Hydrogels and methods of making the same

Abstract

Described herein are collagen substrates or hydrogels that may be used for the production of grafts comprising cultured cells. Also described herein are molds for the production of collagen substrates, optionally with cultured cells.

Description

[0001] PATENT APPLICATION

[0002] Docket No. OCCE-OOl-WOl

[0003] -1-

[0004] HYDROGELS AND METHODS OF MAKING THE SAME

[0005] RELATED APPLICATION^ )

[0006] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 703,744, filed October 4, 2024. The entire teachings of the above application are incorporated herein by reference.

[0007] BACKGROUND OF THE INVENTION

[0008] Corneal disease or damage is typically treated via a cornea transplant. Examples of different corneal transplant methods include Penetrating Keratoplasty (PK), Descemet Stripping Endothelial Keratoplasty (DSEK), and Descemet Membrane Endothelial Keratoplasty (DMEK). However, there is a scarcity of corneal tissue donors and even in cases in which corneal tissue is available, problems frequently remain, such as damage during the transplantation procedure.

[0009] Although attempts have been made to produce bioengineered corneal grafts that comprise cultured corneal cells there continue to be limitations regarding the culturing of the cells on a curved corneal graft. Specifically, there continue to be difficulties in producing a cell cultured corneal graft having a curvature that matches that of an eye, where the cells are distributed appropriately throughout the corneal graft.

[0010] SUMMARY OF THE INVENTION

[0011] Disclosed herein is a mold for the preparation of three-dimensional hydrogels. The mold includes a bottom plate comprising one or more wells, wherein each well has a curved surface; and a top plate comprising one or more plungers, wherein the one or more plungers align with the one or more wells. In some embodiments, the mold further comprises a lid. Docket No. OCCE-OOl-WOl

[0012] -2-

[0013] In some embodiments, the mold is formed of a medical grade polymer, such as a resin material, such as a photopolymer resin, optionally a medical grade photopolymer resin. In some embodiments, the mold is formed of a material that is resistant to temperatures ranging from 4° to 40° C, a material that can withstand autoclaving without deformation to structure or material properties, and / or a material that exhibits limited water absorption. In some embodiments, the mold is fabricated via resin-based three-dimensional printing process, digital light processing (DLP), stereolithography, microprinting, casting, thermoforming, or metal subtractive manufacturing process.

[0014] The mold may be rectangular or circular in shape. In some embodiments, the mold comprises 500 wells or less, for example, the mold may comprise 1 to 100 wells, 50 to 100 wells, 1 to 10 wells, or 1 to 5 wells. The mold may also comprise a corresponding number of plungers, e.g., 500 plungers or less, 1 to 100 plungers, 50 to 100 plungers, 1 to 10 plungers, or 1 to 5 plungers. In some embodiments, the mold comprises five wells and five plungers or one well and one plunger. Alternatively, the mold may comprise three wells and three plungers. Alternatively, the mold comprises seven wells and seven plungers. In some embodiments, the one or more wells each have a diameter greater than 7.5 mm. In some embodiments, the one or more wells each have a diameter of 7.5- 12mm, e.g., a diameter of about 10.5 mm. In some embodiments, the one or more wells each have a curved radius of 4.5 to 8 mm, e.g., a curved radius of 6.5 mm. In some embodiments, the one or more wells optionally each have a rim around each well. In some embodiments, each rim for the one or more wells has a height of 3 to 4 mm, e.g., has a height of about 3.4 mm.

[0015] In some embodiments, the one or more plungers each have a diameter that is 1 mm less than the diameter of the one or more wells. In some embodiments, the one or more plungers each have a bottom arc curve of 4.5 to 8 mm, e.g., a bottom arc curve of 6.5 mm. In some embodiments, the one or more plungers each have a length of 5 to 10 mm, e.g., a length of about 7.7 mm.

[0016] Also disclosed herein is a method of using the mold described herein to manufacture a three-dimensional curved hydrogel. Further disclosed herein is a method of using the mold described herein to manufacture a three-dimensional curved hydrogel and culture endothelial cells or epithelial cells.

[0017] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0018] -3-

[0019] In some embodiments, the method includes forming a cross-linked collagen substrate in the one or more wells of the mold. In some embodiments, the crosslinked collagen substrate is formed by inducing gelation of collagen, vitrifying the collagen after completion of gelation, and rehydrating the vitrified collagen thereby forming a crosslinked collagen substrate. In some embodiments, the top plate is placed on the bottom plate, thereby inserting the one or more plungers into the one or more wells against the collagen substrate forming the three-dimensional curved hydrogel.

[0020] In some embodiments, the methods further include culturing cells on the three- dimensional curved hydrogel. The cells may be endothelial cells, e.g., corneal endothelial cells. In some embodiments, the cells are mammal cells, e.g., human cells.

[0021] In some embodiments, the methods further include cutting or punching out a graft from the hydrogel. The graft may comprise cultured cells. In some embodiments, the methods further include inserting the graft into a surgical applicator or injector. The graft may be applied to a cornea of a subject.

[0022] Also disclosed herein is a method of manufacturing a cross-linked collagen substrate. In some embodiments, the methods include inducing gelation of collagen; vitrifying the collagen after completion of gelation; and rehydrating and crosslinking the vitrified collagen thereby forming a crosslinked collagen substrate.

[0023] In alternative embodiments, a method of manufacturing a cross-linked collagen substrate comprises inducing gelation of collagen; washing the collagen after completion of gelation; vitrifying the washed collagen; and rehydrating the vitrified collagen thereby forming a crosslinked collagen substrate.

[0024] In some embodiments, the gelation of collagen comprises mixing collagen with EDCM / NHS crosslinking solution to form a collagen / EDCM / NHS mixture; mixing the collagen / EDCM / NHS mixture with NaOH to form a neutralized collagen solution; and incubating the neutralized collagen for a period of time to induce the gelation of the collagen. The EDCM / NHS crosslinking solution may comprise 0.1- 1% (w / v) EDCM and 0.1-1% (w / v) NHS in IX PBS. In some embodiments, the collagen is an acidic collagen. In some aspects, the collagen is a recombinant collagen (e.g., synthesized by fermentation or fibroblasts). In some aspects, the

[0025] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0026] -4- collagen is isolated from a native tissue (e.g., bovine, porcine, rat or human tissue). In some embodiments, the collagen is mixed with the EDCM / NHS crosslinking solution at a ratio of 1:0.8-1:1 (vokvol). The NaOH may be added to the collagen / EDCM / NHS mixture in an amount to achieve a neutralized collagen solution having a pH of 7-8. In some embodiments, the neutralized collagen solution is deposited into a well of a plate or a mold. The neutralized collagen may be incubated at 37C + 5% CO2 for 30minutes to 2 hours to induce gelation of the collagen.

[0027] Optionally, the gelled collagen may be washed with an EDCM / NHS crosslinking solution. In some embodiments, the EDCM / NHS wash is a 0.485% EDCM / NHS solution. In some embodiments, the gelled collagen is washed for about 5 minutes.

[0028] In some embodiments, vitrifying the collagen after completion of gelation comprises transferring a plate of collagen to a climate chamber and maintaining the collagen in the climate chamber until completely dry. In some embodiments, the collagen is maintained in the climate chamber for 3-5 days. In some embodiments, the climate chamber is set at 5-12° C and 40-60% relative humidity.

[0029] In some embodiments, the vitrified collagen is rehydrated with HEPES buffer. The vitrified collagen may be rehydrated by washing the collagen with 20 mM HEPES buffer for 1-5 washes, optionally for 3 washes. Each wash may occur for 5- 30 minutes, or for about 10 minutes.

[0030] Optionally, in some embodiments, rehydrating and crosslinking the vitrified collagen comprises rehydrating the vitrified collagen; incubating the rehydrated collagen with riboflavin 5 ’-phosphate to form an incubated collagen / riboflavin 5’- phosphate mixture; and crosslinking the incubated collagen / riboflavin 5’-phosphate mixture under a UV light. In some embodiments, the vitrified collagen is rehydrated with HEPES buffer. The vitrified collagen may be rehydrated by washing the collagen with 20 mM HEPES buffer for 1-5 washes, optionally for 3 washes. Each wash may occur for 5-30 minutes, or for about 10 minutes. In some embodiments, the rehydrated collagen is incubated with the riboflavin 5 ’phosphate in 20% dextran in the dark, e.g., for 20-60 minutes or for about 30 minutes. In some embodiments, excess riboflavin solution is aspirated from the incubated collagen / riboflavin 5’- phosphate mixture. In some embodiments, the incubated collagen / riboflavin 5’-

[0031] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0032] -5- phosphate mixture is crosslinked under UV light for 3-10 minutes, or for about 5 minutes. In some embodiments, the crosslinked collagen is washed with HEPES buffer, e.g., with 20 mM HEPES buffer for 3-6 washes or for about 5 washes. Each wash may occur for about 3-10 minutes or for about 5 minutes.

[0033] Also disclosed herein is a crosslinked collagen substrate manufactured using the methods described herein. Further disclosed herein a method of culturing cells on a crosslinked collagen substrate manufactured using the methods described herein. In some embodiments, the cells are endothelial cells, e.g., corneal endothelial cells. In some embodiments, the cells are epithelial cells, e.g., corneal epithelial cells. In some embodiments, the cells are mammalian cells, e.g., human cells.

[0034] The practice of the present invention will typically employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi) which are within the skill of the art. Nonlimiting descriptions of certain of these techniques are found in the following publications: Ausubel, F., et al., (eds.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.Y., edition as of December 2008; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Harlow, E. and Lane, D., Antibodies - A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1988; Freshney, R.I., “Culture of Animal Cells, A Manual of Basic Technique”, 5th ed., John Wiley & Sons, Hoboken, NJ, 2005. Non-limiting information regarding therapeutic agents and human diseases is found in Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill / Appleton & Lange; 10th ed. (2006) or 11th edition (July 2009). Non-limiting information regarding genes and genetic disorders is found in McKusick, V.A.: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition) or the more recent online database: Online Mendelian Inheritance in Man, OMIM™. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD) and

[0035] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0036] -6-

[0037] National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), as of May 1, 2010, World wide Web URL: ncbi.nlm.nih.gov / omim / and in Online Mendelian Inheritance in Animals (OMIA), a database of genes, inherited disorders and traits in animal species (other than human and mouse), at omia.angis.org.au / contact.shtml. All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In case of a conflict between the specification and any of the incorporated references, the specification (including any amendments thereof, which may be based on an incorporated reference), shall control. Standard art-accepted meanings of terms are used herein unless indicated otherwise. Standard abbreviations for various terms are used herein.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0040] FIG. 1A provides a diagrammatic illustration of a rectangular mold of the present invention.

[0041] FIG. IB provides a schematic of an exemplary use of the rectangular mold of the present invention.

[0042] FIG. 1C provides a diagrammatic illustration of a circular mold of the present invention.

[0043] FIG. ID provides a schematic of an exemplary use of the circular mold of the present invention.

[0044] FIG. 2 provides a flowchart exemplifying the production of a curved collagen graft.

[0045] FIG. 3 demonstrates cultured human corneal endothelial cells (cHCEC) using Endo-Tek™.

[0046] FIG. 4A provides a representative image of confluent endothelial cells cultured in vitro at passage 3 (P3).

[0047] FIG. 4B shows cell density of cells in FIG. 4A.

[0048] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0049] -7-

[0050] FIG. 4C shows high expression of endothelial markers in cHCEC by immunofluorescence staining.

[0051] FIG. 5A shows a vitrigel with a thickness of 15-25 pm displays an organized collagen fiber structure. The collagen structure exhibits improved mechanical properties and tensile strength relative to descemet membrane endothelial keratoplasty (DMEK). It may be designed to optimize for cell compatibility.

[0052] FIG. 5B demonstrates that a vitrigel exhibits high transparency. The vitrigel may exhibit transparency over 90% (e.g., 93-98% transparency) in the visible wavelength range.

[0053] FIG. 5C shows a ready-to-ship Endo-Tek™ graft labeled with an ‘S’ mark, loaded in an applicator. The vitrigel may be suitable for loading and transportation.

[0054] FIG. 5D provides a rendering of the bottom plate of a mold to produce a curved gel.

[0055] FIG. 6 demonstrates the gelation of the collagen solution in a mold of the present invention followed by the culture of the cells in the mold. Yellow = bottom mold; red = top mold; and blue = lid.

[0056] FIG. 7A provides a representative image of cHCEC (P4) on Endo-Tek™.

[0057] FIG. 7B shows cell density of cells in FIG. 7A.

[0058] FIG. 7C shows high expression of endothelial markers in cHCEC on Endo- Tek™ demonstrated by immunofluorescence staining.

[0059] FIG. 8A provides images of a representative eye with Endo-Tek™ show corneal clearing at various time points.

[0060] FIGS. 8B-8C show optical coherence tomography (OCT) at 10 weeks postsurgery indicates 99% restoration of baseline pachymetry.

[0061] FIG. 8D shows specular microscopy verifies the presence of cHCECs on Endo-Tek™.

[0062] FIG. 9A provides histology images demonstrating the presence of Endo-Tek™ on the posterior surface of the cat cornea (red arrow).

[0063] FIG. 9B provides immunofluorescence staining reveals the human origin of the transplanted cHCECs indicated by staining for human- specific nucleoli markers and corneal endothelial markers.

[0064] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0065] -8-

[0066] FIG. 10 demonstrates a single chemical crosslinked solutions method. Post gelation: vitrification at 10°C + 40% relative humidity (rh) and rehydration.

[0067] FIG. 11A shows percent transparency, through the visible spectrum, of single chemical crosslinked gels without cells after the gel has gone through vitrification and rehydration.

[0068] FIG. 1 IB provides a representative image of a single chemical crosslinked gel in a tissue culture plate to demonstrate optical clarity of the gel.

[0069] FIG. 11C provides a representative optical coherence tomography image displaying the thickness of a single chemical crosslinked gel.

[0070] FIG. 12 provides a schematic of a bottom plate of a mold described herein.

[0071] FIG. 13 provides a schematic of a lid of a mold described herein.

[0072] FIG. 14 provides a schematic of a top plate of a mold described herein.

[0073] FIG. 15 provides a schematic of a lid of a mold described herein.

[0074] FIG. 16 provides a schematic of a top plate of a mold described herein.

[0075] FIG. 17 provides a schematic of a bottom plate of a mold described herein.

[0076] DETAILED DESCRIPTION OF THE INVENTION

[0077] Disclosed herein are hydrogels (e.g., three-dimensional crosslinked collagen substrates) for use in culturing cells (e.g., corneal endothelial cells). The hydrogels may be referred to herein as a collagen substrate or a vitrigel. An exemplary hydrogel is identified throughout the figures and Examples as Endo-Tek™. The hydrogel produced herein may provide high density cHCECs. In addition, the hydrogel may be a clear, biocompatible, vitrigel substrate with improved biomechanical properties over DMEK. The hydrogels may be used to form curved grafts (e.g., curved grafts for transplant). In some aspects, the grafts may provide for delivery of a controlled dose and location of cHCECs. The grafts described herein may provide improved quality control over deceased donor tissue and, in some aspects, may replace pathologic Descemet membrane for improved corneal optics and enhanced cell adhesion and survival. Also disclosed herein are molds for the formation of the hydrogels and, in some aspects, culture of the cells.

[0078] FIGS 1A-1D, wherein like parts are designated by like reference numerals throughout, illustrate an example embodiment or embodiments of the mold, according

[0079] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0080] -9- to the present invention. Although the present invention will be described with reference to the example embodiment or embodiments illustrated in the figures, it should be understood that many alternative forms can embody the present invention. One of skill in the art will additionally appreciate different ways to alter the parameters of the embodiment(s) disclosed, such as the size, shape, or type of elements or materials, in a manner still in keeping with the spirit and scope of the present invention.

[0081] Mold

[0082] Aspects of the invention are directed to molds for the preparation of hydrogels, such as three-dimensional hydrogels. In some embodiments, the mold includes a bottom plate and a top plate, optionally the mold further includes a lid. The mold may have any shape. For example, the mold may be rectangular or circular in shape. In some embodiments, the mold is formed of a resin material, such as a photopolymer resin. In some embodiments, the resin is a medical grade photopolymer resin. In some embodiments, the resin is a biocompatible resin. In some embodiments, the mold is formed of a material that is resistant to temperatures ranging from 4° to 40° C. In some embodiments, the mold is formed of a material that can withstand autoclaving without deformation to structure or material properties. In some embodiments, the mold is formed of a material that exhibits limited water absorption. An exemplary material for forming the mold is LOCTITE® MED412™ medical grade photopolymer resin. In some embodiments, the mold is formed of a material that passes ISO 10993-5, ISO 10993-10, and / or ISO 10993-23.

[0083] In some embodiments, the mold is designed using computer aided design (CAD) software, such as Autodesk Fusion360. The mold may be formed or fabricated using any known three-dimensional printing process, such as Digital Light Projection (DLP), stereolithography, casting, thermoforming, or microprinting. In one embodiment, the mold is fabricated using an additive manufacturing means, such as Digital Light Projection (DLP) manufacturing. In some embodiments, the mold is fabricated through metal subtractive manufacturing techniques.

[0084] In some embodiments, the bottom plate includes one or more wells. Each well may have a curved surface, e.g., a curved bottom surface of the well. In some

[0085] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0086] -10- embodiments, the bottom plate has 1 to 500, 1 to 300, 1 to 100, 1 to 75, 1 to 50, 1 to 25, 1 to 10, 10 to 500, 10 to 300, 10 to 100, 25 to 500, 25 to 300, 25 to 100, 50 to 500, 50 to 300, or 50 to 100 wells. In some aspects, the bottom plate has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 wells. In some embodiments, each well has a diameter of greater than 7.5 mm. In some embodiments, each well has a diameter of about 7.5 to 12 mm or 9 to 11 mm, e.g., about 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, or 12 mm. In some embodiments, each well has a curved radius of 4.5 to 8 mm or 5.5 to 7 mm, e.g., about 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 mm. In some embodiments, each well includes a rim. The rim for each well may have a height of 3 to 4 mm or 3.2 to 3.8 mm, e.g., about 3, 3.1, 3.2, 3.3, 3.4,

[0087] 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0 mm. In some embodiments, the wells do not include a rim. The dimensions of the wells, such as the diameter and curved radius, may be determined by the type of grafts being produced and may be adjusted accordingly.

[0088] In some embodiments, the top plate includes one or more plungers. Each plunger may coincide or align with a well in the bottom plate. In some embodiments, the top plate has 1 to 500, 1 to 300, 1 to 100, 1 to 75, 1 to 50, 1 to 25, 1 to 10, 10 to 500, 10 to 300, 10 to 100, 25 to 500, 25 to 300, 25 to 100, 50 to 500, 50 to 300, or 50 to 100 plungers. In some aspects, the top plate has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 plungers. In some embodiments, the number of plungers in the top plate is the same as the number of wells in the bottom plate. In some embodiments, each plunger has a diameter that is about 1 mm less than the diameter of the corresponding well. For example, each plunger may have a diameter of about 6.5 to 11 mm or 8 to 10 mm, e.g., about 6.5, 7,

[0089] 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, or 11.5 mm. In some embodiments, each plunger has a bottom arc curve of 4.5 to 8 mm or 5.5 to 7 mm, e.g., about 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8 mm. In some embodiments, each plunger has a length of 5 to 10 mm or 7 to 8 mm, e.g., about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mm. The plunger length may be adjusted to achieve different thicknesses of the collagen substrate. The dimensions of the plungers, such as the diameter and arc curve, may be determined by the type of grafts being produced and may be adjusted accordingly.

[0090] In one embodiment, the mold is rectangular in shape. In some aspects, the mold has a width of 15 to 25 mm or 18 to 22 mm, e.g., about 15, 15.5, 16, 16.5, 17,

[0091] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0092] -11-

[0093] 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 22, 22.5, 23, 23.5, 24, 24.5, or 25 mm. In some embodiments, the mold has a length of 15 to 60 mm, 15 to 25 mm, 18 to 21 mm, 35 to 45 mm, 38 to 42 mm, 50 to 60 mm, 52 to 58 mm, e.g., about 15, 15.5, 16,

[0094] 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 50, 50.5, 51, 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58,

[0095] 58.5, 59, 59.5, or 60 mm. In some embodiments, the mold having the top plate on the bottom plate has a height of 10 to 20 mm or 13 to 18 mm, e.g., about 15, 15.5, 16,

[0096] 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mm. In an exemplary embodiment, a mold has a length of 50 to 60 mm or about 55 mm, and a width of 15 to 25 mm or about 20.5 mm. In an alternative embodiment, a mold has a length of 15 to 25 mm or about 20.5 mm and a width of 15 to 25 mm or about 20.5 mm. In another embodiment, a mold has a length of 35 to 45 mm or about 41 mm and a width of 15 to 25 mm or about 20.5 mm. The size of the mold may be adjusted based on the number of wells present within the mold, for example, a mold having a single well may have a size of about 17 to 20 mm width and 17 to 20 mm length, and as additional wells are added the size of the mold will increase incrementally based on the number of wells added.

[0097] In another embodiment, the mold is circular in shape. In some aspects, the mold has a diameter of 40 to 60 mm or 47 to 53 mm, e.g., about 40, 40.5, 50, 50.5, 51,

[0098] 51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5 or 60 mm. In some embodiments, the mold having the top plate on the bottom plate has a height of 3 to 15 mm or 7 to 11 mm, e.g., about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8,

[0099] 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 mm. In an exemplary embodiment of a mold having seven wells, the mold has a diameter of 45 to 55 mm or about 50 mm. The size of the mold may be adjusted based on the number of wells present within the mold and as additional wells are added or removed the size of the mold will increase or decrease incrementally based on the change to the number of wells.

[0100] In some embodiments, a punch guide is placed around the rim of a well on the bottom plate. In some embodiments, a biopsy punch is inserted into a well on the bottom plate. The biopsy punch may be inserted through a punch guide located around a well on the bottom plate. In some embodiments, the punch guide and / or biopsy punch are manufactured of the same material as the mold, e.g., a resin such as

[0101] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0102] -12- a photopolymer resin. In one embodiment, the biopsy punch produces a 7.5 mm graft from the collagen substrate.

[0103] As shown in FIG. 1, a mold 10 may have at least two primary elements, a top plate 20 and a bottom plate 30. Optionally, the mold further includes a lid 40. In some aspects, top plate 20 has one or more plungers 22. In some aspects, bottom plate 30 has one or more wells 32. Each well may optionally have a rim 34 around each well. Rim 34 may create a space between bottom plate 30 and top plate 20 when top plate 20 is located or nested on bottom plate 30. In some aspects, a punch guide 50 is placed around well 32 and / or rim 34. In some aspects, a biopsy punch 60 is inserted into a well 32, optionally through punch guide 50.

[0104] Hydrogel

[0105] Aspects of the invention include hydrogels, such as collagen substrates. In some embodiments, a collagen substrate is a crosslinked collagen substrate. In some embodiments, a collagen substrate is formed or manufactured using a 3-step process. In some embodiments, the 3-step process comprises 1) gelation, 2) vitrification, and 3) rehydration. In some embodiments, the process utilizes chemical crosslinking during the gelation phase. In some embodiments, the process utilizes chemical crosslinking during the rehydration phase.

[0106] In some embodiments, the methods of forming the hydrogel comprise inducing the gelation of collagen. In one embodiment, a gelation phase comprises mixing collagen with a crosslinking solution. A crosslinking solution may comprise l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCM) and N- hydroxysuccinimide (NHS). In alternative aspects, a crosslinking solution comprises l-(3-Dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxy succinimide (NHS). In some embodiments, a crosslinking solution comprises 0.1-1% or 0.5% (w / v) EDCM and 0.1-1% or 0.5% (w / v) NHS in phosphate buffered saline (PBS), e.g., IX PBS. In some embodiments, the crosslinking solution is mixed with a collagen. The collagen may be acidic or pepsin soluble. In some embodiments, the collagen is a recombinant collagen. A recombinant collagen may be synthesized by fermentation or fibroblasts. In alternative embodiments, the collagen is isolated from a native tissue, such as bovine, porcine, rate or human native tissue. In some

[0107] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0108] -13- embodiments, the collagen is a telo collagen (e.g., acid soluble telocollagen) or atelo collagen (e.g., pepsin soluble atelocollagen). In some embodiments, the collagen is mixed with the crosslinking solution at up to a 1:1 (vokvol) ratio, or optionally at 1:0.8 (vokvol) ratio.

[0109] In some embodiments, a collagen / crosslinking solution is neutralized, e.g., with NaOH. In some embodiments, the collagen / crosslinking solution is neutralized to a pH of 7 to 8. In one embodiment, the collagen / crosslinking solution is neutralized on ice with IM NaOH. In some embodiments, the neutralized collagen is incubated for a period of time to induce the gelation of the collagen. In some embodiments, the neutralized collagen is incubated at 37° C + 5% CO2. In some embodiments, the neutralized collagen is incubated for 30 minutes to 2 hours, or for 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 minutes. In some embodiments, a neutralized collagen solution is deposited into the center of a tissue culture plate, petri dish, or mold prior to incubation. In one embodiment, a neutralized collagen solution is deposited in a well of a mold, such as a mold described herein. The mold may be rotated to allow for the neutralized collagen solution to evenly cover the bottom of the well. In certain aspects, the mold having the neutralized collagen is incubated to induce gelation.

[0110] In an optional step, the collagen may be washed after gelation and prior to vitrification. For example, the collagen may be washed with a 1 to 5 ml, or about 2 ml, 0.1-1% or 0.485% EDCM and 0.1-1% or 0.485% NHS wash. In some embodiments, the wash step occurs for 3 to 7 minutes or for about 5 minutes. In some embodiments, the wash step assists with the cross linking of the collagen. The wash step may be completed prior to the collagen entering the climate chamber for vitrification.

[0111] In some embodiments, upon completion of the gelation of the collagen, the collagen is transferred to a climate chamber. For example, the collagen may be transferred to the climate chamber after gelation in the mold. In some embodiments, the climate chamber is set at 5°-12° C, or at 5°, 6°, 7°, 8°, 9°, 10°, 11°, or 12° C, and at 40-60% relative humidity, or 40%, 45%, 50%, 55%, or 60% relative humidity. In some embodiments, the collagen is maintained in the climate chamber until the collagen matrix is fully dried. In some embodiments, the collagen is maintained in

[0112] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0113] -14- the climate chamber for 3 to 5 days or for about 3 days. In some embodiments, the collagen is vitrified.

[0114] In some embodiments, the vitrified collagen is rehydrated. In certain aspects, the vitrified collagen is rehydrated in the mold. The vitrified collagen may be rehydrated with HEPES buffer. For example, the vitrified collagen may be rehydrated with 20 mM HEPES. In some embodiments, the vitrified collagen is washed 1 to 5 times, e.g., 1, 2, 3, 4, or 5 times. In some embodiments, the vitrified collagen is washed for a period of 5 to 30 minutes or 10 to 20 minutes, e.g., 5, 10, 15, 20, 25, or 30 minutes. In some embodiments, the vitrified collagen is washed at room temperature.

[0115] In an optional step, the rehydrated collagen may be incubated with riboflavin 5’ phosphate and contacted with a UV light. The rehydrated collagen may be incubated with riboflavin 5’ phosphate in 20% dextran. For example, the rehydrated collagen may be incubated with riboflavin 5’ phosphate in the mold. In some embodiments, the rehydrated collagen is incubated with the riboflavin 5’ phosphate in the dark. In some embodiments, the rehydrated collagen is incubated with the riboflavin 5’ phosphate for 20 to 40 minutes or 25 to 35 minutes, e.g., 20, 25, 30, 35, or 40 minutes, optionally in the dark. Excess riboflavin solution may be aspirated from the collagen after incubation.

[0116] In some embodiments, the collagen is contacted with a UV light after incubation with the riboflavin 5’ phosphate. The UV light may cause the crosslinking of the collagen. In some embodiments, the UV light is applied for at least 5 minutes or from 5 to 10 minutes. In some embodiments, the crosslinked collagen is washed with HEPES buffer, e.g., 20 mM HEPES buffer. In some embodiments, the crosslinked collagen is crosslinked at least 3 or at least 6 times. In some embodiments, the collagen is crosslinked in the mold.

[0117] Cultured Cells

[0118] Aspects of the invention include cells cultured on a collagen substrate. In some embodiments, the cells are endothelial cells or epithelial cells. In some embodiments, the cells are corneal cells. In some aspects, the cells are keratocytes, corneal endothelial cells, corneal epithelial cells, or corneal limbal epithelial stem

[0119] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0120] -15- cells. In other aspects, the cells may be any cell type that could be cultured on a curved surface, such as cardiomyocytes, skeletal muscle stem cells, skin cells (e.g., fibroblasts, keratinocytes, and the like), pancreatic cells, cartilage, neurons, osteoblasts, chondrocytes, or the like. The cells may be obtained from any mammalian species, e.g., human, equine, bovine, porcine, canine, feline, rodent, e.g., mice, rats, hamster, primate, etc.

[0121] In some embodiments, the cells are cultured on a collagen substrate within a mold, such as a mold described herein. The cells may be cultured on a collagen substrate located in one or more wells of the mold. In some embodiments, a lid is placed over the bottom plate of the mold, e.g., while the cells are being cultured in the one or more wells of the mold. In some aspects, the cells are cultured on the collagen substrate with a culture media. In some embodiments, the cells are cultured in the mold for a cell culture period, e.g., a full cell culture period. In one aspect, the cell culture period is about 9 to 14 days. In some embodiments, the cells are cultured at 37° C + 5% CO2.

[0122] For example, confluent cells may be isolated (e.g., by TripLE Select) and cell numbers may be quantified by an automated cell counter. In some aspects, a predetermined number of cells are seeded onto the collagen substrate in each well of the mold. The number of cells to be seeded onto the collagen substrate may be determined based on the diameter of the well of the mold. In one embodiment, approximately 2.6 x 105cells are seeded onto the collagen substrate. The cells may be maintained in a first culture medium in a humidified atmosphere at 37° C in 5% CO2with the first culture medium being refreshed or changed every 2 or 3 days for 4 to 5 days. The first medium may then be replaced with a second medium which may be refreshed or changed every 2 to 3 days for 5 to 7 days (e.g., until the day of surgery).

[0123] In some embodiments, upon completion of a culture cycle, the lid is removed from the mold. In some embodiments, a portion of the substrate is punched out or removed using a punch, such as a biopsy punch. The portion of the substrate punched out may comprise cultured cells. In some embodiments, the substrate with the cultured cells is punched out to form a cell laden graft. In certain aspects, the cell

[0124] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0125] -16- laden graft is formed with a 7.5 mm punch. In some aspects, the cell laden graft is inserted into a surgical applicator, e.g., for administration to a subject.

[0126] Methods of Treatment

[0127] In one embodiment, the cells cultured on a collagen substrate described herein are transplantable, e.g., a graft of the collagen substrate with cultured cells can be administered to a subject in need thereof. In some embodiments, the subject who is administered a graft with cultured cells is the same subject from whom the cells were obtained. In some embodiments, the subject is a different subject. The cells for transplantation (e.g., a graft comprising the cultured cells) can be a form suitable for transplantation.

[0128] The method can further include administering the cells to a subject in need thereof, e.g., a mammalian subject, e.g., a human subject. The source of the cells can be a mammal, preferably a human. The source or recipient of the cells can also be a non-human subject, e.g., an animal model. The term “mammal” includes organisms, which include mice, rats, cows, sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats, and preferably humans. Likewise, transplantable cells can be obtained from any of these organisms, including a non-human transgenic organism. In one embodiment, the transplantable cells are genetically engineered, e.g., the cells include an exogenous gene or have been genetically engineered to inactivate or alter an endogenous gene.

[0129] Additional aspects of the invention include methods of treating a disease by administering to a subject a graft comprising cultured cells. In some embodiments, the graft comprises a collagen substrate. In some embodiments, the cultured cells for administration via a graft are endothelial cells. In some embodiments, the cultured cells for administration via a graft are epithelial cells. In some embodiments, the cultured cells for administration via a graft are corneal cells. In some embodiments, the cultured cells are corneal endothelial cells or corneal epithelial cells. In some embodiments, a graft comprises cultured cells. In some embodiments, a graft comprising cultured cells is applied to skin, bone and / or cartilage. For example, cultured grafts comprising skin cells may be used for skin tissue engineering, cultured grafts comprising osteoblasts may be used for bone tissue engineering, and / or cultured grafts comprising chondrocytes may be used for treating cartilage diseases.

[0130] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0131] -17-

[0132] In some aspects, a graft comprising cultured corneal cells may be administered to a subject to treat an ophthalmological disease. In some aspects, a graft comprising cultured corneal cells may be administered to a subject to treat a corneal endothelial disease. In some embodiments, the graft is a corneal graft for administration to the eye of a subject. A corneal graft may be administered to a subject via a corneal transplant surgery. For example, a corneal graft may be inserted into the eye of a subject during surgery and may be used to replace a defective layer or cell type of the cornea to restore corneal function and clarity, thereby improving the subject’s vision. The corneal graft may be administered to the subject, such as through corneal transplant surgery, using methods known to those of skill in the art.

[0133] A subject can be one who has been previously diagnosed with or identified as suffering from or having an ophthalmological disease or disorder. A subject can be one who has been previously diagnosed with or identified as suffering from or having a corneal endothelial disease or disorder. For example, a corneal graft may be administered to a subject to treat an ophthalmological disease or disorder and / or a corneal endothelial disease. Non-limiting examples of diseases or disorders that may be treated by a corneal graft include Fuchs corneal dystrophy or pseudophakic bullous keratopathy, corneal endothelial dystrophies such as posterior polymorphous dystrophy or congenital hereditary endothelial dystrophy, and iridocorneal endothelial syndrome.

[0134] As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient” and “subject” are used interchangeably herein. The terms, “patient” and “subject” are used interchangeably herein. A subject can be male or female.

[0135] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0136] -18-

[0137] Preferably, the subject is a mammal. The mammal can be a human, nonhuman primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of disorders associated with ophthalmological disease. In addition, the methods and compositions described herein can be used to treat domesticated animals and / or pets.

[0138] As applied to a subject, the terms “treat,” “treating,” “treatment,” etc. refer to providing medical or surgical attention, care, or management to an individual. The individual is usually ill or injured, or at increased risk of becoming ill relative to an average member of the population and in need of such attention, care, or management. It may include administering to a subject a graft of cultured cells so that the subject exhibits a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. The term “treatment” includes prophylaxis. Those in need of treatment include those already diagnosed with a condition, as well as those likely to develop a condition due to genetic susceptibility or other factors.

[0139] The terms “decrease,” “reduced,” “reduction,” “decrease” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced,” “reduction,” “decrease” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0140] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0141] -19-

[0142] The terms “increased,” “increase,” “enhance” or “activate” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased,” “increase,” “enhance” or “activate” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold, or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0143] The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) below normal, or lower, concentration of the marker. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject the null hypothesis when the null hypothesis is actually true. The decision is often made using the p-value.

[0144] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0145] As used herein the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0146] The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0147] The articles “a” and “an” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention

[0148] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0149] -20- includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention provides all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. It is contemplated that all embodiments described herein are applicable to all different aspects of the invention where appropriate. It is also contemplated that any of the embodiments or aspects can be freely combined with one or more other such embodiments or aspects whenever appropriate. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity, those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. For example, any one or more active agents, additives, ingredients, optional agents, types of organism, disorders, subjects, or combinations thereof, can be excluded.

[0150] Where the claims or description relate to a composition of matter, it is to be understood that methods of making or using the composition of matter according to any of the methods disclosed herein, and methods of using the composition of matter for any of the purposes disclosed herein are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where the claims or description relate to a method, e.g., it is to be understood that methods of making compositions useful for

[0151] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0152] -21- performing the method, and products produced according to the method, are aspects of the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0153] Where ranges are given herein, the invention includes embodiments in which the endpoints are included, embodiments in which both endpoints are excluded, and embodiments in which one endpoint is included and the other is excluded. It should be assumed that both endpoints are included unless indicated otherwise. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also understood that where a series of numerical values is stated herein, the invention includes embodiments that relate analogously to any intervening value or range defined by any two values in the series, and that the lowest value may be taken as a minimum and the greatest value may be taken as a maximum. Numerical values, as used herein, include values expressed as percentages. For any embodiment of the invention in which a numerical value is prefaced by “about” or “approximately,” the invention includes an embodiment in which the exact value is recited. For any embodiment of the invention in which a numerical value is not prefaced by “about” or “approximately,” the invention includes an embodiment in which the value is prefaced by “about” or “approximately.”

[0154] “Approximately” or “about” generally includes numbers that fall within a range of 1% or in some embodiments within a range of 5% of a number or in some embodiments within a range of 10% of a number in either direction (greater than or less than the number) unless otherwise stated or otherwise evident from the context (except where such number would impermissibly exceed 100% of a possible value). It should be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one act, the order of the acts of the method is not necessarily limited to the order in which the acts of the method are recited, but the invention includes embodiments in which the order is so limited. It should also be

[0155] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0156] -22- understood that unless otherwise indicated or evident from the context, any product or composition described herein may be considered “isolated.”

[0157] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description.

[0158] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0159] All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present invention. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or prior publication, or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0160] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0161] -23-

[0162] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The details of the description and the examples herein are representative of certain embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention. It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0163] EXEMPLIFICATION

[0164] EXAMPLE 1

[0165] Collagen Substrate

[0166] The following details the methods to produce single crosslinked collagen substrates through a 3-step process 1) gelation, 2) vitrification, and 3) rehydration. Post substrate fabrication, human corneal endothelial cells are cultured on the substrate. The single crosslinked method utilizes chemical crosslinking during the gelation phase through the addition of l-(3-Dimethylaminopropyl)-3- ethylcarbodiimide Methiodide (EDCM) and N-hydroxysuccinimide (NHS).

[0167] Gelation

[0168] 1. Prepare 0.5% (w / v) EDCM and 0.5% (w / v) NHS solutions in IX PBS.

[0169] 2. In a conical tube stored on ice, mix 6 mg / ml acidic collagen with EDCM / NHS crosslinking solution, 1:0.8 (vokvol)

[0170] 3. On ice, neutralize collagen with IM NaOH to achieve a pH = 7-8.

[0171] 4. Pipette 1.8mL neutralized collagen solution into the center of a well of curved mold.

[0172] 5. Gently rotate mold to ensure collagen solution evenly covers the bottom of the well.

[0173] 6. Incubate plate at 37C + 5% CO2 for 30 minutes to 1 hour to induce gelation.

[0174] Vitrification

[0175] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0176] -24-

[0177] 7. Once gelation is complete, transfer plate to climate chamber set at 10°C + 40% relative humidity for 3 days until matrix is completely dry.

[0178] Rehydration and Crosslinking

[0179] 8. Once dry, remove from climate chamber and rehydrate matrix with 20mM HEPES by washing gel 3x for 10 minutes each wash cycle at room temperature.

[0180] 9. Incubate rehydrated gel with 1.56mg / ml riboflavin 5’-phosphate in 20% dextran for 30 minutes at room temperature in the dark (cover with aluminum foil) to crosslink gel.

[0181] 10. Aspirate excess riboflavin solution.

[0182] 11. Crosslink under UV light for 5 minutes.

[0183] 12. Wash the gel with 20mM HEPES 5x for 5 minutes.

[0184] Curved Mold

[0185] A three-piece disposable curved mold, seen in FIG. 1A, was parametrically designed using a computer aided design (CAD) software, Autodesk Fusion360. One mold was designed to produce 3 curved collagen gels with a diameter of 10.5mm and a curve radius of 6.58mm. The mold was designed for fabrication through additive manufacturing means, specifically Digital Light Projection (DLP) manufacturing with biocompatible LOCTITE® MED412™ medical grade photopolymer resin, and was designed to be used during collagen substrate production through cell culture of human corneal endothelial cells.

[0186] Lid

[0187] A lid of the curved mold is used during Endo-Tek™ production post gelation during cell culture on human endothelial cell on the collagen substrate. The dimensions for the lid can be seen in FIG. 13. The outer width and length dimensions of the lid could increase or decrease dependent on the number of curved molds designed in a single part. The lid was designed to fit on the bottom plate with a depth to allow clearance for the protruding circular surface of plate plus a gap for air flow.

[0188] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0189] -25-

[0190] Top Plate Plunger

[0191] A top plate plunger of the mold is used during the gelation phase of the collagen substrate. The dimensions for the top plate plunger can be seen in FIG. 14. The outer width and length dimensions of the top plate plunger could increase or decrease dependent on the number of curved molds designed in a single part with the smallest dimension being 19 mm X 19 mm for a single curved mold and increasing in size for each additional curved surface added. The diameter of the plunger is 9.5 mm, designed to fit inside the 10.5 mm curved surface with a 1 mm gap to allow excess liquid collagen to be expelled during molding. The diameter of the plunger and gap could decrease or increase depending on size of curved surface. The bottom curve of the plunger was designed with an arc curve of 6.58mm based on literature findings for the average arc length of the posterior cornea. The arc curve of the designed plunger could range from 4.5 - 8 mm based on minimum and maximum literature findings. The length of the plunger was designed to be 7.78 mm leaving a specified gap for the collagen solution resulting in a final substrate thickness of about 20 pm. The length of the plunger could be adjusted to achieve different substrate thicknesses.

[0192] Bottom Plate

[0193] A bottom plate of the curved mold is used during the collagen substrate fabrication, cell culture, and cutting out the cell laden graft. The dimensions for the bottom plate can be seen in FIG. 12. The outer width and length dimensions of the bottom plate could increase or decrease dependent on the number of curved molds designed in a single part with the smallest dimension being 19 mm X 19 mm for a single curved mold and increasing in size for each additional curved surface added. The diameter of the curved surface is 10.5 mm which allows a 3 mm edge from the desired 7.5 mm graft diameter. The curvature of the curved surface was designed with an arc length of 6.58mm based on literature findings for the average arc length of the posterior cornea. The arc curve of the designed plunger could range from 4.5 - 8 mm based on minimum and maximum literature findings. A rim height 3.39 mm was designed to extend above the top planer surface of the bottom plate to act as a spacer between the bottom plate and top plate. The rim, along with the length of the plunger of the top plate, allows for a specific gap space which results in a 20 pm final

[0194] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0195] -26- substrate thickness. The rim height of 3.39 mm could be increased or decreased to change the thickness of the substrate. The female curvature of the bottom plate matches that of the male plunger of the top plate.

[0196] Endo-Tek™ fabrication process

[0197] The mold was designed to be utilized from the initial injection of liquid collagen, through substrate fabrication, culturing of human endothelial cells on the collagen substrate, cutting a 7.5mm cell laden graft, and inserting the cell-laden substrate into a surgical applicator / injector. The mold was designed in a three-part building block fashion to allow for modularity throughout the production of Endo- Tek™. FIG. IB shows a diagram of the modularity of the design through each step of the fabrication process.

[0198] EXAMPLE 2

[0199] Collagen Substrate

[0200] The following details the methods to produce single crosslinked collagen substrates through a 3-step process 1) gelation, 2) vitrification, and 3) rehydration. Post substrate fabrication, human corneal endothelial cells are cultured on the substrate. The single crosslinked method utilizes chemical crosslinking during the gelation phase through the addition of l-(3-Dimethylaminopropyl)-3- ethylcarbodiimide Methiodide (EDCM) and N-hydroxysuccinimide (NHS).

[0201] Gelation

[0202] 1. Prepare 0.485% (w / v) EDCM and 0.485% (w / v) NHS solutions in IX PBS.

[0203] 2. In a conical tube stored on ice, mix 6 mg / ml acidic collagen with EDCM / NHS crosslinking solution, 1:0.8 (vokvol)

[0204] 3. On ice, neutralize collagen with IM NaOH to achieve a pH = 7-8.

[0205] 4. Pipette 150pl neutralized collagen solution into the center of a well of curved mold.

[0206] 5. Gently rotate mold to ensure collagen solution evenly covers the bottom of the well.

[0207] 6. Incubate plate at 37C + 5% CO2 for 30 minutes to 1 hour to induce gelation.

[0208] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0209] -27-

[0210] 7. Remove plate from incubator and wash gelled collagen with 2 ml 0.485% (w / v) EDCM and 0.485% (w / v) NHS solution for 5 minutes.

[0211] 8. Remove solution from gelled collagen.

[0212] Vitrification

[0213] 9. Once gelation is complete, transfer plate to climate chamber set at 11.5°C + 43% relative humidity for 3 days until matrix is completely dry.

[0214] Rehydration and Crosslinking

[0215] 10. Once dry, remove from climate chamber and rehydrate matrix with 20mM HEPES by washing gel 3x for 10 minutes each wash cycle at room temperature.

[0216] Curved Mold

[0217] A three-piece disposable curved mold, seen in FIG. 1C, was parametrically designed using a computer aided design (CAD) software, Autodesk Fusion360. One mold was designed to produce 7 curved collagen gels with a diameter of 10.5mm and a curve radius of 6.58mm. The mold was designed for fabrication through additive manufacturing means, specifically Digital Light Projection (DLP) manufacturing with biocompatible LOCTITE® MED412™ medical grade photopolymer resin, and was designed to be used during collagen substrate production through cell culture of human corneal endothelial cells.

[0218] Lid

[0219] A lid of the curved mold is used during Endo-Tek™ production post gelation during cell culture on human endothelial cell on the collagen substrate. The dimensions for the lid can be seen in FIG. 16. The diameter dimensions of the lid could increase or decrease dependent on the number of curved molds designed in a single part. The lid was designed to fit on the bottom plate with a depth to allow clearance for the protruding circular surface of plate plus a gap for air flow.

[0220] Top Plate Plunger

[0221] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0222] -28-

[0223] A top plate plunger of the mold is used during the gelation phase of the collagen substrate. The dimensions for the top plate plunger can be seen in FIG. 14. The dimensions of the top plate could increase or decrease dependent on the number of curved molds designed in a single part with a diameter being about 50 mm for a mold having seven wells. The diameter of the plunger is 9.5 mm, designed to fit inside the 10.5 mm curved surface with a 1 mm gap to allow excess liquid collagen to be expelled during molding. The diameter of the plunger and gap could decrease or increase depending on size of curved surface. The bottom curve of the plunger was designed with an arc curve of 6.58mm based on literature findings for the average arc length of the posterior cornea. The arc curve of the designed plunger could range from 4.5 - 8 mm based on minimum and maximum literature findings. The length of the plunger was designed to be 7.78 mm leaving a specified gap for the collagen solution resulting in a final substrate thickness of about 20 pm. The length of the plunger could be adjusted to achieve different substrate thicknesses.

[0224] Bottom Plate

[0225] A bottom plate of the curved mold is used during the collagen substrate fabrication, cell culture, and cutting out the cell laden graft. The dimensions for the bottom plate can be seen in FIG. 17. The dimensions of the bottom plate could increase or decrease dependent on the number of curved molds designed in a single part with a diameter being about 50 mm for a mold having seven wells. The diameter of the curved surface is 10.5 mm which allows a 3 mm edge from the desired 7.5 mm graft diameter. The curvature of the curved surface was designed with an arc length of 6.58mm based on literature findings for the average arc length of the posterior cornea. The arc curve of the designed plunger could range from 4.5 - 8 mm based on minimum and maximum literature findings. The female curvature of the bottom plate matches that of the male plunger of the top plate.

[0226] Endo-Tek™ fabrication process

[0227] The mold was designed to be utilized from the initial injection of liquid collagen, through substrate fabrication, culturing of human endothelial cells on the collagen substrate, cutting a 7.5mm cell laden graft, and inserting the cell-laden

[0228] 4902-2338-7752, v. 1 Docket No. OCCE-OOl-WOl

[0229] -29- substrate into a surgical applicator / injector. The mold was designed in a three-part building block fashion to allow for modularity throughout the production of Endo- Tek™. FIG. ID shows a diagram of the modularity of the design through each step of the fabrication process.

[0230] 4902-2338-7752, v. 1

Claims

CLAIMSWhat is claimed is:

1. A method of manufacturing a cross-linked collagen substrate comprising a) inducing gelation of collagen; b) vitrifying the collagen after completion of gelation; c) rehydrating and crosslinking the vitrified collagen thereby forming a crosslinked collagen substrate.

2. The method of claim 1, wherein the gelation of collagen comprises a) mixing collagen with EDCM / NHS crosslinking solution to form a collagen / EDCM / NHS mixture; b) mixing the collagen / EDCM / NHS mixture with NaOH to form a neutralized collagen solution; and c) incubating the neutralized collagen for a period of time to induce the gelation of the collagen.

3. The method of claim 2, wherein the EDCM / NHS crosslinking solution comprises 0.1-1% (w / v) EDCM and 0.1-1% (w / v) NHS in IX PBS.

4. The method of claim 2 or claim 3, wherein the collagen is an acidic collagen.

5. The method of claim 2 or claim 3, wherein the collagen is a recombinant collagen.

6. The method of claim 2 or claim 3, wherein the collagen is isolated from a native tissue.

7. The method of claim 6, wherein the collagen is isolated from bovine, porcine, rat, or human native tissue.

8. The method of claim 6 or claim 7, wherein the collage is telo collagen or atelo collagen.

9. The method of any one of claims 2-8, wherein the collagen is mixed with the EDCM / NHS crosslinking solution at a ratio of 1:0.8-1:1 (vol: vol).Docket No. OCCE-OOl-WOl-31-10. The method of any one of claims 2-9, wherein the NaOH is added to the collagen / EDCM / NHS mixture in an amount to achieve a neutralized collagen solution having a pH of 7-8.

11. The method of any one of claims 2-10, wherein the neutralized collagen solution is deposited into a well of a plate or a mold.

12. The method of any one of claims 2-11, wherein the neutralized collagen is incubated at 37C + 5% CO2 for 30minutes to 1 hour to induce gelation of the collagen.

13. The method of any one of claims 1-12, wherein vitrifying the collagen after completion of gelation comprises transferring a plate of collagen to a climate chamber and maintaining the collagen in the climate chamber until completely dry.

14. The method of claim 13, wherein the collagen is maintained in the climate chamber for 3-5 days.

15. The method of claim 13 or claim 14, wherein the climate chamber is set at 5- 10 C and 40-60% relative humidity.

16. The method of any one of claims 1-15, wherein rehydrating and crosslinking the vitrified collagen comprises a) rehydrating the vitrified collagen; b) incubating the rehydrated collagen with riboflavin 5 ’-phosphate to form an incubated collagen / riboflavin 5 ’-phosphate mixture; and c) crosslinking the incubated collagen / riboflavin 5’-phosphate mixture under a UV light.

17. The method of claim 16, wherein the vitrified collagen is rehydrated with HEPES buffer.

18. The method of claim 16 or claim 17, wherein the vitrified collagen is rehydrated by washing the collagen with 20 mM HEPES buffer for 1 to 5 cycles.4902-2338-7752, v. 1Docket No. OCCE-OOl-WOl-32-19. The method of any one of claims 16-18, wherein the vitrified collagen is rehydrated by washing the collagen with 20 mM HEPES buffer for 3 cycles.

20. The method of any one of claims 16-19, wherein the vitrified collagen is rehydrated by washing the collagen for 5 to 30 minutes per wash cycle.

21. The method of any one of claims 16-20, wherein the vitrified collagen is rehydrated by washing the collagen for 10 minutes per wash cycle.

22. The method of any one of claims 16-21, wherein the rehydrated collagen is incubated with the riboflavin 5’phosphate in 20% dextran in the dark.

23. The method of any one of claims 16-22, wherein the rehydrated collagen is incubated with the riboflavin 5’phosphate in 20% dextran in the dark for 20 to 60 minutes.

24. The method of any one of claims 16-23, wherein the rehydrated collagen is incubated with the riboflavin 5’phosphate in 20% dextran in the dark for 30 minutes.

25. The method of any one of claims 16-24, wherein excess riboflavin solution is aspirated from the incubated collagen / riboflavin 5’ -phosphate mixture.

26. The method of any one of claims 16-25, wherein the incubated collagen / riboflavin 5 ’-phosphate mixture is crosslinked under UV light for 3 to 10 minutes.

27. The method of any one of claims 16-26, wherein the incubated collagen / riboflavin 5 ’-phosphate mixture is crosslinked under UV light for 5 minutes.

28. The method of any one of claims 16-27, further comprising washing the crosslinked collagen with HEPES buffer.

29. The method of any one of claims 16-28, further comprising washing the crosslinked collagen with 20 mM HEPES buffer for 3 to 6 cycles.

30. The method of any one of claims 16-29, further comprising washing the crosslinked collagen with 20 mM HEPES buffer for 5 cycles.4902-2338-7752, v. 1Docket No. OCCE-OOl-WOl-33-31. The method of any one of claims 16-30, further comprising washing the crosslinked collagen with 20 mM HEPES buffer for 3 to 10 minutes per wash cycle.

32. The method of any one of claims 16-30, further comprising washing the crosslinked collagen with 20 mM HEPES buffer for 5 minutes per wash cycle.

33. A method of manufacturing a cross-linked collagen substrate comprising a) inducing gelation of collagen; b) crosslinking and vitrifying the collagen after completion of gelation; c) rehydrating the vitrified collagen thereby forming a crosslinked collagen substrate.

34. The method of claim 33, wherein the gelation of collagen comprises a) mixing collagen with EDCM / NHS crosslinking solution to form a collagen / EDCM / NHS mixture; b) mixing the collagen / EDCM / NHS mixture with NaOH to form a neutralized collagen solution; and c) incubating the neutralized collagen for a period of time to induce the gelation of the collagen.

35. The method of claim 34, wherein the EDCM / NHS crosslinking solution comprises 0.1-1% (w / v) EDCM and 0.1-1% (w / v) NHS in IX PBS.

36. The method of claim 34 or claim 35, wherein the collagen is an acidic collagen.

37. The method of claim 34 or claim 35, wherein the collagen is a recombinant collagen.

38. The method of claim 34 or claim 35, wherein the collagen is isolated from a native tissue.

39. The method of claim 38, wherein the collagen is isolated from bovine, porcine, rat, or human native tissue.

40. The method of claim 38 or claim 39, wherein the collage is telo collagen or atelo collagen.4902-2338-7752, v. 1Docket No. OCCE-OOl-WOl-34-41. The method of any one of claims 34-40, wherein the collagen is mixed with the EDCM / NHS crosslinking solution at a ratio of 1:0.8-1:1 (vol: vol).

42. The method of any one of claims 34-41, wherein the NaOH is added to the collagen / EDCM / NHS mixture in an amount to achieve a neutralized collagen solution having a pH of 7-8.

43. The method of any one of claims 34-42, wherein the neutralized collagen solution is deposited into a well of a plate or a mold.

44. The method of any one of claims 34-43, wherein the neutralized collagen is incubated at 37C + 5% CO2 for 30 minutes to 1 hour to induce gelation of the collagen.

45. The method of any one of claims 33-44, wherein washing the collagen after completion of gelation comprises rinsing the collagen with an EDCM / NHS crosslinking solution for 5 minutes.

46. The method of claim 45, wherein the gelled collagen is washed with 0.485% EDCM and 0.485% NHS crosslinking solution.

47. The method of any one of claims 33-46, wherein vitrifying the collagen after completion of gelation comprises transferring a plate of collagen to a climate chamber and maintaining the collagen in the climate chamber until completely dry.

48. The method of claim 47, wherein the collagen is maintained in the climate chamber for 3-5 days.

49. The method of claim 47 or claim 48, wherein the climate chamber is set at 5- 10 C and 40-60% relative humidity.

50. The method of any one of claims 33-49, wherein the vitrified collagen is rehydrated with HEPES buffer.

51. The method of claim 50, wherein the vitrified collagen is rehydrated by washing the collagen with 20 mM HEPES buffer for 1 to 5 cycles.4902-2338-7752, v. 1Docket No. OCCE-OOl-WOl-35-52. The method of claim 50 or claim 51, wherein the vitrified collagen is rehydrated by washing the collagen with 20 mM HEPES buffer for 3 cycles.

53. The method of any one of claims 50-52, wherein the vitrified collagen is rehydrated by washing the collagen for 5 to 30 minutes per wash cycle.

54. The method of any one of claims 50-53, wherein the vitrified collagen is rehydrated by washing the collagen for 10 minutes per wash cycle.

55. A crosslinked collagen substrate manufactured using the method of any one of claims 1-54.

56. A method of culturing cells on a crosslinked collagen substrate manufactured using the method of any one of claims 1-54.

57. The method of claim 56, wherein the cells are endothelial cells.

58. The method of claim 56 or claim 57, wherein the cells are corneal endothelial cells.

59. The method of any one of claims 56-58, wherein the cells are mammal cells.

60. The method of any one of claims 56-59, wherein the cells are human cells.

61. A mold for the preparation of three-dimensional hydrogels comprising a) a bottom plate comprising one or more wells, wherein each well has a curved surface; and b) a top plate comprising one or more plungers, wherein the one or more plungers align with the one or more wells.

62. The mold of claim 61, wherein the mold is formed of a medical grade polymer.

63. The mold of claim 61 or claim 62, wherein the mold is formed of a resin material.

64. The mold of any one of claims 61-63, wherein the mold is formed of a photopolymer resin.4902-2338-7752, v. 1Docket No. OCCE-OOl-WOl-36-65. The mold of any one of claims 61-64, wherein the mold is formed of a material that is resistant to temperatures ranging from 4 to 40 C.

66. The mold of any one of claims 61-65, wherein the mold is formed of a material that can withstand autoclaving without deformation to structure or material properties.

67. The mold of any one of claims 61-66, wherein the mold is formed of a material that exhibits limited water absorption.

68. The mold of any one of claims 61-67, wherein the mold is fabricated via resinbased three-dimensional printing process, digital light processing (DLP), stereolithography, microprinting, casting, thermoforming, or metal subtractive manufacturing process.

69. The mold of any one of claims 61-68, wherein the mold comprises 1 to 100 wells and 1 to 100 plungers.

70. The mold of any one of claims 61-69, wherein the mold comprises 50 to 100 wells and 50 to 100 plungers.

71. The mold of any one of claims 61-69, wherein the mold comprises 1 to 10 wells and 1 to 10 plungers.

72. The mold of any one of claims 61-69, wherein the mold comprises 3 to 7 wells and 3 to 7 plungers.

73. The mold of any one of claims 61-69, wherein the mold comprises one well and one plunger.

74. The mold of any one of claims 61-69, wherein the mold comprises three wells and three plungers.

75. The mold of any one of claims 61-69, wherein the mold comprises seven wells and seven plungers.

76. The mold of any one of claims 61-75, wherein the one or more wells each have a diameter greater than 7.5 mm.4902-2338-7752, v. 1Docket No. OCCE-OOl-WOl-37-77. The mold of any one of claims 61-76, wherein the one or more wells each have a diameter of 7.5- 12mm.

78. The mold of any one of claims 61-77, wherein the one or more wells each have a diameter of 10.5 mm.

79. The mold of any one of claims 61-78, wherein the one or more wells each have a curved radius of 4.5 to 8 mm.

80. The mold of any one of claims 61-79, wherein the one or more wells each have a curved radius of 6.5 mm.

81. The mold of any one of claims 61-80, wherein the mold further comprises a rim around the one or more wells.

82. The mold of claim 81, wherein each rim for the one or more wells has a height of 3 to 4 mm.

83. The mold of claim 81 or claim 82, wherein each rim for the one or more wells has a height of about 3.4 mm.

84. The mold of any one of claims 61-83, wherein the one or more plungers each have a diameter that is 1 mm less than the diameter of the one or more wells.

85. The mold of any one of claims 61-84, wherein the one or more plungers each have a bottom arc curve of 4.5 to 8 mm.

86. The mold of any one of claims 61-85, wherein the one or more plungers each have a bottom arc curve of 6.5 mm.

87. The mold of any one of claims 61-86, wherein the one or more plungers each have a length of 5 to 10 mm.

88. The mold of any one of claims 61-87, wherein the one or more plungers each have a length of about 7.7 mm.

89. The mold of any one of claims 61-87, wherein the one or more plungers each have a length of about 6.9 mm.

90. The mold of any one of claims 61-89, further comprising a lid.4902-2338-7752, v. 1Docket No. OCCE-OOl-WOl-38-91. A method of using the mold of any one of claims 61-90 to manufacture a three-dimensional curved hydrogel.

92. The method of claim 91, comprising forming a cross-linked collagen substrate in the one or more wells of the mold.

93. The method of claim 92, wherein the cross-linked collagen substrate is formed by inducing gelation of collagen, vitrifying the collagen after completion of gelation, and rehydrating and crosslinking the vitrified collagen thereby forming a crosslinked collagen substrate.

94. The method of claim 92 or claim 93, wherein the top plate is placed on the bottom plate, thereby inserting the one or more plungers into the one or more wells against the collagen substrate forming the three-dimensional curved hydrogel.

95. The method of any one of claims 91-94, further comprising culturing cells on the three-dimensional curved hydrogel.

96. The method of claim 95, wherein the cells are endothelial cells.

97. The method of claim 95 or claim 96, wherein the cells are corneal cells.

98. The method of any one of claims 95-97, wherein the cells are mammal cells.

99. The method of any one of claims 95-98, wherein the cells are human cells.

100. The method of any one of claims 91-99, further comprising cutting out a graft from the hydrogel.

101. The method of claim 100, wherein the graft comprises cultured cells.

102. The method of claim 100 or claim 101, further comprising inserting the graft into a surgical applicator.4902-2338-7752, v. 1

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