NZ745900A - Methods of tissue generation - Google Patents

Abstract

The present invention relates to a method of forming a three-dimensional tissue comprising bioprinting insulin-producing cells, such as beta TC6 cells, and placental extracellular matrix (ECM) onto a surface or scaffold that is to be transplanted onto or into a subject. The placental ECM is prepared through incubating the placental tissue in a solution of high osmotic potential. Also provided herein are methods for forming three-dimensional tissues in vivo. In one embodiment, provided herein is a method for forming a three-dimensional tissue in vivo, comprising depositing on a surface that is in or on a subject at least one composition that comprises cells. In another embodiment, provided herein is a method for forming a three-dimensional tissue in vivo, comprising depositing on a surface that is in or on a subject at least one composition that comprises cells and at least one composition that comprises an extracellular matrix (ECM). In another embodiment, provided herein is a method for forming a three-dimensional tissue in vivo, comprising depositing on a surface that is in or on a subject at least one composition that comprises cells, at least one composition that comprises an extracellular matrix (ECM), and at least one other additional components.

Description

METHODS OF TISSUE TION This application is a divisional application of New Zealand application number 705477 and claims priority to U.S. provisional patent application No. 61/696,487, filed ber 4, 2012, the disclosure of both of which is herein incorporated by reference in their entirety.

INTRODUCTION Provided herein are methods of generating three-dimensional tissues in vivo comprising the deposition and/or administration of cells and extracellular matrix components to a subject.

BACKGROUND 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 materials.

The logy involves the rapid creation and release of liquid ts comprising cells followed by their precise deposition on a substrate. Tissues and organs engineered using basic cellular materials by means of bioprinting represent a promising alternative to donorderived tissues and organs used today in standard transplantation approaches.

SUMMARY The present invention ularly relates to: (1) A method of forming a three-dimensional tissue comprising bioprinting at least one cellular composition and placental ellular matrix (ECM) onto a surface that is to be lanted into or onto a human or animal subject, wherein the cellular composition comprises insulin-producing cells and the placental ECM is prepared using a process that comprises ting placental tissue in a solution of high osmotic potential. (2) The method of (1), wherein said cellular composition and said ECM are both ted onto said surface. (3) The method of (1), wherein said cellular composition and said ECM are both printed onto said surface. (4) The method of any of ), wherein said surface is an cial surface. (followed by 1A) (5) The method of (4), wherein said artificial surface is a prosthetic device or ure. (6) The method of any one of (1)-(3), wherein said surface is decellularized tissue or a decellularized organ. (7) The method of any of (1)-(6), wherein said ECM comprises telopeptide placental collagen. (8) The method of (7), wherein said telopeptide placental en comprises basetreated Type I telopeptide placental collagen. (9) The method of (7) or (8), n said collagen has not been chemically modified or contacted with a protease. (10) The method of any of (1)-(9), n said tal ECM comprises basetreated and/or detergent treated Type I telopeptide placental en 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 ; between % and 92% Type I collagen by weight; and 2% to 50% Type III collagen or 2% to 50% type IV collagen by . (11) The method of any of (1)-(10), wherein said placental ECM comprises basetreated , detergent treated Type I telopeptide placental collagen that has not been chemically modified or ted 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. (12) The method of any of (1)-(11), further comprising deposition of a hydrogel. (13) The method of (12), wherein said hydrogel is a thermosensitive hydrogel. (14) The method of (12), wherein said hydrogel is a photosensitive hydrogel. (15) The method of any one of 14), wherein said ECM and said hydrogel are combined in a ratio of about 10:1 to 1:10 by weight. (16) The method of any one of (1)-(15), further comprising deposition of a synthetic polymer. (17) The method of (16), wherein said synthetic polymer is thermosensitive. (18) The method of (16), wherein said synthetic polymer is photosensitive. (19) The method of (16), wherein said tic polymer comprises a thermoplastic. (followed by 1B) (20) The method of (19), n said synthetic polymer is poly(L-lactide-coglycolide ) (PLGA). (21) The method of (19), n said thermoplastic is polycaprolactone, polylactic acid, polybutylene terephthalate, polyethylene terephthalate, polyethylene, polyester, polyvinyl acetate, or polyvinyl chloride. (22) The method of (16), wherein said synthetic polymer is polyacrylamide, polyvinylidine chloride, poly(o-carboxyphenoxy)-p-xylene) (poly(o-CPX)), actide- anhydride) , n-isopropyl acrylamide, pent erythritol diacrylate, thyl acrylate, carboxymethylcellulose, or poly(lactic-coglycolic acid) (PLGA). (23) The method of any of (1)-(22), wherein said surface is prepared by ting a composition comprising a biomolecule on said surface prior to said printing. (24) The method of (23), wherein said biomolecule is or comprises a type of collagen, a type of ectin, a type of laminin, or a tissue adhesive. (25) The method of any of (1)-(24), wherein said surface is ed by covering all or a portion of said surface with a decellularized tissue. (26) The method of (25), wherein said decellularized tissue is decellularized amniotic membrane, decellularized diaphragm, decellularized skin, or decellularized fascia.

These and other embodiments are described further herein. Certain ments not the subject matter of the present application are described herein for completeness. [0004a] Provided herein are methods for g three-dimensional tissues in vivo. In one embodiment, provided herein is a method for forming a three-dimensional tissue in vivo, comprising depositing on a surface that is in or on a subject at least one composition that comprises cells. In another embodiment, provided herein is a method for forming a threedimensional tissue in vivo, comprising depositing on a e that is in or on a subject at least one composition that comprises cells and at least one ition that comprises an extracellular matrix (ECM). In another embodiment, provided herein is a method for forming a three-dimensional tissue in vivo, comprising ting on a surface that is in or on a subject at least one composition that comprises cells, at least one composition that comprises an extracellular matrix (ECM), and at least one other additional components. Cells that may be used in (followed by 2) accordance with the methods described herein are described in Section 4. l . 1, below. Tissues and Organs onto which cells, ECM, and/or other additional components can be deposited in accordance with the methods described herein are described in n 4.1.2, below. ECM that may be used in accordance with the methods 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 described herein are described in Section 4.1.4, below.

In one embodiment, the cells and ECM used in the methods for forming three- dimensional tissues in Vivo described herein are deposited as part of the same composition. In another embodiment, the cells and ECM used in the methods for forming three-dimensional tissues in Vivo described herein are deposited as part of different compositions. In a specific embodiment, the ECM used in the methods for forming three-dimensional tissues in Vivo described herein comprises ?owable ECM. In another specific embodiment, the cells and ECM used in the s for g three-dimensional tissues in Vivo described herein are deposited as part of different compositions, for example, wherein the ECM is ted te from, e.g., , 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.

In certain embodiments, the cells and/or ECM used in the methods for forming three- dimensional tissues in Vivo bed herein are deposited onto a surface in or on a subject rently, 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 one or more additional components are bioprinted onto said surface in ance with the methods bed herein.

In certain embodiments, the cells and/or ECM used in the methods for forming three- dimensional tissues in Vivo described herein are ted onto a surface in or on a subject rently, before, or after deposition of a biomolecule, wherein said biomolecule comprises a type of en, a type of ?bronectin, a type of laminin, or a tissue adhesive. In certain embodiments, the biomolecule(s) is nted onto said surface in accordance with the methods described herein.

In certain embodiments, the cells and/or ECM used in accordance with the methods described herein are not bioprinted onto a surface in or on a subject, e.g., the cells and ECM are not bioprinted but, rather, are applied to said surface by a method that does not se bioprinting. In certain embodiments, the cells and/or ECM that are not nted onto a surface in or on a subject are applied to a surface that has been bioprinted, e.g., the cells and/or ?owable ECM are applied to a scaffold, e. g., a synthetic scaffold, such as a synthetic matrix, that has been bioprinted in or on the subject. In a c embodiment, the cells and/or ECM are applied to only part, e. g., one side, of the ld (e.g., the e). In another speci?c embodiment, the cells and/or ECM are applied to all sides of the ld, i.e., the entire scaffold has cells and/or ECM applied to it. In another speci?c embodiment, the scaffold is polycaprolactone (PCL).

In certain embodiments, the cells and/or ?owable ECM (as well as additional components) used in the methods for forming three—dimensional tissues in Vivo described herein are printed onto said surface, e.g., the cells and ECM are bioprinted (e.g., with an inkj et printer).

In certain embodiments, the cells and ?owable ECM (as well as additional ents) used in the methods for forming three-dimensional tissues in Vivo described herein are sprayed onto said surface. In certain embodiments, the cells and/or ?owable ECM (as well as additional components) used in the methods for forming three-dimensional s in VlVO described herein are deposited onto said surface Via lization.

In certain embodiments, the surface onto which cells, ECM, and/or additional components may be deposited comprises an arti?cial surface, i.e., a surface that has been man- made. In another speci?c embodiment, the surface onto which cells, ECM, and/or additional components may be deposited comprises tissue or an organ (or portion thereof) of a subject (e.g., a human subject). In n embodiments, the surface of said tissue or organ 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 speci?c embodiment, the surface onto which cells, ECM, and/or additional components may be deposited in accordance with the methods described herein is a e that has been bioprinted, e.g., bioprinted in accordance with the methods bed herein. In a speci?c embodiment, the surface is polycaprolactone (PCL).

In speci?c ments, the methods described herein are used for therapeutic purposes, e. g., the methods are used to deposit cells corresponding to a speci?c tissue type, or cells corresponding to multiple tissue types, onto a surface (i.e., a tissue or organ) of said subject that will bene?t from the deposition of said cells. Such methods may onally comprise the deposition ofECM (e.g., a e ECM) and/or additional ents onto said surface of said t.

In another aspect, provided herein are compositions comprising cells and ECM (e. g., a ?owable ECM), wherein said compositions are suitable for use in the methods described . 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 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.

Fig. 2 depicts multiple view of bioprinted lds onto which extracellular matrix (ECM) has been d to both sides ofthe scaffold and subsequently dehydrated.

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.

Fig. 4 depicts the results of a cell viability assay. Placental stem cells cultured on a hybrid scaffold comprising bioprinted PCL and dehydrated ECM erated and ed viable over an 8-day e period.

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).

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

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.

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.

Fig. 9 depicts the results of a cell proliferation assay. tal stem cells cultured in a three-dimensional hybrid ld that was generated by nting PCL, ECM, and placental stem cells proliferate over a 7-day culture period.

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

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

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

Fig. 13 depicts levels of insulin production from bioprinted scaffolds comprising PCL, placental ECM, and insulin-producing cells (B—TC—6 cells) ing exposure to glucose challenge (A) or under control conditions (B, C). 4. DETAILED DESCRIPTION Provided herein are s for forming three-dimensional tissues in vivo. In one embodiment, provided herein is a method for g a three-dimensional tissue in vivo, comprising depositing on a surface that is in or on a subject at least one composition that comprises cells. In another embodiment, provided herein is a method for forming a three- dimensional tissue in vivo, comprising ting on a surface that is in or on a subject at least one composition that comprises cells and at least one composition that comprises an extracellular matrix (ECM). In another embodiment, provided herein is a method for forming a three- dimensional tissue in vivo, comprising depositing on a surface that is in or on a subject at least one composition that comprises cells, at least one composition that comprises an extracellular matrix (ECM), and at least one other onal components. Cells that may be used in accordance with the methods described herein are described in Section 4.1.], below. Tissues and Organs onto which cells, ECM, and/or other additional components can be deposited in accordance with the methods described herein are bed in Section 4.1.2, below. ECM that may be used in accordance With the methods described herein is described in Section 4.1.3, below. Surfaces onto which cells, ECM, and/or additional ents may be deposited in ance with the methods described herein are described in Section 4.1.4, below.

In one embodiment, provided herein is a method for forming a three-dimensional tissue in vivo, comprising depositing on a surface that is in or on a subject at least one composition that comprises cells. In a speci?c ment, said cells are deposited using a bioprinter. In another speci?c ment, said cells comprise a single type of cell. In another speci?c embodiment, said cells comprise more than one type of cell.

In another embodiment, provided herein is a method for forming a three-dimensional tissue in vivo, comprising depositing on a surface that is in or on a subject at least one ition that comprises cells and at least one composition that comprises an extracellular matrix (ECM), wherein said ECM comprises ?owable ECM. In a speci?c embodiment, said cells and said ECM are deposited using a bioprinter. In another speci?c embodiment, said cells comprise a single type of cell. In another speci?c embodiment, said cells comprise more than one type of cell.

In another embodiment, ed herein is a method for forming a three-dimensional tissue in vivo, comprising depositing on a surface that is in or on a subject at a composition that ses cells and extracellular matrix (ECM), wherein said ECM comprises ?owable ECM.

In a c embodiment, said cells and said ECM are deposited using a bioprinter. In another c embodiment, said cells comprise a single type of cell. In another speci?c embodiment, said cells se more than one type of cell.

In another embodiment, provided herein is a method for forming a three-dimensional tissue in vivo, comprising depositing on a surface that is in or on a subject cells, extracellular matrix (ECM), and at least one additional component. In a speci?c ment, said cells, said ECM, and said one or more additional components are deposited using a bioprinter. In another speci?c embodiment, said cells comprise a single type of cell. In another speci?c embodiment, said cells comprise more than one type of cell. In another speci?c embodiment, said cells, said ECM, and said one or more onal components are ated as part of the same composition. In another speci?c embodiment, said cells, said ECM, and said one or more additional components are formulated as part te compositions. In r speci?c embodiment, said one or more additional components is a growth factor, a polymerizable monomer, a cross-linker, a polymer, or a hydrogel.

In certain embodiments, the surface in or on a subject onto which cells, ECM, and/or onal components may be deposited in accordance with the methods described herein comprises an arti?cial surface, i.e., a surface that has been man-made (e.g., a prosthetic). In another speci?c embodiment, the surface in or on a t onto which cells, ECM, and/or additional components may be deposited in ance with the methods described herein comprises tissue or an organ (or portion thereof) in a subject (e.g., a human subject). In certain embodiments, the surface of said tissue or organ in a subject may be decellularized, e.g., d so as to remove cells from all or part of the surface of the tissue or organ. In a speci?c embodiment, the surface onto which cells, ECM, and/or additional components may be deposited in accordance with the methods described herein is a surface that has been bioprinted, e.g., bioprinted in ance with the methods described herein. In a speci?c embodiment, the surface comprises a synthetic material, e.g., a synthetic polymer. In another speci?c embodiment, the tic polymer is PCL.

In certain embodiments, the cells and ECM (e.g., a ?owable ECM) are not ted concurrently, but are deposited in layers. In a c embodiment, a layer of cells is deposited on a surface in or on a t, followed by the tion of a layer ofECM on the surface in or on the subject. In another speci?c embodiment, a layer ofECM is deposited on a surface in or on a subject, followed by the deposition of a layer of cells on the surface in or on the subject. In certain embodiments, multiple layers ofECM can be deposited on a surface in or on a subject followed by the tion of multiple layers of cells on the surface in or on the subject, and Vice versa. Likewise, additional components that are deposited concurrently with, before, or after the deposition of cells and/or ECM may be layered among cells and ECM in accordance with the methods described herein.

In certain embodiments, the cells and ECM (e.g., a ?owable ECM) are deposited such that the surface in or on a subject being deposited on is wholly covered by both cells and ECM.

In other embodiments, the cells and ECM (e.g., a ?owable ECM) are deposited such that the surface in or on a subject being deposited on is lly covered by both cells and ECM.

In certain embodiments, the cells and ECM (e.g., a ?owable ECM) are deposited such that the surface in or on a subject being deposited on is covered by cells in speci?c, d areas; and covered by ECM in speci?c, desired areas, n such speci?c areas may or may not overlap.

In certain embodiments, the cells and ECM (e.g., a ?owable ECM) may be ted/printed onto a surface three dimensionally. As used herein “three-dimensional printing” or “three-dimensional deposition” refers to the process of printing/depositing such that, e.g., the print heads of a bioprinter move below, above, and around a three-dimensional surface (e.g., an organ or bone in a subject), e.g., the printer heads are mechanically controlled so as to rotate along a speci?ed path. As used herein, three—dimensional printing and three-dimensional tion is in st to standard methods of bioprinting that are known in the art, where the printing is performed by starting to build tissue on a ?at/planar/two-dimensional surface. 4.1 BIOPRINTING “Bioprinting,” as used herein, lly refers to the deposition of living cells, as well as other components (e.g., a ?owable ECM; synthetic matrices) onto a surface using standard or modi?ed 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 208577, the disclosures of each of which are incorporated by nce herein their entirety. Additionally, nters suitable for the methods described herein are commercially available, e. g., the plotterTM from Envisiontec GmbH (Gladbeck, Germany); and the NovoGen MMX BioprinterTM from Organovo (San Diego, CA).

The bioprinter used in the methods described herein may include mechanisms and/or software that s l 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 re preferably may be constructed and/or set to maintain a cell temperature of about 37°C during printing.

In certain embodiments, the inkjet ng 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 a ?owable composition, and/or ECM (e.g., a ?owable ECM) prior to printing, e.g., connected to the inkjet valve. The nter 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.

In certain ments, the nter 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 g 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 eters in er, or between 10 and 70 micrometers in diameter, or n 20 and 60 micrometers in diameter.

In further embodiments, the nozzles are each independently about 40 or 50 micrometers in er. 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, gle, etc., without departing from the spirit of the invention.

In certain embodiments, the bioprinter used in accordance with the methods described herein comprises a plurality of print heads and/or a plurality of print jets, wherein said plurality of print heads or print jets may, in certain embodiments, be separately controllable. In certain embodiments, each of said print heads or print jets es ndently from the ing said print heads or print jets. In certain embodiments, at least one of said plurality of print heads or plurality of print jets may print compositions comprising cells, and at least one of said plurality of print heads or plurality of print jets may print compositions comprising ECM. In certain embodiments, at least one of said plurality of print heads or plurality of print jets may print compositions sing cells, at least one of said plurality of print heads or plurality of print jets may print compositions comprising ECM, and at least one of said plurality of print heads or plurality of print jets may print compositions comprising an additional component.

In certain embodiments, one or more print heads or print jets of a bioprinter used in accordance with the methods described herein may be modi?ed so that it is suitable for printing on certain e. For example, a print head or print jet may be modified by attaching it to a certain surgical instrument, e.g., a laparoscope. In accordance with such embodiments, the surgical instrument may be ?tted with one or more other components that aid in the printing procedure, e.g., a camera.

In certain ments, an anatomical image of the tissue or organ to be printed on may be ucted 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 on. For example, if it is desired to print on a bone, an anatomical image of the bone may be constructed and a program may be generated that s the printer heads of the bioprinter to rotate around the three-dimensional bone inside the subject surface during printing.

In certain embodiments, the surface of the tissue or organ to be printed on is scanned in or on said the subject so as to form a surface map, and said surface map is used to guide the depositing of the cells, ECM, and/or any additional components to be printed. Such scanning may comprise, without limitation, the use of a laser, on beam, magnetic resonance imaging, microwave, or computed tomography. The scan of said surface may comprise a resolution of least 1000, 100, 10, l, or 0.1 microns.

In certain embodiments, the methods of bioprinting provided herein comprise the delivery/deposition of dual droplets of cells (e.g., itions comprising single cells or compositions comprising multiple cells) and ?owable extracellular matrix (ECM) on a surface in or on a subject.

In certain embodiments, the methods of bioprinting provided herein comprise the tion of a single cell type and ?owable ECM on a surface in or on a subject. Exemplary cell types that can be used in accordance with such methods are provided in n 4.1.1, below.

ECM, including ?owable ECM, is described in Section 4.1.3, below.

In other ments, the methods of bioprinting provided herein se the deposition of multiple (e.g., two, three, four, ?ve or more) cell types and ?owable ECM on a surface in or on a subject. In a speci?c 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 le cell types. In r speci?c embodiment, the le 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 speci?c 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 n 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.

In a speci?c embodiment, the cells to be deposited and the ?owable ECM are deposited on a surface in or on a subject together (e.g., simultaneously) as part of the same composition. In another speci?c embodiment, the cells to be deposited and the ?owable ECM are deposited on a surface in or on a subject together as part of different compositions. In another speci?c embodiment, the cells to be deposited and the ?owable ECM are deposited on a surface in or on a t separately (e.g., at different times).

In certain embodiments, the cells and ?owable ECM are deposited on a surface in or on a t with one or more additional components. In one embodiment, the one or more onal 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 ition comprises the cells, the ?owable ECM, and the one or more additional components). In another embodiment, the one or more onal 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 or on a subject. In a speci?c embodiment, the one or more additional components promote the survival, differentiation, proliferation, etc. of the cell(s). In another speci?c ment, the one or more additional components comprise a cross-linker (see Section 4.1.3.2). In another speci?c embodiment, the one or more additional components comprise a hydrogel. In another speci?c embodiment, the one or more additional components comprise a synthetic polymer.

Those of skill in the art will recognize that the cells and ?owable ECM, as well as any additional components used in accordance with the methods described herein, may be printed from te nozzles of a r, or through the same nozzle of a printer in a common composition, ing upon the ular tissue or organ being formed. It also will be ized by those of skill in the art that the printing may be simultaneous or sequential, or any combination f and that some of the components (e.g., cells, ?owable ECM, or additional ents) may be printed in the form of a ?rst 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 in a subject that is being printed on.

In certain embodiments, the cells, ECM, and/or any other materials (e. g., synthetic matrices, e.g., PCL) may be bioprinted in a speci?ed pattern so as to yield a desired result. For example, bioprinted materials (e. g., cells, ECM, matrices, and other components described herein) may be nted in layers at varying angles so as to generate speci?c desirable patterns, such as three-dimensional ures having speci?c pore sizes. In a speci?c embodiment, bioprinted materials (e.g., cells, ECM, matrices, and other components described herein) are printed in a criss-cross fashion so as to generate a bioprinted ure with pores of desired sizes that appear box-like. In another speci?c embodiment, bioprinted materials (e. g., cells, ECM, matrices, and other components described herein) are printed 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) at angles of speci?c degrees, e. g., degree angles, 45 degree angles, 60 degree angles, in order to generate desired ns. In accordance with such methods of printing, bioprinted structures having desirable qualities, e.g., the ability to foster cellular growth and proliferation, can be generated. See Example 1, below.

In a speci?c embodiment, es, e.g., tic matrices are bioprinted in speci?c patterns that are ive to supporting the growth and proliferation of cells on said bioprinted matrices.

In speci?c ments, the synthetic matrix is PCL. 4.1.1 CELLS Any type of cell known in the art can be used in accordance with the methods described herein, including prokaryotic and eukaryotic cells.

The cells used in accordance with the methods described herein may be syngeneic (i.e., cally cal or closely related to the cells of the recipient t, so as to minimize tissue lant 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 ent 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 r living or cadaveric) or d from an established cell strain or cell line. For example, cells may be harvested from a donor (e. g., a ial recipient) using standard biopsy techniques known in the art.

In certain embodiments, the cells used in accordance with the methods described herein are contained within a ?owable 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, modi?ed Kreb’s on, Eagle’s medium, modi?ed Eagle's medium (MEM), Dulbecco’s Modi?ed Eagle’s Medium (DMEM), Hank’s Balanced Salts, etc.), and the like.

In certain embodiments, the cells used in accordance with the methods described herein may se 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, ?ltering, 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 neous or substantially homogeneous cell populations, to select for ular cell types, or the like.

In one embodiment, the cell type(s) used in the methods described herein comprises stem cells. A non-limiting list of stem cells that can be used in accordance with the s described herein includes: embryonic stem cells, embryonic germ cells, d otent stem cells, mesenchymal stem cells, bone marrow-derived hymal stem cells Cs), tissue plastic-adherent placental stem cells (PDACs), umbilical cord stem cells, ic ?uid stem cells, amnion derived adherent cells (AMDACs), osteogenic placental nt 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 nt cells, or side population stem cells.

In a speci?c 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 speci?c embodiment, said placental stem cells are PDACs®. In one embodiment, said PDACs are CD34—, CDlO+, CD105+, and CD200+. In another embodiment, said PDACs are CD34—, CDlO+, CD105+, and CD200+ and additionally are CD45—, CD80—, CD86—, and/or CD90+.

In another speci?c embodiment, the methods described herein comprise the use of AMDACs (e.g., the AMDACs bed in international application publication no.

WON/059828). In one embodiment, said AMDACs are Oct4-. In another embodiment, said AMDACs are CD49f+. In another embodiment, said AMDACs are Oct4- and CD49f+.

In another c embodiment, the methods described herein comprise the use of PDACs and AMDACs.

In another speci?c embodiment, the methods described herein comprise the use of BM-MSCs.

In another embodiment, the cell type(s) used in the methods described herein comprise differentiated cells. In a speci?c embodiment, the differentiated ) used in accordance with the methods described herein comprise endothelial cells, epithelial cells, dermal cells, endodermal cells, mesodermal cells, asts, osteocytes, chondrocytes, natural killer cells, dendritic cells, c cells, pancreatic cells, and/or stromal cells. In another speci?c embodiment, the cells are insulin-producing cells, e.g., pancreatic cells (e. g., islet cells) or an insulin-producing cell line, e.g., B-TC-6 cells.

In r speci?c ment, 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, nous 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 c cells, pancreatic acinar cells, paneth cells, type II pneumocytes, and/or clara cells.

In another speci?c ment, the differentiated cell(s) used in accordance with the methods bed herein comprise somatotropes, lactotropes, ropes, 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, chromaf?n cells, Leydig cells, theca interna cells, corpus luteum cells, osa lutein cells, theca lutein cells, juxtaglomerular cell, macula densa cells, peripolar cells, and/or mesangial cells.

In another speci?c 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 elial continuous cells, blood vessel and lymphatic vascular endothelial c 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 aris marginal cell (lining endolymphatic space of ear), cells of Claudius, cells of her, choroid plexus cells, pia-arachnoid squamous cells, pigmented ciliary epithelium cells, nonpigmented ciliary epithelium cells, corneal elial 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.

In another speci?c embodiment, the differentiated cell(s) used in accordance with the methods described herein se epidermal keratinocytes, epidermal basal cells, nocyte of ?ngernails 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 ?ed squamous epithelium, basal cell of epithelia, and/or urinary epithelium cells.

In another speci?c 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 ory epithelium, cold-sensitive primary sensory s, heat-sensitive primary sensory neurons, Merkel cells of epidermis, olfactory receptor neurons, pain-sensitive primary sensory neurons, photoreceptor rod cells, photoreceptor ensitive cone cells, photoreceptor green-sensitive cone cells, photoreceptor nsitive cone cells, proprioceptive primary sensory s, touch-sensitive primary y 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 tus of ear, type I taste bud cells, cholinergic neural cells, rgic neural cells, and/or peptidergic neural cells.

In another speci?c 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 ?ber 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.

In another speci?c embodiment, the differentiated ) 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 ?broblasts, corneal cytes, tendon ?broblasts, bone marrow reticular tissue ?broblasts, nonepithelial ?broblasts, pericytes, nucleus us cells, cementoblast/cementocytes, odontoblasts, odontocytes, hyaline cartilage chondrocytes, rtilage chondrocytes, elastic cartilage chondrocytes, osteoblasts, osteocytes, osteoclasts, osteoprogenitor cells, hyalocytes, te cells (ear), hepatic stellate cells (Ito cells), pancreatic stelle cells, red skeletal muscle cells, white skeletal muscle cells, intermediate al muscle cells, nuclear bag cells ofmuscle spindle, nuclear chain cells of muscle spindle, satellite cells, ry heart muscle cells, nodal heart muscle cells, Purkinj e ?ber cells, mooth muscle cells, myoepithelial cells of iris, and/or myoepithelial cells of exocrine glands.

In another speci?c embodiment, the differentiated cell(s) used in accordance with the methods bed herein comprise locytes, megakaryocytes, monocytes, connective tissue hages. 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, cytes, retinal pigmented epithelial cells, oogonia/oocytes, spermatids, spermatocytes, spermatogonium cells, spermatozoa, ovarian follicle cells, Sertoli cells, thymus lial cell, and/or interstitial kidney cells.

The cells used in ance 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 ition 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.

In certain embodiments, the cells used in accordance with the methods described herein are formulated in itions that additionally comprise ?owable ECM (see Section 4.1.3). Alternatively, said le ECM may be deposited as part of a separate composition in accordance with the methods described herein concurrently with, , or after the deposition of said cells. In n 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 ?owable ECM and one or more synthetic monomers or polymers. In certain embodiments, the cells used in accordance with the methods bed 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 ance with the methods described herein concurrently with, before, or after the deposition of said cells.

In certain embodiments, the cells used in ance with the methods described herein are formulated in compositions that additionally comprise one or more additional ents, e.g., components that promote the survival, differentiation, eration, etc. of the cell(s). Such components may include, without limitation, nutrients, salts, sugars, al s, 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-l), transforming growth factor-beta (TGF-beta), bone-morphogenetic n, ?broblast growth factor, et derived growth factor (PDGF), vascular endothelial growth factor , connective tissue growth factor (CTGF), basic ?broblast growth factor (bFGF), epidermal growth factor, ?broblast growth factor (FGF) (numbers 1, 2 and 3), osteopontin, bone morphogenetic protein-2, growth hormones such as somatotropin, cellular tants 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 ance with the methods bed herein concurrently with, before, or after the deposition of said cells.

In certain embodiments, the cells used in accordance with the s described herein are primary culture cells, e.g., cells that have been cultured in vitro. Such primary cells can be passaged once or multiple times, e.g. twice, three times, four times, ?ve times, six times, seven times, eight times, nine times, ten times, or more than ten times. In a speci?c ment, said primary cells have been passaged no more than six times. In another c embodiment, said primary cells are derived from the subject to be d in or on.

In certain embodiments, the cells used in accordance with the methods described herein are genetically engineered to produce a protein(s) or polypeptide(s) that is not naturally produced by the cell, or have been genetically engineered to produce a protein(s) or polypeptide(s) in an amount that is greater than that naturally produced by the cell. In a c embodiment, such cells comprise or consist of differentiated cells. In another speci?c embodiment, said cells comprise a plasmid that directs the production of said protein or polypeptide. Such cells may be cultured in such a manner that the amount of protein or polypeptide can be controlled and/or optimized. For example, said cells may be engineered so that approximately 1 x 106 of said cells produces about or at least 0.01 to 0.1, 0.1 to 1.0, 1.0 to .0, or 10.0 to 100.0 uM of said protein or polypeptide in an in vitro culture in growth medium over approximately 24 hours.

In a c embodiment, the cells used in accordance with the methods described herein are genetically engineered to produce a cytokine or a peptide comprising an active part thereof. Exemplary cytokines that may be produced by such ered cells include, without limitation, adrenomedullin (AM), angiopoietin (Ang), bone morphogenetic protein (BMP), brain-derived rophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (Epo), ?broblast growth factor (FGF), glial cell line-derived neurotrophic factor , granulocyte colony stimulating factor (G—CSF), granulocyte—macrophage colony ating factor (GM- CSF), growth differentiation factor (GDP—9), hepatocyte growth factor (HGF), ma derived growth factor (HDGF), insulin-like growth factor (IGF), migration-stimulating factor, myostatin (GDP-8), myelomonocytic growth factor (MGF), nerve growth factor (NGF), placental growth factor (PlGF), platelet-derived growth factor , thrombopoietin (Tpo), transforming growth factor alpha ), TGF-B, tumor necrosis factor alpha (TNF-u), vascular endothelial growth factor (VEGF), or a Wnt n.

In another speci?c embodiment, the cells used in accordance with the s described herein are genetically engineered to produce a soluble receptor for a protein or polypeptide, e. g., a soluble receptor for a cytokine. Exemplary soluble receptors that may be produced by such cells include, without limitation, soluble receptors for AM, Ang, BMP, BDNF, EGF, Epo, FGF, GNDF, G-CSF, GM-CSF, GDP-9, HGF, HDGF, IGF, migration-stimulating factor, GDP-8, MGF, NGF, PlGF, PDGF, Tpo, TGF—a, TGF—B, TNF-u, VEGF, and Wnt protein.

In r c embodiment, the cells used in accordance with the methods described herein are genetically ered to produce an interleukin. Exemplary interleukins that may be produced by such cells include, without limitation, interleukin-l alpha (IL-let), IL- IB,IL-1F1,IL-1F2,IL-1F3,IL-1F4,IL—1F5,IL-1F6,IL—1F7,IL-1F8,IL-1F9,IL-2,IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL—1 1, IL-12 35 kDa alpha subunit, IL-12 40 kDa beta subunit, both IL- 12 alpha and beta ts, IL-1 3, IL—l4, IL- 1 5 , IL- 1 6, IL- 1 7A, IL- 1 7B, IL- 17C, IL-17D, IL-17E, IL-17F isoform 1, IL—17Fisoform 2, IL-l8, IL-19, IL-20, IL-21, IL-22, IL-23 p19 subunit, IL-23 p40 subunit, IL-23 p19 subunit and IL-23 p40 subunit together, IL-24, IL-25, IL-26, IL-27B, ILp28, IL-27B and IL—27-p28 together, IL-28A, IL-28B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-360t, IL—36B, and IL-36y.

In another speci?c embodiment, the cells used in accordance with the methods bed herein are genetically engineered to produce a soluble receptor for an interleukin.

Exemplary soluble ors for interleukins that may be produced by such cells include, without limitation, soluble receptors for IL-lu, IL-lB, , IL-lF2, IL—lF3, IL-lF4, IL-lFS, IL-lF6, IL-lF7, IL-1F8, , IL-2, IL-3, IL—4, IL-5, IL—6, IL—7, IL-8, IL-9, IL—10, IL-1 1, IL-12 35 kDa alpha subunit, IL-12 40 kDa beta subunit, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17B, IL- l7C, IL-17D, IL-l7E, IL-17F isoform 1, IL-17F isoform 2, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23 p19 subunit, IL-23 p40 subunit, IL-24, IL-25, IL-26, IL-27B, ILp28, IL-28A, IL-28B, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL—360L, IL—36B, and IL-36y.

In another speci?c embodiment, the cells used in accordance with the s described herein are genetically engineered to produce an eron. Exemplary interferons that may be produced by such cells include, without limitation, IFN-u, IFN-B, IFN—y, IFN—M, IFN- k2, IFN—k3, IFN—K, IFN—s, IFN-K, IFN—r, IFN—S, IFN—C, IFN-(D, and IFN-V.

In another speci?c embodiment, the cells used in accordance with the methods described herein are genetically engineered to produce a soluble receptor for an interferon.

Exemplary e receptors for interferons that may be produced by such cells include, without limitation, soluble receptors for IFN-oc, IFN—B, IFN-y, IFN—Al, IFN-XZ, IFN—k3, IFN—K, IFN-S, IFN-K, IFN-‘C, IFN-5, IFN-C, , or IFN-v.

In another speci?c embodiment, the cells used in accordance with the methods described herein are genetically engineered to produce insulin or ulin. In another speci?c embodiment, the cells used in accordance with the methods described herein are genetically engineered to produce a or for insulin. In certain embodiments, said cells genetically engineered to produce n or proinsulin, and/or a receptor for insulin, are additionally genetically engineered to produce one or more of prohormone convertase l, prohormone tase 2, or carboxypeptidase E.

In r speci?c embodiment, the cells used in accordance with the methods described herein are cally engineered to produce leptin. In another c embodiment, the cells used in accordance with the methods bed herein are genetically engineered to produce erythropoietin (EPO). In another speci?c embodiment, the cells used in accordance with the s described herein are genetically ered to produce thrombopoietin.

In r speci?c embodiment, the cells used in accordance with the methods described herein are genetically engineered to produce tyrosine 3-monooxygenase, a n capable of producing L-DOPA from l-tyrosine. In certain embodiments, said cells are further engineered to express aromatic L-amino acid decarboxylase, which produces dopamine from L- DOPA.

In another speci?c embodiment, the cells used in accordance with the methods described herein are genetically engineered to produce a hormone or prohormone. Exemplary es that may be produced by such cells include, without limitation, antimullerian hormone (AMH), adiponectin (Acrp30), corticotropic hormone (ACTH), angiotensin (AGT), angiotensinogen (AGT), antidiuretic hormone (ADH), vasopressin, atrial-natriuretic peptide (ANP), calcitonin (CT), cholecystokinin (CCK), corticotrophin-releasing hormone (CRH), erythropoietin (Epo), follicle-stimulating hormone (FSH), testosterone, estrogen, gastrin (GRP), ghrelin, glucagon (GCG), gonadotropin-releasing hormone (GnRH), growth hormone (GH), growth e releasing hormone (GHRH), human chorionic gonadotropin (hCG), human placental lactogen (HPL), inhibin, leutinizing hormone (LH), melanocyte stimulating e (MSH), orexin, oxytocin (OXT), parathyroid e (PTH), prolactin (PRL), relaxin (RLN), secretin (SCT), somatostatin (SRIF), thrombopoietin (Tpo), thyroid-stimulating hormone (Tsh), and thyrotropin-releasing hormone (TRH).

In another speci?c embodiment, the cells used in accordance with the methods described herein are genetically engineered to e cytochrome P450 side chain cleavage enzyme (P450SCC).

In another speci?c embodiment, the cells used in accordance with the s described herein are genetically engineered to produce low density lipoprotein or (LDLR).

In another speci?c embodiment, the cells used in accordance with the methods described herein are genetically engineered to produce polycystin-l (PKDl), PKD-2 or PKD3.

In another speci?c embodiment, the cells used in accordance with the methods described herein are genetically engineered to produce phenylalanine hydroxylase.

In certain embodiments, the cells used in accordance with the methods described herein are formulated in compositions that additionally comprise a polymerizable monomer(s).

In such embodiments, for example, a rization catalyst may be added immediately prior to bioprinting, such that once the cells are d, 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 um persulfate, or ribo?avin, are added to the composition immediately prior to bioprinting. Upon deposition of the cells in the composition onto a surface, the mide polymerizes, sequestering and supporting the cells.

In certain embodiments, the cells used in ance with the methods described herein are formulated in compositions that additionally comprise ves. In a speci?c 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, ?brin t, and/or gelatin-resorcinol-formaldehyde glues. In another speci?c 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.

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

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 in or on a subject 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, ?ve or more cell types.

In certain ments, the cells used in accordance with the methods bed herein are formulated in compositions such that the cells form a tissue as part of the composition, wherein said tissue can be ted on a surface in or on a subject using the s described herein. Such tissues may se cells of single type, or may se multiple cell types, e.g., two, three, four, ?ve or more cell types.

In certain embodiments, the cells used in accordance with the methods described herein are deposited onto a e in or on a subject as dual droplets of cells and/or compositions having small volumes, e.g., from 0.5 to 500 picoliters per droplet. In various ments, the volume of cells, or composition comprising the cells, is about 0.5, l, 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 Provided herein are methods of bioprinting on tissues and organs in or on a subject using one or more of the methods ed herein. Also provided herein are methods of bioprinting new tissue in or on a subject. Also provided herein are methods of bioprinting tissue in or on existing organs in a subject, as well as the generation of new organs in a subject.

Any type of tissue known in the art can be printed in or on a subject using the methods described herein. In certain embodiments, the tissue printed in or on a subject comprises a single cell type. In other ments, the tissue printed in or on a subject multiple cell types. In certain embodiments, the tissue printed in or on a subject comprises more than one type of tissue.

In certain embodiments, the methods described herein comprise deposition of cells, ECM, and/or other components on a surface in or on a subject, wherein said surface comprises tissue from a subject. In certain embodiments, the methods described herein comprise deposition of cells, ECM, and/or other components on a surface in or on a subject, wherein said surface comprises an organ of the subject.

In a c embodiment, the cells printed in or on a subject comprise connective tissue cells. In certain embodiments, said connective tissue cells are printed on connective tissue of said subject (i.e., connective tissue in or on the subject).

In another speci?c embodiment, the cells printed in or on a subject comprise muscle tissue cells. In n ments, said muscle tissue cells are printed on muscle tissue of said subject (i.e., muscle tissue in or on the subject). The muscle tissue can comprise visceral h) muscle tissue, skeletal muscle tissue, or cardiac muscle tissue.

In another speci?c embodiment, the cells printed in or on a subject comprise neural tissue cells. In certain embodiments, said neural tissue cells are printed on neural tissue of said t (i.e., neural tissue in or on the subject). The neural tissue can comprise l nervous system tissue (e.g., brain tissue or spinal cord tissue) or peripheral nervous system tissue (e.g., cranial nerves and spinal nerves).

In another speci?c embodiment, the cells printed in or on a t se epithelial tissue cells. In certain embodiments, said epithelial tissue cells are printed on epithelial tissue of said subject (i.e., epithelial tissue in or on the subject). The epithelial tissue can comprise endothelium.

In n embodiments, the cells, ECM, and/or other components printed in or on a subject are printed on an organ of the subject. The cells, ECM, and/or other components can be printed in or on an organ of a subject that is 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, s system, reproductive system, respiratory system, and/or skeletal system. ary 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, te, , ovaries, spinal cord, pharynx, larynx, a, bronchi, diaphragm, ureter, urethra, esophagus, colon, thymus, and spleen. In a speci?c embodiment, a as or portion thereof is generated or formed in accordance with the methods described herein.

In a speci?c embodiment, the cells, ECM, and/or other components printed in or on a t are printed on bone. In another speci?c embodiment, the cells, ECM, and/or other components printed in or on a subject are printed on skin. In another speci?c embodiment, the cells, ECM, and/or other ents printed in or on a subject are not printed on skin. In another c embodiment, the cells, ECM, and/or other components printed in or on a subject are printed on lung tissue, or a lung or portion thereof. In another speci?c embodiment, the cells, ECM, and/or other components printed in or on a subject are printed on liver tissue, or a liver or n thereof. In another speci?c embodiment, the cells, ECM, and/or other components printed in or on a subject are printed on neural tissue, or a nerve or portion thereof. 4.1.3 EXTRACELLULAR MATRIX (ECM) The methods described herein comprise the deposition of cells (e. g., compositions comprising single cells and/or itions comprising multiple cells) and extracellular matrix (ECM), including ?owable ECM, on a surface in or on a t. The ECM can be derived from any known source of ECM, and can be made e using any method known in the art. In speci?c embodiments, the ECM comprises ?owable ECM. The ECM can be made ?owable 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.

The ECM (e. g., a ?owable 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.

] In certain embodiments, the ECM used in accordance with the methods described herein ses ian ECM, plant ECM, molluscan ECM, and/or piscine ECM.

In a speci?c embodiment, the ECM used in accordance with the methods described herein comprises mammalian ECM. In r speci?c embodiment, the ECM used in accordance with the s described herein ses mammalian ECM, wherein said mammalian ECM is derived from a placenta (e.g., a human placenta). In another speci?c embodiment, said placental—derived ECM comprises telopeptide en.

In another speci?c embodiment, said placental—derived ECM comprises base-treated and/or detergent treated Type I telopeptide placental en that has not been chemically modi?ed or ted with a protease, wherein said ECM comprises less than 5% ?bronectin 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 en by weight.

In another speci?c ment, said placental-derived ECM comprises base-treated, detergent treated Type I telopeptide placental collagen that has not been chemically modi?ed or contacted with a se, wherein said ECM comprises less than 1% ?bronectin or less than 1% n by ; between 74% and 92% Type I collagen by ; and 4% to 6% Type III collagen or 2% to 15% type IV collagen by weight.

Placental ECM, e. g., ECM comprising placental telopeptide collagen, used in accordance with the methods bed herein, may be prepared using methods known in the art, or may be prepared as follows. First, tal 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, 6.g. The tal tissue can be from any part of the placenta , aseptically. 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 t the umbilical cord. The placenta may be stored at room temperature, or at a temperature of about 20 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 ed prior to the time of birth, for, e.g., HIV, HBV, HCV, HTLV, syphilis, CMV, and other Viral pathogens known to inate placental tissue.

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 US. Patent Application Publication Nos. 20040048796 and 20030187515, the contents of which are hereby incorporated by reference in their entireties.

The placental tissue may be subjected to an c shock. The osmotic shock can be in addition to any clari?cation step or it can be the sole clari?cation step ing 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 ofNaCl (e.g., 02-10 M or 02-20 M), KCl (e.g., 02-10 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 on, 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 on is about 0.25-5M, about 0.5-4M, about 0.75-3M, or about 1.0-2.0M NaCl. The low osmotic potential on can be any low c 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 n 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 ed by a water wash.

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 ic 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 ° C, about 5° C to about 15° C, about 0° C, about 5° C, about 10° C, about ° 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.

The composition resulting from the detergent ent may then, in certain embodiments, be incubated under basic conditions. Particular bases for the basic treatment e 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 ofNH4OH, 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° 0, 5° C to 25° 0, 5° C to 20° C., 5° C to 15° C, about 0° C., about 5° 0, about 10° 0, about 15° 0, about 20° 0, 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.

The ECM can be produced without treatment by a base; on of a base ent step typically results in an ECM composition comprising relatively higher amounts of elastin, ?bronectin and/or laminin than the ECM composition ed with inclusion of the basic treatment.

Typically, the process described above for human placental tissue results in tion of placental ECM comprising base-treated and/or ent treated Type I telopeptide placental collagen that has not been chemically modi?ed or contacted with a protease, wherein said ECM comprises less than 5% ?bronectin or less than 5% laminin by weight; n 25% and 92% Type I collagen by weight; between 2% and 50% Type III collagen; between 2% and 50% type IV collagen by ; and/or less than 40% elastin by weight. In a more speci?c embodiment, the process results in production of base-treated, detergent treated Type I telopeptide tal en, wherein said collagen has not been chemically modi?ed or contacted with a protease, and wherein said composition comprises less than 1% ?bronectin by weight; less than 1% laminin by weight; n 74% and 92% Type I collagen by weight; n 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.

In certain embodiments, compositions provided herein that comprise ?owable ECM may additionally comprise other ents. In certain embodiments, the compositions provided herein that comprise ?owable ECM additionally comprise one or more cell types, e.g., one or more of the cell types ed 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.

In certain embodiments, the compositions provided herein that comprise ?owable ECM additionally comprise a hydrogel (e.g., a thermosensitive hydrogel and/or a photosensitive el). Alternatively, a hydrogel may be deposited as part of a separate composition in accordance with the methods bed herein concurrently with, before, or after the deposition of said ECM.

In certain embodiments, the compositions provided herein that comprise ?owable 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 speci?c embodiment, the compositions provided herein that comprise ?owable ECM and a hydrogel (e.g., a thermosensitive hydrogel and/or a photosensitive hydrogel) are formulated such that the ratio of ECMzhydrogel ranges from about :1 to about 1:10 by weight.

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 itions include self-assembling es, such as RAD16. Hydrogel-forming materials include polysaccharides such as alginate and salts thereof, peptides, polyphosphazines, and rylates, which are crosslinked ionically, or block polymers such as polyethylene oxidepolypropylene glycol block copolymers which are crosslinked by temperature or pH, respectively. In some embodiments, the hydrogel or matrix may be biodegradable.

In n embodiments, the compositions provided herein that comprise ?owable ECM additionally comprise a synthetic polymer. In a speci?c ment, 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, thyl acrylate, carboxymethylcellulose, and/or poly(lactic-co-glycolic acid) (PLGA). In another speci?c embodiment, the synthetic polymer comprises a thermoplastic, e.g., polycaprolactone (PCL), polylactic acid, polybutylene terephthalate, hylene terephthalate, polyethylene, polyester, polyvinyl acetate, and/or polyvinyl chloride. Alternatively, one or more synthetic rs 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 speci?c embodiment, the synthetic polymer is PCL.

In certain embodiments, the compositions provided herein that comprise le ECM additionally se tenascin C, a human protein known to interact with ?bronectin, or a nt f. Alternatively, tenascin C may be deposited as part of a separate composition in ance with the methods described herein concurrently with, before, or after the deposition of said ECM.

In certain embodiments, the compositions ed herein that comprise ?owable ECM additionally se titanium-aluminum-vanadium (Ti6Al4V). Alternatively, Ti5Al4V 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 certain embodiments, the ECM in a composition provided herein and/or an onal component of the composition, such as a tic polymer, may be derivatized.

Methods for derivatization of ECM and synthetic rs are known in the art, and include, without limitation, derivatization using cell attachment es (e.g., a e 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 The ECM used in accordance with the s described herein can be made ?owable using methods known in the art and described herein.

In one embodiment, the ECM used in accordance with the methods described herein is made ?owable by contacting the ECM with an acid or base, e.g., an acidic or basic solution sing an amount of said acid or base that is suf?cient to lize said ECM. Once the ECM has been made ?owable, if desired, the ECM containing composition can be made l, or brought to a d pH, using methods known in the art.

In another embodiment, the ECM used in accordance with the methods described herein is made ?owable 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 ?owable, if desired, the enzymes can be inactivated using methods known in the art.

In another embodiment, the ECM used in accordance with the methods described herein is made ?owable using physical approaches. In a specific embodiment, the ECM used in accordance with the methods described herein is made ?owable by milling the ECM, i.e., grinding the ECM so as to overcome of the interior bonding forces. In another speci?c embodiment, the ECM used in accordance with the methods described herein is made ?owable by shearing the ECM, e.g., with a blender or other source. In r c embodiment, the ECM used in accordance with the methods described herein is made ?owable by cutting the ECM. In certain embodiments, when ECM is made more ?owable 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). 2 Methods of Cross-linking ECM The ECM used in accordance with the methods described herein can be cross-linked using s known in the art and described herein.

In certain embodiments, the ECM is cross-linked before it is applied to a surface in or on a subject, i.e., the ECM may be linked before printing. In accordance with such embodiments, a cross-linker may be included in a ition 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 ng of the ECM.

In other embodiments, the ECM is cross-linked after it is applied to a surface in or on a subject, i.e., the ECM may be cross-linked after ng. In one embodiment, the ECM is cross-linked after it is applied to a surface in or on a subject by ?rst 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 ary, the ECM can subsequently be linked by treating the ECM and cross-linker under conditions that give rise to the cross-linking of the ECM.

] In another embodiment, the ECM is cross—linked after it is applied to a e in or on a subject 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.

] In a speci?c embodiment, the ECM is cross-linked by chemical cross-linking of hyaluronic acid, an ECM component. In another speci?c embodiment, the ECM is cross-linked by chemical cross-linking ofECM proteins. Exemplary means of chemical cross-linking hyaluronic acid and ECM ents include those described in, e.g., Burdick and Prestwich, 2011, Adv. Mater. 232H4l-H56.

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

In another speci?c embodiment, the ECM is cross-linked by the use of enzymes.

Enzymes suitable for cross-linking ofECM include, without limitation, lysyl oxidase (see, e. g., Levental et al., 2009, Cell 1392891—906) and tissue type lutaminases (see, e. g., Grif?n et al., 2002, J. Biochem. 368:377—96).

Those of skill in the art will understand the need to select biocompatible chemical cross-linkers, i.e., cross-linkers that have been deemed safe for use in subjects (e.g., human subjects). 4.1.4 SURFACES Any le e in or on a t can be used as the surface upon which the cells, ?owable ECM, and/or any additional components can be deposited (e.g., printed) on in accordance with s described herein.

In one embodiment, the surface in or on a subject upon which the cells, ?owable ECM, and/or any additional components are deposited comprises an arti?cial surface that has been transplanted into said subject, i.e., a surface that has been man-made. In a speci?c embodiment, said arti?cial e is a etic. Such arti?cial surfaces may be selected based on their suitability for administration to and/or transplantation in a subject, e.g., a human subject.

For example, an arti?cial surface known not to be immunogenic (Le, a surface that does not elicit a host immune response) may be selected for use. In certain ments, an arti?cial surface may be d so as to render it suitable for administration to and/or transplantation in a subject, e.g., a human t.

In one embodiment, the surface in or on a subject upon which the cells, ?owable 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.

In one embodiment, the e in or on a subject upon which the cells, ?owable ECM, and/or any additional ents are deposited comprises a metal surface. Exemplary types of c 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, y, nickel, platinum, silver, tin, um, tungsten, and zinc.

In certain embodiments, the arti?cial surfaces in or on a subject upon which the cells, ?owable ECM, and/or any additional components are deposited are engineered so that they form a particular shape. For example, an arti?cial 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), ?owable ECM, and/or any additional components may be deposited on and/or in said e.

In another embodiment, said surface in or on a subject comprises a tissue or an organ of a subject (e.g., a human subject). In certain embodiments, the surface in or on a subject of said tissue or organ from a subject may be ularized, e.g., treated so as to remove cells from all or part of the surface of the tissue or organ.

In another embodiment, said surface in or on a subject is ed for a method described herein by depositing a composition comprising a biomolecule on said surface prior to said printing, wherein said biomolecule is or comprises a type of collagen, a type of ?bronectin, a type of laminin, or a tissue ve.

In another embodiment, said surface in or on a subject is prepared for a method described herein by ng all or a portion of said surface with a decellularized tissue, e.g., ularized ic membrane, decellularized diaphragm, decellularized skin, or decellularized fascia.

In accordance with the methods described herein, cells, ?owable ECM, and/or any additional components may be deposited on (e.g., printed on) any suitable tissue or organ of a subject. In a speci?c embodiment, the tissue of the subject that provides the printing surface is connective tissue (including bone), muscle tissue (including al (smooth) muscle tissue, skeletal muscle tissue, and cardiac muscle tissue), neural tissue (including central s system tissue (e.g., brain tissue or spinal cord tissue) or peripheral nervous system tissue (e.g., cranial nerves and spinal nerves)), or lial tissue (including endothelium). In another speci?c embodiment, the organ of the subject 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, mentary system, muscular system, nervous system, reproductive system, atory system, and/or skeletal system. In r speci?c embodiment, the organ of a subject 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 ine, prostate, testes, ovaries, spinal cord, pharynx, larynx, a, bronchi, diaphragm, ureter, urethra, esophagus, colon, thymus, and spleen. In another c embodiment, a pancreas of a subject that provides the printing surface for the s described herein.

In a c embodiment, the cells, e ECM, and/or any additional components are deposited on (e.g., printed on) a surface in or on a subject that comprises or consists of bone.

Exemplary bones that can be printed on include long bones, short bones, ?at bones, irregular bones, and seismoid bones. c 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 ?ngers, bones of the feet and toes, ankle bones, wrist bones, chest bones (e.g., the sternum), and the like.

In a speci?c ment, the cells, ?owable ECM, and/or any onal components are deposited on (e. g., printed on) a surface in or on a subject, wherein the surface is on the exterior of said subject. In a speci?c embodiment, the cells, ?owable ECM, and/or any onal components are deposited on (e.g., printed on) a surface in or on a subject, wherein the surface is within the interior of said individual. In a speci?c embodiment, the cells, ?owable ECM, and/or any additional components are deposited on (e.g., printed on) a surface in or on a t, wherein the surface is within a body cavity or organ of said individual.

] In certain embodiments, the surfaces in or on a subject bed herein that serve as scaffolds for the deposition (e.g., deposition by bioprinting or by other means) of cells, ?owable ECM, and/or any additional components are surfaces that have not been bioprinted. In certain embodiments, the surfaces in or on a subject described herein that serve as lds for the tion (e.g., deposition by bioprinting or by other means) of cells, ?owable ECM, and/or any additional components are surfaces that have been bioprinted, e.g., bioprinted in accordance with the methods described herein. In a speci?c embodiment, the bioprinted surface comprises a synthetic material. In a c embodiment, the synthetic material is PCL. 4.2 COMPOSITIONS Provided herein are compositions that can be used in accordance with the methods described herein. In one embodiment, ed 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 itions comprising ?owable ECM (e. g., the ?owable 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 sing 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. .

In one embodiment, provided herein is a composition comprising cells (e. g., the cells described in Section 4.1.1, above) and ?owable ECM (e.g., the ?owable ECM described in Section 4.1.3, above). In a speci?c 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 speci?c embodiment, the ?owable ECM is derived from placenta (e.g., human placenta).

] In another embodiment, ed herein is a composition comprising ?owable ECM (e.g., the ?owable 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).

In r 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).

In r embodiment, provided herein is a composition comprising cells (e.g., the cells described in n 4.1.1, above), ?owable ECM (e.g., the ?owable ECM described in Section 4.1.3, above), and one or more cross-linkers (e.g., the cross-linkers bed in Section 4.1.3.2, above).

] In a c embodiment, a composition provided herein ses stem cells and ?owable ECM, wherein said stem cells are PDACs and wherein said ?owable ECM is derived from placenta. In another speci?c embodiment, a composition ed herein comprises stem cells and a cross-linker, wherein said stem cells are PDACs. In another speci?c embodiment, a composition ed herein comprises stem cells, ?owable ECM, and a cross-linker, wherein said stem cells are PDACs and wherein said ?owable ECM is derived from placenta.

In another speci?c embodiment, a composition provided herein comprises stem cells and ?owable ECM, wherein said stem cells are AMDACs and wherein said ?owable ECM is derived from ta. In another speci?c embodiment, a composition provided herein comprises stem cells and a cross-linker, wherein said stem cells are . In another speci?c embodiment, a composition provided herein comprises stem cells, ?owable ECM, and a cross-linker, wherein said stem cells are AMDACs and wherein said ?owable ECM is derived from placenta.

] In another speci?c embodiment, a composition provided herein comprises stem cells and ?owable ECM, wherein said stem cells are BM—MSCs and wherein said e ECM is derived from placenta. In r speci?c embodiment, a composition provided herein comprises stem cells and a cross-linker, wherein said stem cells are BM-MSCs. In another speci?c embodiment, a composition provided herein comprises stem cells, ?owable ECM, and a cross-linker, wherein said stem cells are BM-MSCs and wherein said ?owable ECM is derived from placenta.

The compositions provided , in addition to comprising cells (e.g., the cells described in Section 4.1.1, above) and/or ?owable ECM (e.g., the ?owable ECM described in n 4.1.3, above) and/or one or more cross—linkers (e.g., the cross-linkers described in n 4.1.3.2, above) may additionally se other components. In certain embodiments, the compositions provided herein additionally se a hydrogel (e.g., a thermosensitive hydrogel and/or a photosensitive hydrogel. Alternatively, a hydrogel may be formulated in a composition te from the cell and ECM sing 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, nyl acetate, and/or polyvinyl chloride). Alternatively, a synthetic polymer may be ated in a composition separate from the cell and ECM comprising compositions ed herein. In certain embodiments, the compositions provided herein additionally comprise tenascin C or a fragment thereof.

Alternatively, tenascin C or a fragment thereofmay be formulated in a composition separate from the cell and ECM comprising compositions ed . In certain embodiments, the compositions provided herein that additionally comprise titanium-aluminum-vanadium (Ti6Al4V). Alternatively, Ti6Al4V may be formulated in a composition te from the cell and ECM comprising compositions provided .

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 itions. Such components may include, without limitation, nutrients, salts, sugars, survival factors, and growth factors. Exemplary growth s that may be used in ance with the methods described herein include, without limitation, n-like growth factor (e.g., IGF-l), transforming growth factor-beta eta), bone-morphogenetic protein, ?broblast growth factor, platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), basic ?broblast growth factor (bFGF), epidermal growth factor, ?broblast 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 e the survival, differentiation, proliferation, etc. of the ce11(s) may be formulated in a composition separate from the cell and ECM comprising compositions provided herein. 4.3 USES The methods described herein can be used for any suitable purpose. In a speci?c embodiment, the methods described herein are used for therapeutic purposes, e.g., cells, ECM, and/or other additional ents are printed in or on a subject so as to result in a eutic effect in said subject. 4.3.1 Therapeutic Uses In certain ments, the methods described herein are used to treat a subject in need of a transplant (e.g., a tissue or organ transplant). In n embodiments, the methods described herein are used to treat a subject in need of a graft (e.g., a skin graft). Methods of transplantation, including grafting (e. g., skin grafting) and surgical lantation procedures are well-known to those of skill in the art and can be modi?ed to conform to the methods described herein.

In n embodiments, the cells and/or ECM used in the methods described herein are derived are from the lant recipient. In other embodiments, the cells and/or ECM used in the methods described herein are not from the transplant recipient, but are from another subject, e.g., a donor, a cadaver, etc. In certain embodiments, the cells used in the methods described herein are from the transplant recipient, and the ECM used in the methods described herein is not from the transplant recipient, but is from another source. In a speci?c embodiment, the ECM used in the methods described herein is derived is from a placenta (e.g., a human placenta). In certain embodiments, the ECM used in the s described herein is derived is from the transplant recipient, and the cells used in the methods described herein are not from the lant recipient, but are from another .

In a c embodiment, the methods bed herein are used to generate lial tissue (e.g., skin, dermis, or epidermis) on a surface in or on a subject, wherein said subject is in need of such epithelial tissue (e.g., the subject is a burn victim or has or had a form of skin disease). In a speci?c embodiment, said subject is human. In another speci?c embodiment, at least one cellular composition used in the method comprises mal cells. In r speci?c embodiment, at least one cellular composition used in the method comprises dermal cells. In another speci?c embodiment, at least one cellular composition used in the method comprises mesenchymal stem cells. In r speci?c embodiment, the cellular composition(s) used in the method comprise epidermal cells, dermal cells, and mesenchymal stem cells. In another speci?c embodiment, the surface of the subject is a portion of the subject’s skin.

] In another speci?c embodiment, the methods described herein are not used to generate epithelial tissue (e.g., skin, dermis, or epidermis) on a surface in or on a subject.

In another speci?c embodiment, the methods described herein are used to generate tive tissue (e.g., bone) on a surface in or on a subject, wherein said t is in need of such connective tissue (e.g., the t has or had osteoporosis or bone cancer). In a speci?c embodiment, said subject is human. In another speci?c embodiment, the surface of the subject is one or more of the subject’s bones.

In another speci?c embodiment, the methods described herein are used to generate neural tissue (e.g., brain tissue or spinal cord tissue) on a surface in or on a t, wherein said subject is in need of such neural tissue. In a speci?c embodiment, said subject has been diagnosed with a neural disease (i.e., a disease of the central or eral s system). In another speci?c 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 speci?c embodiment, said subject is human. . In another speci?c embodiment, the surface of the subject is the subject’s brain. . In r speci?c embodiment, the surface of the subject is the subject’s spinal cord.

In another speci?c embodiment, the methods described herein are used to generate liver tissue on a surface in or on a subject, wherein said subject is in need of such liver tissue (e.g., the subject has or had cirrhosis of the liver, hepatitis, or liver cancer). In a c embodiment, said subject is human. In another speci?c embodiment, the surface of the t is the subject’s liver.

] In another speci?c embodiment, the methods described herein are used to generate lung tissue on a e in or on a t, wherein said subject is in need of such lung tissue (e.g., the subject has or had lung cancer, ema, or COPD). In a speci?c embodiment, said subject is human. In another speci?c ment, the surface of the subject is the subject’s lung.

In another c embodiment, the methods described herein are used to generate circulatory system tissue (e.g., heart tissue, arteries, or veins) on a surface in or on a subject, wherein said subject is in need of such circulatory system tissue (e.g., the subject has or had heart disease, coronary artery disease, or issues with the valves of the heart). In a speci?c embodiment, said subject is human. In another speci?c embodiment, the surface of the subject is the subject’s heart. In another c embodiment, the surface of the subject is the one or more of the subject’s arteries or veins.

In another speci?c embodiment, the methods described herein are used to te kidney tissue on a surface in or on a subject, wherein said subject is in need of such kidney tissue (e.g., the subject has or had a form of kidney e). In a speci?c embodiment, said subject is human. In another speci?c embodiment, the cellular composition used in the method comprises at least one type of parenchymal cell and one type of stromal cell. In another speci?c embodiment, said one type of parenchymal cell and said one type of stromal cells are present in a population of kidney cells disaggregated from autologous or allogeneic kidney tissue. In another speci?c embodiment, the surface of the subject is one or both of the subject’s kidneys.

In another speci?c embodiment, the methods described herein are used to generate pancreatic tissue on a surface in or on a subject, wherein said subject is in need of such pancreatic tissue (e. g., the subject has or had pancreatic ). In a speci?c embodiment, said subject is human. In another speci?c embodiment, the surface of the subject is the subject’s pancreas.

In another speci?c embodiment, the methods described herein are used to generate prostate tissue on a surface in or on a subject, wherein said subject is in need of such prostate tissue (e. g., the subject has or had prostate cancer). In a speci?c embodiment, said subject is human. In another c embodiment, the surface of the subject is the subject’s te.

In another speci?c embodiment, the methods described herein are used to generate stomach tissue on a surface in or on a subject, wherein said subject is in need of such stomach tissue (e. g., the subject has or had stomach cancer). In a speci?c embodiment, said subject is human. In another speci?c embodiment, the surface of the t is the subject’s stomach.

In another speci?c embodiment, the methods described herein are used to te colon tissue on a e in or on a subject, wherein said subject is in need of such colon tissue (e.g., the subject has or had colon ). In a c embodiment, said subject is human. In r speci?c embodiment, the surface of the subject is the subject’s colon.

In another speci?c embodiment, the methods described herein are used to generate intestinal tissue (e.g., one or more tissues associated with the small or large intestine) on a surface in or on a subject, wherein said subject is in need of such intestinal tissue (e.g., the subject has or had a disease of the intestines). In a speci?c embodiment, said subject is human.

In another speci?c embodiment, the surface of the subject is a portion of the subject’s small intestine. In another speci?c embodiment, the surface of the subject is a portion of the t’s large intestine.

In another speci?c embodiment, the methods described herein are used to te esophageal tissue on a surface in or on a subject, wherein said subject is in need of such esophageal tissue. In a c embodiment, said t is human. In another speci?c embodiment, the surface of the subject is the subject’s esophagus.

In another speci?c embodiment, the methods described herein are used to generate thyroid tissue on a surface in or on a subject, wherein said subject is in need of such thyroid tissue. In a speci?c embodiment, said subject is human. In another speci?c embodiment, the surface of the t is the subject’s thyroid.

In another c embodiment, the methods described herein are used to deposit cells in a subject, wherein said cells are engineered to express one or more s (e.g., proteins or polypeptides), and wherein said subject is in need of said one or more factors, e.g., the subject is de?cient in said one or more factors (e.g., due to genetic mutation or genetic predisposition).

In a speci?c embodiment, the subject is a human, e.g., a human with a genetic e or er, wherein said genetic disease or disorder can be treated with, in whole or in part, said one or more factors. In r speci?c embodiment, the t is a human with a e that can be treated with, in whole or in part, said one or more factors. In accordance with such embodiments, the cells can be deposited on any surface, in or on said subject, so long as the one or more factors are expressed by the cell in amount suf?cient to treat the disease or disorder in question. Exemplary tissues and organs in or on a subject upon which said cells can be deposited are described in Section 4.1.2, above. Exemplary factors that can be produced by said cells are described in Section 4.1.1, above. 4.3.2 Patient Populations The methods described herein can be used to bene?t various patient populations. In one embodiment, the methods described herein are used in subjects that require an organ transplant. In another embodiment, the methods bed herein are used in subjects that require ent of a disease or disorder.

In a speci?c embodiment, the methods described herein are used to engineer tissue 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 speci?c embodiment, the methods described herein are used to er tissue in a subject that has been diagnosed with a bone or connective tissue sarcoma, brain cancer, breast cancer, ovarian cancer, kidney , pancreatic , esophageal cancer, h cancer, esophageal cancer, liver cancer, lung cancer (e.g., small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), throat , and mesothelioma), colon cancer and/or prostate cancer.

] In r speci?c embodiment, the methods described herein are used to engineer tissue in a subject that has been diagnosed with a respiratory disease, e. g., the t has been diagnosed with asthma, chronic obstructive pulmonary disorder (COPD), emphysema, pneumonia, tuberculosis, lung cancer and/or cystic ?brosis.

In another speci?c embodiment, the methods described herein are used to engineer tissue 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.

In another speci?c ment the s described herein are used to engineer tissue in a subject that has been diagnosed with bone cancer (e.g., osteosarcoma), osteonecrosis, metabolic bone disease, Fibrodysplasia ossi?cans progressive, or osteoporosis.

In another c embodiment, the methods described herein are used to engineer tissue in a t 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 le sclerosis.

In r speci?c embodiment, the methods described herein are used to engineer tissue in a subject that has undergone trauma that has damaged the central or eral nervous system of the subject, e.g., the subject has suffered a traumatic brain injury (TBI) or spinal cord injury (SCI).

In another speci?c embodiment, the methods described herein are used to engineer tissue in a t that has been diagnosed with a disease of the circulatory , e.g., the subject has been diagnosed with coronary heart e, cardiomyopathy (e.g., intrinsic or extrinsic cardiomyopathy), heart attack, stroke, in?ammatory heart disease, hypertensive heart disease, or valvular heart disease.

In another speci?c embodiment, the methods described herein are used to engineer tissue in a subject that has been diagnosed with kidney disease. In one embodiment, the subject possesses one kidney that is malfunctioning. In another embodiment, the subject possesses two s that are mal?lnctioning.

In another speci?c embodiment, the s described herein are used to engineer tissue in a subject that has been diagnosed with a genetic disease or disorder, e.g., the subject has been diagnosed with familial hypercholesterolemia, stic kidney disease, or ketonuria.

In r speci?c embodiment, the methods described herein are used to er tissue in a subject that has been diagnosed with diabetes.

In another speci?c embodiment, the methods bed herein are used to engineer tissue in a subject that has been diagnosed with a cleft palate.

In another speci?c embodiment, the methods described herein are used to engineer tissue in a subject that undergoes regular procedures that require piercing of the subject’s skin (e.g., the subject is a dialysis patient).

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 n 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 speci?c embodiment, a subject to which a tissue or organ ted in accordance with the methods described herein is transplanted is a human.

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 ted in accordance with the methods described herein is transplanted is a human child. 4.4 KITS Provided herein is a pharmaceutical pack or kit comprising one or more containers ?lled with one or more of the ients of the compositions described herein. Optionally associated with such ner(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice re?ects approval by the agency of cture, use or sale for human administration.

In a speci?c embodiment, a kit provided herein comprises a composition comprising the cells described herein and the ?owable ECM bed herein. Such a kit may ally se a composition comprising one or more onal components. The kits encompassed herein can be used in accordance with the methods described herein.

. EXAMPLES .1 EXAMPLE 1: BIOPRINTED SCAFFOLDS T ATTACHMENT AND GROWTH OF PLACENTAL STEM CELLS This example demonstrates that synthetic material can be bioprinted to produce scaffolds of controlled ?ber er 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) ent a suitable ate for the attachment and growth of cells, including placental cells, such as placental stem cells. .1.1 Methods ] To fabricate hybrid scaffolds comprising synthetic material and ECM, polycaprolactone (PCL) (Mn 45,000, Sigma) was ?rst 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 s. ECM was isolated from human placenta as previously described (see, e. g., Bhatia MB, Wounds 20, 29, 2008). ed 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/cmz. The cells were cultured over an 8- daytime 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. . 1.2 Results ] By optimizing printing conditions, PCL lds of different ?ber sizes, pore sizes and pore structures were generated (Fig. 1). The printed ?bers formed a stable network for the tion of hybrid scaffolds comprising PCL and ECM. Further, the printing of varying ?ber sizes and pore structures made it possible to make hybrid scaffolds comprising various properties. ation ofECM 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 tion between the PCL and ECM was d when the hybrid scaffolds were manipulated by processing or culturing of the lds, which included rehydration (Fig. 2).

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 signi?cantly 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 n staining (Fig. 4). Together, these data indicate that PCL-ECM hybrid lds support cellular attachment, al, and growth. .1.3 Conclusion 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 lds. .2 EXAMPLE 2: BIOPRINTED SCAFFOLDS SUPPORT MENT AND GROWTH OF PLACENTAL STEM CELLS 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 erate over time in culture with the hybrid scaffolds. .2.1 Methods ECM was prepared as described in Example 1 and mixed with 0.5% te hydrogel containing 1 million/ml placental stem cells. Next, PCL and the cell-containing ECM were bioprinted, in , to generate a hybrid scaffold sing PCL and ECM. In each layer of the scaffold, PCL was first d, then the ECM/cell component was printed to fill the gaps in between the PCL lines. Two or ?ve of such layers were printed and crosslinked with CaClz 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. .2.2 s The bioprinted scaffolds maintained an intact ure throughout the duration of cell culture (Fig. 5). PCL provided a good ural 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 ucts; cells were found throughout the depth of the scaffolds during culture (Fig. 6).

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 ng (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 te 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 ted cell growth. .2.3 Conclusion This example demonstrates that hybrid scaffolds comprising ECM and synthetic material (PCL) can be generated by methods that comprise aneous bioprinting ofECM 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 s. 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. .3 EXAMPLE 3: FUNCTIONAL BIOPRINTED LDS This example demonstrates that synthetic material and ECM comprising cells can be bioprinted to produce functional scaffolds.

] B-TC-6 cells, an n producing cell line, were bioprinted with human placenta derived extracellular matrix (ECM) into a bioprinted ld. The ld 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 ning 15% fetal calf serum in a cell e incubator in 6 well plates (3 to ml ofmedium per well). At different time points, the scaffolds were harvested for n staining and MTS proliferation assays, to characterize cell viability and cell eration, respectively. Figure 10 shows the structure of the bio-printed scaffolds.

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 ed alive in each layer (see Figure 10).

The MTS assay con?rmed 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).

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 ld 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 ined the y to produce and secrete insulin after being bioprinted.

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 on by exposing the scaffolds to a glucose surge challenge (see Figure 13). One ld (“A” of Figure 13) was exposed to glucose starvation conditions (IMDM medium without e, 10% FCS) for two days and then challenged with an insulin producing ion (50 mM glucose/ 1 mM IBMX).

As controls, bioprinted lds 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 ld (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 ions followed by nge 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 ing media change as compared to the level of insulin production at 0.5 hour post-media change).

This Example demonstrates that bioprinted scaffolds comprising synthetic material, cells, and ECM can be generated and that the cells of the bioprinted lds remain both Viable and functional.

The compositions and methods disclosed herein are not to be limited in scope by the speci?c embodiments described herein. , various modi?cations 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 ?gures. Such modi?cations are intended to fall Within the scope of the appended claims.

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

Claims (27)

WHAT IS CLAIMED IS:

1. A method of forming a three-dimensional tissue comprising bioprinting at least one cellular composition and placental extracellular matrix (ECM) onto a surface that is to be transplanted into or onto a human or animal subject, wherein the cellular composition comprises insulin-producing cells and the tal ECM is prepared using a process that comprises incubating placental tissue in a solution of high osmotic potential.

2. The method of claim 1, wherein said cellular composition and said ECM are both deposited onto said surface.

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

4. The method of any preceding claim, wherein said surface is an artificial surface.

5. The method of claim 4, wherein said artificial surface is a prosthetic device or structure.

6. The method of any one ofclaims 1-3, wherein said e is decellularized tissue or a decellularized organ.

7. The method of any preceding claim, n said ECM comprises telopeptide tal collagen.

8. The method of claim 7, wherein said telopeptide placental collagen comprises basetreated Type I telopeptide placental collagen.

9. The method of claim 7 or claim 8, wherein said collagen has not been chemically modified or contacted with a protease.

10. The method of any preceding claim, wherein said placental ECM comprises base-treated and/or ent treated Type I telopeptide placental collagen that has not been chemically modified or contacted with a protease, n 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.

11. The method of any preceding claim, wherein said placental ECM comprises base-treated, detergent treated Type I telopeptide placental en that has not been chemically modified or ted with a se, 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.

12. The method of any preceding claim, further sing deposition of a hydrogel.

13. The method of claim 12, wherein said hydrogel is a thermosensitive hydrogel.

14. The method of claim 12, n said hydrogel is a photosensitive hydrogel.

15. The method of any one of claims 12-14, wherein said ECM and said hydrogel are combined in a ratio of about 10:1 to 1:10 by weight.

16. The method of any one of claims 1-15, further comprising deposition of a synthetic polymer.

17. The method of claim 16, wherein said synthetic polymer is thermosensitive.

18. The method of claim 16, n said synthetic r is ensitive.

19. The method of claim 16, wherein said synthetic polymer comprises a thermoplastic.

20. The method of claim 19, wherein said synthetic polymer is poly(L-lactide-co-glycolide)

21. The method of claim 19, wherein said thermoplastic is polycaprolactone, polylactic acid, polybutylene terephthalate, hylene terephthalate, polyethylene, polyester, polyvinyl acetate, or polyvinyl chloride.

22. The method of claim 16, wherein said synthetic polymer is polyacrylamide, polyvinylidine chloride, poly(o-carboxyphenoxy)-p-xylene) (poly(o-CPX)), poly(lactideanhydride ) (PLAA), n-isopropyl acrylamide, pent erythritol diacrylate, polymethyl acrylate, carboxymethylcellulose, or poly(lactic-co-glycolic acid) (PLGA).

23. The method of any preceding claim, wherein said surface is prepared by depositing a ition sing a biomolecule on said surface prior to said printing.

24. The method of claim 23, wherein said biomolecule is or comprises a type of collagen, a type of fibronectin, a type of laminin, or a tissue ve.

25. The method of any preceding claim, wherein said surface is prepared by covering all or a portion of said surface with a decellularized tissue.

26. The method of claim 25, wherein said decellularized tissue is decellularized ic membrane, decellularized diaphragm, decellularized skin, or decellularized fascia.

27. A method according to claim 1, substantially as herein described or exemplified we. mm. M M. ?g? N3; Nu m www Hmmmw Na aw .N H Ea mmm 3H MW 3* «3% «a: Kim gma? .Eam 2/