Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0684—Cells of the urinary tract or kidneys
  • C12N5/0685—Bladder epithelial cells
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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0688—Cells from the lungs or the respiratory tract
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5082—Supracellular entities, e.g. tissue, organisms
  • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
  • C12N2502/1347—Smooth muscle cells
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  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/28—Vascular endothelial cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2503/00—Use of cells in diagnostics
  • C12N2503/02—Drug screening
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2510/00—Genetically modified cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/30—Synthetic polymers
  • C12N2533/40—Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• 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.
• 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 are then identified as lead compounds for further testing in vivo.
• 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.
• arrays of isolated polypeptides have been used to determine protein expression profiles, and to analyze interactions between polypeptides and test compounds such as drugs.
• 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.
• the present invention provides a platform for predicting in vivo responses to a variety of external stimuli.
• an organoid microarray comprises a plurality of miniaturized organ systems at different locations on one or more substrates.
• miniature organ systems (organoids) of the invention are arrayed on a substrate at a plurality of locations.
• the substrate may be a microtiter plate and the locations may be wells of the microtiter plate.
• 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.
• 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.
• the scaffold comprises at least one component of an extracellular matrix (either naturally or synthetically derived).
• each location on the substrate contains at least two different cell types.
• the resulting miniature organ system mimics at least one aspect of a functional organ system.
• the organ system at each location mimics substantially all of the physiological functions of an organ.
• 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.
• a particular genetic background e.g., individual(s) having a congenital disease
• 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.
• Organ system microarrays are also used in methods to determine the system- wide effects of a particular test compound.
• organoids are provided which represent a plurality of different organ systems from a single individual or from a representative population of individuals.
• 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.
• 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.
• 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.
• 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.
• 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).
• the desired therapeutic result is one that mimics results obtained using a known therapeutic treatment.
• the invention also provides a platform for identifying, confirming, and evaluating disease markers, such as genetic polymorphisms.
• 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.
• an organoid mimics substantially all of the functions of an organ in its natural environment.
• 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.
• 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.
• 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.
• 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.
• a location comprises at least five cell types.
• 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 substrate may be any solid support that permits cells to be exposed to liquid culture media and suitable supplies of oxygen without substantial contamination.
• the substrate is a microtiter plate and individual locations of the microarrays are formed within the wells of the microtiter plates.
• 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.
• 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.
• the substrate is treated so that cell attachment at sites other than a location for organoid growth is inhibited.
• 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.
• Scaffolds used in the invention preferably are formed of either natural or synthetic polymers.
• 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).
• Synthetic polymers that are useful include bioerodible polymers such as poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide)
• PLA 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.
• 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.
• pieces of polyvinyl alcohol sponges e.g., Ivalon,TM from Unipoint Industries
• alkylated or acylated derivatives thereof are used as scaffold materials.
• 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.
• 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.
• 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.
• 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.
• 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.
• a solution of polymer in an appropriate solvent such as methylene chloride, is cast as a branching pattern relief structure.
• an appropriate solvent such as methylene chloride
• a thin film is obtained and can be compression molded (e.g., at 30,000 psi) into an appropriate pattern.
• molten polymers are drawn into filaments and a mesh is formed by compressing fibers together.
• Meshes can be solitary or entwined with other fibers.
• 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 NICRYLTM braided absorbable suture (Ethicon Co., Somerville, ⁇ .J.).
• Polyglycolide fibers can be used as supplied by the manufacturer.
• 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).
• stromal cells such as fibroblast cells
• cell adhesion molecules are deposited directly on the scaffold.
• cell adhesion molecules include, but are not limited to, fibronectin, laminin, chondronectin, epinectin, uromorulin, and the like.
• 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.
• ECM-like matrix materials such as Matrigel ® may also be used.
• 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.
• the scaffold material is a heterogeneous material derived from the organ tissue used to form a particular location on the array.
• the architecture of the scaffold is substantially as it is found in vivo and can include attached cells.
• 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).
• radioisotopes e.g., tritiated thymidine
• vital stains e.g., tetrazolium dye, trypan blue
• cells are obtained by biopsy from an organ of a patient or patient population.
• an organ comprises an extracellular matrix component and at least one cell type.
• 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).
• 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.
• 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).
• 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).
• 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).
• cells 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.
• serum-containing media e.g., especially fetal bovine serum or calf serum
• serum-free media e.g., especially fetal bovine serum or calf serum
• 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.
• 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.
• the cells are unmodified after isolation from a biological source such as the body of a mammal.
• cells are genetically or phenotypically altered to provide some additional function or to provide a normal function that the cell was previously lacking.
• 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.
• 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.
• a reporter gene is introduced into specific "control cells" at each location.
• a gene which produces an easily assayable phenotype e.g., fluorescence
• 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.
• 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.
• culture conditions e.g., providing growth factors, such as cytokines, interferons, or by providing other bioactive compounds, including drugs.
• phenotypic modifications may also be caused by the genetic modifications discussed above.
• 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.
• a defined function e.g., bladder, kidney, or other organ function
• scaffolds comprising cells are formed directly at individual locations by seeding cells on previously microarrayed scaffolds.
• 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.
• cells are seeded on scaffolds or matrices derived from the same organ that is the source of the cells.
• 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.
• the microarray comprises 1 to 40 locations with 10,000 to 10,000,000 cells at each location.
• the microarray comprises at least 1-3 locations.
• cells are kept oxygenated using bioreactors which may be incorporated into the culture plates.
• a microarray according to the invention may be kept indefinitely.
• the microarray is frozen, and may be stored frozen and thawed for future use.
• the microarray is maintained by changing the culture medium every 1-3 days.
• the culture medium comprises tissue specific nutrients (e.g. cytokines).
• 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.
• the microarray reflects the genetic diversity of a defined population of individuals (e.g. Askhenazi Jews, individuals with sickle cell anemia, a pedigree).
• the locations represent organ systems from a variety of organs from individuals with a congenital disease.
• 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.
• an aliquot of the cell type is obtained to test for the expression of organ-specific markers.
• 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).
• 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.
• 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).
• 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 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.
• samples are obtained from three individuals, and each sample is preferably represented at 1-3 locations on a microarray.
• organs from different individuals are pooled at a given location to generate a representative organ at that location.
• the population represented by the microarray is a population of individuals having a particular congenital disease.
• 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.
• 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).
• microarrays are generated which represent organ systems from individuals within a pedigree, sharing a heritable defect (e.g., familial breast cancer, familial Alzheimer's).
• 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).
• apolygenic disease e.g., bipolar disorder, and schizophrenia
• a particular environment e.g., a geographic area associated with a high risk of cancer
• Organ system microarrays provide a platform for determining a genetic profile associated with a disease state.
• 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.
• 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.
• 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.
• 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.
• 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.
• the effect of test compounds on the expression profiles of an organ system exposed to a disease is evaluated.
• 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.
• 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.
• organ system microarray provides a plurality of locations, with subsets of locations representing different grades of a single type of tumor.
• each subset represents a population of individuals.
• 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.
• 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.
• microarrays also include locations representing normal organ tissue.
• the microarrays may be used to identify test compounds which specifically target cancer cells and have mimmal toxic effects on non-cancer cells.
• organ systems which represent a plurality of different types of cancerous organs are arrayed.
• the microarray is used to identify drugs capable of targeting many different types of cancers.
• 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).
• 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.
• the "body microarray” may be used to determine system-wide effects of a particular test compound.
• 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).
• the organ system microarrays comprise locations, each location representing a different developmental stage in the development of a specific organ.
• 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).
• 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.
• the microarrays are used to identify teratogenic effects of compounds prior to testing in vivo.
• the microarray is used as a bank of transplantable organ cells which are stored for future implantation
• 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.
• 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).
• the locations are used to generate small blood vessels suitable for creating larger vascular networks in vivo.
• the microarrays provide a bank of genetically modified organ systems.
• 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.
• 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
• Genetic material within a cell may be introduced or modified prior to or
• 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.
• At least one location comprises an organ system representative of human bladder tissue.
• 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.
• transport media e.g., PBS, commercial media
• individual tissue components of the bladder e.g., smooth muscle cells, urothelial cells
• the individual tissue components may themselves be deposited at locations on the microarray to generate smooth muscle cell locations and urothelial cell locations.
• 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.
• DMEM Dulbecco's Modified Eagle's medium
• tissue fragments are removed and the media is changed.
• 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 fetal calf serum
• Ten ml of cells at approximately 10,000 to 100,000 cells/ml are plated onto 10 cm plates.
• 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.
• cells are resuspended in 5 ml of medium.
• Cells are assayed for viability by exposing a 100 ⁇ l 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% CO 2 .
• a bladder specimen is obtained to provide a source of urothelial cells.
• 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.
• 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 ⁇ g/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.
• 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.
• 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.
• an enzyme solution e.g., a mild collagenase solution from about 0.05 to about 0.40 percent collagenase.
• 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% CO 2 . 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.
• 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.
• PBS/EDTA solution followed by Trypsin/EDTA as above.
• 70 ⁇ l 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.
• scaffolds e.g., in microtiter plates
• bladder organ system locations comprise biodegradable polymer scaffolds.
• 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.
• the scaffold is seeded with the cultured bladder muscle cells and/or urothelial cells.
• 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.
• 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.
• 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.
• cold gas e.g., ethylene oxide
• 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.
• 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.
• 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
• the substrate e.g., 1-3 wells of a microtiter dish.
• a single scaffold matrix is first seeded in a culture dish and cut into sections for depositing at different locations on a substrate.
• 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% CO 2 .
• Attachment of cells to the scaffold may be determined microscopically.
• 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).
• a solid support e.g., a nylon membrane
• RNA expression is monitored (e.g., by dot blotting) to determining the expression of organ- specific RNAs.
• 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.
• tissue components are isolated individually for later reconstitution of an organ system on a scaffold.
• 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.
• 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 cm 2 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 ⁇ g/ml streptomycin sulfate, 0.25 ⁇ g/ml amphoterocin B (fungizone)), and lmg/ml collagenase/dispase) in a 50 ml tube and incubated in a 37°C shaker for 1 hour.
• digestion solution comprising DMEM, 3.1 g HEPES (Sigma H-9136); 10 ml of PSF (100 U/ml penicillin G sodium, 100 ⁇ g/ml streptomycin sulfate, 0.25 ⁇ g/ml amphoterocin B (fungizone)
• PSF 100 U/ml penicillin G sodium, 100 ⁇ g/ml streptomycin sulfate, 0.25 ⁇ g/ml amphot
• 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.
• 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%
• the digested kidney sample is filtered through a 200 micron sieve, 3-4 times, to remove any undigested tissue fragments.
• 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
• 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 .
• 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.
• HBSS Hanks balanced salt solution
• 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).
• M-199 Medium- 199
• the cannula is gently flushed with several ml of M-199/heparin to remove blood and clots.
• 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).
• 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.
• 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).
• 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).
• complete medium Medium- 199, FBS, 100 microgram/ml ECGF, L-glutamine, 17.5 u/ml Heparin (porcine intestinal mucosa, Sigma,) and PSF
• 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.
• 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.
• 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.
• 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.
• a skin system is generated from a cell culture of human foreskin.
• 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.
• 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 ⁇ g/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.
• Segments are shaken from the tube into a sterile 100 cm culture dish and cut into 4 mm 2 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 CaCl 2 x 2H 2 O, 0.8 mM MgSO x 7H 2 O, 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.
• 20 ml wash solution 50 ml 10X HBSS, 1.26 mM CaCl 2 x 2H 2 O, 0.8 mM MgSO x 7H 2 O, 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.
• 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.
• 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.
• 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.
• Dynabeads 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.
• subconfluent cell cultures e.g., grown for about 7-8 days
• Trypsin EDTA e.g., grown for about 7-8 days
• 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- ⁇ l 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 ⁇ l 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.
• MCP-1 magnetic particle concentrator
• 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% CO 2 . 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.
• endothelial cells are cultured in growth medium B (Endothelial basal medium, IX GPS (Antibiotics), 10% FBS, 2 ng/ml bFGF (25 ⁇ g/ml stock solution) (Scios Nova)) until used for seeding the scaffolds.
• growth medium B Endothelial basal medium, IX GPS (Antibiotics), 10% FBS, 2 ng/ml bFGF (25 ⁇ g/ml stock solution) (Scios Nova)
• biodegradable polymer scaffolds or acellular collagen scaffolds may be prepared essentially as described for the bladder cell microarrays.
• skin cells preferably 10,000 to 10,000
• 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).
• a substrate e.g., the wells of a microtiter plate.
• 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.
• an organ system representing the cell relationships of the trachea is provided at at least one location on a substrate.
• 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.
• 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 Ca 2+ and Mg 2+ ).
• Cells are resuspended in 10 ml of medium and the viability of a 100 ⁇ l 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% CO 2 .
• 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
• biodegradable polymer scaffolds or acellular collagen scaffolds may be prepared essentially as described for the bladder cell microarrays.
• tracheal chondrocyte cells (at a concentration of 50 x 10 6 cells/ml) are seeded on a scaffold at a plurality of locations on a substrate (e.g., the wells of a microtiter plate).
• a substrate e.g., the wells of a microtiter plate.
• 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.

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