Electrospun nanofiber membrane for corneal cell transplantation

Abstract

Electrospun nanofiber membranes are provided. The membranes are electrospun from a suitable biocompatible and biodegradable biopolymer, e.g. gelatin, collagen, etc., to generate a membrane, which is then cross-linked. The membrane may be seeded with cells. The compositions find use tissue regeneration, particularly in repair, regeneration, and / or reconstruction of lamellar or partial defects of wounded corneal tissue.

Description

ELECTROSPUN NANOFIBER MEMBRANE FOR CORNEAL CELL TRANSPLANTATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No.63 / 614,383, filed December 22, 2023, the disclosure of which application is herein incorporated by reference. GOVERNMENT SUPPORT RESEARCH

[0002] This invention was made with Government support under contract EY026877 awarded by the National Institutes of Health. The Government has certain rights in the invention. BACKGROUND

[0003] The cornea must remain transparent to ensure clear vision. It is comprised of five layers: three cell-laden layers, and two made primarily of proteins. The corneal endothelium, the innermost layer of the cornea, is comprised of a single layer of corneal endothelial cells (CECs) maintaining a high cell density. It is anchored to Descemet’s Membrane (DM) which is approximately 10 µm in thickness. Human CECs are inherently unable to proliferate within the body and are thus incapable of regeneration in the event of cell loss. The paramount roles of CECs is to transport nutrients from the anterior chamber while maintaining precise hydration levels, thereby preserving the thickness and transparency of the cornea. Disruptions in this function, known as pump-leakage, may result in edema of the corneal stoma and surface epithelium. Given the global scarcity of corneal donations, their remains an urgent need for the development of cultured CECs transplantation methods.

[0004] Current methods for transplanting CECs include Descemet’s stripping automated endothelial keratoplasty (DSAEK) and descemet’s membrane endothelial keratoplasty (DMEK) as current clinical gold standards, while approaches undergoing clinical evaluation include direct injection and transplantation as sheets on fabricated carriers, mimicking DMEK transplant surgery. While direct cell injection approaches have shown promising results in clinical trials, they are currently not yet FDA-cleared and widely available for use in patients. A need remains to develop transplantation methods that overcome the severe shortage of graft tissue worldwide. This may be achieved by creating a minimally immunogenic transient carrier to deliver cells to the inner cornea. The material used for this purpose should be thin to ensure the functionality of the cornea and CECs, porous to facilitate permeability, possess appropriate mechanical properties, and be transparent.

[0005] Electrospinning is a valuable tools for creating thin membranes with high permeability. It is capable of producing various forms of nanofiber scaffolds, and has been actively utilized in the field of tissue engineering, particularly in wound healing, vascular graft, and cardiactissue engineering. In a DMEK-like corneal transplantation procedure, a 7 to 8 mm circular disc of Descemet’s membrane and attached endothelial cells are rolled into a cylinder before they are injected into the anterior chamber through a 2.4 mm-4.0 mm incision. The transplant graft is then unfurled and properly placed endothelial side down before adhering to the posterior cornea with the aid of a gas bubble. DMEK is currently employed successfully by corneal surgeons with very good visual outcomes, but is limited by a higher learning curve and higher rates of post-operative re-bubbling to fully attach the graft compared to DSAEK. SUMMARY

[0006] Electrospun nanofiber membranes are provided. The membranes are electrospun from a suitable biocompatible and biodegradable biopolymer, e.g. gelatin, collagen, etc., to generate a membrane that is then cross-linked. The membrane thus generated has adjustable mechanical properties, and the thickness of nanofiber membrane. It features high permeability of biological factors and transparency in visible light wavelength. The membrane compositions find use tissue regeneration, particularly in repair, regeneration, and / or reconstruction of lamellar or partial defects of wounded corneal tissue. In some embodiments the nanofibers are formed of proteins such as collagen, or derivatives of collagen such as gelatin. In some embodiments the nanofibers are cross-linked, e.g. with glutaraldehyde. In some embodiments, the membrane is a composite of collagen or gelatin protein, with additional biomolecule(s), e.g. glycosaminoglycans such as hyaluronic acid, chondroitin sulfate, heparan sulfate, and dermatan sulfate. The protein and glycosaminoglycans can be physically or chemically crosslinked to each other.

[0007] A feature of the membranes of the disclosure is that it provides a scaffold with a short waiting time for cell attachment, e.g. from about 5 to about 15 minutes. In an embodiment, the nanofiber membrane is seeded with cells, e.g. corneal endotheial cells, etc.

[0008] In an embodiment the nanofiber membrane provides a delivery vehicle for corneal endothelial cell transplantation, which may be implanted after removal of diseased or damaged Descemet’s membrane and endothelial cells.

[0009] In an embodiment the nanofiber membrane provides a transplantable scaffold for corneal regeneration.

[0010] In an embodiment the nanofiber membrane provides a transplantable scaffold for corneal regeneration through the delivery of corneal cells.

[0011] In an embodiment the nanofiber membrane provides a carrier for ocular drug delivery.

[0012] The nanofiber membranes may have a thickness ranging from about 5 µm, about 10 µm, about 20 µm to about 150 μm. Transparency is at least about 75% compared to glass, at least about 80% compared to glass, and may be at least about 85% compared to glass. Thecalculated fiber diameter may be from about 200 to about 400 nm, or from about 250 to about 300 nm. The elastic modulus may be similar to that of the human Descemet’s membrane, e.g. from about 0.2 to about 0.6 Mpa, and may be around about 0.3 to about 0.5 Mpa.

[0013] In an embodiment, an electrospun membrane composition for use in treating or reconstructing a surgically incised or wounded corneal area in a mammalian subject in need thereof. The electrospun membrane may comprise one or both of cells and therapeutic agents that aid in treating or reconstructing a surgically incised or wounded area, where the cells or agent are entrapped or encapsulated in the defined hydrogel structure. Cells of interest include regenerative cells, such as a stem cell, including without limitation corneal stem cells, corneal stromal stem cells, corneal mesenchymal stromal cells, corneal limbal epithelial cells, corneal epithelial cells, corneal endothelial cells, keratinocytes, etc. Cells suitable for treating corneal tissue may include, for example, one or more of corneal stromal stem cells, mesenchymal cells, keratocytes, keratinocytes, endothelial cells, and epithelial cells, and limbal epithelial cells, and transient amplifying cells.

[0014] In another aspect, a method of treating or reconstructing a surgically incised or wounded corneal site in a mammalian subject is provided, by administering a electrospun membrane to deliver cells, drugs, factors, etc. In some embodiments a cavity is debrided to eliminate scarred, fibrotic, and / or necrotic material and create fresh wound edges. In some embodiments a cavity of specific shape and dimensions created, e.g. with surgical instruments, or a laser, for example to remove tissue that is scarred, fibrotic, opacified, etc.

[0015] In another aspect, a biocompatible hydrogel electrospun membrane is provided that optionally comprises cells, therapeutic agents, etc. The biocompatible hydrogel structure is suitable for use in tissue repair or regeneration.

[0016] The membranes in the present invention can serve as, but are not be limited to, tissue scaffolds, tissue substitutes, optical elements (e.g. corneal or lens tissue), tissue fillers, tissue spacers, or as delivery vehicles for cells, tissues, and / or pharmaceutical agents.

[0017] These and other embodiments of the subject invention will readily occur to those of skill in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0019] FIGS.1A-1B. Optimization of spinning time and crosslinking time based on thickness and transparency of the gelNF membrane. (A) Macroscopic image of gelNF membrane undervaried spinning and crosslinking times. The numbers on the top right of the image represent spinning time on the left of the slash and crosslinking time on the right of the slash. The number in the top right corner indicates spinning time / GA crosslinking time. (B) Transparency measurements of gelNF membrane in the wavelengths of the visible light.

[0020] FIGS. 2A-2C. Analysis in nanofiber diameter and mechanical property of gelNF membrane. (A) SEM images of the gelNF membrane in before / after GA crosslinking and (B) calculation of the nanofiber diameter. (C) Tensile stress test of gelNF membrane and calculated elastic modulus, elongation at break, and ultimate tensile strength.

[0021] FIGS.3A-3C. Permeability and degradability of the gelNF membrane. (A) Permeability test of the gelNF membrane using FITC-dextran and cell insert. (B) Measurement of permeability changes in the gelNF membrane over incubation time. Day 0 scale bar: 40 µm and day 14 scale bar: 10 µm (C) Measurement of the gelNF degradation using changes in thickness.

[0022] FIGS.4A-4C. IhCEC culture on top of the gelNF membrane. (A) Cytotoxicity of the gelNF membrane relative to the TCP and (B) comparison of the morphology and cell density of the IhCEC cultured on TCP and the gelNF membrane on day 3 and 7. Scale bar: 200 µm. (C) Immunofluorescence staining of the tight junctional (ZO-1), pump protein (Na+ / K+-ATPase) and nucleus (DAPI) of the IhCEC on day 3 and 7. Scale bars: 50 µm.

[0023] FIGS.5A-5B. PrCEC culture on top of the gelNF membrane. (A) Morphology and Live & Dead staining of the PrCEC cultured on top of the gelNF membrane on day 1. Scale bar in phase contrast image: 100 µm and for the Live & Dead staining: 50 µm. (B) Immunofluorescence staining of the junctional protein (ZO-1 and N-cadherin), water pump ion (aquaporin-1), and nucleus (DAPI) on day 3 and 7. Scale bars: 50 µm.

[0024] FIGS.6A-6F. Ex vivo study for transplantation of PrCEC cultured on top of the gelNF membrane using geuder and artificial anterior chamber. (A) Macro images for gelNF membrane placed inside of the geuder and schematic images for PrCEC cultured on top of the gelNF membrane transplantation. (B) Macro images of (1) preparation of DM and endothelium removed artificial anterior chamber, (2) after the graft injection, (3) air bubble apply to unfold the membrane with 5 min incubation, and (4) addition of culture media. (C) Macro image of the posterior of the cornea indicates that gelNF membrane remained at the cornea (after 72 h culture). (D) Immunofluorescence staining of transplanted PrCEC and gelNF membrane after 72 h. GelNF membrane showed autofluorescence to green and the nuclei of the PrCEC were labeled with DAPI. Scale bar: 50 µm (left), and 100 µm (right). I Immunofluorescence staining of ZO-1 (green), phalloidin (red), and nuclei (blue) transplanted PrCEC cultured gelNF membrane. The image is obtained from the edge of the gelNF membrane. (F) Partially Z-stacked images to visualize the ZO-1 expression (White arrows) that was obscured by autofluorescence of the gelNF membrane. Scale bars 50 µm.

[0025] FIGS.7A-7C. Cytotoxicity of gelNF. Live & Dead assay of the IHECE cultured on top of the gelNF membrane on day (A) 3 and (B) 7. Scale bars : 50 µm. (C) Quantified ratio of the live cell;

[0026] FIGS.8A-8D. Isolation of primary rabbit corneal endothelial cell and characterization in shape and junctional protein epression. (A) Phase-contrast image of isolated PrCEC cultured on TCP at day 4. Scale bar: 100 µm. (B) Calculation of elongation ratio of PrCEC in different time (day). (C) Immunofluorescence staining of the ZO-1 (green), and nuclei (blue) and (D) N-cadherin (green) and nuclei (blue) on day 3. Scale bars: 50 µm;

[0027] FIGS.9A-9B. Functional protein expression of PrCEC cultured on top of the gelNF membrane. Immunofluorescence staining of the Na+ / K+-ATPase (red), ZO-1 (green), and nucleus (DAPI) on (A) day 3 and (B) 7. Scale bars: 50 µm. DETAILED DESCRIPTION

[0028] The invention described below relates to electrospin nanofiber membrane compositions and methods that find use in partial or total repair, regeneration, and / or reconstruction of wounded corneal tissue in a mammalian subject or host organism. Other purposes of the instant disclosure include, but are not limited to, the use for effective transplantation of cells into the host organism to encourage recellularization of the cornea.

[0029] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. Although a number of methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described herein.

[0030] In describing embodiments of the present invention, the following terms will be employed, and are intended to be defined as indicated below. As used in this specification and the appended claims, the singular for“s ”a“" ”an" a“d "”he" include plural referents unless the content clearly dictates otherwise.

[0031] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0032] The practice of the present invention will employ, unless otherwise indicated, conventional methods of medicine, pharmacology, chemistry, biochemistry, molecular biology and recombinant DNA techniques, within the skill of the art. Such techniques are explained fully in the literature. See, e.g. S.S. Wong and D.M. Jameson Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation (CRC Press, 2Supnd / Sup edition, 2011); G.T. Hermanson Bioconjugate Techniques (Academic Press, 3Suprd / Sup edition, 2013); B. Bowling Clinical Ophthalmology: A Systematic Approach, 8e (Saunders Ltd., 8Supth / Sup edition, 2015); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition). All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entireties.

[0033] As used herein, about, or approximately mean within 50 percent, preferably within 20 percent, more preferably within 5 percent, of a given value or range.

[0034] A value which “substantially different” from another value can mean that there is a statistically significant difference between the two values. Any suitable statistical method known in the art can be used to evaluate whether differences are significant or not“

[0035] "Statistically significant" difference means a significance is determined at a confidence interval of at least 90%, more preferably at a 95% confidence interval.

[0036] The terms "treatment”, “treating”, “treat” and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and / or adverse effect attributable to the disease.

[0037] The terms "reconstructing" and "reconstruction," and the like are used herein to generally refer to rebuilding, healing and regenerating an injured matter or tissue.

[0038] The term "subject" or "mammalian subject" refers to any mammalian subject for whom treatment or therapy is desired, particularly humans. "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as non-human primates, dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is a human.

[0039] The term "therapeutically effective amount" or "effective amount" means the amount of a compound, agent, composition, construct that when administered to a mammalian subject for treatment is sufficient, in combination with another agent, or alone in one or more doses or administrations, to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, agent, composition, construct, the defect or disease to be treated, and its severity and the age, weight, etc., of the mammalian subject to be treated.

[0040] As used herein, a “biocompatible” substance (for example polymer or cross-linking agent) is one that does not generally cause significant adverse reactions (e.g. toxic or antigenic responses) to cells, tissues, organs or the organism as a whole, of example, whether it is in contact with the cells, tissues, organs or the organism as a whole, for example, whether it is in contact with the cells, tissues, organs or localized within the organism, whether it degrades within the organism, remains for extended periods of time, or is excreted whole. A biocompatible substance (e.g., a biocompatible polymer) may be selectively compatible in that it exhibits biocompatibility with certain cells, tissues, organs or even certain organisms. For example, the biocompatible substance may be selectively biocompatible with vertebrate cells, tissues and organs but toxic to cells from pathogens or pathogenic organisms. In some circumstances, the biocompatible substance may also be toxic to cells derived from tumors and / or cancers.

[0041] As used herein, “nanofiber” refers to a fiber with a diameter no more than 1000 nanometers, and may be less than about 500 nm, less than about 350 nm, less than about 300 nm, and may be from about 200 to about 35 nm in diameter.

[0042] The term "biopolymer" refers to a biocompatible polymers comprising polymers that can be found naturally in organisms, as well as chemical and physical modifications of such polymers, and include, but are not limited to, proteins, fibrins, fibrinogen, collagens, gelatins, elastins, laminin, fibronectin, extracellular matrix constituents, glycosaminoglycans, chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, hyaluronic acid, albumin, alginates, chitosans, cellulose, thrombin, heparin,polysaccharides, synthetic polyamino acids, prolamines, combinations thereof, and other such molecules. Collagen and gelatin are of particular interest. Polyethylene glycol, polylactic acid, polyvinyl alcohol, and / or other synthetic polymers may also be included in the membrane. In some embodiments, the biopolymer is a conjugated version of the native biopolymer, where the conjugate confers additional functionality such as crosslinkability. Such conjugations include functionalization with, for instance, a photocrosslinkable moiety such as a methacrylate, acrylate, or methacrylamide, or a functional group involved in non-photochemical crosslinking reactions. Other functional groups that can be conjugated to the biopolymers include but are not limited to thiols, norboronenes, vinyl sulfones, azide, alkynes, and the like.

[0043] The term “polymer,” as used herein, refers to a molecule consisting of individual monomers joined together. Polymers that are contemplated herein can be naturally occurring, synthetically produced, or produced using recombinant methodologies.

[0044] The term "transparent," as used herein, refers to at least 70%, 80, or 90% transmission of white light.

[0045] Electrospinning is a versatile method of fabricating polymeric fibers. The polymer fibers are typically characterized by fiber diameters ranging from several microns down to 100 nmor less. These polymeric fibers may be used to further fabricate products of varying complexity and different three-dimensional shapes. Electrospun (ES) fibers can be cross-linked, e.g. by chemical methods with glutaraldehyde, heating, UV treatment etc. Other cross-linking agents include formaldehyde, glutaraldehyde, glyceraldehyde, (1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide) (EDC) together with N-hydroxysuccinimide (NHS), genipin, etc.

[0046] As used herein, the term “electrospinning” refers to a process in which a high voltage is used to create an electrically charged jet of polymer fluid, such as a polymer solution, which dries or solidifies to generate polymer fibers. Systems for electrospinning generally include a syringe, a nozzle, a pump, a high-voltage power supply, and a grounded collector. A high voltage power supply is connected to the orifice of the needle at one end and to the grounded collector on the other end.

[0047] The methods generally include the use of an external electric field for atomization of the polymeric solution during the spinning process. When the external electrostatic field is applied to the solution, a suspended conical droplet is formed at the solution source (e.g., a needle used for injection of the solution into the spinning apparatus). Initially, the surface tension of the droplet is in equilibrium with the electric field. Electrostatic atomization occurs when the electrostatic field is strong enough to overcome the surface tension of the liquid. The liquid droplet then becomes unstable and a tiny jet is ejected from the surface of the droplet. The material eventually reaches a grounded target, where it is collected as an interconnected web containing fine fibers.

[0048] Any suitable electric field can be used in the methods of the invention. Typically, electric fields ranging from around 100 V to around 100 kV are used in the methods of the invention. The electric field can range, for example, from 500 V to 50 kV, or from 1 kV to 25 kV, or from 5 kV to 15 kV. The electric field can be 5 kV, 5.5 kV, 6 kV, 6.5 kV, 7 kV, 7.5 kV, 8 kV, 8.5 kV, 9 kV, 9.5 kV, 10 kV, 10.5 kV, 11 kV, 11.5 kV, 12 kV, 12.5 kV, 13 kV, 13.5 kV, 14 kV, or 15 kV. Other field strengths can be used depending on the composition of the particular solution used for the electrospinning process.

[0049] Any suitable flow rate can be used for introducing the solution from the source into the electric field. Typically, the flow rate will range from about 0.1 mL / hr to about 5 mL / hr. The flow rate can range, for example from 0.1 mL / hr to 0.5 mL / hr, or from 0.5 mL / hr to 1 mL / hr, or from 1 mL / hr to 1.5 mL / hr. The flow rate can range from 0.5 mL / hr to 1.5 mL / hr, or from 0.5 mL / hr to 1 mL / hr. The flow rate can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 mL / hr. Other flow rates can be used depending on factors including the composition of the solution and the strength of the electric field used in the process.

[0050] The target surface used for fiber collection can be placed at any suitable position with respect to the source of the solution. The distance between the solution source and the target surface will typically range from about 5 cm to 50 cm. The distance can range, for example,from 5 to 10 cm, or from 10 cm to 15 cm, or from 15 cm to 20 cm, or from 20 cm to 25 cm. The distance can range from 5 cm to 30 cm, or from 10 cm to 20 cm. The distance between the solution source and the target surface can be around 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 cm. Other distances can be employed depending on factors including the composition of the solution, the strength of the electric field, and the solution flow rate used in the process.

[0051] The polymer product comprises a fiber mat, or membrane, which can provide a substrate for cell and / or tissue culture.

[0052] As used herein, the term “cell” in the context of the in-vivo and in-vitro applications of the present invention encompasses mammalian cells of any genus or species, particularly human cells. The types of cells that may be incorporated into the polymeric biomaterial include progenitor cells of the same type as those from the tissue site, and progenitor cells that are histologically different from those of the tissue site such as embryogenic or adult stem cells, that can act to accelerate the healing, regenerative or reconstructive process. The compositions comprising cells can be administered in the form of a solution or a suspension of the cells mixed with the polymeric biomaterial solution, such that the cells are substantially immobilized within the application site upon gelation. This serves to concentrate the effect of the cells at the site of application; and may provide for release of the cells over a course of time

[0053] Corneal cells may used, for example limbal epithelial stem cells, corneal stromal stem cells, keratinocytes, keratocytes, corneal endothelial cells, etc. may be isolated, harvested, and / or propagated from cadaveric donor corneal tissue, from small limbal biopsies from patients (either autologous from a patient’s healthy eye, or from another living patient's eye as a donation); generated by in vitro culture, etc. Corneal stem cells also include corneal stromal stem cells, which are quiescent, mesenchymal cells. It has been suggested that corneal stromal stem cells are a subpopulation of stromal cells that can differentiate into keratocytes. Stromal corneal stem cells are also positive for ABcG2 expression.

[0054] Corneal endothelial cells (CECs) play a pivotal role in maintaining the clarity and function of the cornea by regulating fluid balance and preventing edema. The unique nature of human corneal endothelial cells makes them a crucial element in the success of corneal transplantation procedures. Human corneal endothelial cells are a monolayer of hexagonal- shaped cells located at the posterior surface of the cornea. Despite their low proliferative capacity in vivo, these cells exhibit remarkable functional efficiency in maintaining corneal transparency. Morphologically, human corneal endothelial cells form a tightly packed hexagonal mosaic, ensuring optimal coverage of the corneal surface.

[0055] Among the crucial markers associated with CECs is Na+ / K+-ATPase, an enzyme responsible for actively pumping ions across the cell membrane. The presence of Na+ / K+- ATPase is essential for maintaining the osmotic balance of the cornea, preventing fluidaccumulation and subsequent corneal edema. Another key marker is ZO-1, a tight junction protein that plays a pivotal role in cell-cell adhesion and the formation of the endothelial cell monolayer. ZO-1 not only contributes to the structural integrity of the endothelium but also participates in the regulation of paracellular permeability, ensuring the maintenance of corneal transparency. The success of corneal transplantation critically relies on the preservation, isolation, and transplantation of viable human corneal endothelial cells. In addition to these markers, N-cadherin is a cell adhesion molecule that contributes to the hexagonal mosaic pattern characteristic of corneal endothelial cells.

[0056] Therapeutically effective amounts of the cells seeded on a membrane of the instant disclosure will vary depending e.g., on the condition to be treated, typical survival of the particular cell type within the hydrogel construct (e.g., including the average lifespan of cells of the particular cell type), etc.

[0057] In some embodiments, a therapeutically effective amount of cells on a membrane of the disclosure is 1x103or more cells / cm2, including e.g., 5 x l03cells / cm2or more, 1 x l04cells / cm2or more, 5 x l04cells / cm2or more, 1 x l05cells / cm2or more, 5 x l05cells / cm2or more, 1 x l06cells / cm2or more, 5 x l06cells / cm2or more, 1 x l07cells / cm2or more, 5 x l07cells / cm2or more.

[0058] As used herein, the term “under physiological conditions” encompasses those conditions that are compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, osmolarity, osmolality etc.

[0059] The nanofiber membranes may comprise suitable therapeutic factors. Suitable growth factors and cytokines include, but are not limited to stem cell factor (SCF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage stimulating factor (GM-CSF), stromal cell-derived factor- 1, steel factor, vascular endothelial growth factor (VEGF), transforming growth factor-beta (TGFP), platelet derived growth factor (PDGF), angiopoeitins (Ang), epidermal growth factor (EGF), fibroblast growth factor (FGF) hepatocyte growth factor, nerve growth factor, keratinocyte growth factor, insulin-like growth factor (IGF-1), interleukin (IL)-3, IL-la, IL-Ιβ, IL-6, IL-7, IL-8, IL-11, and IL-13, colony- stimulating factors, thrombopoietin, erythropoietin, fit3-ligand, and tumor necrosis factor α. Examples of growth factors include EGF, bFGF, HNF, NGF, PDGF, IGF-1 and TGF. These growth factors can be mixed with the membrane materials comprising the compositions. The bioactive agents can also have pro- angiogenic activities, e.g., VEGF, PDGF, prominin-1 polypeptide, and variants thereof that have pro-angiogenic activities, i.e., promote neovascularization and angiogenesis.

[0060] In some embodiments, one or more factors is provided at a concentration ranging from about 0.01 mg / mL to about 10 mg / ml in the membrane hydrogel, including any concentration in this range such as about 0.01 mg / ml, 0.1 mg / ml, 1 mg / ml, 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml, 3 mg / ml, 3.5 mg / ml, 4 mg / ml, 4.5 mg / ml, 5 mg / ml, 5.5 mg / ml, 6 mg / ml, 6.5 mg / ml, 7 mg / ml,7.5 mg / ml, 8 mg / ml, 8.5 mg / ml, 9.0 mg / ml, 9.5 mg / ml, or 10 mg / ml. In another embodiment, the factor(s) is at a concentration of at least 1 mg / mL in the membrane.

[0061] The factors from can be crosslinked to the biopolymer in the membrane using SPAAC. For example, factors can be conjugated for SPAAC with an azide-N-hydroxysuccinimide (NHS) crosslinker to produce an azide-conjugated secreted factor. The biopolymer can be conjugated with an alkyne-NHS crosslinker to produce an alkyne conjugated protein, which is subsequently reacted with the azide conjugated factor, thereby crosslinking the biopolymer and the factor within the membrane. Alternatively, a factor can be reacted with an alkyne-NHS crosslinker to produce an alkyne-conjugated factor. A biopolymer can be reacted with an azide-NHS crosslinker to produce an azide-conjugated biopolymer, which is subsequently reacted with the alkyne-conjugated factor thereby crosslinking the biopolymer and the factor within the hydrogel.

[0062] Photochemical or non-photochemical bioconjugation methods can also be used for direct covalent linkage of factors to biopolymers. Factors may include more than one functional group that can be crosslinked to allow formation of bonds among factors and the membrane.

[0063] The nanofiber membranes may comprise drugs for deliver to the eye. Suitable drugs include, without limitation, Glaucoma Medications such as Latanoprost (Xalatan), Timolol (Timoptic), Dorzolamide (Trusopt), Brimonidine (Alphagan), Travoprost (Travatan), Bimatoprost (Lumigan); anti-inflammatory agents such as Prednisolone (Pred Forte, Pred Mild), Dexamethasone (Maxidex), Fluorometholone (FML), Loteprednol (Lotemax); antibiotics for ocular infections such as Tobramycin (Tobrex), Ciprofloxacin (Ciloxan), Ofloxacin (Ocuflox), Besifloxacin (Besivance), Moxifloxacin (Vigamox); allergy medications such as Olopatadine (Patanol, Pataday), Ketotifen (Zaditor, Alaway), Azelastine (Optivar), Cyclosporine (Restasis, Cequa); Lifitegrast (Xiidra); Corticosteroids such as Loteprednol (Lotemax), Prednisolone (Pred Forte, Pred Mild), Dexamethasone (Maxidex); Mydriatics / Cycloplegics, such as Atropine, Tropicamide (Mydriacyl), Cyclopentolate; anti- VEGF agents such as Bevacizumab (Avastin), Ranibizumab (Lucentis), Aflibercept (Eylea), and the like. Cellularized Or Acellular Compositions

[0064] For each type of tissue being replaced, the electrospun biomaterial compositions can be seeded with cells, i.e. cellularized. Such cells can be somatic / differentiated cells, pluripotent stem cells, or progenitor / stem cells. Utility

[0065] The electrospun membrane compositions and methods of the present invention can be applied to any clinical situation where tissue engineering, regeneration or reconstruction ina mammalian host or subject is necessary. Tissue engineering is a rapidly growing field encompassing a number of technologies aimed at replacing or restoring tissue and organ function. The key objective in tissue engineering is the regeneration of a defective tissue through the use of materials that can integrate into the existing tissue so as to restore normal tissue function. Such compositions can comprise cells that settle in the host and encourage recellularization of the wounded tissue. Furthermore, such compositions can also serve as a three-dimensional tissue model for the in-vitro study of cellular responses and interplay. Application to corneal transplant

[0066] The compositions of the disclosure address a need for effective compositions and methodologies to treat cornal transplantation. Ocular and corneal defects may be caused by, e.g., neurotrophic keratopathy, recurrent corneal erosion, corneal ulcer, corneal burns, exposure keratopathy, physical trauma, retinal disease, retinal degeneration, optic nerve damage, optic nerve degeneration, and other disorders. Corneal endothelial cell disorders include Fuchs’ Endothelial Dystrophy and Pseudophakic Bullous Keratopathy.

[0067] Delivering (cultured) cells such as corneal limbal epithelial cells, corneal stromal stem cells (CSSCs) or corneal endothelial cells to the site of corneal injury may minimize the fibrotic response and enhance the regeneration of the corneal tissue. Delivery of corneal cells, such as keratocytes and keratinocytes, and other cells, seeded on nanofiber membranes, as described herein, may be instrumental in repairing and regenerating corneal tissue. KITS

[0068] The present invention also provides kits comprising separate containers holding compositions comprising nanofiber membranes, and optionally seeded with living cells to be delivered to the wounded tissue site. Compositions can be lyophilized. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. The kit can further comprise a container comprising pharmaceutically acceptable excipients or formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery devices. The kit can also comprise a package insert containing written instructions describing methods for care of a corneal wound as described herein. ADMINISTRATION

[0069] The electrospun membrane compositions of the present invention can be administered in the form of pharmaceutical compositions, comprising an isotonic excipient prepared under sufficiently sterile conditions for administration to a mammalian subject, particularly to a human being.

[0070] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. It is also understood that the terminology used herein is for the purposes of describing particular embodiments

[0071] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or only and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

[0072] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims. EXPERIMENTAL Example 1

[0073] The corneal endothelium, comprised of densely packed corneal endothelial cells (CECs) adhering to Descemet's membrane (DM), plays a critical role in maintaining corneal transparency by regulating water and ion movement. CECs have limited regenerative capacity within the body and globally there is an insufficiency of donated corneas to replace damaged corneal endothelium. Thus, the development of a carrier for cultured CECs transplantation is imperative. In this study we successfully manufactured a gelatin nanofiber membrane (gelNF membrane) using electrospinning, followed by crosslinking with glutaraldehyde (GA). The fabricated gelNF membrane exhibited approximately 80% transparency compared to glass and maintained a thickness of 20 µm. Moreover, the gelNF membrane demonstrated desirable permeability and degradability. Importantly, CECs cultured on the gelNF membrane at high densities showed no cytotoxic effects, and the expression of cellular functional proteins was verified. To assess the potential of this gelNF membrane as a carrier for cultured CEC transplantation, we conducted Descemet’s membrane endothelial keratoplasty (DMEK) on rabbit eyes. The outcomes demonstrate this gelNF membrane is suitable as a carrier for cultured CEC transplantation, offering advantages in terms of transparency, permeability, and sufficient mechanical properties required for successful transplantation.

[0074] In this study, we developed a carrier for CEC transplantation by electrospinning gelatin solution into nanofiber membranes. Gelatin, derived from collagen, has been applied acrossvarious disciplines, including wound healing and bone regeneration, leveraging its notable biocompatibility. Furthermore, it manifests characteristics conducive to electrospinning, such as heightened solubility in non-organic solvents. This property not only mitigates toxicity but also allows for facile modification of the solvent in the preparation of electrospinning solutions. Our objective was to minimize toxicity and immune reactions by reducing the use of other synthetic polymers and organic solvents. The gelNF membrane has been utilized in various fields, but to achieve enhanced properties, they are often blended with a variety of synthetic polymers. To address challenges related to transparency and toxicity issues associated with organic solvent use, we sought to explore the potential use of electrospun gelNF membranes as carriers for CECs transplantation. After confirming protein expression through the transplantation of human and rabbit CECs onto the fabricated gelNF membrane, we conducted ex vivo tests to validate the transplantation of cells into freshly harvested rabbit corneas. Materials and Methods

[0075] Fabrication of electrospun gelatin nanofiber (gelNF). To prepare the gelatin solution for the electrospinning, gelatin type A (G1890 Sigma Aldrich) was dissolved in 70% acetic acid solution (695092, Sigma Aldrich) and 30% distilled water. The gelatin solution (25%, w / v) was then placed into a 5 ml syringe and connected to a 23-gauge needle. The Electrospinning machine (TL-01, Tong Li Tech) fabricated the nanofiber membrane using a flow rate of 1 ml / h, 17 kV of voltage, and a 15 cm distance from needle to collector. The nanofibers were collected at the cover glass, covered with aluminum foil, and evaporated overnight at RT to remove residual solvent. The samples were then crosslinked with glutaraldehyde (G6257, Sigma Aldrich) under a vapor crosslink system and evaporated overnight at RT to remove residual solvent. The nanofiber membranes were soaked in PBS, then separated from the foil. The fabricated samples were sterilized under UV for 30 min for in vitro and ex vivo study.

[0076] Characterization of gelNF membrane. To characterize the properties of the gelNF membrane, thickness light transmittance, permeability, degradability, and tensile strength was assessed. To measure the light transmittance, gelNF membranes were cut into 96 well size using biopsy punch and placed into the plate reader (Spark®, Tecan Life Sciences) at the wavelength of visible light. The transparency of each gelNF membrane is evaluated in comparison to a cover slip with a reference transparency of 100%. To analyze the morphology of the nanofibers and calculate the diameter of the nanofibers, scanning electron microscopy (SEM, Thermo Fisher Scientific Apreo, n = 100) was conducted. The fiber diameter was calculated from the 100 fibers of each group using image J. The thickness of the gelNF membranes were measured using a vernier caliper (Fowler, n = 3) and SEM. The FITC- dextran (FD10S and FD70, Sigma Aldrich) and cell crowns (CellCrownTM, Scaffdex) were usedto test the permeability (n = 3). First, 12.5 ug / ml of the dextran dissolved in the cell culture media were added at the top of the cell crown. After 24h incubation, the media from the bottom part of the cell crown were measured to calculate the penetrated dextran. For degradability testing, the gelNF membranes were incubated at 37°C incubation in PBS for 14 and 28 days and SEM employed to evaluate changes in the nanofiber. To measure the tensile strength of the membrane, Instron testing instrument (INSTRON 5560, n = 9) was conducted and the specimens were prepared in rectangular shape (5 cm x 5 cm). The 30 mm / min strain was assessed with 1 kN load cell until break point. The elastic modulus was calculated from the stress-strain curve.

[0077] Immortalized human corneal endothelial cell (IhCEC) culture. IhCEC (T0577) purchased from Applied Biological Materials (ABM) and cultivated as followed by manufacture’s protocol. Briefly, the culture media (TM001) was supplements with 10% FBS (Gibco), 5 ug / ml human insulin (TM058), 10 µg / ml human transferrin, 3 ng / ml sodium selenite, 10 nM hydrocortisone, 10 nM β-estradiol, 10 ng / ml rhVEGF 165aa (Z100895), 10 ng / ml rhEGF (Z100135), 10 ng / ml Heparin, 2 mM L-glutamine (G275), and 1% Penicillin / Streptomycin Solution (15140122, Gibco). The IhCECs were cultivated on substrate coated with FNC coating mix (Athena (0407, Athena)) 37°C in a humidified incubator with 5% CO2 and subcultured using 0.05% Trypsin-EDTA (Gibco). The culture medium was changed every 2-3 days.

[0078] Isolated primary rabbit corneal endothelial cell (PrCEC) culture. Rabbit eyeballs were purchased from Visiontech inc. To isolate the CECs, descemet’s membrane was separated and placed in 2 mg / ml collagenase (C0130, Sigma Aldrich) solution to incubate at 37°C for 1 h. It was centrifuged at 1000 rpm for 3 min and resuspended. FNC coating mix, Opti-MEM, and supplements (11058021, Gibco). To analyze the elongation ratio of PrCEC, we drew horizontal and vertical lines meeting perpendicularly within the cell membrane and compared the length ratio of these two lines. To culture the CECs on top of the gelNF membrane, the cells were seeded at a density of 8 x 104cm2. Phase contrast microscope (EVOS, Thermo Fisher Scientific) was used for analysis morphology of the CECs.

[0079] Cytotoxicity of the gelNF membrane and proliferation of CECs cultured on top of the gelNF membrane. To evaluate the cytotoxicity of the gelNF membrane, Cell Counting Kit-8 (CK04, Dojindo) was assessed following the manufacturer’s protocol (n = 6). Briefly, 10% of the working reagents total volume of the culture media was added and incubated for 90 min at 37°C. 100 μl were transferred into the 96 well for absorbance measurements at 450 nm wavelength. To test the viability of the CECs cultured on top of the gelNF membrane, we performed a Live & Dead assay kit following the manufacture’s protocol. Briefly, the CECs were cultured on top of the gelNF membrane for 24 h. The culture media was then changed to Live & Dead solution mixed with fresh culture media. After a 15 min incubation, the live anddead cells were observed via confocal microscope. The live cell ratio was determined by dividing the number of live cells by the total cell count (n = 5).

[0080] Ex vivo study of cultured CECs transplantation using artificial anterior chamber. The rabbit cornea (n = 5) was dissected from eyeballs, followed by Descemet's membrane stripping to remove the endothelium. An artificial anterior chamber (K20-2125, Corza Ophthalmology) was utilized to mimic the eyeball shape. Three points were marked to distinguish the direction of cell cultivation: one point at the 1 o'clock direction and two points at the 2 o'clock direction on the gelNF membrane. The rabbit CECs were seeded at the density of 8 x 104 / cm2and cultured on the gelNF membrane until they became confluent and prepared into a circular shape with a diameter of 8 mm using a biopsy punch. To transplant the gelNF membrane, an incision was made using a 2.5 mm (8065921501, Alcon) knife, followed by injection into the anterior chamber using a glass Geuder (CorneaGen). Subsequently, an air bubble was introduced using a cannula and allowed to wait for 5 minutes. After removing the air bubble, cell culture medium was added. After 3 days of incubation, the cells and gelNF membrane were examined using a confocal microscope.

[0081] Immunofluorescence staining. To investigate the proteins expression of CECs, the samples were rinsed with Phosphate-Buffered Saline (PBS, Thermo Fisher Scientific) and fixed using 4% paraformaldehyde (15710, Electron Microscopy Sciences) for 30 min at RT followed by 3 times wash using PBS. The samples were then permeabilized using 0.2% triton X-100 (93443, Sigma Aldrich) for 5 min followed by 3 times washing with PBS. The samples were subsequently incubated with 3% Bovine serum albumin (BSA, A2153, Sigma Aldrich) dissolved in PBS for 1 h at RT, then incubated primary antibody dissolved in 1% BSA solution for overnight at 4°C. The following day, samples were rinsed 3 times with PBS and incubated with secondary antibody dissolved in 1% BSA solution for 1 h at RT. For DAPI staining, the DAPI solution was diluted in PBS and incubated for 5 min at RT. The images were photographed using confocal microscope (LSM T-PMT, ZEISS). The primary and secondary antibodies used in this study are ZO-1(339188, Thermo Fisher Scientific), Na+ / K+-ATPase (sc- 48345, Santa Cruz Biotechnology), N-cadherin (ab98952, Abcam), Aquaporin-1(ab219055, Abcam), phalloidin-555 (ab176756, Abcam), and DAPI (62248, Thermo Fisher Scientific).

[0082] Statistical analysis. All the data are presented in mean ± standard deviation. Statistical analysis was performed using two-tailed (α = 0.05) student t test for two experimental groups. One-way ANOVA with a post hoc Tukey’s multiple comparison test was applied for more than three test groups. A statistically significant difference is denoted as ** (p< 0.01), or *** (p< 0.001). Results

[0083] Characterization of gelNF membrane. First, we established the electrospinning conditions to impart the necessary characteristics for utilizing the produced gelNF membrane as a corneal material. The transparency of the gelNF membrane, measured in the visible light wavelength, showed percentages relative to glass at different spinning times: 45-70% for 120 min, 35-73% for 30 min, 65-72% for 10 min, 73-92% for 5 min, and 69-88% for 3 min, respectively (FIG. 1B). When each gelNF membrane was placed above text, the text was visible through the membrane, however, it was more clearly visible with the 3, 5, and 10 min spun gelNF membranes compared to the 30 and 120 min spun ones (FIG.1A). The thickness, measured with a Vernier caliper, increased proportionally with spinning time. Specifically, the thickness was approximately 150 μm for 120 min, less than 20 μm for 10 min, and less than 10 μm for 5 min spinning times. In addition to thickness and transparency, the sample subjected to 5 minutes of spinning and 5 minutes of crosslinking exhibited better shape maintenance in the liquid, in contrast to the sample spun for 3 minutes, which displayed a contracted shape (FIG.1A).

[0084] To confirm the porosity essential for securing the functionality of the corneal endothelium, as well as to elucidate the advantages of using electrospinning, we utilized SEM to examine the morphology of the fibers and membrane. The results revealed a nanofiber morphology with interconnected pores between nanofibers. Additionally, we observed minimal aggregation of the gelatin solution, predominantly resulting in fine nanofibers (FIG.2A). The calculated fiber diameter was 295 ± 82 nm before crosslinking and slightly increased to 304 ± 62 nm after crosslinking, with no significant differences (FIG. 2B). To assess whether the produced gelNF membrane possesses a tensile strength similar to DM and suitable mechanical properties for transplantation, we measured the tensile strength and calculated the elastic modulus by converting the data into a stress-strain curve. Fabricated gelNF membranes showed enough mechanical property to measure the tensile strength. The elastic modulus of the gelNF membrane showed 0.4 ± 0.15 MPa which has no significant differences with human DM (FIG.2C). The permeability of the gelNF membrane, another critical function, was assessed using FITC dextran. In the gelNF membrane cultured with CECs, FITC-dextran permeated through the membrane at concentrations of 0.286 ± 0.002 μg / ml for 10 kDa and 0.13 ± 0.003 μg / ml for 70 kDa (FIG.3A). Analyzing the degradability of the gelNF membrane through changes in thickness and permeability, when incubated in PBS for 14 days, the initial 6 μm thickness of the gelNF membrane decreased to less than 1 μm, showing an 88.6% thickness reduction after 14 days (FIG. 3C). Additionally, beginning at 7 days, there was a significant increase in permeability for 10 kDa dextran (FIG.3B).

[0085] IhCEC culture on top of the gelNF membrane. The cytotoxicity assessment of the gelNF membrane through the Live & Dead assay on IhCEC demonstrated predominant cell attachment in a viable state on both day 3 and 7 (FIG.7A and B). The calculated live cell ratioindicated that 93% and 97% of IhCECs remained viable on days 3 and 7, respectively (FIG. 7C). Following the verification of suitable characteristics of gelNF as a corneal graft material, CECs were cultured to assess cellular compatibility. IhCECs cultured in gelNF showed comparable proliferation to TCP after 24 hours (FIG. 4A), and it was confirmed that high- density culture was possible up to day 7 (FIG. 4B). Furthermore, the immunofluorescence staining of the similar morphology and expression levels of functional proteins such as ZO-1 and Na+ / K+-ATPase in IhCECs cultured in gelNF were comparable to those cultured in TCP (FIG.4C).

[0086] PrCEC isolation and cultivation on top of the gelNF membrane. After confirming appropriate cultivation of immortalized cells in gelNF, PrCEC were isolated from rabbit eyeballs to confirm the primary cell-compatibility. We investigated whether the cells exhibited a hexagonal shape, a characteristic feature of CECs, and expressed junctional proteins. Utilizing phase contrast imaging on day 4, it was observed that PrCECs were forming a monolayer at high cell density (FIG.8A). Additionally, PrCECs were observed to proliferate initially, and from day 3, they became tightly packed with a decreased elongation ratio, forming a normal hexagonal shape and junctional proteins (FIG.8B-D). Furthermore, when PrCECs were cultured in gelNF for one day, phase-contrast imaging revealed the absence of detached cells, and Live & Dead staining indicated that most cells were viable (FIG. 5A). Immunofluorescence staining for functional protein expression showed that, comparable to the control FNC coating mix-coated cover slips, gelNF gradually formed tight junctions from day 3, becoming more pronounced by day 7. Adherent junctional proteins and aquaporin-1 were also expressed in a similar pattern to the cover slip substrate (FIG.5B). The expression of the sodium-potassium ion pump appeared to increase with the progression of culture (FIG. 9).

[0087] Cultured CECs transplantation to the ex vivo rabbit eye. The membrane type-gelNF, exhibits mechanical properties comparable to DM and demonstrates a folding shape akin to that observed in the glass geuder (FIG. 6A). In the CECs cultured gelNF membrane transplantation test, the geuder was used to insert an 8mm disc of rolled cell-substrate into an artificial anterior chamber. A marking system of peripheral lines was employed to ensure correct orientation of the cells upon unrolling. It was observed that the membrane unfolded with ease. Air was then injected underneath the membrane to facilitate its adherence to the posterior corneal stroma. Following 5-minutes of air bubble incubation, stable attachment of the membrane to the cornea after addition of culture media was confirmed (FIG. 6B). Throughout a 3-day culture period and subsequent fixation and washing steps, the membrane remained firmly adhered to the cornea (FIG.6C). Immunofluorescence staining of phalloidin, ZO-1, and nuclei in the transplanted CECs cultured gelNF membrane, demonstrated a lower density of stromal cells in the cornea with DM removed. Conversely, in the region where thegelNF membrane was transplanted, a higher density of CECs (nuclei and phalloidin) were confirmed. (FIG.6D and E). The gelNF membrane exhibited autofluorescence, allowing the confirmation of ZO-1 expression (white arrows) through partial z-stacked imaging (FIG.6F).

[0088] The primary role of corneal endothelium is to maintain cornea clarity and thickness by regulating the movement of nutrients and water through the cornea via cell-cell tight junctions and the action of the Na+ / K+-ATPase pump. To preserve this critical function, any material used for CEC transplantation must be permeable. The aim of our research is to design a carrier for cultivated CECs that optimizes transparency and thus visual acuity without reducing efficient permeability, a vital aspect of CEC functionality. Consequently, we analyzed the thickness for maintaining CEC functionality, permeability, cell-compatibility, and transplantability to assess their suitability. First, we compared the thickness and transparency of gelNF membranes produced at different spinning and crosslinking times to optimize electrospinning parameters. We found thin membranes undergo excessive crosslinking, which influences transparency. Membranes that are too thick also impact transparency. In our work, the ideal transparency was achieved by utilizing gelNF membranes crafted by 5 minutes spinning followed by 5 minutes crosslinking procedure (FIG. 1). The glutaraldehyde (GA) induces crosslink formation between the amine and lysine moieties of gelatin, impacting not only color but also transparency. GA crosslinking is an efficient crosslinking method for electrospun gelNF membrane with a short crosslinking time. However, there is still a risk of toxicity associated with using GA. In studies utilizing the GA vapor crosslink system, exposure for several hours did not pose toxicity issues. In current research, after performing GA crosslinking for only 5 minutes, a process was carried out to remove residual reagent over a sufficient period, suggesting a reduced risk of cytotoxicity (FIG.4A, 5A, and FIG.7).

[0089] Gelatin is a substance derived from collagen and is utilized in various research fields due to its high biocompatibility. There have been studies on producing nanofibers using electrospinning, mostly blending them with synthetic polymers to enhance mechanical properties. However, existing studies using natural polymer have highlighted a potential residual toxic solvent for the increased crosslink level due to inadequate biomechanical. While this approach can achieve high tensile strength, the use of organic solvents may lead to toxicity issues. In the gelNF membrane used in this experiment, vapor crosslinking using GA solution which is the least toxic method for gelatin crosslinking. In the tensile strength test, although there is approximately an 8-fold difference in properties compared to the DM of the human cornea (FIG.2 B), when comparing with the overall tensile strength of the cornea (15.8 MPa), it can be considered similar to DM and deemed suitable as a gelNF membrane to recapitulate the role of Descemet's Membrane. The mechanical property of the endothelium graft is required be appropriate to endure the stretching forces and maintain integrity during surgery and post-transplantation as the cornea goes through various movements andstresses. Moreover, an optimal balance of tensile strength is necessary to ensure that the material is strong enough to maintain its structural integrity and support the endothelial cells, while also allowing for necessary flexibility and compliance within the corneal environment. In this study, the gelNF membrane demonstrated suitable properties for transplantation (FIG.2B and FIG. 6A) and appropriate degradation rates (FIG. 3C), addressing the limitations of materials that degrade too rapidly during cell cultivation while maintaining desired properties for CEC delivery.

[0090] The thickness is one of the crucial considerations for the material intended for transplantation into the corneal endothelium. It is important to have a thickness comparable to the actual DM while not hindering the pump-leakage phenomenon. The gelNF membrane used in this study was optimized at less than 20 μm after 5 minutes of spinning. Considering that carriers utilized for DMEK should be less than 50 μm, the gelNF membrane fabricated in this study is adequate for use as a CEC transplantation material. The other advantages of an electrospun nanofiber membrane are its ability to ensure permeability through pore structures, as reported through various studies. In this study, the porosity of the gelNF membrane produced can be observed through the pore structures between nanofibers in the top view image obtained via SEM (FIG.2A). Additionally, a porous structure can be confirmed through the cross-sectioned view image (FIG.3C, day 0). The permeability test using Dextran is an analytical method primarily utilized in membranes that function as barriers. In conjunction with SEM analysis, permeability experiments were conducted using FITC-labeled dextran to ascertain the extent to which actual molecules penetrate the gelNF membrane. In this study, both 10 kDa and 70 kDa dextran were observed to permeate the gelNF membrane, indicating its permeability property (FIG. 3A). Permeability was also associated with degradation. A significant difference in permeability was noted seven days after incubating the membrane at body temperature (FIG. 3B). Analysis of the thickness changes in the gelNF membrane revealed that over 88% of the membrane had degraded after incubation at body temperature for 14 days (FIG.3C). The rapid degradation of the gelNF membrane may be advantageous in terms of eliminating potential factors that can induce an inflammatory response within the eye. It is presumed that CECs would have begun migrating from the gelNF membrane to the cornea, considering that the gelNF membrane had already attached to the posterior cornea after 5 minutes of incubation following graft transplantation (FIG.6B).

[0091] To maintain the thickness and transparency of the cornea, preserving the pump function of CECs is crucial. To achieve this, it is important to maintain a high cell density of CECs while forming tight junctions. In this study, two types of CECs were tested. Firstly, it was confirmed that gelNF membrane allows for high cell density cultivation of IhCECs without cytotoxicity (FIG. 4A and B). Functional protein expression confirmed using immunofluorescence staining revealed patterns comparable to the control group of this study,represented by the cover slip, showing expression of ZO-1 and sodium-potassium pump ions. Likewise, PrCEC cultured on gelNF membrane showed high cell attachment without cytotoxicity from day 1, as well as a clearer hexagonal cell shape (FIG.5A). Furthermore, the functional protein expression was comparable to that of CECs cultured on cover slips (FIG. 5B). These results of high CEC cell density and functional protein expression demonstrate the gelNF membrane is suitabile for CEC cultivation.

[0092] Lastly, an ex vivo study was conducted to evaluate the practical application of CEC- cultured gelNF membrane. The artificial anterior chamber, considered an excellent tool for DMEK surgery practice, was utilized in this experiment to assess the material prior to transplantation in an animal model. Grafts inserted into the anterior chamber through a glass geuder cannula were affixed to the posterior of the cornea using an air bubble for only 5- minutes (FIG.6B). This attachment occurred much faster than the several hours or days of gas bubble use with supine positioning in current DMEK procedures. While the reasons for this relatively rapid adhesion are not fully known, it is likely a result of high surface contact area as a result of the mesh-like surface topology of the gelatin nanofibers. The reduced adhesion time presents an advantage of reduced patient discomfort with strict supine positioning in the post-operative period and eliminating the need for re-bubbling procedures. Subsequent examination of cytoskeleton (phalloidin) and tight junctional protein expression in CECs post-transplantation revealed sustained expression for three days when in contact with both the gelNF membrane and the cornea (FIG.6E and F). Despite the autofluorescence of the gelNF membrane, the clear visualization of ZO-1 expression was aided by employing partial z-stack imaging to confirm tight junctional protein formation (FIG.6F, white arrows). In this study, an engraftment test was conducted using rabbit corneas over a period of three days. It was observed that corneal endothelial cells migrated to the decellularized cornea within three days of cultivation. Consequently, it is anticipated that, during the 14-day degradation period of the membrane, there is ample time for corneal endothelial cells to migrate to the cornea. To validate this accurately, transplantation results need confirmation through subsequent studies. However, considering that the membrane adhered to the cornea during the washing and fixation processes after three days of cultivation, it is believed that interactions may have occurred between cells and corneal tissue or between the gelNF membrane and corneal tissue.

[0093] The objective of this study was to develop a material for transplanting cultured CECs. A gelNF membrane was developed using electrospinning that maintained sufficient thickness, transparency, and permeability for proper corneal functionality. We also confirmed that CECs expressed functional proteins effectively. Additionally, through ex vivo studies, it was verified that the CECs cultured on the gelNF membrane exhibited properties suitable fortransplantation and successfully migrated towards the cornea. Therefore, the gelNF membrane is useful for CECs transplantation. References

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[0126] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

What is Claimed is 1. A method of treating a cornea, the method comprising contacting a corneal surface with an electrospun membrane containing a biocompatible biopolymer.

2. The method of claim 1, wherein the biopolymer is collagen or a derivative of collagen such as gelatin.

3. The method of claim 1, wherein the membrane contains a composite of biocompatible biopolymers.

4. The method of claim 3, wherein the composite of biocompatible biopolymers comprises collagen or a derivative of collagen such as gelatin, and hyaluronic acid.

5. The method of any of the previous claims, wherein the biopolymer is cross-linked 6. The method of any of the previous claims, wherein the membrane is at least 75% transparent.

7. The method of any of the previous claims, wherein the membrane is from about 5 µm to about 150 µm thickness.

8. The method of any of the previous claims, wherein the membrane has an elastic modulus of from about 0.2 to 0.6 MPa.

9. The method of any of the previous claims, wherein the nanofibers of the membrane are from about 200 to 500 nm in diameter.

10. The method of any of the previous claims, wherein the membrane is seeded with cells.

11. The method of claim 10, wherein the cells are corneal endothelial cells.

12. The method of any of the previous claims, wherein the membrane comprises a therapeutic drug or factor.

13. The method of any of claims 1-12, wherein the corneal surface is treated for repair, regeneration, and / or reconstruction of lamellar or partial defects of wounded corneal tissue.

14. The method of claim 13, wherein the corneal surface comprises a surgically incised or wounded corneal area.

15. The method of claim 13 or 14, wherein a cavity in the cornea is debrided to eliminate scarred, fibrotic, and / or necrotic material.

16. The method of claim 15, wherein a surgical instrument or laser is used to remove tissue.

17. A composition for use in the method of any of the preceding claims.

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