Support for culturing corneal cells, comprising graphene

Abstract

The present invention relates to: a support for culturing corneal cells, the support comprising graphene; and a use thereof. The support which is for culturing corneal cells and comprises graphene according to the present invention is non-toxic to corneal cells, is optimized for the cell proliferation rate, adhesion rate, and survival rate of corneal cells, enhances the expression of corneal cell-specific genes, and can maintain an optimized corneal cell state through cytoskeleton remodeling and mitochondrial activation, and thus has the effect of being applicable to cell therapy products, graft materials in the field of regenerative medicine, and platforms for tissue engineering.

Description

A scaffold for corneal cell culture containing graphene

[0001] The present invention relates to a support for corneal cell culture comprising graphene and its uses.

[0002] The cornea is the outermost structure of the eyeball and is one of the primary organs responsible for refracting light. It consists of a total of six layers: the corneal epithelium, Bowman's layer, corneal stroma, Dua's layer, Descemet's membrane, and corneal endothelium. The corneal epithelium is composed of 5 to 6 cell layers; base cells are generated from peripheral stem cells, migrate to the center, and slough off after 7 days. Bowman's layer consists of cellless collagen fibers and is colorless and transparent. However, it cannot regenerate and leaves scars in the event of surgery or trauma. The corneal stroma accounts for 90% of the corneal thickness, and the direction and size of its cells are uniform. Descemet's membrane is composed of 3 to 4 layers of cells and is presumed to be the basement membrane of the endothelium. The corneal endothelium consists of a single layer of cells; the composition of these cells is fixed, regeneration is extremely limited, and the number of cells decreases with age. When the number of cells decreases, surrounding cells enlarge to fill the space. The corneal endothelium is a single-layer structure of hexagonal cells located on the posterior surface of the cornea and contains embedded physiological ion pumps. Corneal endothelial cells (CECs) maintain mechanical strength by controlling hydration through the pumping of water from the stroma into the aqueous humor. At birth, the average density of human corneal endothelial cells is approximately 5,000 cells / mm³. 2It is known that... However, because mitotic potential is limited, the total number of cells decreases with age. When corneal endothelial cells are damaged, the cells repair themselves through cell expansion and migration rather than mitosis; consequently, the cornea swells to several times its normal thickness, becomes opaque, and loses its function. The damage is permanent. Currently, the established treatment for corneal endothelial cell damage is corneal transplantation. Recovery can be achieved through the transplantation of donor tissue via penetrating keratoplasty (PK) or lamellar keratoplasty. However, due to the severe shortage of corneal donors, there is an urgent need to develop technology to culture corneal cells in vitro with high proliferation and survival rates to overcome this issue.

[0003] New studies on stem cell differentiation, such as the induction of induced pluripotent stem cells (iPSCs) and mesenchymal stem cells into corneal endothelial cells, have recently been conducted for clinical treatment. In particular, the ability of induced pluripotent stem cells (iPSCs) to differentiate into corneal endothelial-like cells (iCECs) is a promising patient-specific approach that eliminates the need for postoperative immunosuppression. A study reported in 2016 described the derivation of corneal endothelial-like cells (CECs) from induced pluripotent stem cells (iPSCs) derived from BJ human foreskin fibroblasts (Zhao and Afshari. 2016). Similarly, another study devised a procedure for adult fibroblast-derived induced pluripotent stem cells (iPSCs) to induce corneal endothelial-like cells (CECs) (Wagoner et al. 2018). Although research related to such stem cells has been actively conducted, it is subject to limitations such as reduced cell adhesion and viability, making it necessary to research optimal scaffolds for clinical use.

[0004] The objective of the present invention is to provide a support for corneal cell culture.

[0005] In addition, the present invention provides a composition for implantation.

[0006] In addition, the present invention provides an implant material.

[0007] In addition, the present invention provides a method for manufacturing a support for corneal cell culture.

[0008] In addition, the present invention provides a support for corneal cell culture.

[0009] In addition, the present invention provides a corneal cell transplantation method.

[0010] To achieve the above objective, the present invention provides a corneal cell culture support comprising graphene.

[0011] In addition, the present invention provides a transplantation composition comprising corneal cells cultured on the corneal cell culture support.

[0012] In addition, the present invention provides an implant material comprising the above-mentioned culture support and corneal cells cultured on the support.

[0013] In addition, the present invention provides a method for manufacturing a support for corneal cell culture.

[0014] In addition, the present invention provides a use of a corneal cell culture support for use in the manufacture of the implantable composition or implant material of the present invention.

[0015] In addition, the present invention provides a corneal cell transplantation method comprising the step of transplanting a transplantation composition or a transplantation material to an individual requiring corneal cell transplantation.

[0016] The graphene-containing scaffold of the present invention is non-toxic to corneal cells, is optimized for the cell proliferation rate, adhesion rate, and survival rate of corneal cells, enhances the expression of corneal cell-specific genes, and can maintain an optimized corneal cell state through cytoskeletal remodeling and mitochondrial activation; therefore, the corneal cell culture scaffold has the effect of being utilized in cell therapies, implant materials in the field of regenerative medicine, and tissue engineering platforms.

[0017] Figure 1 is a schematic diagram showing the manufacturing of a GO plate.

[0018] Figures 2a and 2b are diagrams analyzing the surface roughness (a) and thickness (b) of a GO plate according to the number of GO coatings.

[0019] Figures 3a and 3b show the cell proliferation-promoting effect of GO plates on corneal endothelial cells (a) and IHCE (immortalized human corneal endothelial cell line) (b):

[0020] Control: GO uncoated control group;

[0021] GO: 3-times coated GO plate

[0022] Figure 4 shows the cell adhesion rate and viability of GO plates according to the number of GO coatings:

[0023] VTN only: Standard cell culture plate;

[0024] GO 50nm: 12-times coated GO plate; and

[0025] GO 20 nm: 3-times coated GO plate,

[0026] Figure 5 is a figure confirming the expression of corneal endothelial cell-specific markers in corneal endothelial cells cultured on a GO plate.

[0027] Figures 6a and 6b show the expression of intercellular adhesion-related genes, cell adhesion-related genes, and cell growth-promoting genes in corneal endothelial cells (iPSC-CEC or IHCE) cultured on a GO plate.

[0028] Figure 7 shows the expression of F-actin, a signaling factor regulating mechanical properties, in corneal endothelial cells cultured on a GO plate.

[0029] Figure 8 shows the expression of cell adhesion signal-related proteins in corneal endothelial cells cultured on a GO plate.

[0030] Figure 9 shows the nuclear localization of YAP1, mitochondrial activation, and expression of corneal cell-specific markers in corneal endothelial cells cultured on a GO plate.

[0031] Hereinafter, the present invention will be described in detail with reference to the attached drawings for embodiments thereof. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited by this. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom. Furthermore, in order to clearly explain the present invention in the drawings, parts unrelated to the description have been omitted, and similar parts throughout the specification have been given similar reference numerals.

[0032] Throughout the specification, when it is stated that a part is "connected (connected, in contact, combined)" with another part, this includes not only cases where they are "directly connected," but also cases where they are "indirectly connected" with other members interposed between them. Furthermore, when it is stated that a part "includes" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but rather allows for the inclusion of additional components.

[0033] The terms used herein are merely for describing specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as “comprising” or “having” are intended to indicate the presence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0034] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

[0035]

[0036] In one aspect, the present invention relates to a support for corneal cell culture comprising graphene.

[0037] In one embodiment, graphene oxide (GO) may be coated on at least a portion of the surface of a support, and spin-coating a graphene oxide dispersion 1 to 5 times on the surface of the support where corneal cells are to be cultured can most enhance the growth and survival rate of the corneal cells.

[0038] In one embodiment, the thickness of the coated graphene oxide may be 10 to 60 nm, and it is more preferable that it be 10 to 30 nm.

[0039] In one embodiment, the surface roughness of the coated graphene oxide may be 1 to 10 nm, and is more preferably 1 to 3 nm.

[0040] In one embodiment, the corneal cell may be a corneal cell progenitor cell, a corneal epithelial cell, a corneal endothelial cell, or a corneal stem cell, most preferably a corneal endothelial cell, and may be an iPSC-derived corneal endothelial cell or an immortalized human corneal endothelial cell line.

[0041] In one embodiment, the corneal cells may be corneal cells derived from embryonic stem cells (ESC), adult stem cells (ASC), or induced pluripotent stem cells (iPSC).

[0042] In one embodiment, the adult stem cells may be mesenchymal stem cells (MSCs), and the mesenchymal stem cells may be umbilical cord blood-derived mesenchymal stem cells (UCB-MSC), umbilical cord-derived mesenchymal stem cells (UC-MSC), adipose-derived mesenchymal stem cells (AD-MSC), or bone marrow-derived mesenchymal stem cells (BM-MSC).

[0043] In one embodiment, the material of the support is not particularly limited but may include glass, extracellular matrix (ECM), biodegradable polymer, metal (e.g., aluminum, iron, gold, and stainless steel alloy, etc.) on which graphene oxide can be coated, decellularized ECM, vitronectin, collagen, fibronectin, laminin, Matrigel, fibrin, gelatin, polylysine, heparan sulfate proteoglycan, hyaluronic acid, alginate, agarose, chitosan, dextran, methacrylated gelatin (GelMA), cellulose, Pectin, Chondroitin Sulfate, Polydimethylsiloxane (PDMS), Polycaprolactone, Polycarbonate (PC), Propylene Carbonate, Ethylene Carbonate, Dimethyl Carbonate, Diethyl Carbonate, Poly(lactate-co-glycolate) (PLGA), Poly Lactic Acid (PLA), Polyurethane (PU), Poly(lactide-co-caprolactone) (PLCL), Polydioxanone (PDO), Polystyrene (PS),Poly(ethylene glycol, PEG), Poly(vinyl acetate, PVA), Polyacrylates, Polycyclic olefins, Poly(1-trimethylsilyl-1-propyne, PTMSP), Polytetrafluoroethylene (PTFE), Urethane, Polyethylene terephthalate (PET), Polyvinylidene fluoride (PVDF), Polyethylene naphthalate (PEN), Polysulfones, Cyclic olefin copolymer (COC), Polycaprolactone (PL), Polypropylene glycol (PPG), Polyacrylamide (PAAm), Polyglycolic acid (PGA), polymethylmethacrylate (PMMA), polyhydroxybutyrate (PHB), polyvinylpyrrolidone (PVP), polyester, polyether sulfone (PES), polyvinylidene fluoride (PVDF), polyamide, polyimide (PI), polyethylene (PE), polypropylene (PP), agarose, silicone, polyurethane acrylate, polystyrene block copolymer,It may include materials of styrene-butadiene-styrene block copolymer (SBS), styrene-isoprene-styrene block copolymer (SIS), styrene-ethylene-butylene-styrene block copolymer (SEBS), styrene-butadiene block copolymer (SBR), styrene-ethylene-propylene-styrene block copolymer (SEPS), styrene-acrylate copolymer, styrene-methyl methacrylate copolymer (PSMMA), styrene-acrylonitrile copolymer (PSAN), derivatives thereof, or copolymers thereof.

[0044] In one embodiment, the support may be a cell culture vessel and may be in the form of a flask, dish, well plate, sheet, substrate, microfluidic chip or mold, and the well plate may be a micro-well plate, a 96-well plate, a 24-well plate or a 6-well plate.

[0045] In one embodiment, the support may be non-toxic to corneal cells.

[0046] In one embodiment, the support can improve the adhesion or proliferation rate of corneal cells.

[0047] In one embodiment, the support can increase the expression of Ki-67 or pH3 (phospho-Histone H3) in corneal cells.

[0048] In one embodiment, the support can increase the expression of a corneal cell-specific marker (e.g., Phalloidin) in corneal cells.

[0049] In one embodiment, the support can increase the expression of a corneal endothelial cell-specific marker (e.g., N-cadherin or CD166).

[0050] In one embodiment, the support can increase the expression of cell adhesion or growth-related genes (e.g., CDH2, ITGB1, or EGFR) in corneal endothelial cells.

[0051] In one embodiment, the support can activate mechanical signal transmission in corneal endothelial cells.

[0052] In one embodiment, the support can increase the expression of F-actin in corneal endothelial cells.

[0053] In one embodiment, the support can increase the phosphorylation of ERK protein or FAK protein in corneal endothelial cells and decrease the phosphorylation of YAP.

[0054] In one embodiment, the support can induce nuclear localization of YAP1 or mitochondrial activation in corneal endothelial cells.

[0055] In the present invention, the terms "derived" and "derived" refer to components obtained from a source mentioned by a useful method.

[0056] In the present invention, the term "pluripotent stem cell (PSC)" refers to a stem cell capable of induced differentiation into any form of cell constituting the body, and pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Specifically, embryonic stem cells are induced from the inner cell mass of a blastocyst in the pre-implantation stage. The induced cells are maintained in a specific environment and are capable of unlimited culture and pluripotent differentiation. Furthermore, induced pluripotent stem cells may refer to pluripotent differentiated cells created by dedifferentiation from somatic cells, and are formed by making somatic cells very similar to embryonic stem cells through a process called reprogramming, such as cell fusion, nuclear transfer, and overexpression of pluripotency regulatory factors. Furthermore, pluripotent stem cells are not limited to embryonic stem cells and induced pluripotent stem cells, but may include all cells possessing both pluripotency and self-replicating ability. However, preferably, the pluripotent stem cells may be mammalian cells, and more preferably, human-derived pluripotent stem cells.

[0057] In the present invention, the term "biodegradable polymer" refers to a polymer that spontaneously and gradually degrades in vivo after a certain period and possesses one or more characteristics among biocompatibility, hemoaffinity, anti-calcification properties, cellular nutrient component, and intercellular matrix formation ability. The types of such biodegradable polymers are not limited thereto, but include fibrin, collagen, gelatin, cellulose, pectin, chondroitin sulfate, chitosan, alginate, hyaluronic acid, dextran, polylactic acid, poly(glycolic acid), poly(lactic-co-glycolic acid), poly(lactic-co-glycolic acid), poly-ε-(caprolactone), polyanhydride, polyorthoester, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly-N-isopropylacrylamide, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) copolymers, copolymers thereof, mixtures thereof, etc.

[0058] The above support can be manufactured by molding a biodegradable polymer according to existing known methods, such as the solvent-casting and particle-leaching technique, the gas forming technique, the fiber extrusion and fabric forming process, the thermally induced phase separation technique, the emulsion freeze drying method, the high pressure gas expansion method, but is not limited thereto.

[0059] In one aspect, the present invention relates to a transplantable composition comprising corneal cells cultured on a corneal cell culture support of the present invention.

[0060] In one embodiment, the corneal cells may be corneal endothelial cells.

[0061] In one embodiment, the composition may be a cell therapy agent for preventing or treating corneal damage disease.

[0062] In the present invention, the term "cell therapy agent" refers to a pharmaceutical product used for the purposes of treatment, diagnosis, and prevention, consisting of cells and tissues produced by isolation, culture, and special processing from a human being. It refers to a pharmaceutical product used for the purposes of treatment, diagnosis, and prevention through a series of actions such as proliferating, selecting, or otherwise altering the biological characteristics of living autologous, allogeneic, or xenogeneic cells in vitro to restore the function of cells or tissues.

[0063] The cell therapy agent according to the present invention can be injected into the body of an individual, for example, by using the clinical method published by Lindvall et al. (1989, Arch. Neurol. 46: 615-31) or Douglas Kondziolka (Pittsburgh, 1998). The formulation may include a pharmaceutically acceptable conventional carrier in addition to corneal cells, which are the active ingredient, and in the case of an injectable formulation, may include a preservative, an analgesic, a solubilizing agent, or a stabilizer, and in the case of a formulation for local administration, may include a base, an excipient, a lubricant, or a preservative.

[0064] The cell therapy product according to the present invention may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person with ordinary knowledge in the ordinary art. The pharmaceutically acceptable carrier included in the cell therapy product of the present invention is one that is commonly used in formulation and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil. The cell therapy composition of the present invention may additionally include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., in addition to the above components.

[0065] The cell therapy product according to the present invention can be administered parenterally, intravenously, subcutaneously, intraperitoneally, or topically. Suitable dosages of the cell therapy product composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate, and response responsiveness.

[0066] In the present invention, the terms “administering,” “introducing,” and “implanting” are used interchangeably and may refer to the placement of a composition according to one embodiment into an individual by a method or route that results in at least partial localization of the composition according to one embodiment to a desired site. At least a portion of the cells or cellular components of the composition according to one embodiment may be administered by any suitable route to deliver them to a desired location within a living individual.

[0067] In one aspect, the present invention relates to a support for corneal cell culture according to the present invention; and an implant material comprising corneal cells cultured on said support.

[0068] In one embodiment, the corneal cell may be a corneal cell progenitor cell, a corneal epithelial cell, a corneal endothelial cell, or a corneal stem cell, most preferably a corneal endothelial cell, and may be an iPSC-derived corneal endothelial cell or an immortalized human corneal endothelial cell line.

[0069] In one embodiment, corneal cells can be cultured on graphene oxide coated on a support.

[0070] In one aspect, the present invention relates to a method for preparing a corneal cell culture support comprising the steps of: ultrasonically treating an aqueous graphene oxide dispersion; coating an aqueous graphene oxide dispersion onto a support; and drying.

[0071] In one embodiment, the graphene oxide aqueous dispersion can be coated on the surface of a support 1 to 5 times, and the coating can be done such that the thickness of the graphene oxide is 10 to 30 nm and the surface roughness is 1 to 3 nm.

[0072] In one aspect, the present invention relates to the use of a corneal cell culture support for use in the manufacture of the implantable composition or implant material of the present invention.

[0073] In one aspect, the present invention relates to a corneal cell transplantation method comprising the step of transplanting a transplantation composition or a transplant material to an individual requiring corneal cell transplantation.

[0074] In one aspect, the present invention relates to a method for preventing or treating corneal damage, comprising the step of implanting an implantable composition or implant material into an individual requiring corneal cell transplantation.

[0075] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0076]

[0077] Example 1. Preparation of a support for cell culture

[0078] 1-1. Fabrication of Graphene Oxide (GO) Plates

[0079] A graphene oxide aqueous dispersion with a concentration of 2.5 mg / mL, obtained by purifying to approximately pH 6 through repeated centrifugation, was ultrasonically treated for 30 minutes at a temperature below 30°C to uniformly adjust the average particle size of the graphene oxide to approximately 10 μm, thereby preparing a GO aqueous dispersion (Graphene Oxide Solution) ((S10, Grapheneol Co., Ltd.). The GO aqueous dispersion was spin-coated 3, 6, and 12 times, respectively, onto a silicon oxide substrate to produce GO films with various surface thicknesses and roughness. Subsequently, the prepared films were detached from the substrate using hydrofluoric acid vapor and simultaneously transferred onto a polydimethylsiloxane (PDMS) plate surface under conditions of 80°C and nitrogen to maintain the physical properties and hydrophilicity of the surface (Fig. 1).

[0080]

[0081] 1-2. Analysis of Surface Characteristics of GO Plates

[0082] To confirm the synthesis of GO on the GO plate prepared in Example 1-1 above and to identify defects (D peak) in the graphene structure, analysis was performed using Raman spectroscopy. Additionally, the wrinkles and roughness characteristics of the surface after GO coating were confirmed using a Scanning Electron Microscope (SEM), and the surface thickness and height distribution were precisely measured using an Atomic Force Microscope (AFM). Surface roughness was calculated based on the standard deviation of height values ​​obtained from the AFM image, and the GO film thickness was derived by measuring the height difference between the region where the film is present and the region where it is not present at the edge of the film.

[0083] Number of Coatings Surface Thickness Surface Roughness 3 approx. 20 nm approx. 2 nm 6 approx. 30 nm approx. 3 nm 12 approx. 50 nm approx. 6 nm

[0084] As a result, GO was successfully synthesized and was found to be free of defects in the graphene structure. The surface roughness showed a tendency to increase with increasing GO coating cycles (3 times: 2 nm; 6 times: 3 nm; 12 times: 6 nm) (Table 1, Fig. 2a), confirming that as the coating was repeated, the fine undulations of the surface became rougher and wrinkles formed. In addition, AFM analysis results showed that the coating thickness of the graphene oxide increased with increasing GO coating cycles (Fig. 2b) (3 times: 16.574 nm; 6 times: 31.396 nm; 12 times: 50.114 nm), indicating that a uniform layer was formed with each coating, resulting in a cumulative increase in film thickness.

[0085]

[0086] Example 2. Analysis of the effect of iPSC-derived corneal endothelial cell culture

[0087] 2-1. Analysis of Cell Adhesion and Proliferation Rates According to GO Coating

[0088] To analyze the effects of GO coating on corneal endothelial cells prepared in Example 1 above, iPSC-derived corneal endothelial cells (CECs) were prepared and cultured on standard cell culture plates and on GO plates coated with GO 3, 6, or 12 times, respectively, for analysis. Specifically, differentiated iPSC-derived corneal endothelial cells (Endo day 3) were cultured at 5,000 cells / cm² in 10 cm plates (100 mm TC-treated Culture Dish, Corning, USA). 2 They were cultured by aliquoting, and on Endo day 9, undifferentiated iPSCs and immature corneal endothelial cell precursors were removed by the Wash-out Method. They were then passedaged and cultured until Endo day 15 in Human Endothelial-SFM Medium (Gibco) supplemented with B431542 (1μM), H-1152 (2.5μM), 2-phospho-L-ascorbic acid (0.02mg / mL), ITS (1%), hEGF (10ng / mL), CaCl₂ (0.2mg / mL), and Fasudil (10μM). At Endo day 15, immortalized human corneal endothelial cell lines (IHCE) for comparison with iPSC-derived corneal endothelial cells were plated at 5,000 cells / cm² on GO plates (6-well or 24-well plates) prepared by GO coating 3 times, 6 times, or 12 times in Example 1 above. 2The cells were subcultured at a cell density and set as Day 0, and cultured for 4 days while replacing the medium every 2 days (Day 0-4, Total Endo Day 15-19). While these high-purity and high-efficiency corneal endothelial cells were cultured on GO plates for 4 days, the morphology of the corneal endothelial cells according to the number of culture days was observed and compared under a microscope. In addition, to analyze the cell proliferation rate by confirming the expression of Ki-67 (proliferation marker protein Ki-67) and pH3 (phospho-Histone H3, phosphorylated histone H3 at Ser10), which are markers indicating cell division activity, corneal endothelial cells cultured for 4 days were fixed with 4% paraformaldehyde at room temperature for 20 minutes, and then treated with a blocking buffer (5% NDS or NGS-based) containing 0.3% Triton X-100 for 1 hour to allow permeation of the cell membrane. Subsequently, primary antibodies against Ki-67 and phospho-Histone H3 were applied, and the cells were incubated overnight at 4°C. After washing three times with PBS-T, the cells were treated with an antibody buffer containing the fluorescently labeled Alexa Fluor 488 secondary antibody and DAPI, and incubated at room temperature for one hour. Stained cells were observed using confocal scanning electron microscopy while immersed in 1X PBS without mounting. The acquired fluorescence images were analyzed by quantifying the fluorescence intensity of each marker using ImageJ to analyze cell proliferation rates. For viability analysis, viable and non-viable cells were quantified using an automated cell counter after staining with 0.4% Trypan blue solution. This allowed for a quantitative comparison of changes in proliferative activity under GO scaffold culture conditions.

[0089] As a result, corneal endothelial cells derived from induced pluripotent stem cells cultured on GO plates coated with GO three times exhibited a higher proliferation rate and cell viability compared to standard cell culture plates (Fig. 3a), and a similar trend was observed in IHCE cells cultured under the same conditions (Fig. 3b). These results, in which the group cultured on GO plates maintained higher proliferation and viability compared to standard plates, suggested that the GO surface can promote cell activity in various cell types. Additionally, an analysis of cell proliferation according to the number of GO coatings revealed a relatively high cell density on GO plates coated three times (GO 20 nm). Although a certain level of cell proliferation was maintained on GO plates coated twelve times (GO 50 nm), the cell density was relatively lower compared to GO plates coated three times (Fig. 4). Consequently, cell culture supports coated with GO three times were used for subsequent experiments. These results demonstrate that graphene oxide is non-toxic to corneal endothelial cells and that cell adhesion and proliferation efficiency can be affected by the number of coating layers and thickness.

[0090]

[0091] 2-2. Analysis of Corneal Endothelial Cell-Specific Marker Expression

[0092] In the above Example 2-1, the expression of the corneal cell marker Phalloidin, the corneal endothelial cell-specific marker N-cadherin, and CD166 was analyzed by immunocytochemistry (ICC) in corneal endothelial cells (Day 4, Endo Day 19) cultured on a GO plate or a standard culture plate. Specifically, iPSC-derived corneal endothelial cells were cultured for 4 days on a GO plate coated with GO three times, then fixed in 4% paraformaldehyde at room temperature for 20 minutes, and then treated with a blocking buffer (5% NDS or NGS-based) containing 0.3% Triton X-100 for 1 hour to allow cell membrane penetration. Subsequently, primary antibodies against CD166 (1:500) and N-cadherin were applied and incubated overnight at 4°C. After washing three times with PBS-T, the cells were treated with an antibody buffer containing fluorescently labeled Alexa Fluor 488 secondary antibody and DAPI, and incubated at room temperature for 1 hour. Images were acquired using confocal scanning microscopy while the stained cells were kept immersed in 1X PBS without mounting.

[0093] As a result, when cultured on GO plates, the expression of corneal endothelial cell-specific markers N-cadherin and CD166 was found to be significantly increased compared to the control group (Fig. 5).

[0094]

[0095] 2-3. Analysis of Cell Adhesion and Growth-Related Marker Expression

[0096] In Example 2-1 above, the expression of the cell-cell adhesion-related gene CDH2 (Cadherin-2), the cell adhesion-related gene ITGB1 (Integrin Subunit Beta 1), and the cell growth-promoting gene EGFR (Epidermal Growth Factor Receptor) was confirmed by quantitative PCR (qPCR) analysis in corneal endothelial cells (iPSC-CEC) or IHCE (Day 4, Endo Day 19) cultured on GO plates or standard culture plates. Specifically, total RNA was extracted from cultured corneal endothelial cells or IHCE using Trizol™ reagent (Thermo Fisher Scientific, USA), and the extracted RNA was reverse transcribed into cDNA using a cDNA Reverse Transcription Kit. Subsequently, SYBR Green-based quantitative PCR analysis was performed using the QuantStudio™ real-time PCR system (Applied Biosystems). The expression of each gene was quantitatively analyzed using the ΔΔCt method with GAPDH as the endogenous control.

[0097] As a result, when cultured on GO plates, not only corneal endothelial cells derived from iPSCs (iPSC-CEC) (Fig. 6a) but also IHCE (Fig. 6b) showed an increased expression of genes related to cell adhesion and growth, such as CDH2, ITGB1, and EGFR, compared to the control group cultured on a general culture plate.

[0098]

[0099] 2-4. Analysis of Mechanotransduction Activation

[0100] To determine whether mechanical signal transduction changed in corneal endothelial cells (Day 4, Endo Day 19) cultured on GO plates or general culture plates in Example 2-1 above, F-actin expression was analyzed by immunocytochemistry, and the expression of ERK (Extracellular signal-regulated kinase), p-ERK (phosphorylated ERK), FAK (Focal adhesion kinase), p-FAK (phosphorylated FAK), N-Cadherin (Neural cadherin), YAP (Yes-associated protein), and p-YAP (phosphorylated YAP) was confirmed by Western blot analysis. Total protein was extracted using a lysis buffer containing protease and phosphatase inhibitors, and protein quantification was performed via BCA (bicinchoninic acid) analysis. An equal amount of protein was quantified for each sample, loaded onto 10% SDS-PAGE for electrophoresis, and then transferred to a PVDF membrane. After blocking the transferred membrane with 5% skim milk (in TBS-T) solution at room temperature for 1 hour, the primary antibody was diluted in 3% BSA and incubated overnight at 4°C. Subsequently, the reaction was carried out with an HRP-conjugated secondary antibody at room temperature for 1 hour, and protein expression was detected using an ECL detection reagent.

[0101] As a result, when cultured on GO plates, the expression of F-actin, a signaling factor regulating the mechanical properties of cells, was significantly increased in corneal endothelial cells (Fig. 7), and the phosphorylation of ERK and FAK proteins was significantly increased compared to the control group, while the phosphorylation of YAP was decreased (Fig. 8). Through this, it was confirmed that the coating of GO activates ERK and FAK proteins in corneal endothelial cells and effectively regulates cell adhesion signals through cytoskeletal reconstruction.

[0102]

[0103] 2-5. Analysis of Mitochondrial Activation

[0104] To analyze changes in the activation of YAP1 signaling involved in the regulation of cell growth and fibrosis in corneal endothelial cells (Day 4, Endo Day 19) cultured on GO plates or general culture plates in Example 2-1 above, the intracellular expression site of YAP1, mitochondrial activation, and Phalloidin expression were analyzed by immunocytochemistry. Specifically, to confirm the YAP1 protein expression and intracellular distribution and structural characteristics of mitochondria in corneal endothelial cells, the cells were fixed in 4% paraformaldehyde at room temperature for 20 minutes, and then permeated through the cell membrane by treating with a blocking buffer (5% NDS or NGS-based) containing 0.3% Triton X-100 for 1 hour. Subsequently, primary antibodies against YAP1 (1:500) and Mitochondria (MAB1237) (1:200) were applied, and the cells were incubated overnight at 4°C. After washing three times with PBS-T, the cells were incubated with antibody buffer containing fluorescently labeled Alexa Fluor 488 secondary antibody and DAPI at room temperature for 1 hour. Images were acquired using confocal microscopy while the stained cells were kept immersed in 1X PBS without mounting.

[0105] As a result, when cultured on a GO plate, it was found that YAP1 was localized to the nuclei of corneal endothelial cells and mitochondria were activated compared to the control group, and the expression of Phalloidin was increased (Fig. 9).

[0106] Through this, it was confirmed that coating a cell culture support with GO at an optimized specific number of times promotes cell growth specifically in corneal endothelial cells, activates mechanical signaling, promotes cell energy metabolism, and enhances cell structural stability and adhesion.

Claims

1. A support for corneal cell culture comprising graphene.

2. A corneal cell culture support according to claim 1, wherein at least a portion of the surface of the support is coated with graphene oxide (GO).

3. A corneal cell culture support according to claim 2, wherein the thickness of the coated graphene oxide is 10 to 60 nm.

4. A support for corneal cell culture according to claim 2, wherein the surface roughness of the coated graphene oxide is 1 to 10 nm.

5. A corneal cell culture support, wherein the corneal cells are corneal cell progenitor cells, corneal epithelial cells, corneal endothelial cells, or corneal stem cells.

6. A corneal cell culture support, wherein the corneal cells are corneal cells derived from embryonic stem cells (ESC), adult stem cells (ASC), or induced pluripotent stem cells (iPSC).

7. A corneal cell culture support comprising a material of glass, an extracellular matrix (ECM), a biodegradable polymer, or a metal, as described in claim 1.

8. A support for corneal cell culture in the form of a flask, dish, well plate, sheet, substrate, microfluidic chip, or mold according to claim 1.

9. A composition for transplantation comprising corneal cells cultured on a corneal cell culture support according to claim 1.

10. A support for corneal cell culture according to claim 1; and A graft material comprising corneal cells cultured on the above support.

11. In Clause 10, an implant material in which corneal cells are cultured on graphene oxide. 12.1) A step of ultrasonically treating the graphene oxide aqueous dispersion; 2) a step of coating a graphene oxide aqueous dispersion onto a support; and 3) A method for preparing a corneal cell culture support comprising a drying step.

13. A method according to claim 12, wherein the graphene oxide aqueous dispersion is coated 1 to 5 times.

14. A method according to claim 12, wherein the graphene oxide is coated such that its thickness is 10 to 30 nm and its surface roughness is 1 to 3 nm.

15. Use of a corneal cell culture support for use in the manufacture of an implantable composition or implant material.

16. A corneal cell transplantation method comprising the step of transplanting a transplantation composition or a transplantation material to an individual requiring corneal cell transplantation.

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