WO2014039427A1 - Methods of tissue generation - Google Patents

Abstract

Provided herein are methods of generating tissues and organs in vitro or ex vivo comprising depositing cells and extracellular matrix onto a surface, as well as methods of using such tissues and organs. In one embodiment, the cells and ECM used in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein are deposited as part of the same composition. In another embodiment, the cells and ECM used in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein are deposited as part of different compositions.

Description

METHODS OF TISSUE GENERATION

[0001] This application claims priority to U.S. provisional patent application No.

61/696,479, filed September 4, 2012, the disclosure of which is herein incorporated by reference in its entirety.

1. INTRODUCTION

[0002] Provided herein are methods of generating tissues and organs in vitro or ex vivo comprising depositing cells and/or an extracellular matrix onto a surface, as well as methods of using such tissues and organs.

2. BACKGROUND

[0003] Bioprinting (e.g., organ printing) is an area of research and engineering that involves printing devices, such as modified ink-jet printers, that deposit biological material. The technology involves the rapid creation and release of liquid droplets comprising cells followed by their precise deposition on a surface. Tissues and organs engineered using basic cellular materials by means of bioprinting represent a promising alternative to the donor-derived tissues and organs that are used today in standard transplantation approaches.

3. SUMMARY

[0004] In one aspect, provided herein is a method for generating a tissue (e.g., a three- dimensional tissue) or an organ comprising depositing cells and/or an extracellular matrix (ECM) onto a surface in vitro or ex vivo so as to form said tissue. Cells that may be used in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein are described in Section 4.1.1, below. Tissues and Organs that may be engineered in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein are described in Section 4.1.2, below. ECM that may be used in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein is described in Section 4.1.3, below. Surfaces onto which cells, ECM, and/or additional components may be deposited in accordance with the methods for generating tissues (e.g., three- dimensional tissues) and organs described herein are described in Section 4.1.4, below.

[0005] In one embodiment, the cells and ECM used in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein are deposited as part of the same composition. In another embodiment, the cells and ECM used in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein are deposited as part of different compositions. In a specific embodiment, the ECM used in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein comprises flowable ECM. In another specific embodiment, the cells and ECM used in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein are deposited as part of different compositions, for example, wherein the ECM is deposited separate from, e.g., before the deposition of the cells, and/or wherein the ECM is dehydrated prior to the deposition of the cells. In embodiments where the ECM is dehydrated, it may later be rehydrated at a desired time, e.g., at the time cells are deposited onto the surface that the ECM and cells have been deposited on.

[0006] In certain embodiments, the cells and ECM used in the methods for forming three- dimensional tissues in vivo described herein are deposited onto a surface concurrently, before, or after deposition of one or more additional components, e.g., a growth factor(s), a cross-linker(s), a polymerizable monomer(s), a polymer, a hydrogel(s), etc. In certain embodiments, the surface onto which said cells and ECM are deposited is a surface that has been bioprinted in accordance with the methods described herein.

[0007] In certain embodiments, the cells and flowable ECM (as well as additional components) used in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein are printed onto said surface, e.g., the cells and ECM are bioprinted. In certain embodiments, the surface onto which said cells and ECM are bioprinted is a surface that has been bioprinted in accordance with the methods described herein.

[0008] In certain embodiments, the cells and/or flowable ECM (as well as additional components) used in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein are not printed onto said surface, e.g., the cells and ECM are not bioprinted but, rather, are applied to said surface by a method that does not comprise bioprinting. In certain embodiments, the cells and/or flowable ECM (as well as additional components) that are not bioprinted onto a surface are applied to a surface that has been bioprinted, e.g., the cells and/or fiowable ECM (as well as additional components) are applied to a scaffold, e.g., a synthetic scaffold, such as a synthetic matrix. In a specific embodiment, the cells and/or fiowable ECM (as well as additional components) are applied to only part, e.g., one side, of the scaffold (e.g., the surface). In another specific embodiment, the cells and/or fiowable ECM (as well as additional components) are applied to all sides of the scaffold, i.e., the entire scaffold has cells and/or fiowable ECM applied to it. In another specific embodiment, the scaffold is polycaprolactone (PCL).

[0009] In certain embodiments, the surface onto which cells, ECM, and/or additional components may be deposited in accordance with the methods for generating tissues (e.g., three- dimensional tissues) and organs described herein comprises an artificial surface, i.e., a surface that has been man-made. In another specific embodiment, the surface onto which cells, ECM, and/or additional components may be deposited in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein comprises tissue or an organ (or portion thereof) that has been removed from a subject (e.g., a human subject). In certain embodiments, the surface of said tissue or an organ that has been removed from a subject may be decellularized, e.g., treated so as to remove cells from all or part of the surface of the tissue or organ. In certain embodiments, the surface onto which cells, ECM, and/or additional components may be deposited in accordance with the methods for generating tissues (e.g., three- dimensional tissues) and organs described herein is two-dimensional. In certain embodiments, the surface onto which cells, ECM, and/or additional components may be deposited in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein is three-dimensional. In a specific embodiment, the surface onto which cells, ECM, and/or additional components may be deposited in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein is a surface that has been bioprinted, e.g., bioprinted in accordance with the methods described herein. In a specific embodiment, the surface is polycaprolactone (PCL).

[0010] In another aspect, provided herein are tissues and organs generated using the methods described herein, as well as methods of using such tissues and organs. [0011] In certain embodiments, the tissues (e.g., three-dimensional tissues) and organs engineered in accordance with the methods described herein are used in transplantation procedures, including skin grafts and surgical transplantation procedures.

[0012] In certain embodiments, the tissues (e.g., three-dimensional tissues) and organs engineered in accordance with the methods described herein are used in experimental procedures, e.g., to assess the effect of a drug or compound on said tissue or organ.

[0013] In another aspect, provided herein are compositions comprising cells and ECM (e.g., a flowable ECM), wherein said compositions are suitable for use in the methods described herein. Also provided herein are kits comprising, in one or more containers, said compositions, as well as instructions for using said compositions in accordance with one or more of the methods described herein.

3.1 BRIEF DESCRIPTION OF DRAWINGS

[0014] Fig. 1 depicts scaffolds comprising polycaprolactone (PCL) that were bioprinted at various angles and in such a way that scaffolds of various pore sizes were generated.

[0015] Fig. 2 depicts multiple view of bioprinted scaffolds onto which extracellular matrix

(ECM) has been applied to both sides of the scaffold and subsequently dehydrated.

[0016] Fig. 3 depicts the results of a cell proliferation assay. Placental stem cells cultured on a hybrid scaffold comprising bioprinted PCL and dehydrated ECM proliferate over an 8-day culture period.

[0017] Fig. 4 depicts the results of a cell viability assay. Placental stem cells cultured on a hybrid scaffold comprising bioprinted PCL and dehydrated ECM proliferated and remained viable over an 8-day culture period.

[0018] Fig. 5 depicts an intact three-dimensional hybrid scaffold comprising PCL, ECM, and placental stem cells, each of which were bioprinted as layers (layers of PCL and layers of ECM/cells).

[0019] Fig. 6 demonstrates that placental stem cells distribute throughout three-dimensional bioprinted scaffolds over a 7-day culture period.

[0020] Fig. 7 depicts the results of a cell viability assay. Placental stem cells bioprinted with ECM and PCL to form a three-dimensional hybrid scaffold proliferate and remain viable over a 7-day culture period. [0021] Fig. 8 demonstrates that stem cells bioprinted with ECM and PCL to form a three- dimensional hybrid scaffold spread throughout the ECM in the hybrid scaffolds over a 7-day culture period.

[0022] Fig. 9 depicts the results of a cell proliferation assay. Placental stem cells cultured in a three-dimensional hybrid scaffold that was generated by bioprinting PCL, ECM, and placental stem cells proliferate over a 7-day culture period.

[0023] Fig. 10 depicts a bioprinted scaffold comprising PCL, placental ECM, and insulin- producing cells (B-TC-6 cells).

[0024] Fig. 11 depicts the results of a cell proliferation assay. Numbers of insulin-producing cells (B-TC-6 cells) in a bioprinted scaffold comprising PCL, placental ECM, and insulin- producing cells remained steady over a 14-day culture period.

[0025] Fig. 12 depicts levels of insulin production from bioprinted scaffolds comprising PCL, placental ECM, and insulin-producing cells (B-TC-6 cells).

[0026] Fig. 13 depicts levels of insulin production from bioprinted scaffolds comprising PCL, placental ECM, and insulin-producing cells (B-TC-6 cells) following exposure to glucose challenge (A) or under control conditions (B, C).

4. DETAILED DESCRIPTION

[0027] In one aspect, provided herein is a method for generating a tissue (e.g., a three- dimensional tissue) or an organ comprising depositing cells and extracellular matrix (ECM) onto a surface in vitro or ex vivo so as to form said tissue. Cells that may be used in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein are described in Section 4.1.1, below. Tissues and Organs that may be engineered in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein are described in Section 4.1.2, below. ECM that may be used in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein is described in Section 4.1.3, below. Surfaces onto which cells, ECM, and/or additional components may be deposited in accordance with the methods for generating tissues (e.g., three- dimensional tissues) and organs described herein are described in Section 4.1.4, below. [0028] In a specific embodiment, provided herein is a method for generating a tissue (e.g., a three-dimensional tissue) or an organ comprising depositing cells and ECM onto a surface in vitro or ex vivo so as to form said tissue or organ.

[0029] In another specific embodiment, provided herein is a method for generating a tissue (e.g., a three-dimensional tissue) or an organ comprising depositing cells and ECM onto a surface in vitro or ex vivo so as to form said tissue or organ, wherein said ECM comprises flowable ECM, and wherein said cells and said flowable ECM are formulated as part of the same composition. In a specific embodiment, said cells and said ECM are deposited using a bioprinter. In another specific embodiment, said cells comprise a single type of cell. In another specific embodiment, said cells comprise more than one type of cell.

[0030] In another specific embodiment, provided herein is a method for generating a tissue (e.g., a three-dimensional tissue) or an organ comprising depositing cells and ECM onto a surface in vitro or ex vivo so as to form said tissue or organ, wherein said ECM comprises flowable ECM, and wherein said cells and said flowable ECM are formulated as part separate compositions. In a specific embodiment, said cells and said ECM are deposited using a bioprinter. In another specific embodiment, said cells comprise a single type of cell. In another specific embodiment, said cells comprise more than one type of cell.

[0031] In another specific embodiment, provided herein is a method for generating a tissue (e.g., a three-dimensional tissue) or an organ comprising depositing cells and ECM onto a surface in vitro or ex vivo so as to form said tissue or organ, wherein said ECM comprises flowable ECM, and wherein said cells and said flowable ECM are formulated as part separate compositions. In a specific embodiment, said cells and said ECM are deposited using a bioprinter. In another specific embodiment, said cells comprise a single type of cell. In another specific embodiment, said cells comprise more than one type of cell.

[0032] In another specific embodiment, provided herein is a method for generating a tissue (e.g., a three-dimensional tissue) or an organ comprising depositing cells, ECM, and one or more additional components onto a surface in vitro or ex vivo so as to form said tissue or organ.

[0033] In another specific embodiment, provided herein is a method for generating a tissue (e.g., a three-dimensional tissue) or an organ comprising depositing cells, ECM, and one or more additional components onto a surface in vitro or ex vivo so as to form said tissue or organ. In a specific embodiment, said cells, said ECM, and said one or more additional components are deposited using a bioprinter. In another specific embodiment, said cells comprise a single type of cell. In another specific embodiment, said cells comprise more than one type of cell. In another specific embodiment, said cells, said ECM, and said one or more additional components are formulated as part of the same composition. In another specific embodiment, said cells, said ECM, and said one or more additional components are formulated as part separate compositions. In another specific embodiment, said one or more additional components is a growth factor, a polymerizable monomer, a cross-linker, a polymer, or a hydrogel.

[0034] In certain embodiments, the surface onto which cells, ECM, and/or additional components may be deposited in accordance with the methods for generating tissues (e.g., three- dimensional tissues) and organs described herein comprises an artificial surface, i.e., a surface that has been man-made. In another specific embodiment, the surface onto which cells, ECM, and/or additional components may be deposited in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein comprises tissue or an organ (or portion thereof) that has been removed from a subject (e.g., a human subject). In certain embodiments, the surface of said tissue or an organ that has been removed from a subject may be decellularized, e.g., treated so as to remove cells from all or part of the surface of the tissue or organ. In certain embodiments, the surface onto which cells, ECM, and/or additional components may be deposited in accordance with the methods for generating tissues (e.g., three- dimensional tissues) and organs described herein is two-dimensional. In certain embodiments, the surface onto which cells, ECM, and/or additional components may be deposited in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein is three-dimensional. In a specific embodiment, the surface onto which cells, ECM, and/or additional components may be deposited in accordance with the methods for generating tissues (e.g., three-dimensional tissues) and organs described herein is a surface that has been bioprinted, e.g., bioprinted in accordance with the methods described herein. In a specific embodiment, the surface comprises a synthetic material, e.g., a synthetic polymer. In another specific embodiment, the synthetic polymer is PCL.

[0035] In certain embodiments, the cells and ECM (e.g., a flowable ECM) are not printed concurrently, but are printed in layers. In a specific embodiment, a layer of cells is printed on a surface, followed by the printing of a layer of ECM. In another specific embodiment, a layer of ECM is printed on a surface, followed by the printing of a layer of cells. In certain embodiments, multiple layers of ECM can be printed on a surface followed by the printing of multiple layers of cells, and vice versa. Likewise, additional components that are printed concurrently with, before, or after the printing of cells and/or ECM may be layered among cells and ECM in accordance with the methods described herein.

[0036] In certain embodiments, the cells and ECM (e.g., a flowable ECM) are printed such that the surface being printed on is wholly covered by both cells and ECM. In other

embodiments, the cells and ECM (e.g., a flowable ECM) are printed such that the surface being printed on is partially covered by both cells and ECM.

[0037] In certain embodiments, the cells and ECM (e.g., a flowable ECM) are printed such that the surface being printed on is covered by cells in specific, desired areas; and covered by ECM in specific, desired areas, wherein such specific areas may or may not overlap.

[0038] In certain embodiments, the cells and ECM (e.g., a flowable ECM) may be printed onto a surface three dimensionally. As used herein "three-dimensional printing" refers to the process of printing such that the print heads of bioprinter move below, above, and around a three-dimensional surface, e.g., the printer heads are mechanically controlled so as to rotate along a specified path. As used herein, three-dimensional printing is in contrast to standard methods of bioprinting that are known in the art, where the printing is performed by starting to build tissue on a flat/planar/two-dimensional surface.

[0039] In one embodiment, ECM is printed on a surface (e.g., a prosthetic or a bone) in vitro or ex vivo, and cells are later seeded on said surface that comprises ECM using standard cell culturing approaches. Such a surface may then be transplanted into a subject. In another embodiment, ECM is printed on a surface (e.g., a prosthetic or bone) in vitro or ex vivo, and said surface that comprises ECM is transplanted into a subject, wherein cells of subject attach to and/or grow on said surface.

[0040] In a specific embodiment, provided herein is a method for generating a tissue comprising depositing cells and ECM onto a surface in vitro or ex vivo so as to form said tissue, wherein said surface comprises a bone having an inner face and an outer face, and wherein a first cellular composition comprising a first type of cell is printed on said inner face, and a second cellular composition comprising second type of cell is printed on said outer face. As used herein, the "inner face" of the bone represents the face of the bone intended to lie against stromal and muscle tissue, and the "outer face" of the bone represents the face of the bone intended to be exposed to the exterior of the recipient's body. In accordance with this embodiment, the inner face may be covered partially or wholly by, e.g., stromal cells, fatty tissue, mesenchymal stem cells, myocytes, or combinations of the like, and the outer face may be covered partially or wholly by, e.g., dermal cells. In a specific embodiment, said method additionally comprises the deposition of one more additional components (e.g., a cross-linker). In another specific embodiment, said printing is performed three-dimensionally.

[0041] In a specific embodiment, provided herein is a method for generating a liver comprising depositing cells and ECM onto a surface in vitro or ex vivo so as to form said liver, wherein said surface comprises liver tissue. In a specific embodiment, the liver tissue is obtained from the subject for which the liver generated is intended to be transplanted. In another specific embodiment, the liver tissue is not obtained from the subject for which the liver generated is intended to be transplanted (e.g., the liver tissue is obtained from a living or cadaveric donor). In another specific embodiment, said cells that are deposited are liver cells (e.g., hepatocytes). In a specific embodiment, said method additionally comprises the deposition of one more additional components (e.g., a cross-linker). In another specific embodiment, said printing is performed three-dimensionally.

[0042] In a specific embodiment, provided herein is a method for generating a skin comprising depositing cells and ECM onto a surface in vitro or ex vivo so as to form said skin, wherein said surface comprises skin tissue. In a specific embodiment, the skin tissue is obtained from the subject for which the skin generated is intended to be transplanted. In another specific embodiment, the skin tissue is not obtained from the subject for which the skin generated is intended to be transplanted (e.g., the skin tissue is obtained from a living or cadaveric donor). In another specific embodiment, said cells that are deposited are skin cells (e.g., epidermal cells). In a specific embodiment, said method additionally comprises the deposition of one more additional components (e.g., a cross-linker). In another specific embodiment, said printing is performed three-dimensionally.

4.1 BIOPRINTING

[0043] "Bioprinting," as used herein, generally refers to the deposition of living cells, as well as other components (e.g., a flowable ECM; synthetic matrices) onto a surface using standard or modified printing technology, e.g., ink jet printing technology. Basic methods of depositing cells onto surfaces, and of bioprinting cells, including cells in combination with hydrogels, are described in Warren et al. US 6,986,739, Boland et al. US 7,051,654, Yoo et al. US

2009/0208466 and Xu et al. US 2009/0208577, the disclosures of each of which are incorporated by reference herein their entirety. Additionally, bioprinters suitable for production of the tissues and organs provided herein are commercially available, e.g., the 3D-Bioplotter™ from

Envisiontec GmbH (Gladbeck, Germany); and the NovoGen MMX Bioprinter™ from Organovo (San Diego, CA).

[0044] The bioprinter used in the methods described herein may include mechanisms and/or software that enables control of the temperature, humidity, shear force, speed of printing, and/or firing frequency, by modifications of, e.g., the printer driver software and/or the physical makeup of the printer. In certain embodiments, the bioprinter software and/or hardware preferably may be constructed and/or set to maintain a cell temperature of about 37°C during printing.

[0045] In certain embodiments, the inkjet printing device may include a two-dimensional or three-dimensional printer. In certain embodiments, the bioprinter comprises a DC solenoid inkjet valve, one or more reservoir for containing one or more types of cells, e.g., cells in the flowable composition, and/or ECM (e.g., a flowable ECM) prior to printing, e.g., connected to the inkjet valve. The bioprinter may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more reservoirs, e.g., one for each cell type or each ECM used to construct the tissues and organs described herein. The cells may be delivered from the reservoir to the inkjet valve by air pressure, mechanical pressure, or by other means. Typically, the bioprinter, e.g., the print heads in the bioprinter, is/are computer-controlled such that the one or more cell types, and said ECM, are deposited in a predetermined pattern. Said predetermined pattern can be a pattern that recreates or recapitulates the natural arrangement of said one or more types of cells in an organ or tissue from which the cells are derived or obtained, or a pattern that is different from the natural arrangement of said one or more types of cells.

[0046] In certain embodiments, the bioprinter used in the methods provided herein may be a thermal bubble inkjet printer, see, e.g., Niklasen et al. US 6,537,567, or a piezoelectric crystal vibration print head, e.g., using frequencies up to 30 kHz and power sources ranging from 12 to 100 Watts. Bioprinter print head nozzles, in some embodiments, are each independently between 0.05 and 200 micrometers in diameter, or between 0.5 and 100 micrometers in diameter, or between 10 and 70 micrometers in diameter, or between 20 and 60 micrometers in diameter. In further embodiments, the nozzles are each independently about 40 or 50 micrometers in diameter. Multiple nozzles with the same or different diameters may be used. In some embodiments the nozzles have a circular opening; in other embodiments, other suitable shapes may be used, e.g., oval, square, rectangle, etc., without departing from the spirit of the invention.

[0047] In certain embodiments, an anatomical image of the tissue or organ to be bioprinted may be constructed using software, e.g., a computer-aided design (CAD) software program. In accordance with such embodiments, programs can be generated that allow for three-dimensional printing on a three-dimensional surface that is representative of the structure of the tissue or organ to be printed. For example, if it is desired to print a bone, an anatomical image of the bone may be constructed and a program may be generated that directs the printer heads of the bioprinter to rotate around the three-dimensional bone surface during printing.

[0048] In certain embodiments, the methods of bioprinting provided herein comprise the delivery/deposition of individual droplets of cells (e.g., compositions comprising single cells or compositions comprising multiple cells) and flowable extracellular matrix (ECM) on a surface.

[0049] In certain embodiments, the methods of bioprinting provided herein comprise the deposition of a single cell type and flowable ECM on a surface. Exemplary cell types that can be used in accordance with such methods are provided in Section 4.1.1, below. ECM, including flowable ECM, is described in Section 4.1.3, below.

[0050] In other embodiments, the methods of bioprinting provided herein comprise the deposition of multiple (e.g., two, three, four, five or more) cell types and flowable ECM on a surface. In a specific embodiment, the multiple cell types are deposited as part of the same composition, i.e., the source of the cells is a single composition that comprises the multiple cell types. In another specific embodiment, the multiple cell types are deposited as part of different compositions, i.e., the source of the cells are distinct compositions that comprise the multiple cell types. In another specific embodiment, a portion of the multiple cell types are deposited as part of one composition (e.g., two or more cell types are in a single composition) and another portion of the multiple types are deposited as a different composition (e.g., one or more cell types are in a single composition). Exemplary cell types that can be used in accordance with such methods are provided in Section 4.1.2, below.

[0051] In a specific embodiment, the cells to be deposited and the flowable ECM are deposited on a surface together (e.g., simultaneously) as part of the same composition. In another specific embodiment, the cells to be deposited and the flowable ECM are deposited on a surface together as part of different compositions. In another specific embodiment, the cells to be deposited and the flowable ECM are deposited on a surface separately (e.g., at different times).

[0052] In certain embodiments, the cells and flowable ECM are deposited with one or more additional components. In one embodiment, the one or more additional components are formulated in the same composition as the cells. In another embodiment, the one or more additional components are formulated in the same composition as the ECM. In another embodiment, the one or more additional components are formulated in the same composition as the cells and the ECM (i.e., a single composition comprises the cells, the flowable ECM, and the one or more additional components). In another embodiment, the one or more additional components are formulated in a composition that is separate from the compositions comprising the cells and/or ECM, and is deposited concurrently with, before, or after the deposition of the cells and/or ECM on a surface. In a specific embodiment, the one or more additional components promote the survival, differentiation, proliferation, etc. of the cell(s). In another specific embodiment, the one or more additional components comprise a cross-linker (see Section 4.1.3.2). In another specific embodiment, the one or more additional components comprise a hydrogel. In another specific embodiment, the one or more additional components comprise a synthetic polymer.

[0053] Those of skill in the art will recognize that the cells and flowable ECM, as well as any additional components used in accordance with the methods described herein, may be printed from separate nozzles of a printer, or through the same nozzle of a printer in a common composition, depending upon the particular tissue or organ being formed. It also will be recognized by those of skill in the art that the printing may be simultaneous or sequential, or any combination thereof and that some of the components (e.g., cells, flowable ECM, or cross- linkers) may be printed in the form of a first pattern and some of the components may be printed in the form of a second pattern, and so on. The particular combination and manner of printing will depend upon the particular tissue or organ being printed.

[0054] In certain embodiments, the cells, ECM, and/or any other materials (e.g., synthetic matrices, e.g., PCL) may be bioprinted in a specified pattern so as to yield a desired result. For example, bioprinted materials (e.g., cells, ECM, matrices, and other components described herein) may be bioprinted or otherwise deposited in layers at varying angles so as to generate specific desirable patterns, such as three-dimensional structures having specific pore sizes. In a specific embodiment, bioprinted materials (e.g., cells, ECM, matrices, and other components described herein) are printed or otherwise deposited in a criss-cross fashion so as to generate a bioprinted structure with pores of desired sizes that appear box-like. In another specific embodiment, bioprinted materials (e.g., cells, ECM, matrices, and other components described herein) are printed or otherwise deposited at angles, so as to generate pores of desired sizes that appear triangular or diamond-like. For example, bioprinted materials (e.g., cells, ECM, matrices, and other components described herein) can be printed or otherwise deposited at angles of specific degrees, e.g., 30 degree angles, 45 degree angles, 60 degree angles, in order to generate desired patterns. In accordance with such methods, structures having desirable qualities, e.g., the ability to foster cellular growth and proliferation, can be generated. See Example 1, below. In a specific embodiment, matrices, e.g., synthetic matrices, are bioprinted in specific patterns that are conducive to supporting the growth and proliferation of cells on said bioprinted matrices. In specific embodiments, the synthetic matrix is PCL.

4.1.1 CELLS

[0055] Any type of cell known in the art can be used in accordance with the methods described herein, including eukaryotic cells.

[0056] The cells used in accordance with the methods described herein may be syngeneic (i.e., genetically identical or closely related to the cells of the recipient subject, so as to minimize tissue transplant rejection), allogeneic (i.e., from a non-genetically identical member of the same species of the recipient subject) or xenogeneic (i.e., from a member of a different species than the recipient subject). Syngeneic cells include those that are autogeneic (i.e., from the recipient subject) and isogeneic (i.e., from a genetically identical but different subject, e.g., from an identical twin). Cells may be obtained from, e.g., a donor (either living or cadaveric) or derived from an established cell strain or cell line. For example, cells may be harvested from a donor (e.g., a potential recipient) using standard biopsy techniques known in the art.

[0057] In certain embodiments, the cells used in accordance with the methods described herein are contained within a flowable physiologically-acceptable composition, e.g., water, buffer solutions (e.g., phosphate buffer solution, citrate buffer solution, etc.), liquid media (e.g., 0.9N saline solution, Kreb's solution, modified Kreb's solution, Eagle's medium, modified Eagle's medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Hank's Balanced Salts, etc.), and the like.

[0058] In certain embodiments, the cells used in accordance with the methods described herein may comprise primary cells that have been isolated from a tissue or organ, using one or more art-known proteases, e.g., collagenase, dispase, trypsin, LIBERASE, or the like. Organ tissue may be physically dispersed prior to, during, or after treatment of the tissue with a protease, e.g., by dicing, macerating, filtering, or the like. Cells may be cultured using standard, art-known cell culture techniques prior to use of the cells in the methods described herein, e.g., in order to produce homogeneous or substantially homogeneous cell populations, to select for particular cell types, or the like.

[0059] In one embodiment, the cell type(s) used in the methods described herein comprise stem cells. A non-limiting list of stem cells that can be used in accordance with the methods described herein includes: embryonic stem cells, embryonic germ cells, induced pluripotent stem cells, mesenchymal stem cells, bone marrow-derived mesenchymal stem cells (BM-MSCs), tissue plastic-adherent placental stem cells (PDACs), umbilical cord stem cells, amniotic fluid stem cells, amnion derived adherent cells (AMDACs), osteogenic placental adherent cells (OPACs), adipose stem cells, limbal stem cells, dental pulp stem cells, myoblasts, endothelial progenitor cells, neuronal stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells, amnion derived adherent cells, or side population stem cells.

[0060] In a specific embodiment, the methods described herein comprise the use of placental stem cells (e.g., the placental stem cells described in US 7,468,276 and US 8,057,788). In another specific embodiment, said placental stem cells are PDACs®. In one embodiment, said PDACs are CD34-, CD 10+, CD105+, and CD200+. In another embodiment, said PDACs are CD34-, CD 10+, CD105+, and CD200+ and additionally are CD45-, CD80-, CD86-, and/or CD90+.

[0061] In another specific embodiment, the methods described herein comprise the use of AMDACs (e.g., the AMDACs described in international application publication no.

WO 10/059828). In one embodiment, said AMDACs are Oct4-. In another embodiment, said AMDACs are CD49f+. In another embodiment, said AMDACs are Oct4- and CD49f+. [0062] In another specific embodiment, the methods described herein comprise the use of PDACs and AMDACs.

[0063] In another specific embodiment, the methods described herein comprise the use of BM-MSCs.

[0064] In another embodiment, the cell type(s) used in the methods described herein comprise differentiated cells. In another specific embodiment, the differentiated cell(s) used in accordance with the methods described herein comprise endothelial cells, epithelial cells, dermal cells, endodermal cells, mesodermal cells, fibroblasts, osteocytes, chondrocytes, natural killer cells, dendritic cells, hepatic cells, pancreatic cells, and/or stromal cells. In another specific embodiment, the cells are insulin-producing cells, e.g., pancreatic cells (e.g., islet cells) or an insulin-producing cell line, e.g., P-TC-6 cells.

[0065] In another specific embodiment, the differentiated cell(s) used in accordance with the methods described herein comprise salivary gland mucous cells, salivary gland serous cells, von Ebner's gland cells, mammary gland cells, lacrimal gland cells, ceruminous gland cells, eccrine sweat gland dark cells, eccrine sweat gland clear cells, apocrine sweat gland cells, gland of Moll cells, sebaceous gland cells, bowman's gland cells, Brunner's gland cells, seminal vesicle cells, prostate gland cells, bulbourethral gland cells, Bartholin's gland cells, gland of Littre cells, uterus endometrium cells, isolated goblet cells, stomach lining mucous cells, gastric gland zymogenic cells, gastric gland oxyntic cells, pancreatic acinar cells, paneth cells, type II pneumocytes, and/or clara cells.

[0066] In another specific embodiment, the differentiated cell(s) used in accordance with the methods described herein comprise somatotropes, lactotropes, thyrotropes, gonadotropes, corticotropes, intermediate pituitary cells, magnocellular neurosecretory cells, gut cells, respiratory tract cells, thyroid epithelial cells, parafollicular cells, parathyroid gland cells, parathyroid chief cell, oxyphil cell, adrenal gland cells, chromaffin cells, Leydig cells, theca interna cells, corpus luteum cells, granulosa lutein cells, theca lutein cells, juxtaglomerular cell, macula densa cells, peripolar cells, and/or mesangial cells.

[0067] In another specific embodiment, the differentiated cell(s) used in accordance with the methods described herein comprise blood vessel and lymphatic vascular endothelial fenestrated cells, blood vessel and lymphatic vascular endothelial continuous cells, blood vessel and lymphatic vascular endothelial splenic cells, synovial cells, serosal cell (lining peritoneal, pleural, and pericardial cavities), squamous cells, columnar cells, dark cells, vestibular membrane cell (lining endolymphatic space of ear), stria vascularis basal cells, stria vascularis marginal cell (lining endolymphatic space of ear), cells of Claudius, cells of Boettcher, choroid plexus cells, pia-arachnoid squamous cells, pigmented ciliary epithelium cells, nonpigmented ciliary epithelium cells, corneal endothelial cells, peg cells, respiratory tract ciliated cells, oviduct ciliated cell, uterine endometrial ciliated cells, rete testis ciliated cells, ductulus efferens ciliated cells, and/or ciliated ependymal cells.

[0068] In another specific embodiment, the differentiated cell(s) used in accordance with the methods described herein comprise epidermal keratinocytes, epidermal basal cells, keratinocyte of fingernails and toenails, nail bed basal cells, medullary hair shaft cells, cortical hair shaft cells, cuticular hair shaft cells, cuticular hair root sheath cells, hair root sheath cells of Huxley's layer, hair root sheath cells of Henle's layer, external hair root sheath cells, hair matrix cells, surface epithelial cells of stratified squamous epithelium, basal cell of epithelia, and/or urinary epithelium cells.

[0069] In another specific embodiment, the differentiated cell(s) used in accordance with the methods described herein comprise auditory inner hair cells of organ of Corti, auditory outer hair cells of organ of Corti, inner pillar cells of organ of Corti, outer pillar cells of organ of Corti, inner phalangeal cells of organ of Corti, outer phalangeal cells of organ of Corti, border cells of organ of Corti, Hensen cells of organ of Corti, vestibular apparatus supporting cells, taste bud supporting cells, olfactory epithelium supporting cells, Schwann cells, satellite cells, enteric glial cells, basal cells of olfactory epithelium, cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, Merkel cells of epidermis, olfactory receptor neurons, pain-sensitive primary sensory neurons, photoreceptor rod cells, photoreceptor blue-sensitive cone cells, photoreceptor green-sensitive cone cells, photoreceptor red-sensitive cone cells, proprioceptive primary sensory neurons, touch-sensitive primary sensory neurons, type I carotid body cells, type II carotid body cell (blood pH sensor), type I hair cell of vestibular apparatus of ear (acceleration and gravity), type II hair cells of vestibular apparatus of ear, type I taste bud cells, cholinergic neural cells, adrenergic neural cells, and/or peptidergic neural cells.

[0070] In another specific embodiment, the differentiated cell(s) used in accordance with the methods described herein comprise astrocytes, neurons, oligodendrocytes, spindle neurons, anterior lens epithelial cells, crystallin-containing lens fiber cells, hepatocytes, adipocytes, white fat cells, brown fat cells, liver lipocytes, kidney glomerulus parietal cells, kidney glomerulus podocytes, kidney proximal tubule brush border cells, loop of Henle thin segment cells, kidney distal tubule cells, kidney collecting duct cells, type I pneumocytes, pancreatic duct cells, nonstriated duct cells, duct cells, intestinal brush border cells, exocrine gland striated duct cells, gall bladder epithelial cells, ductulus efferens nonciliated cells, epididymal principal cells, and/or epididymal basal cells.

[0071] In another specific embodiment, the differentiated cell(s) used in accordance with the methods described herein comprise ameloblast epithelial cells, planum semilunatum epithelial cells, organ of Corti interdental epithelial cells, loose connective tissue fibroblasts, corneal keratocytes, tendon fibroblasts, bone marrow reticular tissue fibroblasts, nonepithelial fibroblasts, pericytes, nucleus pulposus cells, cementoblast/cementocytes, odontoblasts, odontocytes, hyaline cartilage chondrocytes, fibrocartilage chondrocytes, elastic cartilage chondrocytes, osteoblasts, osteocytes, osteoclasts, osteoprogenitor cells, hyalocytes, stellate cells (ear), hepatic stellate cells (Ito cells), pancreatic stelle cells, red skeletal muscle cells, white skeletal muscle cells, intermediate skeletal muscle cells, nuclear bag cells of muscle spindle, nuclear chain cells of muscle spindle, satellite cells, ordinary heart muscle cells, nodal heart muscle cells, Purkinje fiber cells, mooth muscle cells, myoepithelial cells of iris, and/or myoepithelial cells of exocrine glands.

[0072] In another specific embodiment, the differentiated cell(s) used in accordance with the methods described herein comprise reticulocytes, megakaryocytes, monocytes, connective tissue macrophages, epidermal Langerhans cells, dendritic cells, microglial cells, neutrophils, eosinophils, basophils, mast cell, helper T cells, suppressor T cells, cytotoxic T cell, natural Killer T cells, B cells, natural killer cells, melanocytes, retinal pigmented epithelial cells, oogonia/oocytes, spermatids, spermatocytes, spermatogonium cells, spermatozoa, ovarian follicle cells, Sertoli cells, thymus epithelial cell, and/or interstitial kidney cells.

[0073] The cells used in accordance with the methods described herein can be formulated in compositions. In certain embodiments, the cells used in accordance with the methods described herein are formulated in compositions that comprise only a single cell type, i.e., the population of cells in the composition is homogeneous. In other embodiments, the cells used in accordance with the methods described herein are formulated in compositions that comprise more than one cell type, i.e., the population of cells in the composition is heterogeneous. [0074] In certain embodiments, the cells used in accordance with the methods described herein are formulated in compositions that additionally comprise flowable ECM (see Section 4.1.3). Alternatively, said flowable ECM may be deposited as part of a separate composition in accordance with the methods described herein concurrently with, before, or after the deposition of said cells. In certain embodiments, the cells used in accordance with the methods described herein are formulated in compositions that additionally comprise one or more synthetic monomers or polymers. Alternatively, said synthetic monomers or polymers may be deposited as part of a separate composition in accordance with the methods described herein concurrently with, before, or after the deposition of said cells. In certain embodiments, the cells used in accordance with the methods described herein are formulated in compositions that additionally comprise flowable ECM and one or more synthetic monomers or polymers. In certain embodiments, the cells used in accordance with the methods described herein are formulated in compositions that additionally comprise a cross-linking agent. Alternatively, said cross-linking agent may be deposited as part of a separate composition in accordance with the methods described herein concurrently with, before, or after the deposition of said cells.

[0075] In certain embodiments, the cells used in accordance with the methods described herein are formulated in compositions that additionally comprise one or more additional components, e.g., components that promote the survival, differentiation, proliferation, etc. of the cell(s). Such components may include, without limitation, nutrients, salts, sugars, survival factors, and growth factors. Exemplary growth factors that may be used in accordance with the methods described herein include, without limitation, insulin-like growth factor (e.g., IGF-1), transforming growth factor-beta (TGF-beta), bone-morphogenetic protein, fibroblast growth factor, platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), epidermal growth factor, fibroblast growth factor (FGF) (numbers 1, 2 and 3), osteopontin, bone morphogenetic protein-2, growth hormones such as somatotropin, cellular attractants and attachment agents, etc., and mixtures thereof. Alternatively, said one or more additional components that promote the survival, differentiation, proliferation, etc. of the cell(s) may be deposited as part of a separate composition in accordance with the methods described herein concurrently with, before, or after the deposition of said cells. [0076] In certain embodiments, the cells used in accordance with the methods described herein are formulated in compositions that additionally comprise a polymerizable monomer(s). Alternatively, said polymerizable monomer may be deposited as part of a separate composition in accordance with the methods described herein concurrently with, before, or after the deposition of said cells. In such embodiments, for example, a polymerization catalyst may be added immediately prior to bioprinting, such that once the cells are printed, the monomer polymerizes, forming a gel that traps and/or physically supports the cells. For example, the composition comprising the cells can comprise acrylamide monomers, whereupon TEMED and Ammonium persulfate, or riboflavin, are added to the composition immediately prior to bioprinting. Upon deposition of the cells in the composition onto a surface, the acrylamide polymerizes, sequestering and supporting the cells.

[0077] In certain embodiments, the cells used in accordance with the methods described herein are formulated in compositions that additionally comprise adhesives. In a specific embodiment, the cells used in accordance with the methods described herein are formulated in compositions that additionally comprise soft tissue adhesives including, without limitation, cyanoacrylate esters, fibrin sealant, and/or gelatin-resorcinol-formaldehyde glues. In another specific embodiment, the cells used in accordance with the methods described herein are formulated in compositions that additionally comprise arginine-glycine-aspartic acid (RGD) ligands, extracellular proteins, and/or extracellular protein analogs.

[0078] In certain embodiments, the cells used in accordance with the methods described herein are formulated in compositions such that the cells can be deposited on a surface as single cells (i.e., the cells are deposited one cell at a time).

[0079] In certain embodiments, the cells used in accordance with the methods described herein are formulated in compositions such that the cells can be deposited on a surface as aggregates that comprise multiple cells. Such aggregates may comprise cells of single type, or may comprise multiple cell types, e.g., two, three, four, five or more cell types.

[0080] In certain embodiments, the cells used in accordance with the methods described herein are formulated in compositions such that the cells form a tissue as part of the composition, wherein said tissue can be deposited on a surface using the methods described herein. Such tissues may comprise cells of single type, or may comprise multiple cell types, e.g., two, three, four, five or more cell types. [0081] In certain embodiments, the cells used in accordance with the methods described herein are deposited onto a surface as individual droplets of cells and/or compositions having small volumes, e.g., from 0.5 to 500 picoliters per droplet. In various embodiments, the volume of cells, or composition comprising the cells, is about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 20, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 picoliters, or between about 1 to 90 picoliters, about 5 to 85 picoliters, about 10 to 75 picoliters, about 15 to 70 picoliters, about 20 to 65 picoliters, or about 25 to about 60 picoliters.

4.1.2 TISSUES AND ORGANS

[0082] Provided herein are tissues and organs engineered/generated using one or more of the methods provided herein.

[0083] Any type of tissue known in the art can be generated using methods described herein. In certain embodiments, the tissue generated in accordance with the methods described herein comprises a single cell type. In other embodiments, the tissue generated in accordance with the methods described herein comprises multiple cell types. In certain embodiments, the tissue generated in accordance with the methods described herein comprises more than one type of tissue.

[0084] In certain embodiments, the methods described herein comprise deposition of cells on a surface, wherein said surface comprises tissue from a subject, e.g., the tissue is from a donor, from the recipient subject, from a cadaver, or from another source. In certain embodiments, the methods described herein comprise deposition of cells on a surface, wherein said surface comprises tissue that is not from a subject, e.g., the tissue has been synthesized.

[0085] In a specific embodiment, the tissue generated in accordance with the methods described herein is connective tissue.

[0086] In another specific embodiment, the tissue generated in accordance with the methods described herein is muscle tissue. The muscle tissue generated in accordance with the methods described herein can comprise visceral (smooth) muscle tissue, skeletal muscle tissue, or cardiac muscle tissue.

[0087] In another specific embodiment, the tissue generated in accordance with the methods described herein is neural tissue. The neural tissue generated in accordance with the methods described herein can comprise central nervous system tissue (e.g., brain tissue or spinal cord tissue) or peripheral nervous system tissue (e.g., cranial nerves and spinal nerves). [0088] In another specific embodiment, the tissue generated in accordance with the methods described herein is epithelial tissue, including endothelium.

[0089] In certain embodiments, the tissues generated in accordance with the methods described herein can be used to engineer an organ. In certain embodiments, the tissues generated in accordance with the methods described herein can be used to engineer a portion of an organ.

[0090] The tissues and organs engineered in accordance with the methods described herein can be associated with any of the known mammalian organ systems, i.e., the digestive system, circulatory system, endocrine system, excretory system, immune system, integumentary system, muscular system, nervous system, reproductive system, respiratory system, and/or skeletal system. Exemplary organs that can be generated or formed in accordance with the methods described herein include, without limitation, lungs, liver, heart, brain, kidney, skin, bone, stomach, pancreas, bladder, gall bladder, small intestine, large intestine, prostate, testes, ovaries, spinal cord, pharynx, larynx, trachea, bronchi, diaphragm, ureter, urethra, esophagus, colon, thymus, and spleen. In a specific embodiment, a pancreas is generated or formed in accordance with the methods described herein.

[0091] In a specific embodiment, the methods described herein are used to engineer bone. In another specific embodiment, the methods described herein are used to engineer skin. In another specific embodiment, the methods described herein are used to engineer lung tissue, or a lung or portion thereof. In another specific embodiment, the methods described herein are used to engineer liver tissue, or a liver or portion thereof. In another specific embodiment, the methods described herein are used to engineer neural tissue, or a nerve or portion thereof.

[0092] In certain embodiments, a tissue or organ generated in accordance with the methods described herein may additionally comprise components beneficial to the function of said tissue or organ. In certain embodiments, a tissue or organ generated in accordance with the methods described herein may additionally comprise a nerve guidance conduit, i.e. an artificial means of guiding axonal regrowth. In a specific embodiment, the nerve guidance conduit is made of a polyanhydride, e.g., poly(o-carboxyphenoxy)-p-xylene) or poly(lactide-anhydride). In certain embodiments, the nerve guidance conduit can be deposited simultaneously with the printing of the tissue or organ, i.e., the nerve guidance conduit is printed along with the tissue or organ. In other embodiments, the nerve guidance conduit may be prepared prior to the printing of said tissue or organ and placed (e.g., manually placed) into the tissue or organ as it is being printed. In other embodiments, the nerve guidance conduit may be prepared prior to the printing of said tissue or organ and placed (e.g., manually placed) into the tissue or organ after it has been printed.

[0093] In certain embodiments, a tissue or organ generated in accordance with the methods described herein may additionally comprise blood vessels, e.g., blood vessels obtained from a subject (e.g., a donor, the recipient subject, or a cadaver) or blood vessels engineered using the methods described herein.

[0094] In certain embodiments, the tissues and organs generated in accordance with the methods described herein are in the shape of the tissue or organ as it would appear in its natural state, e.g., in the human body. For example, a lung generated in accordance with the methods described herein may resemble a human lung as it appears in the human body.

[0095] In certain embodiments, the tissues and organs generated in accordance with the methods described herein are not in the shape of the tissue or organ as it would appear in its natural state, yet function in the same manner or in a similar manner as does the organ. For example, a lung generated in accordance with the methods described herein may not resemble a human lung as it appears in the human body, but may retain some or all of the functions of the human lung. In such embodiments, the tissues and organs generated in accordance with the methods described herein can be of various shapes including, without limitation, a sphere, a cylinder, rod-like, or cuboidal (i.e., cubes).

4.1.3 EXTRACELLULAR MATRIX (ECM)

[0096] The methods described herein comprise the deposition of cells (e.g., compositions comprising single cells and/or compositions comprising multiple cells) and extracellular matrix (ECM), including flowable ECM, on a surface. The ECM can be derived from any known source of ECM, and can be made flowable using any method known in the art. In specific embodiments, the ECM comprises flowable ECM. The ECM can be made flowable using, e.g., the methods described in Section 4.1.3.1, below. In certain embodiments, the ECM can be cross- linked using, e.g., using the methods described in Section 4.1.3.2, below.

[0097] The ECM (e.g., a flowable ECM) used in accordance with the methods described herein can be formulated as part of a composition for use in accordance with the methods provided herein. [0098] In certain embodiments, the ECM used in accordance with the methods described herein comprises mammalian ECM, plant ECM, molluscan ECM, and/or piscine ECM.

[0099] In a specific embodiment, the ECM used in accordance with the methods described herein comprises mammalian ECM. In another specific embodiment, the ECM used in accordance with the methods described herein comprises mammalian ECM, wherein said mammalian ECM is derived from a placenta (e.g., a human placenta). In another specific embodiment, said placental-derived ECM comprises telopeptide collagen.

[00100] In another specific embodiment, said placental-derived ECM comprises base-treated and/or detergent treated Type I telopeptide placental collagen that has not been chemically modified or contacted with a protease, wherein said ECM comprises less than 5% fibronectin or less than 5% laminin by weight; between 25% and 92% Type I collagen by weight; and 2% to 50%) Type III collagen or 2% to 50%> type IV collagen by weight.

[00101] In another specific embodiment, said placental-derived ECM comprises base-treated, detergent treated Type I telopeptide placental collagen that has not been chemically modified or contacted with a protease, wherein said ECM comprises less than 1% fibronectin or less than 1% laminin by weight; between 74% and 92% Type I collagen by weight; and 4% to 6% Type III collagen or 2% to 15% type IV collagen by weight.

[00102] Placental ECM, e.g., ECM comprising placental telopeptide collagen, used in accordance with the methods described herein, may be prepared using methods known in the art, or may be prepared as follows. First, placental tissue (either whole placenta or part thereof) is obtained by standard methods, e.g., collection as soon as practical after Caesarian section or normal birth, e.g., aseptically. The placental tissue can be from any part of the placenta including the amnion, whether soluble or insoluble or both, the chorion, the umbilical cord or from the entire placenta. In certain embodiments, the collagen composition is prepared from whole human placenta without the umbilical cord. The placenta may be stored at room temperature, or at a temperature of about 2° C to 8° C, until further treatment. The placenta is preferably exsanguinated, i.e., completely drained of the placental and cord blood remaining after birth. The expectant mother, in certain embodiments, is screened prior to the time of birth, for, e.g., HIV, HBV, HCV, HTLV, syphilis, CMV, and other viral pathogens known to contaminate placental tissue. [00103] The placental tissue may be decellularized prior to production of the ECM. The placental tissue can be decellularized according to any technique known to those of skill in the art such as those described in detail in U.S. Patent Application Publication Nos. 20040048796 and 20030187515, the contents of which are hereby incorporated by reference in their entireties.

[00104] The placental tissue may be subjected to an osmotic shock. The osmotic shock can be in addition to any clarification step or it can be the sole clarification step according to the judgment of one of skill in the art. The osmotic shock can be carried out in any osmotic shock conditions known to those of skill in the art. Such conditions include incubating the tissue in solutions of high osmotic potential, or of low osmotic potential or of alternating high and low osmotic potential. The high osmotic potential solution can be any high osmotic potential solution known to those of skill in the art such as a solution comprising one or more of NaCl (e.g., 0.2-1.0 M or 0.2-2.0 M), KC1 (e.g., 0.2-1.0 or 0.2 to 2.0 M), ammonium sulfate, a monosaccharide, a disaccharide (e.g., 20% sucrose), a hydrophilic polymer (e.g., polyethylene glycol), glycerol, etc. In certain embodiments, the high osmotic potential solution is a sodium chloride solution, e.g., at least 0.25 M, 0.5M, 0.75M, 11.0M, 1.25M, 1.5M, 1.75M, 2M, or 2.5M NaCl. In some embodiments, the sodium chloride solution is about 0.25-5M, about 0.5-4M, about 0.75-3M, or about 1.0-2.0M NaCl. The low osmotic potential solution can be any low osmotic potential solution known to those of skill in the art, such as water, for example water deionized according to any method known to those of skill. In some embodiments, the osmotic shock solution comprises water with an osmotic shock potential less than that of 50 mM NaCl. In certain embodiments, the osmotic shock is in a sodium chloride solution followed by a water solution. In certain embodiments, one or two NaCl solution treatments are followed by a water wash.

[00105] The composition resulting from the osmotic shock may then, in certain embodiments, be incubated with a detergent. The detergent can be any detergent known to those of skill in the art to be capable of disrupting cellular or subcellular membranes, e.g., an ionic detergent, a nonionic detergent, deoxycholate, sodium dodecylsulfate, Triton X 100, TWEEN, or the like. Detergent treatment can be carried out at about 0° C to about 30° C, about 5° C to about 25° C, about 5° C to about 20 ⁰ C, about 5° C to about 15° C, about 0° C, about 5° C, about 10° C, about 15° C, about 20° C, about 25° C, or about 30° C. Detergent treatment can be carried out for, e.g., about 1-24 hours, about 2-20 hours, about 5-15 hours, about 8-12 hours, or about 2-5 hours. [00106] The composition resulting from the detergent treatment may then, in certain embodiments, be incubated under basic conditions. Particular bases for the basic treatment include biocompatible bases, volatile bases, or any organic or inorganic bases at a concentration of, for example, 0.2-1.0M. In certain embodiments, the base is selected from the group consisting of NH₄OH, KOH and NaOH, e.g., 0.1M NaOH, 0.25M NaOH, 0.5M NaOH, or 1M NaOH. The base treatment can be carried out at, e.g., 0° C to 30° C, 5° C to 25° C, 5° C to 20° C, 5° C to 15° C, about 0° C, about 5° C, about 10° C, about 15° C, about 20° C, about 25° C, or about 30° C, for, e.g., about 1-24 hours, about 2-20 hours, about 5-15 hours, about 8-12 hours, or about 2-5 hours.

[00107] The ECM can be produced without treatment by a base; omission of a base treatment step typically results in an ECM composition comprising relatively higher amounts of elastin, fibronectin and/or laminin than the ECM composition produced with inclusion of the basic treatment.

[00108] Typically, the process described above for human placental tissue results in production of placental ECM comprising base-treated and/or detergent treated Type I telopeptide placental collagen that has not been chemically modified or contacted with a protease, wherein said ECM comprises less than 5% fibronectin or less than 5% laminin by weight; between 25% and 92% Type I collagen by weight; between 2%> and 50%> Type III collagen; between 2%> and 50%) type IV collagen by weight; and/or less than 40%> elastin by weight. In a more specific embodiment, the process results in production of base-treated, detergent treated Type I telopeptide placental collagen, wherein said collagen has not been chemically modified or contacted with a protease, and wherein said composition comprises less than 1% fibronectin by weight; less than 1%> laminin by weight; between 74%> and 92%> Type I collagen by weight; between 4%> and 6%> Type III collagen by weight; between 2%> and 15%> type IV collagen by weight; and/or less than 12%> elastin by weight.

[00109] In certain embodiments, compositions provided herein that comprise flowable ECM may additionally comprise other components. In certain embodiments, the compositions provided herein that comprise flowable ECM additionally comprise one or more cell types, e.g., one or more of the cell types detailed in Section 4.1.1, above. Alternatively, said cells may be deposited as part of a separate composition in accordance with the methods described herein concurrently with, before, or after the deposition of said ECM. [00110] In certain embodiments, the compositions provided herein that comprise flowable ECM additionally comprise a hydrogel (e.g., a thermosensitive hydrogel and/or a photosensitive hydrogel). Alternatively, a hydrogel may be deposited as part of a separate composition in accordance with the methods described herein concurrently with, before, or after the deposition of said ECM.

[00111] In certain embodiments, the compositions provided herein that comprise flowable ECM additionally comprise one or more cell types, e.g., one or more of the cell types detailed in Section 4.1.1, above, and a hydrogel. In a specific embodiment, the compositions provided herein that comprise flowable ECM and a hydrogel (e.g., a thermosensitive hydrogel and/or a photosensitive hydrogel) are formulated such that the ratio of ECM:hydrogel ranges from about 10 : 1 to about 1 : 10 by weight.

[00112] Exemplary hydrogels may comprise include organic polymers (natural or synthetic) that may be cross-linked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure that entraps water molecules to form a gel. Suitable hydrogels for such compositions include self-assembling peptides, such as RAD 16. Hydrogel-forming materials include polysaccharides such as alginate and salts thereof, peptides, polyphosphazines, and polyacrylates, which are crosslinked ionically, or block polymers such as polyethylene oxide- polypropylene glycol block copolymers which are crosslinked by temperature or pH,

respectively. In some embodiments, the hydrogel or matrix may be biodegradable.

[00113] In certain embodiments, the compositions provided herein that comprise flowable ECM additionally comprise a synthetic polymer. In a specific embodiment, the synthetic polymer comprises polyacrylamide, polyvinylidine chloride, poly(o-carboxyphenoxy)-p-xylene) (poly(o-CPX)), poly(lactide-anhydride) (PLAA), n-isopropyl acrylamide, pent erythritol diacrylate, polymethyl acrylate, carboxymethylcellulose, and/or poly(lactic-co-glycolic acid) (PLGA). In another specific embodiment, the synthetic polymer comprises a thermoplastic, e.g., polycaprolactone (PCL), polylactic acid, polybutylene terephthalate, polyethylene terephthalate, polyethylene, polyester, polyvinyl acetate, and/or polyvinyl chloride. Alternatively, one or more synthetic polymers may be deposited as part of a separate composition in accordance with the methods described herein concurrently with, before, or after the deposition of said ECM. In a specific embodiment, the synthetic polymer is PCL. [00114] In certain embodiments, the compositions provided herein that comprise flowable ECM additionally comprise tenascin C, a human protein known to interact with fibronectin, or a fragment thereof. Alternatively, tenascin C may be deposited as part of a separate composition in accordance with the methods described herein concurrently with, before, or after the deposition of said ECM.

[00115] In certain embodiments, the compositions provided herein that comprise flowable ECM additionally comprise titanium-aluminum- vanadium (T1₆AI₄V). Alternatively, T1₆AI₄V may be deposited as part of a separate composition in accordance with the methods described herein concurrently with, before, or after the deposition of said ECM.

[00116] In certain embodiments, the ECM in a composition provided herein and/or an additional component of the composition, such as a synthetic polymer, may be derivatized. Methods for derivatization of ECM and synthetic polymers are known in the art, and include, without limitation, derivatization using cell attachment peptides (e.g., a peptide comprising one or more RGD motifs), derivatization using cell attachment proteins, derivatization using cytokines (e.g., vascular endothelial growth factor (VEGF), or a bone morphogenetic protein (BMP)), and derivatization using glycosaminoglycans.

4.1.3.1 Methods of Generating Flowable ECM

[00117] The ECM used in accordance with the methods described herein can be made flowable using methods known in the art and described herein.

[00118] In one embodiment, the ECM used in accordance with the methods described herein is made flowable by contacting the ECM with an acid or base, e.g., an acidic or basic solution comprising an amount of said acid or base that is sufficient to solubilize said ECM. Once the ECM has been made flowable, if desired, the ECM containing composition can be made neutral, or brought to a desired pH, using methods known in the art.

[00119] In another embodiment, the ECM used in accordance with the methods described herein is made flowable by contacting the ECM with an enzyme or combination of enzymes, e.g., a protease, such as trypsin, chymotrypsin, pepsin, papain, and/or elastase. Once the ECM has been made flowable, if desired, the enzymes can be inactivated using methods known in the art.

[00120] In another embodiment, the ECM used in accordance with the methods described herein is made flowable using physical approaches. In a specific embodiment, the ECM used in accordance with the methods described herein is made flowable by milling the ECM, i.e., grinding the ECM so as to overcome of the interior bonding forces. In another specific embodiment, the ECM used in accordance with the methods described herein is made flowable by shearing the ECM, e.g., with a blender or other source. In another specific embodiment, the ECM used in accordance with the methods described herein is made flowable by cutting the ECM. In certain embodiments, when ECM is made more flowable by use of physical approaches, the ECM may be manipulated in a frozen state (e.g., the ECM is freeze-dried or frozen in liquid nitrogen).

4.1.3.2 Methods of Cross-linking ECM

[00121] The ECM used in accordance with the methods described herein can be cross-linked using methods known in the art and described herein.

[00122] In certain embodiments, the ECM is cross-linked before it is applied to a surface, i.e., the ECM may be cross-linked before printing. In accordance with such embodiments, a cross- linker may be included in a composition that comprises the ECM and, if necessary, the composition comprising the ECM and cross-linker may be treated under conditions that give rise to the cross-linking of the ECM before the printing of the ECM.

[00123] In other embodiments, the ECM is cross-linked after it is applied to a surface, i.e., the ECM may be cross-linked after printing. In one embodiment, the ECM is cross-linked after it is applied to a surface by first printing the ECM onto said surface, followed by printing of a cross- linker to said surface (i.e., the ECM and the cross-linker are printed as separate compositions). In accordance with this embodiment, if necessary, the ECM can subsequently be cross-linked by treating the ECM and cross-linker under conditions that give rise to the cross-linking of the ECM.

[00124] In another embodiment, the ECM is cross-linked after it is applied to a surface by printing a composition comprising both the ECM and a cross-linker onto a surface and, after said printing, treating the ECM and cross-linker under conditions that give rise to the cross-linking of the ECM.

[00125] In a specific embodiment, the ECM is cross-linked by chemical cross-linking of hyaluronic acid, an ECM component. Exemplary means of chemical cross-linking hyaluronic acid include, without limitation, divinylsulfone cross-linking, bis-epoxide cross-linking, benzyl ester cross-linking, butanediol diglycidyl ether (BDDE) cross-linking, disulfide cross-linking via thiol modification, haloacetate modification of the HA, dihydrazide modification of the HA, tyramine modification of the HA, and the use of Huisgen cycloaddition (i.e., "Click Chemistry"). Such methods are known in the art and further described in, e.g., Burdick and Prestwich, 2011, Adv. Mater. 23:H41-H56.

[00126] In another specific embodiment, the ECM is cross-linked by chemical cross-linking of ECM proteins. Exemplary chemicals capable of cross-linking ECM proteins include, without limitation, glutaraldehyde, hexamethylene diisocyanate (HDMI), genipin, carbodiimide, polyethylene glycol, benzoyl peroxide, BioGlue (a glutaraldehyde based cross-linker; Cryolife Inc.), polyphosphoesters, and hydrolyzable polyrotaxane.

[00127] In another specific embodiment, the ECM is cross-linked by photopolymerization of hyaluronic acid using, e.g., methacrylic anhydride and/or Glycidyl methacrylate (see, e.g., ., Burdick and Prestwich, 2011, Adv. Mater. 23:H41-H56).

[00128] In another specific embodiment, the ECM is cross-linked by the use of enzymes. Enzymes suitable for cross-linking of ECM include, without limitation, lysyl oxidase (see, e.g., Levental et al, 2009, Cell 139:891-906) and tissue type transglutaminases (see, e.g., Griffin et al, 2002, J. Biochem. 368:377-96).

[00129] Those of skill in the art will recognize that the cross-linkers should be selected based on the intended use of the bioprinted product. For example, when a method described herein is used to generate a tissue or organ that is to be administered to a subject, care should be taken to select and use cross-linkers that will be biocompatible, i.e., non-harmful to said subject.

Alternatively, when a method described herein is used to generate a tissue or organ that is not to be administered to a subject, e.g., a tissue or organ to be used in diagnostic assays, then the practitioner of the method may not need to use care in selection of the cross-linker.

4.1.4 SURFACES

[00130] Any suitable surface can be used as the surface upon which the cells, flowable ECM, and/or any additional components can be deposited (e.g., printed) so as to yield the tissues and organs generated in accordance with methods described herein. Such surfaces may be two- dimensional (e.g., flat, planar surfaces) or may be three-dimensional.

[00131] In one embodiment, the surface upon which the cells, flowable ECM, and/or any additional components are deposited comprises an artificial surface, i.e., a surface that has been man-made. In a specific embodiment, said artificial surface is a prosthetic. In certain embodiments, an artificial surface is selected based on its suitability for administration to and/or transplantation in a subject, e.g., a human subject. For example, an artificial surface known not to be immunogenic (i.e., a surface that does not elicit a host immune response) may be selected for use when the tissue or organ to be deposited on the artificial surface is being made with the intent that it be transplanted in a subject. In certain embodiments, an artificial surface may be treated so as to render it suitable for administration to and/or transplantation in a subject, e.g., a human subject.

[00132] In one embodiment, the surface upon which the cells, flowable ECM, and/or any additional components are deposited comprises a plastic surface. Exemplary types of plastic surfaces onto which said cells, ECM, and/or additional components can be deposited include, without limitation, polyester, polyethylene terephtalate, polyethylene, polyvinyl chloride, polyvinylidene chloride, polypropylene, polystyrene, polyamides, polycarbonate, and

polyurethanes.

[00133] In one embodiment, the surface upon which the cells, flowable ECM, and/or any additional components are deposited comprises a metal surface. Exemplary types of plastic surfaces onto which said cells, ECM, and/or additional components can be deposited include, without limitation, aluminum, chromium, cobalt, copper, gold, iron, lead, magnesium, manganese, mercury, nickel, platinum, silver, tin, titanium, tungsten, and zinc.

[00134] In certain embodiments, the artificial surfaces upon which the cells, flowable ECM, and/or any additional components are deposited are engineered so that they form a particular shape. For example, an artificial surface may be engineered so that is the shape of a bone (e.g., an otic bone), and the appropriate cells (e.g., osteocytes, osteoblasts, osteoclasts and other bone- related cells), flowable ECM, and/or any additional components may be deposited on and/or in said surface so as to generate a bone that is suitable for transplantation in a subject.

[00135] In another embodiment, said surface comprises a tissue or an organ from a subject (e.g., a human subject) or a tissue or an organ that is derived from cells of a subject. In certain embodiments, the surface of said tissue or organ from a subject may be decellularized, e.g., treated so as to remove cells from all or part of the surface of the tissue or organ. In a specific embodiment, the subject from which the surface tissue or surface organ is from is the subject that is the intended recipient of the tissue or organ to be generated in accordance with the methods described herein. In another specific embodiment, the subject from which the surface tissue or surface organ is from is not the subject that is the intended recipient of the tissue or organ to be generated in accordance with the methods described herein (e.g., the subject that provides the surface tissue or surface organ to be printed on is a donor or cadaveric subject).

[00136] In accordance with the methods described herein, cells, flowable ECM, and/or any additional components may be deposited on (e.g., printed on) any suitable tissue or organ from a subject. In a specific embodiment, the tissue that provides the printing surface is connective tissue (including bone), muscle tissue (including visceral (smooth) muscle tissue, skeletal muscle tissue, and cardiac muscle tissue), neural tissue (including central nervous system tissue (e.g., brain tissue or spinal cord tissue) or peripheral nervous system tissue (e.g., cranial nerves and spinal nerves)), or epithelial tissue (including endothelium). In another specific embodiment, the organ that provides the printing surface is from any of the known mammalian organ systems, including the digestive system, circulatory system, endocrine system, excretory system, immune system, integumentary system, muscular system, nervous system, reproductive system, respiratory system, and/or skeletal system. In another specific embodiment, the organ that provides the printing surface is all or part of a lung, liver, heart, brain, kidney, skin, bone, stomach, pancreas, bladder, gall bladder, small intestine, large intestine, prostate, testes, ovaries, spinal cord, pharynx, larynx, trachea, bronchi, diaphragm, ureter, urethra, esophagus, colon, thymus, and spleen. In another specific embodiment, the organ that provides the printing surface is a pancreas, or a portion thereof.

[00137] In a specific embodiment, the cells, flowable ECM, and/or any additional components are deposited on (e.g., printed on) a surface that comprises or consists of bone. Exemplary bones that can be printed on include long bones, short bones, flat bones, irregular bones, and seismoid bones. Specific bones that can be printed on include, without limitation, cranial bones, facial bones, otic bones, bones of the phalanges, arm bones, leg bones, ribs, bones of the hands and fingers, bones of the feet and toes, ankle bones, wrist bones, chest bones (e.g., the sternum), and the like.

[00138] In certain embodiments, the surfaces described herein that serve as scaffolds for the deposition (e.g., deposition by bioprinting or by other means) of cells, flowable ECM, and/or any additional components are surfaces that have not been bioprinted. In certain embodiments, the surfaces described herein that serve as scaffolds for the deposition (e.g., deposition by bioprinting or by other means) of cells, flowable ECM, and/or any additional components are surfaces that have been bioprinted, e.g., bioprinted in accordance with the methods described herein. In a specific embodiment, the bioprinted surface comprises a synthetic material. In a specific embodiment, the synthetic material is PCL.

4.2 COMPOSITIONS

[00139] Provided herein are compositions that can be used in accordance with the methods described herein. In one embodiment, provided herein are compositions comprising cells (e.g., the cells described in Section 4.1.1, above) that are suitable for use in accordance with the methods described herein. In another embodiment, provided herein are compositions comprising flowable ECM (e.g., the flowable ECM described in Section 4.1.3, above) that is suitable for use in accordance with the methods described herein. In another embodiment, provided are compositions comprising one or more cross-linkers (e.g., the cross-linkers described in Section 4.1.3.2, above) suitable for use in accordance with the methods described herein. .

[00140] In one embodiment, provided herein is a composition comprising cells (e.g., the cells described in Section 4.1.1, above) and flowable ECM (e.g., the flowable ECM described in Section 4.1.3, above). In a specific embodiment, the cells comprise stem cells, e.g., bone marrow-derived mesenchymal stem cells (BM-MSCs), tissue plastic-adherent placental stem cells (PDACs), and/or amnion derived adherent cells (AMDACs). In another specific embodiment, the flowable ECM is derived from placenta (e.g., human placenta).

[00141] In another embodiment, provided herein is a composition comprising flowable ECM (e.g., the flowable ECM described in Section 4.1.3, above) and one or more cross-linkers (e.g., the cross-linkers described in Section 4.1.3.2, above).

[00142] In another embodiment, provided herein is a composition comprising cells (e.g., the cells described in Section 4.1.1, above) and one or more cross-linkers (e.g., the cross-linkers described in Section 4.1.3.2, above).

[00143] In another embodiment, provided herein is a composition comprising cells (e.g., the cells described in Section 4.1.1, above), flowable ECM (e.g., the flowable ECM described in Section 4.1.3, above), and one or more cross-linkers (e.g., the cross-linkers described in Section 4.1.3.2, above).

[00144] In a specific embodiment, a composition provided herein comprises stem cells and flowable ECM, wherein said stem cells are PDACs and wherein said flowable ECM is derived from placenta. In another specific embodiment, a composition provided herein comprises stem cells and a cross-linker, wherein said stem cells are PDACs. In another specific embodiment, a composition provided herein comprises stem cells, flowable ECM, and a cross-linker, wherein said stem cells are PDACs and wherein said flowable ECM is derived from placenta.

[00145] In another specific embodiment, a composition provided herein comprises stem cells and flowable ECM, wherein said stem cells are AMDACs and wherein said flowable ECM is derived from placenta. In another specific embodiment, a composition provided herein comprises stem cells and a cross-linker, wherein said stem cells are AMDACs. In another specific embodiment, a composition provided herein comprises stem cells, flowable ECM, and a cross-linker, wherein said stem cells are AMDACs and wherein said flowable ECM is derived from placenta.

[00146] In another specific embodiment, a composition provided herein comprises stem cells and flowable ECM, wherein said stem cells are BM-MSCs and wherein said flowable ECM is derived from placenta. In another specific embodiment, a composition provided herein comprises stem cells and a cross-linker, wherein said stem cells are BM-MSCs. In another specific embodiment, a composition provided herein comprises stem cells, flowable ECM, and a cross-linker, wherein said stem cells are BM-MSCs and wherein said flowable ECM is derived from placenta.

[00147] The compositions provided herein, in addition to comprising cells (e.g., the cells described in Section 4.1.1, above) and/or flowable ECM (e.g., the flowable ECM described in Section 4.1.3, above) and/or one or more cross-linkers (e.g., the cross-linkers described in Section 4.1.3.2, above) may additionally comprise other components. In certain embodiments, the compositions provided herein additionally comprise a hydrogel (e.g., a thermosensitive hydrogel and/or a photosensitive hydrogel. Alternatively, a hydrogel may be formulated in a composition separate from the cell and ECM comprising compositions provided herein. In certain embodiments, the compositions provided herein additionally comprise a synthetic polymer, such as polyacrylamide, polyvinylidine chloride, poly(o-carboxyphenoxy)-p-xylene) (poly(o-CPX)), poly(lactide-anhydride) (PLAA), n-isopropyl acrylamide, pent erythritol diacrylate, polymethyl acrylate, carboxymethylcellulose, poly(lactic-co-glycolic acid) (PLGA), and/or a thermoplastic (e.g., polycaprolactone, polylactic acid, polybutylene terephthalate, polyethylene terephthalate, polyethylene, polyester, polyvinyl acetate, and/or polyvinyl chloride). Alternatively, a synthetic polymer may be formulated in a composition separate from the cell and ECM comprising compositions provided herein. In certain embodiments, the compositions provided herein additionally comprise tenascin C or a fragment thereof.

Alternatively, tenascin C or a fragment thereof may be formulated in a composition separate from the cell and ECM comprising compositions provided herein. In certain embodiments, the compositions provided herein that additionally comprise titanium-aluminum- vanadium

(T1₆AI₄V). Alternatively, T1₆AI₄V may be formulated in a composition separate from the cell and ECM comprising compositions provided herein. In certain embodiments, the compositions provided herein additionally comprise a drug (e.g., a small molecule drug). Alternatively, a drug may be formulated in a composition separate from the cell and ECM comprising compositions provided herein. In certain embodiments, the compositions provided herein additionally comprise an antibody (e.g., a therapeutic antibody). Alternatively, an antibody may be formulated in a composition separate from the cell and ECM comprising compositions provided herein.

[00148] In certain embodiments, the compositions provided herein additionally comprise one or more additional components that promote the survival, differentiation, proliferation, etc. of the cell(s) used in the compositions. Such components may include, without limitation, nutrients, salts, sugars, survival factors, and growth factors. Exemplary growth factors that may be used in accordance with the methods described herein include, without limitation, insulin-like growth factor (e.g., IGF-1), transforming growth factor-beta (TGF-beta), bone-morphogenetic protein, fibroblast growth factor, platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), epidermal growth factor, fibroblast growth factor (FGF) (numbers 1, 2 and 3), osteopontin, bone morphogenetic protein-2, growth hormones such as somatotropin, cellular attractants and attachment agents, etc., and mixtures thereof. Alternatively, one or more additional components that promote the survival, differentiation, proliferation, etc. of the cell(s) may be formulated in a composition separate from the cell and ECM comprising compositions provided herein.

4.3 USES

[00149] The tissues and organs generated in accordance with the methods described herein can be used for any suitable purpose. In a specific embodiment, the tissues and organs generated in accordance with the methods described herein are used for therapeutic purposes, e.g., the tissues and/or organs are used in transplants. In another specific embodiment, the organs generated in accordance with the methods described herein are used for experimental purposes, e.g., to assess the effect of one or more compounds and/or surgical procedures on said tissue or organ.

4.3.1 Therapeutic Uses

[00150] In certain embodiments, the tissues and/or organs generated in accordance with the methods described herein are transplanted to a subject in need of such transplantation.

Exemplary tissues and organs that can be transplanted in an individual are described in Section 4.1.2. Methods of transplantation, including grafting (e.g., skin grafting) and surgical transplantation procedures are well-known to those of skill in the art.

[00151] In certain embodiments, the cells and/or ECM from which the transplanted tissue and/or organ is derived are from the transplant recipient. In other embodiments, the cells and/or ECM from which the transplanted tissue and/or organ is derived are not from the transplant recipient, but are from another subject, e.g., a donor, a cadaver, etc.

[00152] In certain embodiments, the cells from which the transplanted tissue and/or organ is derived are from the transplant recipient, and the ECM from which the transplanted tissue and/or organ is derived is not from the transplant recipient, but is from another source. In a specific embodiment, the ECM from which the transplanted tissue and/or organ is derived is from a placenta (e.g., a human placenta).

[00153] In certain embodiments, the ECM from which the transplanted tissue and/or organ is derived is from the transplant recipient, and the cells from which the transplanted tissue and/or organ is derived are not from the transplant recipient, but are from another source.

[00154] In a specific embodiment, the methods described herein are used to generate skin that is suitable for transplantation, and said skin is transplanted (i.e., grafted) in a subject in need of such transplantation (e.g., a burn victim). In a specific embodiment, said subject is human.

[00155] In another specific embodiment, the methods described herein are used to generate a bone that is suitable for transplantation, and said bone is transplanted (e.g., surgically

transplanted) in a subject in need of such transplantation (e.g., someone suffering from osteoporosis or bone cancer). In a specific embodiment, said subject is human. [00156] In another specific embodiment, the methods described herein are used to generate a liver, or portion thereof, that is suitable for transplantation, and said liver or portion thereof is transplanted (e.g., surgically transplanted) in a subject in need of such transplantation (e.g., someone suffering from cirrhosis of the liver, hepatitis, or liver cancer). In a specific

embodiment, said subject is human.

[00157] In another specific embodiment, the methods described herein are used to generate a lung, or portion thereof, that is suitable for transplantation, and said lung or portion thereof is transplanted (e.g., surgically transplanted) in a subject in need of such transplantation (e.g., someone suffering from lung cancer). In a specific embodiment, said subject is human.

[00158] In another specific embodiment, the methods described herein are used to generate a neural tissue that is suitable for transplantation (e.g., brain tissue or spinal cord tissue), and said neural tissue is transplanted (e.g., surgically transplanted) in a subject in need of such

transplantation. In a specific embodiment, said subject has been diagnosed with a neural disease (i.e., a disease of the central or peripheral nervous system). In another specific embodiment, said subject has suffered trauma that has damaged the central or peripheral nervous system of the subject, e.g., the subject has suffered a traumatic brain injury (TBI) or spinal cord injury (SCI). In another specific embodiment, said subject is human.

[00159] In another specific embodiment, the methods described herein are used to generate a circulatory system tissue that is suitable for transplantation (e.g., heart tissue, arteries, or veins), and said circulatory system tissue is transplanted (e.g., surgically transplanted) in a subject in need of such transplantation. In a specific embodiment, said subject is human.

4.3.1.1 Patient Populations

[00160] The tissues and/or organs generated in accordance with the methods described herein can be used to benefit various patient populations. In one embodiment, the tissues and/or organs generated in accordance with the methods described herein are used in subjects requiring transplantation of a tissue and/or organ.

[00161] In a specific embodiment, a tissue(s) and/or organ(s) generated in accordance with the methods described herein is transplanted in a subject that has been diagnosed with cancer, i.e., to replace all or part of one or more of the organs/tissues of said subject that have been affected by the cancer. In a specific embodiment, a tissue(s) and/or organ(s) generated in accordance with the methods described herein is transplanted in a subject that has been diagnosed with a bone or connective tissue sarcoma, brain cancer, breast cancer, ovarian cancer, kidney cancer, pancreatic cancer, esophageal cancer, stomach cancer, liver cancer, lung cancer (e.g., small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), throat cancer, and mesothelioma), and/or prostate cancer.

[00162] In another specific embodiment, a lung tissue(s) and/or organ(s) generated in accordance with the methods described herein is transplanted in a subject that has been diagnosed with a respiratory disease, e.g., the subject has been diagnosed with asthma, chronic obstructive pulmonary disorder (COPD), emphysema, pneumonia, tuberculosis, lung cancer and/or cystic fibrosis.

[00163] In another specific embodiment, a liver tissue(s) and/or organ(s) generated in accordance with the methods described herein is transplanted in a subject that has been diagnosed with a liver disease, e.g., the subject has been diagnosed with hepatitis (e.g., Hepatitis A, B, or C), liver cancer, hemochromatosis, or cirrhosis of the liver.

[00164] In another specific embodiment, a bone tissue(s) and/or organ(s) generated in accordance with the methods described herein is transplanted in a subject that has been diagnosed with a bone disease, e.g., the subject has been diagnosed with bone cancer (e.g., osteosarcoma), osteonecrosis, metabolic bone disease, Fibrodysplasia ossificans progressive, or osteoporosis.

[00165] In another specific embodiment, a neural tissue(s) and/or organ(s) generated in accordance with the methods described herein is transplanted in a subject that has been diagnosed with a neural disease (i.e., a disease of the central or peripheral nervous system), e.g., the subject has been diagnosed with brain cancer, encephalitis, meningitis, Alzheimer's disease, Parkinson's disease, stroke, or multiple sclerosis.

[00166] In another specific embodiment, an epidermal (e.g., skin) tissue(s) generated in accordance with the methods described herein is transplanted in a subject that has been diagnosed with a skin disease (i.e., a disease that affects the skin), e.g., the subject has been diagnosed with skin cancer, eczema, acne, psoriasis, shingles, keratosis; or the subject has scarring.

[00167] In another specific embodiment, a neural tissue(s) and/or organ(s) generated in accordance with the methods described herein is transplanted in a subject that has undergone trauma that has damaged the central or peripheral nervous system of the subject, e.g., the subject has suffered a traumatic brain injury (TBI) or spinal cord injury (SCI).

[00168] In another specific embodiment, a circulatory system tissue(s) and/or organ(s) generated in accordance with the methods described herein is transplanted in a subject that has been diagnosed with a disease of the circulatory system, e.g., the subject has been diagnosed with coronary heart disease, cardiomyopathy (e.g., intrinsic or extrinsic cardiomyopathy), heart attack, stroke, inflammatory heart disease, hypertensive heart disease, or valvular heart disease.

[00169] In some embodiments, a subject to which a tissue or organ generated in accordance with the methods described herein is transplanted is an animal. In certain embodiments, the animal is a bird. In certain embodiments, the animal is a canine. In certain embodiments, the animal is a feline. In certain embodiments, the animal is a horse. In certain embodiments, the animal is a cow. In certain embodiments, the animal is a mammal, e.g., a horse, swine, mouse, or primate, preferably a human. In a specific embodiment, a subject to which a tissue or organ generated in accordance with the methods described herein is transplanted is a human.

[00170] In certain embodiments, a subject to which a tissue or organ generated in accordance with the methods described herein is transplanted is a human adult. In certain embodiments, a subject to which a tissue or organ generated in accordance with the methods described herein is transplanted is a human infant. In certain embodiments, a subject to which a tissue or organ generated in accordance with the methods described herein is transplanted is a human child. 4.3.2 Experimental Uses

[00171] In certain embodiments, the tissues and/or organs generated in accordance with the methods described herein are used for experimental purposes.

[00172] In a specific embodiment, the tissues and/or organs generated in accordance with the methods described herein are used for screening the effect of drugs on said tissues and/or organs. In accordance with such methods, a tissue or organ generated in accordance with the methods described herein can be exposed to a given drug (e.g., a drug to be assessed) and to a control (e.g., a composition that does not comprise the drug), and the effect of the drug on the tissue or organ can be assessed using methods known to those of skill in the art (e.g., by assessing toxicity of the drug as compared to the control; efficacy of the drug to cause a certain result as compared to the control, etc.). [00173] In certain embodiments, the tissues and/or organs generated in accordance with the methods described herein may be transplanted in a non-human animal, and the effect of a drug on said tissue or organ in the non-human animal may be assessed by administering the drug to a first non-human animal that has undergone such a transplant and administering a control (e.g., a composition that does not comprise the drug) to a second non-human animal that has undergone such a transplant, and comparing the results.

[00174] In certain embodiments, the tissues and/or organs generated in accordance with the methods described herein may be transplanted in a non-human animal, and the effect of a surgical procedure on said tissue or organ in the non-human animal may be assessed by performing the surgical procedure on the transplanted tissue/organ of a first non-human animal that has undergone such a transplant and not performing the surgical procedure on a second non- human animal that has undergone such a transplant, and comparing the results.

[00175] In a specific embodiment, the tissues and/or organs generated in accordance with the methods described herein are used for extracorporeal purposes. For example, a tissue or organ generated in accordance with the methods described herein is situated outside of a subject's body yet performs a function from which the subject benefits, e.g., the tissue or organ performs a function normally performed by a tissue or organ that is situated inside a subject's body.

4.4 KITS

[00176] Provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the compositions described herein. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[00177] In a specific embodiment, a kit provided herein comprises a composition comprising the cells described herein and the flowable ECM described herein. Such a kit may optionally comprise a composition comprising one or more additional components (e.g., a cross-linker). In another specific embodiment, a kit provided herein comprises a composition comprising the cells described herein, the flowable ECM described herein, and one or more cross-linkers described herein. The kits encompassed herein can be used in accordance with the methods described herein. 5. EXAMPLES

5.1 EXAMPLE 1: BIOPRINTED SCAFFOLDS SUPPORT ATTACHMENT

AND GROWTH OF PLACENTAL STEM CELLS

[00178] This example demonstrates that synthetic material can be bioprinted to produce scaffolds of controlled fiber diameter and pore size, and that such scaffolds provide a suitable substrate for the application of extracellular matrix (ECM). This example further demonstrates that scaffolds comprising bioprinted synthetic material and ECM (hybrid scaffolds) represent a suitable substrate for the attachment and growth of cells, including placental cells, such as placental stem cells.

5.1.1 Methods

[00179] To fabricate hybrid scaffolds comprising synthetic material and ECM,

polycaprolactone (PCL) (Mn 45,000, Sigma) was first printed into scaffolds (54 x 54 x 0.64 mm) using a bioprinter (EnvisionTEC, Gladbeck, Germany). The printing conditions were as follows: temperature at 90°C, printing pressure 3-5.5 bar, printing speed 2-6 mm/s, with suitable size needles. ECM was isolated from human placenta as previously described (see, e.g., Bhatia MB, Wounds 20, 29, 2008). Isolated ECM was applied to both sides of the bioprinted PCL scaffolds and allowed to dry (dehydrate) so as to generate hybrid scaffolds comprising PCL and ECM. The resultant hybrid PCL-ECM scaffolds were punched into 10 mm diameter disks, pre-wet with media overnight, and seeded with placental stem cells prepared in accordance with the methods described herein (see, e.g., Section 4.1.1) at 12,500 cells/cm². The cells were cultured over an 8- day time period. Calcein staining and MTS proliferation assays were performed in accordance with standard protocols at different time points (n=3) to determine cell viability and proliferation.

5.1.2 Results

[00180] By optimizing printing conditions, PCL scaffolds of different fiber sizes, pore sizes and pore structures were generated (Fig. 1). The printed fibers formed a stable network for the generation of hybrid scaffolds comprising PCL and ECM. Further, the printing of varying fiber sizes and pore structures made it possible to make hybrid scaffolds comprising various properties.

[00181] Dehydration of ECM on both sides of the bioprinted PCL scaffolds resulted in the generation of hybrid scaffolds. Good integration was seen between the PCL and ECM; no separation between the PCL and ECM was noticed when the hybrid scaffolds were manipulated by processing or culturing of the scaffolds, which included rehydration (Fig. 2).

[00182] The placental stem cells spread over the surface of the hybrid scaffolds over time, and covered the majority of the surface of the hybrid scaffolds by day 6 of culture. The MTS cell proliferation assay demonstrated that cell number significantly increased over time (Fig. 3). In addition, the placental stem cells seeded on the hybrid scaffolds demonstrated good viability over the 8 day culture period, as indicated by calcein staining (Fig. 4). Together, these data indicate that PCL-ECM hybrid scaffolds support cellular attachment, survival, and growth.

5.1.3 Conclusion

[00183] This example demonstrates that hybrid scaffolds comprising ECM and synthetic material (PCL) can be generated by methods that comprise bioprinting, and that cells not only attach to such scaffolds, but survive and proliferate when cultured on such scaffolds.

5.2 EXAMPLE 2: BIOPRINTED SCAFFOLDS SUPPORT ATTACHMENT

AND GROWTH OF PLACENTAL STEM CELLS

[00184] This example demonstrates that synthetic material and ECM comprising cells, such as placental cells, e.g., placental stem cells, can be simultaneously bioprinted to produce hybrid scaffolds. As demonstrated by this Example, the bioprinted cells not only survive the bioprinting process, but proliferate over time in culture with the hybrid scaffolds.

5.2.1 Methods

[00185] ECM was prepared as described in Example 1 and mixed with 0.5% alginate hydrogel containing 1 million/ml placental stem cells. Next, PCL and the cell-containing ECM were bioprinted, in layers, to generate a hybrid scaffold comprising PCL and ECM. In each layer of the scaffold, PCL was first printed, then the ECM/cell component was printed to fill the gaps in between the PCL lines. Two or five of such layers were printed and crosslinked with CaCl₂ solution to generate the hybrid scaffolds. The bioprinted, cell-containing scaffolds

(cells/ECM/PCL) were cultured for seven days, and cell proliferation and survival were assessed at various time points via calcein staining and an MTS cell proliferation assay.

5.2.2 Results

[00186] The bioprinted scaffolds maintained an intact structure throughout the duration of cell culture (Fig. 5). PCL provided a good structural support for the ECM hydrogels, which allowed for the generation of three-dimensional constructs. Following bioprinting and throughout culture, the cells were well-distributed throughout the three-dimensional constructs; cells were found throughout the depth of the scaffolds during culture (Fig. 6).

[00187] The placental stem cells survived the bioprinting process and continued to proliferate in the three-dimensional bioprinted hybrid scaffolds throughout culture, as evidenced by calcein staining (Fig. 7). As shown in Fig. 8, most of the cells were found to spread throughout the ECM in the hybrid scaffolds, indicating that the ECM enhanced cell attachment and spreading in the ECM hydrogel. This was confirmed by comparing the location of cells in alginate alone with that of the cells in the scaffolds. Additionally, as shown in Figure 9, an MTS cell proliferation assay demonstrated increases in cell number for both the 2-layer and 5-layer scaffolds, indicating that these hybrid scaffolds supported cell growth.

5.2.3 Conclusion

[00188] This example demonstrates that hybrid scaffolds comprising ECM and synthetic material (PCL) can be generated by methods that comprise simultaneous bioprinting of ECM and PCL. Also demonstrated by this Example is the fact that cells can be bioprinted along with the components of the hybrid scaffold (ECM and PCL), and that the cells survive the bioprinting process. Further, the cells bioprinted along with the components of the hybrid scaffold proliferate when cultured on such scaffolds and intersperse throughout the scaffolds better than when cultured in cellular matrix (alginate) alone.

5.3 EXAMPLE 3: FUNCTIONAL BIOPRINTED SCAFFOLDS

[00189] This example demonstrates that synthetic material and ECM comprising cells can be bioprinted to produce functional scaffolds.

[00190] B-TC-6 cells, an insulin producing cell line, were bioprinted with human placenta derived extracellular matrix (ECM) into a bioprinted scaffold. The scaffold was 15 x 15 x 2.5 mm in dimensions, and contained 5 layers. In each layer, polycaprolactone (PCL) was first printed, followed by printing of B-TC-6 cells, mixed at 15 million cells/ml in alginate-ECM hydrogel (1% alginate and 12% ECM) between the PCL lines. The entire scaffold was immersed in 1% calcium chloride solution to crosslink for 20 minutes. The scaffolds then were cultured in DMEM medium containing 15% fetal calf serum in a cell culture incubator in 6 well plates (3 to 5 ml of medium per well). At different time points, the scaffolds were harvested for calcein staining and MTS proliferation assays, to characterize cell viability and cell proliferation, respectively. Figure 10 shows the structure of the bio-printed scaffolds.

[00191] Calcein staining demonstrated that the B-TC-6 cells survived the printing process and remained viable during culture. A cross-sectional view of the scaffolds showed that the cells distributed evenly throughout the scaffolds, and remained alive in each layer (see Figure 10). The MTS assay confirmed that the insulin producing B-TC-6 cells remained viable for up to 3 weeks, with the overall number of viable cells remaining constant (see Figure 11).

[00192] To determine whether the B-TC-6 cells could function in the bioprinted scaffold, insulin production by the cells was measured. To measure insulin production, the bio-printed scaffolds were exposed to fresh growth medium (3 ml/well in a 6-well plate) for 2 hours and aliquots of the supernatant from each scaffold were measured for insulin concentration using a mouse insulin ELISA kit (Millipore). The highest level of insulin produced was detected at day 0 (see Figure 12). The levels of secreted insulin decreased in culture afterwards (day-3 and day- 6) but remained stable from day 3 to day 6 in the culture (see Figure 12). Thus, the B-TC-6 cells maintained the ability to produce and secrete insulin after being bioprinted.

[00193] A key function of insulin producing cells in the pancreas is to produce insulin in response to increased glucose levels in the blood. It was thus examined whether the bioprinted scaffolds comprising PCL, ECM, and B-TC-6 cells retained this function by exposing the scaffolds to a glucose surge challenge (see Figure 13). One scaffold ("A" of Figure 13) was exposed to glucose starvation conditions (IMDM medium without glucose, 10% FCS) for two days and then challenged with an insulin producing condition (50 mM glucose/ 1 mM IB MX). As controls, bioprinted scaffolds were maintained in normal culture medium with steady glucose levels ("B" and "C" of Figure 13). In the controls, the medium was changed at the same time that the challenge with an insulin producing condition was performed for the test scaffold (i.e., A of Figure 13). The supernatant from each culture (A, B, and C) was sampled every half hour and the insulin concentration from each supernatant was measured by ELISA. Figure 13 shows the levels of insulin production from each culture at the different time points and demonstrates that the bioprinted scaffold exposed to glucose starvation conditions followed by challenge with an insulin producing condition (i.e., A of Figure 13) produced greater than 80-fold more insulin after 3 hours after challenge as compared to its level of insulin production at 0.5 hours post- challenge, while the controls (i.e., B and C of Figure 13) produced much less insulin (only approximately 2-fold more insulin after 3 hours following media change as compared to the level of insulin production at 0.5 hour post-media change).

[00194] This Example demonstrates that bioprinted scaffolds comprising synthetic material, cells, and ECM can be generated and that the cells of the bioprinted scaffolds remain both viable and functional.

[00195] The compositions and methods disclosed herein are not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the compositions and methods in addition to those described will become apparent to those of skill in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[00196] Various publications, patents and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A method of forming a three-dimensional tissue or organ comprising depositing at least one cellular composition comprising cells and extracellular matrix (ECM) onto a surface.

2. The method of claim 1, wherein said ECM comprises flowable ECM.

3. The method of claim 1 or 2, wherein said cellular composition and said ECM are printed onto said surface.

4. The method of any one of claims 1-3, wherein said depositing is accomplished by inkjet printing.

5. The method of any of claims 1-4, wherein said surface is an artificial surface.

6. The method of any of claims 1-4, wherein said surface is decellularized tissue or a decellularized organ.

7. The method of any of claims 1-6, wherein said ECM comprises mammalian ECM, molluscan ECM, piscene ECM, and or plant ECM.

8. The method of claim 7, wherein said mammalian ECM is placental ECM.

9. The method of claim 8, wherein said ECM comprises telopeptide placental collagen.

10. The method of claim 9, wherein said telopeptide placental collagen comprises base- treated, detergent treated Type I telopeptide placental collagen.

11. The method of claim 9 or 10, wherein said collagen has not been chemically modified or contacted with a protease.

12. The method of claim 8, wherein said placental ECM comprises base-treated and/or detergent treated Type I telopeptide placental collagen that has not been chemically modified or contacted with a protease, wherein said ECM comprises less than 5% fibronectin or less than 5% laminin by weight; between 25% and 92% Type I collagen by weight; and 2% to 50%> Type III collagen or 2% to 50%> type IV collagen by weight.

13. The method of claim 8, wherein said placental ECM comprises base-treated, detergent treated Type I telopeptide placental collagen that has not been chemically modified or contacted with a protease, wherein said ECM comprises less than 1% fibronectin or less than 1% laminin by weight; between 74% and 92% Type I collagen by weight; and 4% to 6%> Type III collagen or 2%> to 15%) type IV collagen by weight.

14. The method of any one of claims 1-13, wherein said ECM is derivatized prior to said deposition.

15. The method of claim 14, wherein said ECM is derivatized with one or more of a cell attachment peptide, a cell attachment protein, a cytokine, or a glycosaminoglycan.

16. The method of any of claims 1-15, further comprising deposition of a cell attachment peptide, a cell attachment protein, a cytokine, or a glycosaminoglycan.

17. The method of claim 16, wherein said cytokine is vascular endothelial growth factor (VEGF), or a bone morphogenetic protein (BMP).

18. The method of claim 16, wherein said cell attachment peptide is a peptide comprising one or more RGD motifs.

19. The method of any of claims 1-18, further comprising deposition of a synthetic polymer.

20. The method of claim 19, wherein said synthetic polymer is thermosensitive.

21. The method of claim 19, wherein said synthetic polymer is photosensitive.

22. The method of claim 19, wherein said synthetic polymer comprises a thermoplastic.

23. The method of claim 22, wherein said synthetic polymer is poly(L-lactide-co-glycolide) (PLGA).

24. The method of claim 22, wherein said thermoplastic is polycaprolactone, polylactic acid, polybutylene terephthalate, polyethylene terephthalate, polyethylene, polyester, polyvinyl acetate, or polyvinyl chloride.

25. The method of claim 19, wherein said synthetic polymer is polyacrylamide,

polyvinylidine chloride, poly(o-carboxyphenoxy)-p-xylene) (poly(o-CPX)), poly(lactide- anhydride) (PLAA), n-isopropyl acrylamide, pent erythritol diacrylate, polymethyl acrylate, carboxymethylcellulose, or poly(lactic-co-gly colic acid) (PLGA).

26. The method of any of claims 1-25, further comprising deposition of tenascin C or a fragment thereof.

27. The method of any of claims 1-35, further comprising deposition of a titanium- aluminum-vanadium (T1₆AI₄V) composition.

28. The method of claim 27, wherein said titanium-aluminum- vanadium composition is deposited in the form of an interconnected porous network of fibers.

29. The method of claim 1, wherein said tissue or organ comprises at least one layer of said cellular composition and at least one layer of ECM.

30. The method of claim 1, wherein at least a portion of said tissue or organ comprises at least one layer of said cellular composition and at least one layer of said ECM printed in alternating layers.

31. The method of claim 19, wherein in producing said tissue or organ, at least a portion of said synthetic polymer is printed in the form of a plurality of fibers that are substantially parallel to each other.

32. The method of claim 31 , wherein said plurality of fibers are deposited substantially in parallel to each other are printed so as not to physically contact each other.

33. The method of claim 32, wherein said synthetic polymer is deposited a plurality of times.

34. The method of claim 33, wherein said fibers, when deposited said plurality of times, are printed in a different orientation in at least two of said times.

35. The method of claim 33, wherein said fibers, when deposited said plurality of times, are deposited in a different orientation in each of said times.

36. The method of any of claims 1-35, wherein said tissue comprises at least two layers of said ECM.

37. The method of claim 36, wherein at least a portion of said at least two layers of said substrate are separated from each other by said cellular composition.

38. The method of claim 3, wherein said printing comprises printing an adhesive between said two layers of substrate.

39. The method of any of claims 1-38, wherein said ECM and said cellular composition are deposited onto said surface separately.

40. The method of any of claims 1-38, wherein at least a portion of said ECM is deposited onto said surface prior to printing said cellular composition.

41. The method of any of claims 1-38, wherein said cellular composition and said ECM are combined prior to said depositing.

42. The method of claim 3, wherein said cellular composition is printed onto said ECM during said printing.

43. The method of claim 3, wherein said cellular composition is printed onto said ECM after completion of said printing of said ECM.

44. The method of any of claims 1-43, wherein said ECM is formed into a three-dimensional structure during said depositing.

45. The method of any of claims 1-44, further comprising deposition of a bone substitute.

46. The method of claim 1-45, wherein said surface is or comprises a bone.

47. The method of claim 45, wherein said bone substitute is printed to correspond to a bone in an intended recipient of said tissue.

48. The method of claim 47, wherein the method further comprises generating a three- dimensional map of a bone in an intended recipient of said tissue, wherein said bone substitute is printed to correspond to said three-dimensional map.

49. The method of claim 47 or claim 48, wherein said bone is a cranial bone or a facial bone.

50. The method of claim 47 or claim 48, wherein said bone is an otic bone or a bone of the phalanges.

51. The method of any of claims 47-50, wherein said cellular composition is printed on said bone or bone substitute such that said cellular composition at least partially covers the surface of said bone or bone substitute.

52. The method of claim 1, wherein said tissue comprises (a) a surface consisting of a bone having an inner face and an outer face, and (b) two cellular compositions, wherein said first cellular composition comprises a first type of cell that is printed on said inner face, and a second type of cell that is printed on said outer face.

53. The method of any of claims 1-52, wherein said deposition is performed three- dimensionally.

54. The method of claim 53, wherein said tissue is printed onto a three-dimensional surface.

55. The method of claim 4, wherein said inkjet printing is performed using a printer with a plurality of print heads or a plurality of print jets.

56. The method of claim 55, wherein each of said print heads or print jets is separately controllable.

57. The method of claim 56, wherein each of said print heads or print jets operates independently from the remaining said print heads or print jets.

58. The method of any of claims 55-57, wherein at least one of said plurality of print heads or print jets prints said cellular composition, and at least one other of said plurality of print heads or print jets prints said ECM.

59. The method of claim 1, wherein said cellular composition and said ECM are combined prior to said printing.

60. The method of any of claims 1-59, wherein said cellular composition comprises bone marrow-derived mesenchymal stem cells (BM-MSCs).

61. The method of any of claims 1-59, wherein said cellular composition comprises tissue culture plastic-adherent CD34-, CD 10+, CD105+, CD200+ placental stem cells.

62. The method of claim 61, wherein said placental stem cells are additionally one or more of CD45 , CD80 , CD86 , or CD90⁺.

63. The method of claim 61 or 62, wherein said placental stem cells suppress an immune response in said recipient.

64. The method of claim 63, wherein said placental stem cells suppresses an immune response locally within said recipient.

65. The method of any of claims 1-59, wherein said cellular composition comprises embryonic stem cells, embryonic germ cells, induced pluripotent stem cells, mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, bone marrow-derived mesenchymal stromal cells, tissue plastic-adherent placental stem cells (PDACs), umbilical cord stem cells, amniotic fluid stem cells, amnion derived adherent cells (AMDACs), osteogenic placental adherent cells (OPACs), adipose stem cells, limbal stem cells, dental pulp stem cells, myoblasts, endothelial progenitor cells, neuronal stem cells, exfoliated teeth derived stem cells, hair follicle stem cells, dermal stem cells, parthenogenically derived stem cells, reprogrammed stem cells, amnion derived adherent cells, or side population stem cells.

66. The method of any of claims 1-59, wherein said cellular composition comprises differentiated cells.

67. The method of claim 66, wherein said differentiated cells comprise endothelial cells, epithelial cells, dermal cells, endodermal cells, mesodermal cells, fibroblasts, osteocytes, chondrocytes, natural killer cells, dendritic cells, hepatic cells, pancreatic cells, or stromal cells.

68. The method of claim 66, wherein said differentiated cells comprise salivary gland mucous cells, salivary gland serous cells, von Ebner's gland cells, mammary gland cells, lacrimal gland cells, ceruminous gland cells, eccrine sweat gland dark cells, eccrine sweat gland clear cells, apocrine sweat gland cells, gland of Moll cells, sebaceous gland cells, bowman's gland cells, Brunner's gland cells, seminal vesicle cells, prostate gland cells, bulbourethral gland cells, Bartholin's gland cells, gland of Littre cells, uterus endometrium cells, isolated goblet cells, stomach lining mucous cells, gastric gland zymogenic cells, gastric gland oxyntic cells, pancreatic acinar cells, paneth cells, type II pneumocytes, clara cells, somatotropes, lactotropes, thyrotropes, gonadotropes, corticotropes, intermediate pituitary cells, magnocellular neurosecretory cells, gut cells, respiratory tract cells, thyroid epithelial cells, parafollicular cells, parathyroid gland cells, parathyroid chief cell, oxyphil cell, adrenal gland cells, chromaffin cells, Leydig cells, theca interna cells, corpus luteum cells, granulosa lutein cells, theca lutein cells, juxtaglomerular cell, macula densa cells, peripolar cells, mesangial cell, blood vessel and lymphatic vascular endothelial fenestrated cells, blood vessel and lymphatic vascular endothelial continuous cells, blood vessel and lymphatic vascular endothelial splenic cells, synovial cells, serosal cell (lining peritoneal, pleural, and pericardial cavities), squamous cells, columnar cells, dark cells, vestibular membrane cell (lining endolymphatic space of ear), stria vascularis basal cells, stria vascularis marginal cell (lining endolymphatic space of ear), cells of Claudius, cells of Boettcher, choroid plexus cells, pia-arachnoid squamous cells, pigmented ciliary epithelium cells, nonpigmented ciliary epithelium cells, corneal endothelial cells, peg cells, respiratory tract ciliated cells, oviduct ciliated cell, uterine endometrial ciliated cells, rete testis ciliated cells, ductulus efferens ciliated cells, ciliated ependymal cells, epidermal keratinocytes, epidermal basal cells, keratinocyte of fingernails and toenails, nail bed basal cells, medullary hair shaft cells, cortical hair shaft cells, cuticular hair shaft cells, cuticular hair root sheath cells, hair root sheath cells of Huxley's layer, hair root sheath cells of Henle's layer, external hair root sheath cells, hair matrix cells, surface epithelial cells of stratified squamous epithelium, basal cell of epithelia, urinary epithelium cells, auditory inner hair cells of organ of Corti, auditory outer hair cells of organ of Corti, basal cells of olfactory epithelium, cold-sensitive primary sensory neurons, heat-sensitive primary sensory neurons, Merkel cells of epidermis, olfactory receptor neurons, pain-sensitive primary sensory neurons, photoreceptor rod cells, photoreceptor blue-sensitive cone cells, photoreceptor green- sensitive cone cells, photoreceptor red-sensitive cone cells, proprioceptive primary sensory neurons, touch-sensitive primary sensory neurons, type I carotid body cells, type II carotid body cell (blood pH sensor), type I hair cell of vestibular apparatus of ear (acceleration and gravity), type II hair cells of vestibular apparatus of ear, type I taste bud cells cholinergic neural cells, adrenergic neural cells, peptidergic neural cells, inner pillar cells of organ of Corti, outer pillar cells of organ of Corti, inner phalangeal cells of organ of Corti, outer phalangeal cells of organ of Corti, border cells of organ of Corti, Hensen cells of organ of Corti, vestibular apparatus supporting cells, taste bud supporting cells, olfactory epithelium supporting cells, Schwann cells, satellite cells, enteric glial cells, astrocytes, neurons, oligodendrocytes, spindle neurons, anterior lens epithelial cells, crystallin-containing lens fiber cells, hepatocytes, adipocytes, white fat cells, brown fat cells, liver lipocytes, kidney glomerulus parietal cells, kidney glomerulus podocytes, kidney proximal tubule brush border cells, loop of Henle thin segment cells, kidney distal tubule cells, kidney collecting duct cells, type I pneumocytes, pancreatic duct cells, nonstriated duct cells, duct cells, intestinal brush border cells, exocrine gland striated duct cells, gall bladder epithelial cells, ductulus efferens nonciliated cells, epididymal principal cells, epididymal basal cells, ameloblast epithelial cells, planum semilunatum epithelial cells, organ of Corti interdental epithelial cells, loose connective tissue fibroblasts, corneal keratocytes, tendon fibroblasts, bone marrow reticular tissue fibroblasts, nonepithelial fibroblasts, pericytes, nucleus pulposus cells, cementoblast/cementocytes, odontoblasts, odontocytes, hyaline cartilage chondrocytes, fibrocartilage chondrocytes, elastic cartilage chondrocytes, osteoblasts, osteocytes, osteoclasts, osteoprogenitor cells, hyalocytes, stellate cells (ear), hepatic stellate cells (Ito cells), pancreatic stelle cells, red skeletal muscle cells, white skeletal muscle cells, intermediate skeletal muscle cells, nuclear bag cells of muscle spindle, nuclear chain cells of muscle spindle, satellite cells, ordinary heart muscle cells, nodal heart muscle cells, Purkinje fiber cells, mooth muscle cells, myoepithelial cells of iris, myoepithelial cell of exocrine glands, reticulocytes, megakaryocytes, monocytes, connective tissue macrophages, epidermal

Langerhans cells, dendritic cells, microglial cells, neutrophils, eosinophils, basophils, mast cell, helper T cells, suppressor T cells, cytotoxic T cell, natural Killer T cells, B cells, natural killer cells, melanocytes, retinal pigmented epithelial cells, oogonia/oocytes, spermatids, spermatocytes, spermatogonium cells, spermatozoa, ovarian follicle cells, Sertoli cells, thymus epithelial cell, and/or interstitial kidney cells.

69. The method of any of claims 1-68, wherein said tissue comprises a nerve guidance conduit.

70. The method of claim 69, wherein said nerve guidance conduit is made of a polyanhydride.

71. The method of claim 70, wherein said polyanhydride is poly(o-carboxyphenoxy)-p- xylene) or poly(lactide-anhydride).

72. The method of any of claims 69-71, wherein said nerve guidance conduit is deposited using said polyanhydride into said tissue by said inkjet printing.

73. The method of any of claims 69-71, wherein said nerve guidance conduit is prepared prior to said printing, and is placed into said tissue during printing of said tissue.

74. The method of claim 72 or claim 73, wherein said tissue comprising said nerve guidance conduit is suitable for implantation into a damaged area of the central nervous system (CNS).

75. The method of claim 74, wherein said area of the CNS is the spinal cord.