the invention, a kidney sample is obtained, and adipose tissue, blood vessels, collecting system, and capsule, are removed under a hood using sterile technique. Using sharp tenotomy scissors, the kidney sample is cut into small pieces approximately 1 cm² in size. Kidney tissue fragments are placed in 25 ml of digestion solution (comprising DMEM, 3.1 g HEPES (Sigma H-9136); 10 ml of PSF (100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 0.25 μg/ml amphoterocin B (fungizone)), and lmg/ml collagenase/dispase) in a 50 ml tube and incubated in a 37°C shaker for 1 hour. The digestion solution is diluted 1 : 1 with culture medium (50% DMEM (50%), 50% F-12 HAM (Sigma D6421), 3.1 G/L HEPES (Sigma H-9136), 5 ml 500 ml Pen/Strep, 14 mg/L L-glutamine; and FBS 10%) and the digested kidney sample is filtered through a 200 micron sieve, 3-4 times, to remove any undigested tissue fragments. [077] Cells are centrifuged twice at 1000 RPM for 5 minutes, washing with lx PBX. Cells are resuspended in culture medium at 10,000 to 100,000 cells/ml and plated in 10 - 15 ml of medium/100 mm cell culture plate. Cell medium is changed every 3 days depending on the cell density. Cells are passaged as discussed above using a PBS/EDTA solution, followed by a 5 ml Trypsin/EDTA solution. Cells are plated at 10,000 to 100,000 cells/ml in 10 ml of media per 10 cm plate and incubated until needed in a 37°C incubator in 5% CO .

ii. Endothelial Cell Isolation from Veins

[078] Veins are dissected from a kidney sample and exposed to a heparin/papaverine solution (4 u/ml heparin, 3 mg papaverine HCL in 25 ml Hanks balanced salt solution (HBSS) to prevent spasms by the blood vessel and to improve endothelial cell preservation. A proximal silk loop is placed around the vein in order to distend the vessel and to make cannulation easier. The vessel is secured with a distal silk tie and a small venotomy is made just proximal to the tie with a No. 11 surgical blade. A vein cannula is then inserted into the vein and secured in place with a second silk tie. A second small venotomy is made just beyond the proximal tie to allow for flushing with Medium- 199 (M-199). The cannula is gently flushed with several ml of M-199/heparin to remove blood and clots. [079] The vein is then excised in a proximal to distal fashion from the kidney specimen, using surgical clips on the vein side to insure that any unseen small branches of vein are ligated. The cannula is flushed through with a collagenase solution (0.2% Worthington type I collagenase made by dissolving 200 mg collagenase in 98 ml of M-199, 1 ml of 20% Fetal Bovine Serum (FBS), 1 ml PSF, and filter-sterilized using a 0.22 micron cellulose acetate filter). After allowing approximately 1 ml of the collagenase solution to flush through, a microvascular clamp is applied to an end of the vein, allowing the vein to gently distend with collagenase. The vein is then placed into a 50 ml tube containing HBSS and 4 u/ml heparin as quickly as possible. [080] The tube-containing the vein (filled with collagenase) is placed into water bath at 37°C as soon as possible after excision and incubated for 12 minutes.

During this time, a sterile field is prepared in a laminar flow hood using a paper drape. A solution of 10-12 cc of M-199 is provided in a 15 cc tube which is placed on its side in the hood. Additional instruments required for obtaining cells are place onto the field (e.g., an open 10 cc syringe, a 18 gauge needle and sterile forceps). After the 12 minute incubation period is complete, the tube containing the vein is brought under the hood, and the vein is removed with sterile forceps from the tube. While holding the vein over a new 15 cc tube, the microvascular clamp is carefully removed and the vein is flushed with M-199 which has been loaded into the syringe. The vein is flushed forcefully about 8-10 times, collecting the M-199 which passes through the vein into the tube. Typically, flushes 2 through 10 contain endothelial cells. [081] The vein is then discarded or used for a smooth muscle cell explant. The endothelial cell/M-199 suspension is centrifuged at 125 x g for 10 minutes, and the endothelial cell pellet is resuspended in 2 ml of complete medium (Medium- 199, FBS, 100 microgram/ml ECGF, L-glutamine, 17.5 u/ml Heparin (porcine intestinal mucosa, Sigma,) and PSF) and added to the wells of a 24-well plate pre-warmed at 37°C which has been coated with a gelatin-saline solution (1% Difco gelatin in 0.9% saline, stored at 4°C for at least 24 hours).

[082] On day one of plating, the media is removed and the cells are rinsed once with HBSS which is then replaced with 1 ml of fresh complete media. The plate is examined using a phase contrast microscope to confirm the appearance of clusters of endothelial cells spread throughout each well of the plate. Media is changed 3 times/week, and typically cells isolated by this procedure reach confluence after 3-7 days. Cells are subcultured as they approach confluence (e.g., 70-80% confluence). [083] Cells are passaged as discussed above and cells are diluted to a desired number of wells or plates. Passage ratios from 1 :4 - 1 :6 are well tolerated. Cells are maintained in the incubator until needed to seed scaffolds

iii. Kidney Muscle Cells

[084] Kidney smooth muscle cells may be isolated essentially by the same process described above for bladder muscle cells, using the vein explants obtained in the above procedure.

iv. Forming the Microarray

[085] In one embodiment of the invention, renal cells are seeded on a decellularized kidney scaffold polymer prepared essentially as described above for the bladder cell microarray. Single suspended renal cells are deposited onto a wall of the scaffold and the scaffold-cell(s) are incubated for 2 hours at 37°C to allow the cell(s) to attach. The scaffold is then turned to its opposite side and cell(s) are seeded on this side and also incubated for 2 hours at 37°C. [086] After incubation is completed, medium is slowly added to the cover the scaffold with attached cells, taking care not to disturb the cells within the matrix. Medium is changed daily or more frequently, depending on the level of lactic acid, as determined by monitoring the pH of the medium using a pH indicator stick. Approximately four days after seeding, muscle cells and vascular cells are added. The integrity of the kidney micro-system at a location may be determined by assaying for kidney-specific markers such as or by inspecting cells histologically. [087] Kidney locations may be used in in vitro assays as described above or as a source of cells for future implantation. Cell scaffolds used for implantation may be expanded and cells may be seeded using static and bioreactor systems. C. Skin

[088] In one embodiment of the invention, a skin system is generated from a cell culture of human foreskin. As in the previous embodiment, individual tissue components, e.g., endothelial cells and epithelial cells, are isolated separately to reconstitute a skin system at locations on a microarray using purified components. The individual components may also be deposited at locations on a microarray to be used in assays to characterize/utilize their isolated functions.

i. Human Dermal Microvascular Endothelial cells

[089] In this embodiment, isolated human foreskin is separated into skin and subcutaneous tissue with a sterile scalpel blade in a 100 cm culture dish under a laminar flow hood and washed two or more times with collecting media (450 ml DME, 25 ml 5% FBS, 20 ml PSF, 5 ml 2 mM L-glutamine, 1 ml 100 μg/ml gentamycin sulfate (Whittaker Bioproducts, #17-518Z, 50 mg/ml stock)). The collecting medium is aspirated and discarded and segments of foreskin are transferred to a 50 ml tube in collecting medium to which is added 2 ml of 100X PSF. The tube is shaken on a shaker at room temperature for at least 4-5 hours to kill bacteria and spores that reside on the skin. [090] Segments are shaken from the tube into a sterile 100 cm culture dish and cut into 4 mm² fragments with a sterile scalpel blade and forceps. The segments are then transferred into a sterile tube with 6 ml of digestion medium (6X trypsin (0.3%) and 1% (27 mM) EDTA in HBSS ) and incubated at 37°C, with vigorous shaking, in a water bath for 10 minutes. Skin fragments are allowed to sediment by gravitational force and the digestion medium is aspirated and replaced with 20 ml wash solution (50 ml 10X HBSS, 1.26 mM CaCl₂ x 2H₂O, 0.8 mM MgSO x 7H₂O, 5%FBS, 5 ml PSF, adjusted to 500 ml with glass distilled water, sterile filtered using a 0.2 um filter, and stored at 4°C). [091] After swirling the tube containing the skin fragments vigorously, the wash solution is aspirated and 10 ml of fresh wash solution is added. The fragments are squeezed with a homogenizer and the remaining wash solution, comprising cells disassociated by homogenization, is filtered through 8 layers of sterile gauze into a 50 ml tube. Additional wash solution may be added to the fragments which are squeezed again to collect more cells, each time adding a smaller volume of wash solution. The homogenizer itself may be rinsed with 5 ml wash buffer and the collected wash solution filtered through the gauze. The gauze is rinsed with another 5 ml wash buffer to wash any cells stuck to the gauze into the tube. After this wash and homogenization cycle, there is about 30 ml of cell suspension in the tube. [092] Cells are spun at 1000 rpm or 208 x g for 10 minutes at room temperature and the supernatant aspirated. Cells are resuspended in 10 ml EBM 131 culture medium A (38.5 ml Endothelial basal medium 131 (Clonetics Corp., #CC3121), 10 ml 20% Fetal bovine serum (FBS) (Hyclone, #17-1111-L), 0.5 ml 2 mM L-glutamine (Irvine Scientific, #9317, 100X stock), 0.5 ml PSF, 0.5 ml 0.5mM dibutyryl cyclic AMP (Sigma, #D-0627), and 50 μl 91 μg/ml hydrocortisone (Sigma, #H-0888)) and plated onto a gelatin-coated pi 00 petri dish (coated with autoclaved, filtered 1% Difco Bacto Gelatin in PBS) and incubated at 37°C in the presence of 5% CO₂ overnight. [093] After the overnight incubation, cell cultures are then washed vigorously 3-4 times with 8.0 ml PBS and re-fed with 10 ml culture medium A. After 7-8 days, primary cultures of skin cells grown under these conditions are typically subconfluent. Endothelial cell patches should be clearly visible in the cell cultures without an overlaying network of dendritic cells. Medium is changed approximately every 2 days.

[094] In one embodiment of the invention, endothelial cells are further purified from the primary cultures using Dynabeads (Dynal: 1-800-638-9416, #140.03) conjugated to an endothelial-specific cell marker (e.g., UEA, Vector, #L- 1060)) according to techniques well known in the art. In this embodiment, subconfluent cell cultures (e.g., grown for about 7-8 days) are trypsinized with Trypsin EDTA, and after 1-2 washes in Trypsin/EDTA, cells are recovered in 3 x 1- ml HBSS wash buffer (containing 5% FBS and IX PSF). Trypsinized cells are centrifuged for 10 minutes at 208-x g (1000 rpm) and the cellular pellet is resuspended in 190-μl HBSS wash buffer. The cells are gently pipetted up and down several times to break up cell clusters, avoiding bubbles. The cell suspension is then transferred into a sterile 2 ml screw cap tube to which is added 5 μl UEA-I coated Dynabeads. [095] Cells are incubated with Dynabeads for 3-5 minutes, with gentle rolling to keep the beads in suspension. Endothelial cells and beads form visible tiny clusters. The cell/bead mixture is transferred into a 15 ml Falcon tube, to which 5 ml HBSS wash buffer is added and the cells are pipetted up and down with wash buffer several times. The tube containing bead-bound cells is placed into a magnetic particle concentrator (MCP-1, Dynal, #12001) and beads are collected onto the magnet for about .1 minute. Wash solution is aspirated from the tube using a pasteur pipette while the tube is in the MCP-1.

[096] The tube is then removed and the beads bound to endothelial cells are washed 3 times with 5 ml HBSS wash buffer. After the last wash, cells are resuspended in 6 ml of EBM 131 growth medium A and 3 ml of the cell suspension is plated onto a gelatin coated p60 petri dish. The cells are grown to confluence at 37°C and 5% CO₂. Medium is changed as necessary (e.g., every 3-4 days or twice a week). When the cells become confluent, they are split 1:3 or 1:4 with IX Trypsin/EDTA. Following this passage, the endothelial cells are cultured in growth medium B (Endothelial basal medium, IX GPS (Antibiotics), 10% FBS, 2 ng/ml bFGF (25 μg/ml stock solution) (Scios Nova)) until used for seeding the scaffolds.

ii. Polymer Preparation [097] In this embodiment of the invention, biodegradable polymer scaffolds or acellular collagen scaffolds may be prepared essentially as described for the bladder cell microarrays.

iii. Preparing the Microarray

[098] In one embodiment of the invention, skin cells (preferably 10,000 to

100,000 cells/ml) are seeded on a scaffold at a plurality of locations on a substrate (e.g., the wells of a microtiter plate). Alternatively, a larger scaffold is seeded in a cell culture dish and cut into pieces for placement of a cell-seeded scaffold at individual locations on the substrate.

D. Trachea

[099] In one embodiment of the invention, an organ system representing the cell relationships of the trachea is provided at at least one location on a substrate.

i. Cartilage Tissue Harvest

[0100] In one embodiment of the invention, a cartilage specimen from trachea is obtained and is cut under sterile conditions into 2-3 mm fragments. The fragments are placed in a 3% collagenase solution (300 mg collagenase crystal powder in 10 ml F-12 medium, filter sterilized and frozen in 20 or 50 ml aliquots) and incubated in a 37°C shaking incubator for 8-12 hours or until digested. The size of the fragments is checked frequently to monitor digestion. After digestion, the sample is filtered through nylon mesh to remove undigested cartilage tissue, centrifuged twice at 1000 RPM for 12 minutes, with a wash in IX PBS (without Ca²⁺ and Mg²⁺). Cells are resuspended in 10 ml of medium and the viability of a 100 μl aliquot is determined using trypan blue. Cells are plated on a 100 mm plate in a volume of 10 ml medium (500 ml F-12 medium, 10% Fetal Bovine Serum, 25 mg Vitamin C, PSF) at a concentration of 10,000 to 100,000 cells/ml and placed in an incubator at 37°C in the presence of 5% CO₂. Cells are fed every three days, depending on cell density, by removing and replacing the cell media. [100] Cells are passaged as disclosed above and the cells are placed in the incubator, with further passages, until needed. ii. Polymer Preparation

[101] In this embodiment of the invention, biodegradable polymer scaffolds or acellular collagen scaffolds may be prepared essentially as described for the bladder cell microarrays.

iii. Preparing the Microarray

[102] In one embodiment of the invention, tracheal chondrocyte cells (at a concentration of 50 x 10⁶ cells/ml) are seeded on a scaffold at a plurality of locations on a substrate (e.g., the wells of a microtiter plate). Alternatively, a larger scaffold immersed in F-12 medium is seeded in a cell culture dish and cut into pieces for placement at individual locations on the substrate.

[103] Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention as claimed. Accordingly, the invention is to be defined not by the preceding illustrative description but instead by the spirit and scope of the following claims.

Claims (26)

What is claimed is:CLAIMS

1. An organ system microarray, comprising: a substrate having a plurality of locations, each location comprising a scaffold supporting the growth and proliferation of cells; and an aggregate of cells comprising at least one cell type, wherein said aggregate mimics at least one aspect of a functioning organ system.

2. The microarray of claim 1 , wherein at least two locations comprise cells which are genetically and/or phenotypically distinct.

3. The microarray of claim 1, wherein the locations represent organ systems from a population of individuals.

4. The microarray of claim 1, wherein the locations include an organ system from an individual with a congenital disease.

5. The microarray of claim 1 , wherein at least two locations comprise cells derived from different grades of a single type of cancer.

6. The microarray of claim 1, wherein the locations comprises organ systems at different developmental stages.

7. The microarray of claim 1, wherein the locations comprise cells derived from different types of cancer.

8. The microarray of claim 1 , wherein at least one location comprises genetically modified cells.

9. The microarray of claim 1, wherein the scaffold comprises a natural polymer.

10. The microarray of claim 1, wherein the scaffold comprises at least one extracellular matrix component.

11. The microarray of claim 1 , wherein the scaffold comprises a biodegradable polymer.

12. A method of identifying a lead compound for testing in vivo in the body of a mammal, comprising: a) providing an organ system microarray comprising: a substrate having a plurality of locations, each location comprising a scaffold supporting the growth and proliferation of cells; and at least one cell type; wherein at least two locations comprise cells which are genetically and/or phenotypically distinct; b) adding a test compound to said at least two locations and assaying for an effect; and c) identifying a test compound as a lead compound if said effect is observed at at least two locations.

13. The method of claim 12, wherein the locations represent organ systems from a population of individuals.

14. The method of claim 12, wherein the locations include an organ system from an individual with a congenital disease.

15. The method of claim 12, wherein at least two locations comprise cells derived from different grades of a single type of cancer.

16. The method of claim 12, wherein the locations comprises organ systems at different developmental stages.

17. The method of claim 12, wherein the locations comprise cells derived from different types of cancer.

18. A method of predicting the therapeutic effect of a test compound, comprising:

(a) determining the expression profile of an organ system derived from a patient with a disease a who is being treated with a drug known to be effective, at a time when a therapeutic endpoint is reached;

(b) identifying a test compound which produces a substantially similar expression profile in an organ system derived from a patient having the same disease as the patient in step (a), thereby identifying a compound which may produce the same therapeutic endpoint.

19. The method of claim 18, wherein the organ system in step (a) is treated with the drug in vivo.

20. The method of claim 19, wherein cells from the organ system are sampled at the time the therapeutic endpoint is reached.

21. The method of claim 20, wherein cells from the organ system being tested in step (b) are microarrayed on a substrate at a plurality of locations.

22. The method of claim 20, wherein the therapeutic endpoint is a lack of symptoms.

23. A method of testing a source of genetically modified cells to be implanted into the body of a mammal, comprising: a) providing a substrate having a plurality of locations, each location comprising: a scaffold supporting the growth and proliferation of cells; and at least two cell types, the expression profile of which is known; b) genetically modifying at least one cell type; and d) determining the expression profile of the at least two cell types.

24. The method of claim 23, wherein the at least one cell type is modified prior to placement on the scaffold.

25. The method of claim 23, wherein if a desired expression profile is obtained, the genetically modified cells are implanted into the body of a mammal.

26. The method of claim 25, wherein the genetically modified cells are implanted with at least a portion of the scaffold.