WO2019198086A1

WO2019198086A1 - Bioengineered corneal graft and methods of preparation thereof

Info

Publication number: WO2019198086A1
Application number: PCT/IL2019/050411
Authority: WO
Inventors: Nguyen Khanh; Yan Wu; Amos Eitan; Lior SHAV; Ariel EISENBACH; Yishay Hayardeni; Aryeh Batt
Original assignee: Precise Bio Inc
Priority date: 2018-04-11
Filing date: 2019-04-11
Publication date: 2019-10-17
Also published as: IL277914B1; IL277914A

Abstract

A bioengineered corneal graft and method of making it are disclosed. The corneal bioengineered graft comprises at least one layer selected from the group consisting of: a support layer comprising a hydrogel made from a crosslinked polymer such as collagen methacrylate, polyethylene glycol diacrylate, or gelatin methacrylate, and an endothelial cell layer comprising a hydrogel made from a crosslinked polymer and comprising human endothelial cells. The bioengineered corneal graft can be produced by casting or printing, and can be molded to match the specific geometry of the cornea of the recipient.

Description

BIOENGINEERED CORNEAL GRAFT AND METHODS OF PREPARATION THEREOF

REFERENCE TO RELATED PUBLICATIONS

[0001] This application claims priority from U.S. Provisional Pat. Appl. No. 62/655,832, filed 11 April 2018.

FIELD OF THE INVENTION

[0002] This invention relates in general to bioengineered comeal grafts. It relates particularly to corneal grafts produced by additive manufacturing of biocompatible hydrogels.

BACKGROUND OF THE INVENTION

[0003] Corneal blindness is estimated to affect on the order of 10 million people worldwide. It is further estimated that only about 1% of the people suffering from corneal blindness receive treatment. In part, the low rate of treatment is due to the scarcity of comeal tissue donors. Even in cases in which comeal tissue is available, problems frequently remain, as comeal tissue can easily be damaged during the transplantation procedure, and tends to be less healthy in older people than in younger ones. Furthermore, the time frame for the donation is quite short, as the tissue should be harvested within several hours of death and transplanted within a few days after harvesting.

[0004] One solution to the problem that the supply of corneal tissue from human donors falls far short of the demand is the development of an artificial cornea. While such a proposal was first made in 1789, it was not until the latter half of the twentieth century that a functional artificial cornea was developed.

[0005] Four models of artificial cornea are currently in commercial use: the Boston keratoprosthesis, the Osteo-Odonto-Keratoprosthesis (OOKP), ALPHACOR, and the KERAKLEAR Artificial Cornea. These artificial corneas suffer from various difficulties, primarily expense, although in some cases, long-term stability has been a concern.

[0006] Another problem that affects corneal transplants, whether from human donors or keratoprostheses, is rejection of the transplanted cornea by the body of the recipient.

[0007] Thus, a corneal graft that is easily, rapidly, and inexpensively produced, that has physical, optical, and mechanical properties similar of those to natural corneas, and that is made from biocompatible materials that will lower the likelihood of its rejection by the body of the recipient and reduce the recovery time following the transplantation surgery, thus remains a long-felt, yet unmet need.

SUMMARY OF THE INVENTION

[0008] The bioengineered corneal graft and methods of its preparation disclosed herein are designed to meet this long-felt need. In preferred embodiments, the bioengineered corneal graft comprises two layers: a support layer comprising a biocompatible hydrogel having mechanical properties similar to those of the human cornea and serves in situ to at least partially function as or have the properties of the stroma; and an endothelial cell layer comprising corneal endothelial cells, preferably disposed as a monolayer on the anterior surface of the support layer. In some embodiments of the invention, the bioengineered comeal graft comprises a third layer that is shaped to correct at least partially deficiencies in the vision such as refractive errors of the eye into which the graft is to be implanted.

[0009] It is therefore an object of the present invention to disclose a bioengineered corneal graft, wherein said bioengineered comeal graft comprises an endothelial layer characterized by an anterior surface and a posterior surface, said endothelial layer comprising corneal endothelial cells.

[0010] It is a further object of this invention to disclose such a bioengineered comeal graft, wherein said endothelial layer comprises a biocompatible hydrogel. In some preferred embodiments of the invention, the endothelial layer comprises a first crosslinked polymer. In some particularly preferred embodiments of the invention, said first crosslinked polymer comprises a crosslinked polymer selected from the group consisting of crosslinked collagen, crosslinked collagen methacrylate, crosslinked gelatin, crosslinked gelatin methacrylate, crosslinked polyethylene glycol, crosslinked polyethylene glycol diacrylate, crosslinked polyethylene diacrylamide, crosslinked polyethylene glycol dimethacrylate, mixtures thereof, and combinations thereof. In some especially preferred embodiments of the invention, said first crosslinked polymer comprises a methacrylate or diacrlyate, and said methacrylate or diacrylate is characterized by a degree of acrylation/methacrylation of between 2% and 100%.

[0011] It is a further object of the invention to disclose the bioengineered comeal graft as defined in any of the above, comprising a support layer characterized by an anterior surface and a posterior surface, said support layer comprising a biocompatible hydrogel comprising a second crosslinked polymer and disposed such that said anterior surface of said support layer contacts said posterior surface of said endothelial layer. In some preferred embodiments of the invention, said second crosslinked polymer comprises a crosslinked polymer selected from the group consisting of crosslinked collagen, crosslinked collagen methacrylate, crosslinked gelatin, crosslinked gelatin methacrylate, crosslinked polyethylene glycol, crosslinked polyethylene glycol diacrylate, crosslinked polyethylene diacrylamide, crosslinked polyethylene glycol dimethacrylate, mixtures thereof, and combinations thereof. In some particularly preferred embodiments of the invention, said second crosslinked polymer comprises a methacrylate or diacrlyate, and said methacrylate or diacrylate is characterized by a degree of acrylation/methacrylation of between 2% and 100%.

[0012] It is a further object of this invention to disclose a bioengineered corneal graft comprising an endothelial layer and a support layer as defined in any of the above, comprising at least one additional layer contacting or anterior to said anterior surface of said support layer, said additional layer comprising biocompatible hydrogel comprising a third crosslinked polymer. In some preferred embodiments, said third crosslinked polymer comprises a crosslinked polymer selected from the group consisting of crosslinked collagen, crosslinked collagen methacrylate, crosslinked gelatin, crosslinked gelatin methacrylate, crosslinked polyethylene glycol, crosslinked polyethylene glycol diacrylate, crosslinked polyethylene diacrylamide, crosslinked polyethylene glycol dimethacrylate, mixtures thereof, and combinations thereof. In some particularly preferred embodiments, said third crosslinked polymer comprises a methacrylate or diacrlyate, and said methacrylate or diacrylate is characterized by a degree of acrylation/methacrylation of between 2% and 100%. In some especially preferred embodiments, said additional layer is configured to be in contact with stroma tissue of a human eye.

[0013] It is a further object of this invention to disclose a bioengineered corneal graft comprising an endothelial layer and a support layer as defined in any of the above, wherein said support layer is characterized by a thickness of between 10 and 300 pm. In some preferred embodiments of the invention, said support layer is characterized by a thickness of between 50 and 150 pm.

[0014] It is a further object of this invention to disclose the bioengineered corneal graft as defined in any of the above, wherein said endothelial layer is characterized by a thickness of between 3 and 30 pm. In some preferred embodiments of the invention, said endothelial layer is characterized by a thickness of 3 - 7 pm. [0015] It is a further object of this invention to disclose the bioengineered corneal graft as defined in any of the above, wherein said endothelial layer is characterized by a density of at least 2500 cells mm ².

[0016] It is a further object of this invention to disclose the bioengineered corneal graft as defined in any of the above, wherein said endothelial layer is characterized by a density of between 1500 and 4000 cells mm ².

[0017] It is a further object of this invention to disclose the bioengineered corneal graft as defined in any of the above, wherein said corneal endothelial cells are selected from the group consisting of:

[0018] corneal endothelial cells obtained from a human donor;

[0019] cells proliferated from corneal endothelial cells obtained from a human donor; and,

[0020] endothelial-like cells differentiated from cells selected from the group consisting of stem cells, induced pluripotent stem cells, and endothelial progenitors of autologous tissue.

[0021] It is a further object of this invention to disclose the bioengineered corneal graft as defined in any of the above, wherein at least one surface of said bioengineered comeal graft is characterized by a predetermined non-zero surface curvature. In some preferred embodiments of the invention, said surface curvature is a surface curvature characteristic of an analogous surface of a cornea in a healthy human eye. In some preferred embodiments, said surface curvature is configured to focus light impinging on said comeal graft according to a predetermined pattern. In some preferred embodiments, said surface curvature is configured to compensate for a visual deficiency in a recipient of said comeal graft.

[0022] It is a further object of this invention to disclose the bioengineered corneal graft as defined in any of the above, wherein said comeal graft is characterized by a diameter characteristic of a diameter of a cornea of a human eye.

[0023] It is a further object of this invention to disclose a method for producing a bioengineered comeal graft, wherein said method comprises producing an endothelial layer comprising endothelial cells and characterized by an anterior surface and a posterior surface.

[0024] It is a further object of this invention to disclose such a method, wherein said step of producing an endothelial layer comprises: [0025] depositing a predetermined amount of a bio-ink comprising a first aqueous solution comprising at least one first polymer and a predetermined concentration of comeal endothelial cells (115) atop a first surface;

[0026] placing at least one first spacer (600b) of predetermined height B on said first surface;

[0027] placing a second surface (530) atop said at least one first spacer, thereby forming a preliminary cornea assembly;

[0028] crosslinking at least partially said polymer within said bio-ink, thereby producing an endothelial layer (110) atop said first surface; and,

[0029] removing said first and second surfaces and said at least one first spacer.

[0030] It is a further object of this invention to disclose the method as defined in any of the above, wherein said step of producing an endothelial layer comprises:

[0031] printing, in a layer-by-layer fashion, at least one layer of a bio-ink comprising a second aqueous solution comprising at least one polymer and a predetermined concentration of comeal endothelial cells atop said first surface until said plurality of layers is characterized by a predetermined height B; and, crosslinking at least partially said polymer, thereby forming an endothelial layer; and,

[0032] removing said first surface.

[0033] It is a further object of this invention to disclose the method as defined in any of the above, comprising:

[0034] producing a support layer characterized by an anterior surface and a posterior surface, said support layer comprising a biocompatible hydrogel, at least one additional polymer, and a crosslinker, said step of producing a support layer comprising crosslinking said second polymer; and,

[0035] disposing said support layer and said endothelial layer such that said anterior surface of said support layer contacts said posterior surface of said endothelial layer.

[0036] It is a further object of this invention to disclose the method as defined in any of the above, wherein said method comprises producing a support layer, and said step of producing an endothelial layer comprises: [0037] disposing upon said anterior surface of said support layer a bio-ink comprising a predetermined concentration of endothelial cells suspended in a saline culture medium; and,

[0038] allowing said cells to settle and adhere to said support layer for a predetermined period of time.

[0039] It is a further object of this invention to disclose the method as defined in any of the above, wherein said method comprises producing a support layer, and:

[0040] said step of producing a support layer comprises depositing a predetermined amount of a first aqueous solution comprising at said at least one additional polymer (105) on a third surface (500); placing at least one second spacer (600a) of predetermined height A on said third surface; and, placing a fourth surface (510) atop said at least one second spacer;

[0041] said step of crosslinking comprising crosslinking at least partially said at least one additional polymer within said first aqueous solution, thereby producing a support layer (100); and,

[0042] said method comprises removing said second spacer, said third surface, and said fourth surface subsequent to said step of disposing said support layer such that said anterior surface of said support layer contacts said posterior surface of said endothelial layer.

[0043] In some preferred embodiments, said step of producing an endothelial layer comprises:

[0044] removing said third surface and said at least one second spacer;

[0045] depositing a predetermined amount of a bio-ink comprising a second aqueous solution comprising at least one polymer and a predetermined concentration of corneal endothelial cells (115) atop said support layer;

[0046] placing at least one first spacer (600b) of predetermined height A + B on said second surface;

[0047] placing a second surface (530) atop said at least one first spacer, thereby forming a preliminary cornea assembly; [0048] crosslinking at least partially said polymer within said bio-ink, thereby producing an endothelial layer (110) atop said support layer; and,

[0049] removing said second and fourth surfaces and said at least one first spacer.

[0050] In some other preferred embodiments, said step of producing an endothelial layer comprises:

[0051] disposing upon said anterior surface of said support layer a bio-ink comprising a predetermined concentration of endothelial cells suspended in a saline culture medium; and,

[0052] allowing said cells to settle and adhere to said support layer for a predetermined period of time.

[0053] It is a further object of this invention to disclose such a method, in which said method comprises a said step of producing a support layer comprising:

[0054] printing on a printing surface, in a layer-by-layer fashion, at least one layer of a first aqueous solution comprising said at least one additional polymer until said at least one layer reaches a predetermined height A; and,

[0055] crosslinking at least partially said polymer, thereby forming a support layer.

[0056] In some preferred embodiments, said step of producing an endothelial layer comprises:

[0057] printing atop said support layer, in a layer-by-layer fashion, at least one layer of a bio-ink comprising comeal endothelial cells until said plurality of layers is characterized by a predetermined height B, said bio-ink comprising at least one polymer in a second aqueous solution and a predetermined concentration of comeal endothelial cells; and,

[0058] crosslinking at least partially said polymer, thereby forming an endothelial layer; and,

[0059] said method comprises removing said printing surface subsequent to said steps of producing a support layer and producing an endothelial layer.

[0060] In some other preferred embodiments, said step of producing an endothelial layer comprises: [0061] printing atop said support layer, in a layer-by-layer fashion, at least one layer of a bio-ink comprising comeal endothelial cells until said plurality of layers is characterized by a predetermined height B, said bio-ink comprising a predetermined concentration of endothelial cells suspended in a saline culture medium;

[0062] disposing said bio-ink on said support layer; and,

[0063] allowing said cells to settle and adhere to said support layer for a predetermined period of time; and,

[0064] said method comprises removing said printing surface subsequent to said steps of producing a support layer and producing an endothelial layer.

[0065] It is a further object of this invention to disclose such a method, wherein said method comprises a step of producing a support layer and said step of producing a support layer comprises:

[0066] determining a shape, size, and thickness of a comeal graft;

[0067] preparing a closeable hollow mold from an inert biocompatible material, said mold comprising an upper portion and a lower portion, said upper portion and said lower portion shaped such that when they are placed together the resulting mold is characterized by said shape, size, and thickness;

[0068] placing within said mold said at least one additional polymer and a crosslinker;

[0069] closing said mold;

[0070] performing said step of crosslinking; and,

[0071] removing said support layer from said mold.

[0072] In some preferred embodiments, said step of producing an endothelial layer comprises:

[0073] placing said support layer on said fourth surface;

[0074] placing a first spacer characterized by a height A + B on said second surface, where A is said height of said spacer;

[0075] depositing a predetermined amount of a bio-ink comprising a second aqueous solution comprising at least one polymer and a predetermined concentration of comeal endothelial cells (115) atop said support layer; [0076] placing a second surface (530) atop said at least one first spacer, thereby forming a preliminary cornea assembly;

[0077] crosslinking at least partially said polymer within said bio-ink, thereby producing an endothelial layer (110) atop said support layer; and,

[0078] removing said second and fourth surfaces and said at least one first spacer.

[0079] In some other preferred embodiments, said step of producing an endothelial layer comprises:

[0080] disposing upon said anterior surface of said support layer a bio-ink comprising a predetermined concentration of endothelial cells suspended in a saline culture medium; and,

[0081] allowing said cells to settle and adhere to said support layer for a predetermined period of time.

[0082] In some preferred embodiments of the method in which the method comprises preparation of a preliminary cornea assembly, the method comprises said preliminary cornea assembly between said step of placing a second surface atop said first spacer and said step of crosslinking at least partially said polymer within said bio-ink.

[0083] In some preferred embodiments of the method in which the method requires the use of a second surface and a fourth surface, at least one of said second surface and said fourth surface is characterized by a predetermined non-zero surface curvature. In some particularly preferred embodiments, said surface curvature is characterized by a configuration selected from the group consisting of: a curvature configured to produce a bioengineered corneal graft characterized by a surface curvature matching that of a healthy human cornea; a curvature configured to produce a bioengineered corneal graft that will focus light impinging thereon according to a predetermined pattern; and, a curvature configured to produce a bioengineered comeal graft characterized by a surface curvature that will compensate for a visual defect in a recipient of said corneal graft.

[0084] In some preferred embodiments of the method in which the support layer is prepared using a mold, said shape and thickness are characterized by a configuration selected from the group consisting of: a shape and thickness configured to produce a bioengineered comeal graft characterized by a shape and thickness matching that of a healthy human cornea; a shape and thickness configured to produce a bioengineered corneal graft that will focus light impinging thereon according to a predetermined pattern; and, a shape and thickness configured to produce a bioengineered corneal graft characterized by a shape and thickness that will compensate for a visual defect in a recipient of said corneal graft.

[0085] It is a further object of this invention to disclose the method as defined in any of the above, wherein at least one of said steps of depositing is performed by Laser-Induced Forward Transfer (LIFT) printing.

[0086] It is a further object of this invention to disclose the method as defined in any of the above in which the method comprises a step of allowing said cells to settle and adhere to said support layer for a predetermined period of time, said step of allowing said cells to settle and adhere to said support layer for a predetermined period of time comprises allowing said cells to settle and adhere to said support layer for 0.5 - 5 h.

[0087] It is a further object of this invention to disclose the method as defined in any of the above, wherein said bio-ink comprises a thickener.

[0088] It is a further object of this invention to disclose the method as defined in any of the above in which said method comprises a step of printing said first aqueous solution and said method comprises, prior to said step of printing said first aqueous solution: printing on a surface, in a layer-by-layer fashion, at least one layer of a third aqueous solution comprising at least one third polymer until said at least one layer is characterized by a predetermined height C; and, crosslinking at least partially said third polymer, thereby forming an additional layer; wherein said step of printing a first aqueous solution comprises printing said first aqueous solution atop said additional layer.

[0089] It is a further object of this invention to disclose the method as defined in any of the above in which said method comprises at least one step of printing, and wherein at least one of said steps of printing comprises printing so as to yield a shape at least one surface of which is characterized by a curvature characteristic of a corneal curvature of a human eye.

[0090] It is a further object of this invention to disclose the method as defined in any of the above in which said method comprises at least one step of printing, wherein at least one of said steps of printing is performed by Laser-Induced Forward Transfer (LIFT) printing.

[0091] It is a further object of this invention to disclose the method as defined in any of the above, wherein said method comprises culturing said bioengineered comeal graft in an incubator; if said bio-ink comprises a polymer, said step of culturing following said step of crosslinking polymer within said bio-ink; if said bio-ink does not comprise a polymer, said step of culturing following said step of allowing said cells to settle and adhere to said support layer.

[0092] It is a further object of this invention to disclose the method as defined in any of the above, wherein each of said aqueous solutions comprises an aqueous solution comprising at least one polymer selected from the group consisting of crosslinked collagen, crosslinked collagen methacrylate (COLMA), crosslinked gelatin, crosslinked gelatin methacrylate (GELMA), crosslinked polyethylene glycol, crosslinked polyethylene glycol diacrylate (PEGDA), crosslinked polyethylene diacrylamide, crosslinked polyethylene glycol dimethacrylate, mixtures thereof, and combinations thereof. In some preferred embodiments of the method, at least one of said PEGDA, GELMA, and COLMA is characterized by a degree of acrylation/methacrylation of between 2% and 100%. In some particularly preferred embodiments of the method, at least one of the following conditions is met: (a) said PEGDA is characterized by an average molecular weight of between 200 and 20,000 Da; (b) said GELMA is characterized by an average molecular weight of between 20,000 and 100,000 Da; and, (c) said COLMA is characterized by an average molecular weight of between 50,000 and 350,000. In some particularly preferred embodiments of the invention, at least one of said PEGDA, GELMA, and COLMA is characterized by a polydispersity of between 1 and 3. In some particularly preferred embodiments of the invention, at least one of said solutions is characterized by at least one concentration selected from the group consisting of: a PEGDA concentration of 1 - 40% w/v; a gelatin concentration of 1 - 50% w/v; and a collagen concentration of 0.03 - 15% w/v. In some especially preferred embodiments of the invention, at least one of said solutions is characterized by at least one concentration selected from the group consisting of a gelatin concentration of 20 - 40% w/v and a collagen concentration of 0.8 - 5% w/v.

[0093] It is a further object of this invention to disclose the method as defined in any of the above, wherein at least one of said aqueous solutions comprises phosphate-buffered saline.

[0094] It is a further object of this invention to disclose the method as defined in any of the above, wherein said bio-ink is characterized by a cell concentration of 0.1 - 100 M/ml. IN some preferred embodiments of the invention, said bio-ink is characterized by a cell concentration of 5 - 20 M/ml.

[0095] It is a further object of this invention to disclose the method as defined in any of the above, wherein said method comprises at least one step of crosslinking, and said at least one step of crosslinking comprises irradiating said solution with light characterized by a predetermined wavelength range, thereby photolytically crosslinking said polymer. In some particularly preferred embodiments, said step of irradiating said solution comprises at least one step selected from the group consisting of: irradiating said solution with light characterized by a predetermined power; irradiating said solution with light characterized by a predetermined power per unit area; and, irradiating said solution for a predetermined duration. In some especially preferred embodiments, at least one of said aqueous solutions comprises a photoinitiator. In some especially preferred embodiments, at least one of the following is true: (a) said step of crosslinking comprises crosslinking photolytically under irradiation by ultraviolet light and said photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2- methylpropiophenone; (b) said step of crosslinking comprises crosslinking photolytically under irradiation by blue light and said photoinitiator is lithium phenyl-2, 4,6- trimethylbenzoylphosphinate; and, (c) said step of crosslinking comprises crosslinking photolytically under irradiation by white light and said initiator is a mixture of Eosin Y, triethanolamine and l-vinyl-2-pyrrolidinone.

[0096] It is a further object of this invention to disclose the method as defined in any of the above, wherein at least one of said aqueous solutions comprises an enzymatic crosslinking catalyst, and said step of crosslinking polymer within said aqueous solution comprising an enzymatic crosslinking catalyst is performed enzymatically. In some preferred embodiments, said enzymatic crosslinking catalyst is microbial transglutaminase.

[0097] It is a further object of this invention to disclose the method as defined in any of the above, wherein at least one of said aqueous solutions comprises both a photoinitiator and an enzymatic crosslinking catalyst, and said step of crosslinking said aqueous solution comprising both a photoinitiator and an enzymatic crosslinking catalyst comprises crosslinking at least part of said polymer photolytically and crosslinking at least part of said polymer enzymatically.

[0098] It is a further object of this invention to disclose the method as defined in any of the above in which the method defines a height A, wherein A has a value of between 10 and 300 pm. In some particularly preferred embodiments, A has a value of about 70 pm.

[0099] It is a further object of this invention to disclose the method as defined in any of the above in which the method defines a height B, wherein B has a value of between 3 and 50 pm. In some particularly preferred embodiments, B < 10 pm. [0100] It is a further object of this invention to disclose the bioengineered corneal graft as defined in any of the above, made according to the method as defined in any of the above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0101] The invention will now be described with reference to the drawings, wherein:

[0102] FIG. 1 is a schematic depiction of one non-limiting embodiment of the bioengineered comeal graft of the present invention;

[0103] FIGs. 2A - 21 present schematic depictions of various steps in the preparation of one embodiment of the bioengineered corneal graft of the present invention according to one non limiting embodiment of the method disclosed herein;

[0104] FIG. 3 shows a transmittance spectrum of an bioengineered comeal graft according to one non-limiting embodiment of the present invention;

[0105] FIGs. 4A and 4B show endothelial cell concentrations, as revealed by DAPI staining, of the endothelial cell layer of one non-limiting embodiment of a corneal graft of the present invention, on two different length scales;

[0106] FIGs. 5A and 5B show the expression of the Na⁺/K⁺ ATPase pump and ZO-l markers, respectively, of the endothelial cells in the endothelial cell layer of one non-limiting embodiment of the comeal graft of the present invention; and,

[0107] FIG. 6 presents a flowchart outlining the key steps in some preferred embodiments of the method of preparation of the bioengineered corneal grafts of the present invention.

DETAIFED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0108] In the following description, various aspects of the invention will be described. For the purposes of explanation, specific details are set forth in order to provide a thorough understanding of the invention. It will be apparent to one skilled in the art that there are other embodiments of the invention that differ in details without affecting the essential nature thereof. Therefore the invention is not limited by that which is illustrated in the figure and described in the specification, but only as indicated in the accompanying claims, with the proper scope determined only by the broadest interpretation of said claims. In some cases, for clarity or conciseness, individual components of the invention disclosed herein may be described without reference to other components of the invention. Nonetheless, any combination of components that is not self-contradictory is considered by the inventors to be within the scope of the invention, even if that specific combination is not described explicitly in the specification. In addition, in cases in which an embodiment of the invention is described as "comprising" certain listed elements, embodiments that consist of the listed elements (i.e. contain those elements and no others) are considered by the inventors to be within the scope of the invention.

[0109] The following abbreviations are used herein:

[0110] PEGDA - polyethylene glycol diacrylate

[0111] GELMA - gelatin methacrylate

[0112] COLMA - collagen methacrylate

[0113] PEG - polyethylene glycol

[0114] ECM - extracellular matrix

[0115] hCEC - human corneal endothelial cell

[0116] M - million (10⁶)

[0117] As used herein, the term "comeal graft" refers to a bioengineered construct that is designed to be implantable in a mammalian eye and to have properties of at least part of a cornea.

[0118] As used herein, the term "bio-ink" refers to a liquid printing material comprising living cells, bioactive factors, and/or a polymer material. In bio-inks that contain a polymer material, the polymer material can comprise a single polymer or it can be a mixture of polymers (composite).

[0119] As used herein, with reference to materials that contact body tissue, the term "inert" is used with reference to substances or materials to describe a substance or material that is sufficiently unreactive that it will not react with body tissue with which it comes in contact sufficiently to cause irritation or damage to the tissue; with reference to materials used during the manufacture of the instant invention, the term is used to describe a substance or material that is sufficiently unreactive that it will not mix physically or react chemically with a second substance during the time it contacts the second substance sufficient to make the second substance unusable for its intended purpose.

[0120] Unless otherwise stated, all concentrations expressed as percentages are percent w/v.

[0121] Unless otherwise stated, with reference to numerical quantities, the term "about" refers to a tolerance of ±25% about the stated nominal value. [0122] Unless otherwise stated, all numerical ranges are inclusive of the stated limits of the range.

[0123] The bioengineered comeal grafts of the present invention comprise an endothelial layer, and in preferred embodiments, a support layer. In some embodiments of the invention, the graft additionally comprises a vision-correction layer.

[0124] As described in detail below, the endothelial layer preferably comprises human endothelial cells, most preferably disposed as a monolayer; in typical embodiments of the invention, the cell layer is 3 - 10 pm thick. In some embodiments of the invention, the endothelial cells are encapsulated in a polymer matrix, typically 5 - 30 pm thick. In some other embodiments of the invention, the endothelial layer does not comprise a polymer matrix, but rather the cells are allowed to mature during the formation of the endothelial layer such that an ECM (Descemet membrane) comprising proteins and biological polymers is formed. In some embodiments of the invention, the endothelial layer has properties of a thin hydrogel.

[0125] The support layer comprises a biologically compatible hydrogel, and is typically 10 - 100 pm thick. In preferred embodiments of the invention, the hydrogel comprises a biological polymer such as collagen or gelatin. In particularly preferred embodiments of the invention, the hydrogel comprises at least one other polymer such as PEG, which is added to optimize the support layer's mechanical properties to mimic as closely as possible the mechanical properties of the native cornea. The support layer serves two roles: it supports the growth of the endothelial cells and it allows handling and implantation of the graft. In addition, in some embodiments of the invention, it serves to replace a portion of the comeal stroma of the patient into whom the graft is inserted as well.

[0126] A primary function of the corneal graft is to heal or replace a cornea that has been damaged (e.g. through accident or illness). In some embodiments of the invention, the comeal graft additionally or alternatively serves to correct at least partially the patient's vision (e.g. to correct refractive errors). In these embodiments, the corneal graft may comprise a third layer for vision correction that is shaped to correct at least partially vision deficiencies of the eye into which the graft is implanted. In preferred embodiments, the vision correction layer is made at least partially from synthetic biocompatible materials that are sufficiently stiff and inert such that the vision correction layer will be able to be able to retain its shape and optical properties for a significant period of time, typically on the time scale at least of years.

[0127] Reference is now made to FIG. 1, which shows schematically one embodiment 10 of the comeal graft of the present invention. The corneal graft comprises at least one support layer 100 characterized by a thickness A, and an endothelial cell layer 110 characterized by a thickness B. In typical embodiments of the invention, A is between 10 and 300 pm, and B is between 3 and 50 pm. In the exemplary embodiment illustrated in FIG. 1, A is 180 pm, and B is 20 pm.

[0128] The support layer is made of a biocompatible hydrogel that comprises a polymer matrix. In some embodiments of the invention, the polymer matrix comprises crosslinked polymer. The support layer is designed to have mechanical properties such as compressive and tensile strength similar to those of a native cornea. In preferred embodiments of the invention, the support layer has a compressive strength of between 0.1 kPa - 5 MPa, a tensile strength of 0.1 - 20 MPa, and an elastic modulus of 0.1 - 100 MPa. In typical embodiments, the tensile strength is about 12 MPa and the elastic modulus is about 50 MPa.

[0129] The endothelial cell layer is a functional layer made of a biocompatible hydrogel prepared from a bio-ink, and comprises endothelial cells, preferably human corneal endothelial cells (hCECs) 1110. In some embodiments of the invention, the endothelial layer additionally comprises a crosslinked polymer matrix. The endothelial cell layer helps maintain the transparency of the comeal graft by pumping out water via the stroma of the endothelial cells encapsulated therein. Cells for the endothelial cell layer can be obtained in any way known in the art, e.g. obtained directly from human donors, or by differentiation from stem cells, induced pluripotent stem cells, or endothelial progenitors of autologous tissue. For isolated hCECs from human donors, the cells are isolated, cultured, and expanded according to standard protocols known in the art.

[0130] The cell concentration in the bio-ink typically ranges from 0.1 - 200 M cells/ml. The exact concentration will depend on the specific production conditions. For example, production of a layer characterized by a thickness of 20 pm and a surface density of 3000 cells/mm implies the use of a bio-ink having a concentration of 150 M cells/ml. In practice, it is frequently possible to use a cell concentration that is lower than this theoretical value. For example, if the cells are permitted to proliferate after the deposition of the layer, the cell concentration will not need to be so high; the inventors have found that in some cases, a bio- ink having a cell density of even 1 M cells/ml can produce an endothelial cell layer having 3000 cells/mm . In embodiments of the invention in which the endothelial layer is produced by seeding cells atop the support layer, the concentration of cells in the bio-ink is calculated from the surface area of the graft. As a non-limiting example, seeding 100 pl of a suspension of cells on a graft having a surface area of 100 mm will require that the suspension have a concentration of ~3 M cells/ml in order to produce an endothelial cell layer having 3000 cells/mm .

[0131] In preferred embodiments of the invention, the polymer matrices are derived from aqueous solutions of one or more polymers selected from the group consisting of PEGDA, GELMA, and COLMA, and a crosslinking initiator and/or catalyst. The degree of acrylation and/or methacrylation is between 2 and 100%. In preferred embodiments, the molecular weights of the polymers are from 200 - 20,000 Da for PEGDA; 20,000 - 100,000 Da for GELMA; and 50,000 - 350,000 Da for COLMA. Any source of gelatin (type A or type B) or collagen (any of types I, II, III, IV, or V) known in the art may be used for GELMA-based and COLMA-based polymers, respectively. Non-limiting examples of sources from which gelatin and collagen can be derived for use in the polymer gels used to make the corneas include plants, humans, or non-human animals such as cows, pigs, rodents, and fish.

[0132] The polymers are present in concentrations (w/v) of 1 - 40% for solutions comprising PEGDA; 1 - 50%, preferably 10 - 35% for solutions comprising GELMA; and 0.1 - 20%, preferably 0.2 - 2%, for solutions comprising COLMA. In typical embodiments of the invention, the polydispersity of the polymers is between 1 and 3. In solutions comprising COLMA, the aqueous solution preferably comprises phosphate -buffered saline. The materials used to prepare the comeal graft can comprise any one of the acrylated or methacrylated polymers, or any combination thereof. When the material comprises more than one polymer, the concentration range of each individual polymer within the mixture is the same range of that polymer when it is the only component of the solution. As a non limiting example, a polymer material based on PEGDA and COLMA is prepared from a solution comprising between 1 and 40% PEGDA and between 0.1 and 20% COLMA.

[0133] After the layers of the corneal graft have been deposited, the polymers within each layer are crosslinked to form hydrogels. In some embodiments, crosslinking is performed photolytically. As non-limiting examples, photolytic crosslinking may be performed by irradiation with UV light, preferably in the wavelength range 320 - 390 nm); by irradiation with blue light, preferably in the wavelength range 390 - 450 nm; and by irradiation with white light, preferably having a wavelength range of 400 - 700 nm. For photolytic crosslinking, any appropriate initiator known in the literature can be used. As non-limiting examples, for crosslinking under UV irradiation, a preferred photoinitiator is IRGACURE 2959 (2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone); for crosslinking under irradiation by blue light, a preferred photoinitiator is LAP (lithium phenyl-2, 4,6- trimethylbenzoylphosphinate); for crosslinking under white light irradiation, a preferred initiator is a mixture of Eosin Y, triethanolamine and l-vinyl-2-pyrrolidinone. In typical embodiments, the photoinitiator concentration in the polymer solution is between 0.001 and 5%.

[0134] In some embodiments of the invention, crosslinking of GELMA or COLMA is performed enzymatically. In these embodiments, an enzymatic crosslinking catalyst such as microbial transglutaminase (mTG), typically in a concentration of 3 ng - 30 mg mTG per gram of polymer, is used for the crosslinking. In this crosslinking process, rather than crosslinking pendant acrylate or methacrylate groups, glutamine and/or lysine residues of the protein component of the polymer are crosslinked.

[0135] In some embodiments of the invention in which the polymers are crosslinked, the crosslinking is performed in two stages. In these embodiments, crosslinking is performed both photolytically and enzymatically. Each of the polymeric starting materials incorporate both a photoinitiator and an enzymatic crosslinking catalyst. Photolytic crosslinking is used to construct the support layer and to bind the cell layer to the support. Enzymatic crosslinking is then used to further crosslink each layer and to further enhance the binding of the cell layer to the support layer.

[0136] In preferred embodiments, the corneal graft is produced by casting, printing, or molding. Reference is now made to FIGs. 2A - 21, which present schematically (not to scale) stages of one embodiment of a method of preparing the corneal graft disclosed herein by casting. As shown in FIG. 2A, the process begins with the deposition of a predetermined amount of a polymer solution 105 for formation of the support layer on a suitable surface 500. The amount deposited will be the amount sufficient to provide a support layer of a predetermined thickness (lOOpm being a typical but non-limiting value) and a diameter sufficiently large at least to match that of the cornea of the recipient, typically ~8 mm. Typical polymer solutions are as described above, and are sufficiently viscous that the deposited solution will not spread out significantly during the time that it takes to deposit the materials and crosslink the polymers to form a gel. Any suitably inert, and for embodiments in which crosslinking is performed photolytically, sufficiently transparent flat surface (non limiting examples include a glass or plastic surface such as a petri dish) may be used. At least one spacer 600a of height A is placed on surface 500, separated horizontally from the polymer solution, and a surface 510 is placed on top of the spacer(s). Typically, two spacers are used. Surface 510 may also be made of any suitable inert (and in embodiments in which crosslinking is performed photolytically, transparent) flat material may be used. One non limiting example of a material that can be used for surface 510 is a glass cover slip. The polymer solution is then at least partially crosslinked as described above to form support layer 100, as shown in FIG. 2C.

[0137] One of the surfaces, typically surface 500, is then removed, leaving the support layer on a surface (FIG. 2D). A predetermined amount of bio-ink 115 from which the endothelial cell layer is produced is then placed atop the support layer (FIG. 2E). As shown in FIG. 2F, at least one spacer 600b of height A + B (typically two) is placed on surface 510 separated horizontally from the support layer and bio-ink, and an additional surface 530 made of an inert material such as polydimethylsulfoxane (PDMS) is placed atop the second spacer(s). As shown in FIG. 2F, in preferred embodiments of the method, the assembly is then inverted. The bio-ink is then crosslinked as described above, thereby producing endothelial cell layer 110 (FIG. 2G). Additional crosslinking of the support layer can be performed at this stage as well. In typical embodiments of the invention, this stage of crosslinking provides both additional stabilization of the layers and covalent bonding between them. Surfaces 530 and 510 are then removed sequentially (FIGs. 2H and 21), along with the second spacer(s), to yield corneal graft 10.

[0138] In some embodiments of the invention, the support layer is produced by molding. A two-piece mold is prepared of an inert material. The mold is designed such that when it is closed, it will have the shape, size, and thickness of the comeal graft being prepared. In embodiments in which the preparation of the corneal graft includes at least one step of photolytic or photolytically initiated crosslinking, at least one of the pieces of the mold is sufficiently transparent at the photolysis wavelength for the crosslinking to take place. The mold may be prepared according to any appropriate method known in the art. The mold is filled with polymer solution, the polymer is crosslinked, and the resulting support layer is removed from the mold after the crosslinking has been performed.

[0139] In some embodiments of the invention, the endothelial cell layer does not comprise a polymer matrix. In preferred embodiments of the invention in which the endothelial cell layer does not comprises a polymer matrix, the bio-ink from which it is formed from a saline medium comprising a suspension of endothelial cells. In preferred embodiments of the invention, the saline medium comprises at least one component selected from the group consisting of nutrients, and buffers. In some preferred embodiments of the invention, the bio ink additionally comprises at least one thickening agent. A non-limiting example of a suitable thickener is hyaluronic acid. The nutrients and buffers allow the endothelial cells to remain viable, adhere to the surface of the support, and in embodiments in which the cells proliferate after their deposition, to proliferate. In embodiments in which the endothelial layer does not comprise a polymer matrix, the layer is formed by seeding the cells on the support. Techniques for seeding the cells are well known in the art; as a non-limiting example, the cells are transferred to the support layer by pipetting the cells and medium onto the upper surface of the support layer. In some embodiments of the invention, the cells are deposited on the support layer by printing them onto the support layer. The printing can be performed by any appropriate method known in the art; non-limiting examples include extrusion, inkjet, and laser-induced forward transfer (LIFT) printing.

[0140] After their deposition, the cells then settle onto the support layer, generally over a period of minutes to hours, preferably 0.5 - 5 hours, and adhere to it, thereby forming in preferred embodiments a true monolayer of endothelial cells on the support layer. The cells then mature, and in some embodiments proliferate, generally over a period of days to weeks, during which time the cells connect to one another and form an extra-cellular matrix. In preferred embodiments of the invention, the layer thus produced is 3 - 15 pm thick.

[0141] The cell density in the endothelial cell layer can be controlled by changing deposition parameters such as the cell density in the bio-ink, deposition frequency, laser energy, velocity of movement of the acceptor substrate or of the print head, etc. In typical embodiments of the invention, the cell concentration in the bio-ink is sufficient to produce an endothelial cell layer with a surface density of at least 1500 cells/mm . In preferred embodiments, the conditions are chose to yield a surface density of between 1500 and 4500 cells/mm . In the most preferred embodiments, the conditions are chose to yield a surface density of between 3000 and 4000 cells/mm . While production of an endothelial cell layer containing a literal monolayer of cells is not necessary for the operation of the comeal graft disclosed herein, the method of manufacture described herein generally produces a cell layer that is almost entirely a single layer of cells. Measurements of surface cell density that treat the observed distribution of cells as being a monolayer are therefore sufficiently accurate to confirm that the surface cell density is within predetermined limits.

[0142] Following the casting or printing of the corneal graft, it is cultured in an incubator (37 °C, 5% C0₂) to allow for cell growth and morphology reorganization. The culture period may take anywhere from several days to several weeks, depending on the initial cell density and gel stiffness.

[0143] While in the embodiment of the method depicted in FIG. 2, the corneal graft is produced by casting, in some embodiments of the invention, the materials are deposited to form the cornea via additive manufacturing. While any appropriate form of additive manufcaturing known in the art may be used, in especially preferred embodiments of the invention, the deposition is performed by a Laser Induced Forward Transfer (LIFT) procedure.

[0144] In some embodiments of the invention in which the corneal graft is produced by additive manufacturing.

[0145] In some embodiments of the invention in which additive manufacturing is used to produce it, the support layer is produced by being printed in layer-by-layer fashion on a flat, inert surface, each layer being added until a total height A is reached. In typical embodiments, each layer is approximately 20 pm thick; typical deposition times are 5 s - 5 min, including the time for the layer to dry before the next layer is applied. In some embodiments of the invention, the support layer is crosslinked after all of the layers have been printed. In other embodiments, the material is irradiated while it is being deposited, so the polymers are crosslinked at least partially while the printing is in progress.

[0146] In some other embodiments in which additive printing is used, a method such as LIFT is used, and the sizes of the droplets of the material deposited on the surface are controlled to produce a single layer that may have a non-uniform thickness.

[0147] In embodiments of the invention in which additive manufacturing is used to produce the corneal graft, after the support layer has been printed, the endothelial cell layer is printed atop the support layer, typically in a layer-by-layer fashion, until the endothelial layer reaches height B (total height of the corneal graft = A + B). As with the support layer, the crosslinking of the polymer within the endothelial cell layer can be performed after all of the material has been deposited, or simultaneously with the deposition of the material. The layers are typically deposited with a diameter slightly larger than that of the final graft in order to take into account shrinkage that may occur (e.g. during crosslinking or drying).

[0148] In some embodiments of the invention, the comeal graft comprises a single layer. The single layer can be either a support layer comprising a biocompatible hydrogel comprising a crosslinked polymer or an endothelial cell layer, comprising a biocompatible hydrogel comprising a crosslinked polymer and corneal endothelial cells. The single-layer graft is produced according to the methods disclosed above, excluding the steps necessary to produce the second layer.

[0149] In some embodiments of the invention, the corneal graft comprises an additional crosslinked polymer layer (preferably from GELMA or COLMA) beneath the support layer. This additional layer is designed to improve the mechanical and optical contact of the comeal graft with the stroma of the recipient for better integration after transplantation. In some embodiments, the additional layer is produced by casting or printing it along with the support and endothelial cell layers and crosslinked in vitro. In other embodiments, the additional layer is produced by scraping off the endothelial layer of the recipient's stroma, applying the polymer solution between the stroma and the two -layered comeal graft described above and crosslinking the additional layer, both to itself and to the support layer, by irradiating it with light in vivo during the transplantation process.

[0150] While the embodiments thus far disclosed comprise 1 - 3 layers, embodiments with any arbitrary number of layers are considered by the inventors to be within the scope of the invention. Comeal grafts comprising one or more additional cell or support layers can be produced according to the methods described above. One additional advantage of the instant invention is thus that the corneal graft herein disclosed is modular. That is, in some embodiments of the invention, rather than a standard product, the corneal graft can be produced with the number of layers and the thickness of each layer tailored according to the specific needs of a particular recipient.

[0151] In some non-limiting embodiments of the invention, the comeal graft is manufactured such that at least one surface thereof has a predetermined curvature. In some non-limiting embodiments in which at least one surface of the corneal graft has a predetermined curvature, the comeal graft is manufactured to match the curvature and diameter of the cornea of the recipient. As a non-limiting example of how the required curvature is calculated and produced, Placido reflective imaging and analysis may be used to obtain the keratometric dioptric range and surface curvature of the recipient's cornea. The comeal diameter can be determined by any method known in the art, e.g. with a hand-held ruler combined with a magnifier, or by an instmment such as an autorefractometer, corneal topographer, or optical coherence tomograph. The specific curvature of the corneal graft can be achieved by printing it onto a support mold having the proper curvature. The mold is preferably made of an inert hydrophobic material such as PDMS for easy detachment of the engineered constmct from the mold.

[0152] The natural human cornea has an index of refraction n = 1.376, and focuses light primarily from the front end at the interface with air, but also focuses light from its back end at its interface with internal eye fluid (n = 1.336). Thus, in some non-limiting embodiments, the comeal graft is manufactured to have a curvature that is designed to correct defects such as myopia or presbyopia in the vision of the recipient. In these cases, the shape necessary to correct the visual defect (i.e. to focus correctly an image on the recipient's retina) is calculated according to any method known in the art, a support mold having the proper curvature produces, and the corneal graft printed thereon as described above.

[0153] Reference is now made to FIG. 6, which presents a flowchart outlining the key steps in the preferred embodiments of the method disclosed herein for preparing the bioengineered comeal grafts of the present invention.

Examples

[0154] The following examples are presented in order to assist a person having ordinary skill in the art to make and use the invention disclosed herein, and are not to be considered to be limiting in any way.

EXAMPLE 1

[0155] A comeal graft was made as described above. The support layer was made from a polymer solution comprising 12% w/v PEGDA and 0.5% w/v COLMA, and the endothelial cell layer from a bio-ink comprising 0.5% w/v COLMA. The endothelial cell layer contained >2500 cells mm ².

[0156] Reference is now made to FIG. 3, which shows a transmittance spectrum of the comeal graft taken three days after production. As can be seen in the figure, the transmittance is between 88% and 95% over the entire visible spectrum, demonstrating that the corneal graft of the present invention is effectively transparent. EXAMPLE 2

[0157] A comeal graft was made as described above. The support layer was made from a polymer solution comprising 12% w/v PEGDA and 0.5% w/v COLMA, while the endothelial cell layer was made from a polymer solution comprising 4% w/v PEGDA and 3% w/v GELMA.

[0158] Reference is now made to FIGs. 4A and 4B, which show, on two different length scales, photomicrographs of the endothelial cell layer of the comeal graft, in which DAPI staining has been used to reveal the endothelial cells. The photomicrographs were taken after an incubation period of 10 days following the preparation of the corneal graft. As can be

_2

seen in the photomicrographs, the surface cell density is >3000 mm .

[0159] The corneal graft was also tested for functional protein marker expression. Reference is now made to FIGs. 5A and 5B, which are photomicrographs of the endothelial cell layer of the corneal graft, in which expression of the Na⁺/K⁺ ATPase pump and of the the ZO-l tight junction, respectively, are shown. The photomicrographs were obtained following an incubation period of 10 days following the preparation of the comeal graft. As can be seen from the figures, more than 90% of the cells in the corneal graft express functional protein markers. Tests of cell function were performed by monitoring the transport of fluorescent molecules of Na⁺/K⁺ ions across the comeal grafts in Transwell containing well plates or in an Us sing chamber.

Claims

CLAIMS We claim:

1. A bioengineered comeal graft, wherein said bioengineered corneal graft comprises an endothelial layer characterized by an anterior surface and a posterior surface, said endothelial layer comprising corneal endothelial cells.

2. The bioengineered corneal graft according to claim 1, wherein said endothelial layer comprises a biocompatible hydrogel.

3. The bioengineered corneal graft according to claim 2, wherein said endothelial layer comprises a first crosslinked polymer.

4. The bioengineered corneal graft according to claim 3, wherein said first crosslinked polymer comprises a crosslinked polymer selected from the group consisting of crosslinked collagen, crosslinked collagen methacrylate, crosslinked gelatin, crosslinked gelatin methacrylate, crosslinked polyethylene glycol, crosslinked polyethylene glycol diacrylate, crosslinked polyethylene diacrylamide, crosslinked polyethylene glycol dimethacrylate, mixtures thereof, and combinations thereof.

5. The bioengineered corneal graft according to claim 4, wherein said first crosslinked polymer comprises a methacrylate or diacrlyate, and said methacrylate or diacrylate is characterized by a degree of acrylation/methacrylation of between 2% and 100%.

6. The bioengineered corneal graft according to claim 1, comprising a support layer characterized by an anterior surface and a posterior surface, said support layer comprising a biocompatible hydrogel comprising a second crosslinked polymer and disposed such that said anterior surface of said support layer contacts said posterior surface of said endothelial layer.

7. The bioengineered corneal graft according to claim 6, wherein said second crosslinked polymer comprises a crosslinked polymer selected from the group consisting of crosslinked collagen, crosslinked collagen methacrylate, crosslinked gelatin, crosslinked gelatin methacrylate, crosslinked polyethylene glycol, crosslinked polyethylene glycol diacrylate, crosslinked polyethylene diacrylamide, crosslinked polyethylene glycol dimethacrylate, mixtures thereof, and combinations thereof.

8. The bioengineered comeal graft according to claim 7, wherein said second crosslinked polymer comprises a methacrylate or diacrlyate, and said methacrylate or diacrylate is characterized by a degree of acrylation/methacrylation of between 2% and 100%.

9. The bioengineered corneal graft according to claim 6, comprising at least one additional layer contacting or anterior to said anterior surface of said support layer, said additional layer comprising biocompatible hydrogel comprising a third crosslinked polymer.

10. The bioengineered corneal graft according to claim 9, wherein said third crosslinked polymer comprises a crosslinked polymer selected from the group consisting of crosslinked collagen, crosslinked collagen methacrylate, crosslinked gelatin, crosslinked gelatin methacrylate, crosslinked polyethylene glycol, crosslinked polyethylene glycol diacrylate, crosslinked polyethylene diacrylamide, crosslinked polyethylene glycol dimethacrylate, mixtures thereof, and combinations thereof.

11. The bioengineered corneal graft according to claim 10, wherein said third crosslinked polymer comprises a methacrylate or diacrlyate, and said methacrylate or diacrylate is characterized by a degree of acrylation/methacrylation of between 2% and 100%.

12. The bioengineered corneal graft according to claim 9, wherein said additional layer is configured to be in contact with stroma tissue of a human eye.

13. The bioengineered comeal graft according to claim 6, wherein said support layer is characterized by a thickness of between 10 and 300 pm.

14. The bioengineered corneal graft according to claim 13, wherein said support layer is characterized by a thickness of between 50 and 150 pm.

15. The bioengineered corneal graft according to claim 1, wherein said endothelial layer is characterized by a thickness of between 3 and 30 pm.

16. The bioengineered corneal graft according to claim 15, wherein said endothelial layer is characterized by a thickness of 3 - 7 pm.

17. The bioengineered corneal graft according to claim 1, wherein said endothelial layer is

_2

characterized by a density of at least 2500 cells mm .

18. The bioengineered corneal graft according to claim 1, wherein said endothelial layer is

_2

characterized by a density of between 1500 and 4000 cells mm .

19. The bioengineered comeal graft according to claim 1, wherein said corneal endothelial cells are selected from the group consisting of:

comeal endothelial cells obtained from a human donor;

cells proliferated from corneal endothelial cells obtained from a human donor; and, endothelial-like cells differentiated from cells selected from the group consisting of stem cells, induced pluripotent stem cells, and endothelial progenitors of autologous tissue.

20. The bioengineered corneal graft according to claim 1, wherein at least one surface of said bioengineered corneal graft is characterized by a predetermined non-zero surface curvature.

21. The bioengineered comeal graft according to claim 20, wherein said surface curvature is a surface curvature characteristic of an analogous surface of a cornea in a healthy human eye.

22. The bioengineered corneal graft according to claim 20, wherein said surface curvature is configured to focus light impinging on said comeal graft according to a predetermined pattern.

23. The bioengineered corneal graft according to claim 20, wherein said surface curvature is configured to compensate for a visual deficiency in a recipient of said comeal graft.

24. The bioengineered corneal graft according to claim 1, wherein said corneal graft is characterized by a diameter characteristic of a diameter of a cornea of a human eye.

25. A method for producing a bioengineered comeal graft, wherein said method comprises producing an endothelial layer comprising endothelial cells and characterized by an anterior surface and a posterior surface.

26. The method according to claim 25, wherein said step of producing an endothelial layer comprises:

depositing a predetermined amount of a bio-ink comprising a first aqueous solution comprising at least one first polymer and a predetermined concentration of corneal endothelial cells (115) atop a first surface;

placing at least one first spacer (600b) of predetermined height B on said first surface; placing a second surface (530) atop said at least one first spacer, thereby forming a preliminary cornea assembly;

crosslinking at least partially said polymer within said bio-ink, thereby producing an endothelial layer (110) atop said first surface; and,

removing said first and second surfaces and said at least one first spacer.

27. The method according to claim 25, wherein said step of producing an endothelial layer comprises:

printing, in a layer-by-layer fashion, at least one layer of a bio-ink comprising a second aqueous solution comprising at least one polymer and a predetermined concentration of comeal endothelial cells atop said first surface until said plurality of layers is characterized by a predetermined height B; and, crosslinking at least partially said polymer, thereby forming an endothelial layer; and,

removing said first surface.

28. The method according to claim 25, comprising:

producing a support layer characterized by an anterior surface and a posterior surface, said support layer comprising a biocompatible hydrogel, at least one additional polymer, and a crosslinker, said step of producing a support layer comprising crosslinking said second polymer; and,

disposing said support layer and said endothelial layer such that said anterior surface of said support layer contacts said posterior surface of said endothelial layer.

29. The method according to claim 28, wherein said step of producing an endothelial layer comprises:

disposing upon said anterior surface of said support layer a bio-ink comprising a predetermined concentration of endothelial cells suspended in a saline culture medium; and,

allowing said cells to settle and adhere to said support layer for a predetermined period of time.

30. The method according to claim 28, wherein:

said step of producing a support layer comprises:

depositing a predetermined amount of a first aqueous solution comprising at said at least one additional polymer (105) on a third surface (500);

placing at least one second spacer (600a) of predetermined height A on said third surface; and,

placing a fourth surface (510) atop said at least one second spacer; said step of crosslinking comprising crosslinking at least partially said at least one additional polymer within said first aqueous solution, thereby producing a support layer (100); and, said method comprises removing said second spacer, said third surface, and said fourth surface subsequent to said step of disposing said support layer such that said anterior surface of said support layer contacts said posterior surface of said endothelial layer.

31. The method according to claim 30, wherein said step of producing an endothelial layer comprises:

removing said third surface and said at least one second spacer;

depositing a predetermined amount of a bio-ink comprising a second aqueous solution comprising at least one polymer and a predetermined concentration of corneal endothelial cells (115) atop said support layer;

placing at least one first spacer (600b) of predetermined height A + B on said second surface;

placing a second surface (530) atop said at least one first spacer, thereby forming a preliminary cornea assembly;

crosslinking at least partially said polymer within said bio-ink, thereby producing an endothelial layer (110) atop said support layer; and,

removing said second and fourth surfaces and said at least one first spacer.

32. The method according to claim 30, wherein said step of producing an endothelial layer comprises:

33. The method according to claim 28, wherein said step of producing a support layer comprises:

printing on a printing surface, in a layer-by-layer fashion, at least one layer of a first aqueous solution comprising said at least one additional polymer until said at least one layer reaches a predetermined height A; and,

crosslinking at least partially said polymer, thereby forming a support layer.

34. The method according to claim 33, wherein said step of producing an endothelial layer comprises:

printing atop said support layer, in a layer-by-layer fashion, at least one layer of a bio ink comprising comeal endothelial cells until said plurality of layers is characterized by a predetermined height B, said bio-ink comprising at least one polymer in a second aqueous solution and a predetermined concentration of corneal endothelial cells; and,

crosslinking at least partially said polymer, thereby forming an endothelial layer; and, said method comprises removing said printing surface subsequent to said steps of producing a support layer and producing an endothelial layer.

35. The method according to claim 33, wherein said step of producing an endothelial layer comprises:

printing atop said support layer, in a layer-by-layer fashion, at least one layer of a bio ink comprising corneal endothelial cells until said plurality of layers is characterized by a predetermined height B, said bio-ink comprising a predetermined concentration of endothelial cells suspended in a saline culture medium;

disposing said bio-ink on said support layer; and,

allowing said cells to settle and adhere to said support layer for a predetermined period of time; and,

said method comprises removing said printing surface subsequent to said steps of producing a support layer and producing an endothelial layer.

36. The method according to claim 28, wherein said step of producing a support layer comprises:

determining a shape, size, and thickness of a corneal graft;

preparing a closeable hollow mold from an inert biocompatible material, said mold comprising an upper portion and a lower portion, said upper portion and said lower portion shaped such that when they are placed together the resulting mold is characterized by said shape, size, and thickness;

placing within said mold said at least one additional polymer and a crosslinker; closing said mold;

performing said step of crosslinking; and,

removing said support layer from said mold.

37. The method according to claim 36, wherein said step of producing an endothelial layer comprises:

placing said support layer on said fourth surface;

placing a first spacer characterized by a height A + B on said second surface, where A is said height of said spacer; depositing a predetermined amount of a bio-ink comprising a second aqueous solution comprising at least one polymer and a predetermined concentration of comeal endothelial cells (115) atop said support layer;

removing said second and fourth surfaces and said at least one first spacer.

38. The method according to claim 36, wherein said step of producing an endothelial layer comprises:

39. The method according to any one of claims 26, 31, or 37, comprising inverting said preliminary cornea assembly between said step of placing a second surface atop said first spacer and said step of crosslinking at least partially said polymer within said bio-ink.

40. The method according to any one of claims 30 - 32, wherein at least one of said second surface and said fourth surface is characterized by a predetermined non-zero surface curvature.

41. The method according to claim 40, wherein said surface curvature is characterized by a configuration selected from the group consisting of:

a curvature configured to produce a bioengineered comeal graft characterized by a surface curvature matching that of a healthy human cornea;

a curvature configured to produce a bioengineered comeal graft that will focus light impinging thereon according to a predetermined pattern; and,

a curvature configured to produce a bioengineered comeal graft characterized by a surface curvature that will compensate for a visual defect in a recipient of said corneal graft.

42. The method according to any one of claims 36 - 38, wherein said shape and thickness are characterized by a configuration selected from the group consisting of:

a shape and thickness configured to produce a bioengineered comeal graft characterized by a shape and thickness matching that of a healthy human cornea;

a shape and thickness configured to produce a bioengineered corneal graft that will focus light impinging thereon according to a predetermined pattern; and, a shape and thickness configured to produce a bioengineered corneal graft characterized by a shape and thickness that will compensate for a visual defect in a recipient of said corneal graft.

43. The method according to any one of claims 26 - 38, wherein at least one of said steps of depositing is performed by Laser- Induced Forward Transfer (LIFT) printing.

44. The method according to any one of claims 29, 32, 35, or 38, wherein said step of allowing said cells to settle and adhere to said support layer for a predetermined period of time comprises allowing said cells to settle and adhere to said support layer for 0.5 - 5 h.

45. The method according to any one of claims 29, 32, 35, or 38, wherein said bio-ink comprises a thickener.

46. The method according to any one of claims 27, 33, 34, or 35, comprising, prior to said step of printing said first aqueous solution:

printing on a surface, in a layer-by-layer fashion, at least one layer of a third aqueous solution comprising at least one third polymer until said at least one layer is characterized by a predetermined height C; and,

crosslinking at least partially said third polymer, thereby forming an additional layer; wherein said step of printing a first aqueous solution comprises printing said first aqueous solution atop said additional layer.

47. The method according to any one of claims 27, 33, 34, or 35, wherein at least one of said steps of printing comprises printing so as to yield a shape at least one surface of which is characterized by a curvature characteristic of a corneal curvature of a human eye.

48. The method according to any one of claims 27, 33, 34, or 35, wherein at least one of said steps of printing is performed by Laser-Induced Forward Transfer (LIFT) printing.

49. The method according to any one of claims 26, 32, or 38, comprising culturing said bioengineered corneal graft in an incubator;

if said bio-ink comprises a polymer, said step of culturing following said step of crosslinking polymer within said bio-ink;

if said bio-ink does not comprise a polymer, said step of culturing following said step of allowing said cells to settle and adhere to said support layer.

50. The method according to any one of claims 25 - 38, wherein each of said aqueous solutions comprises an aqueous solution comprising at least one polymer selected from the group consisting of crosslinked collagen, crosslinked collagen methacrylate (COLMA), crosslinked gelatin, crosslinked gelatin methacrylate (GELMA), crosslinked polyethylene glycol, crosslinked polyethylene glycol diacrylate (PEGDA), crosslinked polyethylene diacrylamide, crosslinked polyethylene glycol dimethacrylate, mixtures thereof, and combinations thereof.

51. The method according to claim 50, wherein at least one of said PEGDA, GELMA, and COLMA is characterized by a degree of acrylation/methacrylation of between 2% and 100%.

52. The method according to claim 50, wherein at least one of the following conditions is met:

said PEGDA is characterized by an average molecular weight of between 200 and 20,000 Da;

said GELMA is characterized by an average molecular weight of between 20,000 and 100,000 Da; and,

said COLMA is characterized by an average molecular weight of between 50,000 and 350,000.

53. The method according to claim 50, wherein at least one of said PEGDA, GELMA, and COLMA is characterized by a polydispersity of between 1 and 3.

54. The method according to claim 50, wherein at least one of said solutions is characterized by at least one concentration selected from the group consisting of: a PEGDA concentration of 1

- 40% w/v; a gelatin concentration of 1 - 50% w/v; and a collagen concentration of 0.03 - 15% w/v.

55. The method according to claim 54, wherein at least one of said solutions is characterized by at least one concentration selected from the group consisting of a gelatin concentration of 20

- 40% w/v and a collagen concentration of 0.8 - 5% w/v.

56. The method according to any one of claims 25 - 38, wherein at least one of said aqueous solutions comprises phosphate-buffered saline.

57. The method according to any one of claims 25 - 38, wherein said bio-ink is characterized by a cell concentration of 0.1 - 100 M/ml.

58. The method according to claim 57, wherein said bio-ink is characterized by a cell concentration of 5 - 20 M/ml.

59. The method according to any one of claims 26 - 28, 30, 31, 33, 34, 36, or 37, wherein at least one step of crosslinking comprises irradiating said solution with light characterized by a predetermined wavelength range, thereby photolytically crosslinking said polymer.

60. The method according to claim 59, wherein said step of irradiating said solution comprises at least one step selected from the group consisting of:

irradiating said solution with light characterized by a predetermined power; irradiating said solution with light characterized by a predetermined power per unit area; and,

irradiating said solution for a predetermined duration.

61. The method according to claim 59, wherein at least one of said aqueous solutions comprises a photoinitiator.

62. The method according to claim 61, wherein at least one of the following is true:

said step of crosslinking comprises crosslinking photolytically under irradiation by ultraviolet light and said photoinitiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2- methylpropiophenone ;

said step of crosslinking comprises crosslinking photolytically under irradiation by blue light and said photoinitiator is lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate; and,

said step of crosslinking comprises crosslinking photolytically under irradiation by white light and said initiator is a mixture of Eosin Y, triethanolamine and l-vinyl-2- pyrrolidinone.

63. The method according to either one of claims 24 or 31, wherein at least one of said aqueous solutions comprises an enzymatic crosslinking catalyst, and said step of crosslinking polymer within said aqueous solution comprising an enzymatic crosslinking catalyst is performed enzymatically.

64. The method according to claim 53, wherein said enzymatic crosslinking catalyst is microbial transglutaminase.

65. The method according to any one of claims 25 - 38, wherein at least one of said aqueous solutions comprises both a photoinitiator and an enzymatic crosslinking catalyst, and said step of crosslinking said aqueous solution comprising both a photoinitiator and an enzymatic crosslinking catalyst comprises crosslinking at least part of said polymer photolytically and crosslinking at least part of said polymer enzymatically.

66. The method according to any one of claims 30, 31, 33, or 37, wherein A has a value of between 10 and 300 pm.

67. The method according to claim 66, wherein A has a value of about 70 pm.

68. The method according to any one of claims 26, 27, 31, 34, 35, or 37, wherein B has a value of between 3 and 50 pm.

69. The method according to claim 68, wherein B < 10 pm.

70. A bioengineered corneal graft according to any one of claims 1 - 24, made according to the method of any one of claims 25 - 38.