Tissue-engineered cornea, method for preparing same, and use thereof

Abstract

Disclosed in the present invention are a tissue-engineered cornea, a method for preparing same, and use thereof. The tissue-engineered cornea comprises corneal endothelial cells and a curved membrane. The curved membrane is a chitin membrane crosslinked with gelatin. The corneal endothelial cells are loaded on the surface of the curved membrane. According to the present invention, through the preparation of the chitin membrane, the autologous corneal endothelial cells differentiated from the patient are cultured on the chitin membrane to construct the tissue-engineered cornea, which is then implanted into an animal eye. It is found that the tissue-engineered cornea contributes to the recovery of corneal blindness caused by endothelial dysfunction.

Description

A tissue-engineered cornea, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a tissue-engineered cornea, its preparation method and application, and also to a cell culture medium and a method for inducing iPSC differentiation into corneal endothelial cells using it. Background Technology

[0002] The cornea is the foremost protruding part of the eyeball. The human cornea consists of five layers: the corneal epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. If the cornea becomes diseased due to infection, trauma, or genetic factors, opaque scar tissue will appear. Dense scars located in the central area that obstruct the pupil can cause vision loss in the affected eye, a condition known as corneal blindness. Numerous reports and studies have shown that corneal endothelial cells in adults do not exhibit significant proliferative activity, and their cell density gradually decreases with age. The corneal endothelium is extremely sensitive to many external factors (such as drugs, chemicals, radiation, trauma, and eye diseases). After damage to the corneal endothelium, it can only compensate for and maintain the continuity of its monolayer cells through the expansion and migration of cells around the damaged area, thus maintaining normal corneal function. However, when the density of corneal endothelial cells drops to a certain critical value after damage and cannot be compensated for, blindness will occur; this is called "corneal endothelial blindness." Corneal transplantation is currently the main treatment for restoring corneal transparency. However, allogeneic corneal transplantation has a shortage of donors and is often accompanied by immune rejection. Therefore, the treatment of corneal endothelial disease is an international challenge.

[0003] Currently, replacing damaged corneas with healthy donated corneal tissue is the primary treatment method. However, due to a global shortage of donor corneas, less than 1.5% of patients requiring corneal transplantation can receive allogeneic transplants. Recently, the successful surgical transplantation of in vitro cultured corneal endothelial cells has provided important proof of principle for developing strategies for cell therapies based on corneal endothelial cells; however, obtaining sufficient corneal endothelial cells from healthy donor tissue remains challenging. Furthermore, bio-artificial corneas offer a new solution to the global shortage of donor corneas and most of the limitations encountered in traditional corneal transplantation using donor tissue (such as rejection, surface irregularities, and quality control). However, bio-artificial cornea technology is relatively new, and many unknowns remain to be explored, such as surface coatings that prevent scarring and promote re-epithelialization, and functionalization that specifically interacts with cells, microorganisms, or molecules to better protect the cornea from collagenase damage, infection, and vascularization. Therefore, obtaining sufficient corneal endothelial cells from healthy donor tissue and the safety of bio-artificial corneal materials are two major challenges that urgently need to be addressed.

[0004] Chitin is the second most abundant organic compound in nature after cellulose, widely found in shrimp shells, crab shells, the exoskeletons of arthropods, and the cell walls of lower plants (such as fungi and algae). Recent studies have shown that chitin and its derivatives, due to their porous and hydropermeable structure, ease of chemical modification, and high affinity for macromolecules in vivo, possess great potential as supporting materials in tissue engineering and are biomaterials applicable in various tissue engineering fields. The application of chitin and its derivatives in ophthalmic diseases is still in the research and development stage and has not yet been widely used in clinical practice. Summary of the Invention

[0005] The technical problem this invention aims to solve is the scarcity of donors for allogeneic corneal transplantation, which is often accompanied by immune rejection. This invention provides a tissue-engineered cornea, its preparation method, and its applications. It also provides a cell culture medium and a method for inducing iPSC differentiation into corneal endothelial cells using this medium. The culture medium provided by this invention can:

[0006] (1) Sufficient corneal endothelial cells were obtained by extracting mononuclear cells from the patient’s peripheral blood and reprogramming them into induced pluripotent stem cells, which were then further differentiated into corneal endothelial cells.

[0007] (2) Treat corneal endothelial blindness by in situ transplantation of corneal endothelial cells differentiated from the patient's own body;

[0008] (3) Corneal endothelial cells differentiated from the patient’s own body are used to achieve corneal endothelial blindness repair through tissue-engineered cornea.

[0009] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0010] A first aspect of the present invention provides a tissue-engineered cornea, the tissue-engineered cornea comprising: corneal endothelial cells and a curvature membrane; wherein the curvature membrane is a chitin membrane cross-linked with gelatin; and the corneal endothelial cells are loaded on the surface of the curvature membrane.

[0011] In some technical solutions of the present invention, the chitosan content in the curvature membrane is 3-7%, for example 5%; the gelatin concentration is 5-8%, for example 6.25%; the potassium acetate content is 0.1-1%, for example 0.5%; the crosslinking agent content is 0.2-0.5%, for example 0.375%; the ethanol content is 4%-8%, for example 6.25%; and the radius of curvature is 5-9 mm, for example 7.8 mm; or,

[0012] The curvature membrane is prepared by the following method: chitin membrane is impregnated with gelatin and crosslinking agent solution and then dried by concave pressing.

[0013] In some technical solutions of the present invention, the chitin membrane is obtained by uniformly flattening and drying chitin gel solution.

[0014] In some preferred embodiments of the present invention, the chitin adhesive solution comprises chitin and a formic acid solution of potassium acetate; the gelatin + crosslinking agent adhesive solution is an aqueous solution containing gelatin, a crosslinking agent and ethanol.

[0015] In some preferred embodiments of the present invention, the concentration of chitosan is 3-7%, for example 5%; the concentration of potassium acetate is 0.1-1%, for example 0.5%; the concentration of gelatin is 5-8%, for example 6.25%; the concentration of the crosslinking agent is 2-6 mg / mL, for example 3.75 mg / mL; the concentration of ethanol is 4%-8%, for example 6.25%; and the curvature of the concave pressing is 5-9 mm, for example 7.8 mm.

[0016] In some technical solutions of the present invention, the method for preparing corneal endothelial cells includes: culturing iPSC cells using a cell culture medium; wherein the cell culture medium comprises corneal endothelial culture medium and N-acetylglucosamine.

[0017] In some preferred embodiments of the present invention, the components of the cell culture medium are selected from any one of the following groups:

[0018] (1) DMEM / F12, KnockOut serum substitute, non-essential amino acids, L-glutamine, 2-mercaptoethanol, basic fibroblast growth factor, recombinant human Noggin protein, SB431542 and N-acetylglucosamine;

[0019] (2) DMEM / F12, KnockOut serum substitute, non-essential amino acids, L-glutamine, 2-mercaptoethanol, basic fibroblast growth factor, B27 additive, recombinant human platelet-derived growth factor BB, recombinant human DKK-2 protein and N-acetylglucosamine.

[0020] (3) The cell culture medium contains Opti-MEM TM I-reduced serum culture medium, fetal bovine serum, calcium chloride, chondroitin sulfate, gentamicin, epidermal growth factor, ascorbic acid, rho-associated protein kinase inhibitor Y27632, and N-acetylglucosamine.

[0021] In some more preferred embodiments of the present invention, the concentrations of each component of the cell culture medium are as follows:

[0022] DMEM / F12, 60-90%, for example 80%;

[0023] KnockOut serum substitute, 10-30%, e.g., 20%;

[0024] Non-essential amino acids, 0.5-1.5%, for example, 1%;

[0025] L-glutamine, 0.5-1.5 mM, for example 1 mM;

[0026] 2-Mercaptoethanol, 0.05-0.15 mM, for example 0.1 mM;

[0027] Basic fibroblast growth factor, 6-10 ng / mL, for example 8 ng / mL;

[0028] Recombinant human Noggin protein, 300-700 ng / mL, for example 500 ng / mL;

[0029] SB431542, 5-15μM, e.g., 10μM;

[0030] N-acetylglucosamine, 50-400 μg / mL, for example 50, 100, 200 or 400 μg / mL;

[0031] Additive B27, 0.08-1.5×, for example 0.1×;

[0032] Recombinant human platelet-derived growth factor BB, 5-15 ng / mL, for example 10 ng / mL;

[0033] Recombinant human DKK-2 protein, 5-15 ng / mL, for example 10 ng / mL;

[0034] Opti-MEM TM I. Reduced serum culture medium, 80-95%, for example 92%;

[0035] Fetal bovine serum, 6-10%, for example 8%;

[0036] Calcium chloride, 100-300 mg / L, for example 200 mg / L;

[0037] Chondroitin sulfate, 0.05-0.2%, for example 0.08%;

[0038] Gentamicin, 30-70 mg / mL, for example 50 mg / mL;

[0039] Epidermal growth factor, 3-7 ng / mL, for example 5 ng / mL;

[0040] Ascorbic acid, 10-40 μg / mL, for example 20 μg / mL;

[0041] The rho-related protein kinase inhibitor Y27632, 5-15 μM, e.g., 10 μM.

[0042] In some technical solutions of the present invention, the method for preparing corneal endothelial cells includes the following steps:

[0043] (i) Culture the passaged iPSC cells in mTeSR medium for 3-6 days, for example, 4 days;

[0044] (ii) Replace with the cell culture medium as described in (1) of the first aspect of the present invention and culture for 1-3 days, for example, 2 days;

[0045] (iii) Replace with the cell culture medium described in (2) as described in the first aspect of the present invention and culture for 1-3 days, for example, 1 day;

[0046] (iv) Transfer the cells to a new matrix gel plate and culture them for 4-8 days, for example, 6 days, in a cell culture medium as described in the first aspect of the invention;

[0047] (v) Replace with the cell culture medium described in (3) as in the first aspect of the invention and culture for 5-9 days, for example, 7 days.

[0048] In some preferred embodiments of the present invention, the culture conditions are 37°C and 5% CO2.

[0049] A second aspect of the present invention provides a method for preparing a tissue-engineered cornea as described in the first aspect of the present invention, wherein the corneal endothelial cell suspension is placed on the curvature membrane and cultured at 37°C and 5% CO2.

[0050] A third aspect of the present invention provides a curvature membrane, which is the curvature membrane in the tissue-engineered cornea as described in the first aspect of the present invention.

[0051] A fourth aspect of the present invention provides a method for preparing a curvature film as described in a third aspect of the present invention, the method comprising the method described in a tissue-engineered cornea as described in a first aspect of the present invention.

[0052] The fifth aspect of the present invention provides a method for inducing iPSCs to differentiate into corneal endothelial cells, the method comprising the preparation method in tissue-engineered cornea as described in the first aspect of the present invention.

[0053] The sixth aspect of the present invention provides corneal endothelial cells prepared by the preparation method described in the first aspect of the present invention for tissue-engineered corneas.

[0054] A seventh aspect of the present invention provides a composition comprising corneal endothelial cells as described in the sixth aspect of the present invention and pharmaceutically acceptable excipients.

[0055] In some preferred embodiments of the present invention, the composition further comprises chitosan, such as carboxymethyl chitosan.

[0056] The eighth aspect of the present invention provides a cell culture medium, which is the cell culture medium for tissue-engineered corneas as described in the first aspect of the present invention.

[0057] The ninth aspect of the present invention provides the use of one or more of the following in the preparation of a medicament for treating blindness: a tissue-engineered cornea as described in the first aspect of the present invention, a curvature membrane as described in the third aspect of the present invention, corneal endothelial cells as described in the sixth aspect of the present invention, and a composition as described in the seventh aspect of the present invention.

[0058] In some preferred embodiments of the present invention, the blindness is corneal endothelial blindness.

[0059] The tenth aspect of the present invention provides a method for transplanting corneal endothelial cells, the method comprising: applying an adhesive to the surface of the posterior elastic layer of the eye of a subject in need, and then transplanting a tissue-engineered cornea as described in the first aspect of the present invention and / or corneal endothelial cells as described in the sixth aspect of the present invention.

[0060] In some preferred embodiments of the present invention, the adhesive comprises chitosan, such as carboxymethyl chitosan; the corneal endothelial cells are differentiated from autologous iPSCs.

[0061] The eleventh aspect of the present invention provides a tissue-engineered cornea as described in the first aspect of the present invention, a curvature membrane as described in the third aspect of the present invention, corneal endothelial cells as described in the sixth aspect of the present invention, or a composition as described in the seventh aspect of the present invention for treating blindness.

[0062] In some preferred embodiments of the present invention, the blindness is corneal endothelial blindness.

[0063] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0064] The reagents and raw materials used in this invention are all commercially available.

[0065] The positive and progressive effects of this invention are as follows:

[0066] (1) In the process of differentiation of corneal endothelial cells derived from patient's autologous iPSC, the addition of N-acetylglucosamine to the culture medium can better promote cell adhesion and proliferation.

[0067] (2) In the process of in situ cell transplantation for the treatment of corneal endothelial blindness, the present invention adds an adhesive (carboxymethyl chitosan). Animal experiments have shown that the cells can better adhere to the Descemet's membrane and form a monolayer, which helps the recovery of corneal endothelial blindness.

[0068] (3) This invention prepares a chitin membrane, culturees corneal endothelial cells differentiated from the patient's own body on the chitin membrane, constructs a tissue-engineered cornea, and implants it into the eyes of animals, and finds that it helps to restore corneal endothelial blindness. Attached Figure Description

[0069] Figure 1 shows the CCK8 detection results of corneal endothelial cells cultured with N-acetylglucosamine, chitosan oligosaccharide, and glucosamine hydrochloride added to the corneal endothelial culture medium, respectively.

[0070] Figure 2 shows the CCK8 detection results of corneal endothelial cells cultured with different concentrations of N-acetylglucosamine in the corneal endothelial cell culture medium.

[0071] Figure 3 shows the reprogramming of mononuclear cells extracted from the patient's peripheral blood into iPSCs; where A represents the morphological changes of cells during the reprogramming of peripheral blood into iPSCs; B represents the immunofluorescence staining results of OCT4, Nanog, TRA-1-81, and SSEA4; and C represents the hematoxylin-eosin staining results of teratomas.

[0072] Figure 4 shows the differentiation of patient-derived iPSCs into CECs; where A represents the morphological changes of cells during the differentiation of iPSCs into corneal endothelial cells; B represents the immunofluorescence staining results of first-generation corneal endothelial cells ZO-1; and C represents the immunofluorescence staining results of seventh-generation corneal endothelial cells ZO-1.

[0073] Figure 5 shows bright field images on day 1 and day 11 of corneal endothelial cell orthotopic transplantation for corneal endothelial blindness.

[0074] Figure 6 shows the treatment of corneal endothelial blindness by in situ transplantation of corneal endothelial cells; where A is the result of slit-lamp microscopy; B is the result of analysis and measurement of corneal thickness using optical coherence tomography (OCT); and C is the result of observation of corneal endothelial cell density using corneal endothelial microscopy.

[0075] Figure 7 shows bright field images of corneal endothelial cells loaded with chitosan sheets and chitosan + gelatin sheets on day 1 and day 7.

[0076] Figure 8 shows the treatment of corneal endothelial blindness using tissue-engineered cornea; where A is a chitosan + gelatin curvature film; B is bright-field images of corneal endothelial cells loaded on the chitosan + gelatin curvature film on day 1 and day 14; C is bright-field images on day 1, day 31, and day 66 after tissue-engineered cornea implantation; and D is OCT results on day 31 and day 66 after tissue-engineered cornea implantation. Detailed Implementation

[0077] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0078] The reagents and animals used in this invention are shown in Table 1.

[0079] Table 1. List of Reagents and Animals

[0080] Example 1

[0081] Corneal endothelial cells were cultured by adding N-acetylglucosamine, chitosan oligosaccharide, and glucosamine hydrochloride to the corneal endothelial culture medium, respectively.

[0082] (1) Adjust the corneal endothelial cell density to 9×10 3 Prepare a 96-well plate with cells / mL, add 100 μl of cell suspension to each well, set 5 replicates per group, and incubate in an incubator for 12 h.

[0083] (2) The corneal culture medium containing N-acetylglucosamine, chitosan oligosaccharide and glucosamine hydrochloride was replaced according to the group and incubated in an incubator for 48 hours.

[0084] (3) Add 10 μl of CCK-8 solution to each well, mix well, and then place the culture plate in an incubator for 2 hours.

[0085] (4) The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader. Cell viability was then assessed using a CCK8 assay.

[0086] The results, as shown in Figure 1, indicate that N-acetylglucosamine can better promote cell proliferation.

[0087] Example 2

[0088] Corneal endothelial cells were cultured by adding different concentrations of N-acetylglucosamine to the culture medium.

[0089] (1) Adjust the corneal endothelial cell density to 9×10 3Prepare a 96-well plate with cells / mL, add 100 μL of cell suspension to each well, set 5 replicates per group, and incubate in an incubator for 12 h.

[0090] (2) The corneal culture medium containing different concentrations of N-acetylglucosamine was replaced according to the group and incubated in an incubator for 48 hours.

[0091] (3) Add 10 μL of CCK-8 solution to each well, mix well, and then place the culture plate in an incubator for 2 hours.

[0092] (4) The absorbance (OD value) of each well was measured at a wavelength of 450 nm using an ELISA reader. Cell viability was then assessed using a CCK8 assay.

[0093] The results, as shown in Figure 2, indicate that N-acetylglucosamine at a concentration of 100 μg / mL can better promote cell proliferation.

[0094] Example 3

[0095] 1. Mononuclear cells extracted from the patient's peripheral blood were reprogrammed into induced pluripotent stem cells (iPSCs).

[0096] First, mononuclear cells were extracted from the patient's peripheral blood. Stem cell-related transcription factors (Oct4, Sox2, Klf4, and N-Myc) were introduced into the mononuclear cells using electroporation (1650V, 10ms, 3 pulls). After a period of culture and incubation, the electroporated cells were added to each well of a 6-well plate at a concentration of 3.3 × 10⁻⁶ cells / well. 5 Cells were seeded and cultured in a 37°C, 5% CO2 incubator (day 0 was the day of electroporation). On day 2, 1 mL of erythrocyte expansion medium was added to the electroporated cells, and cultured for another day. On day 3, 1 mL of reprogramming medium was added. On day 5, 1 mL of reprogramming medium was added again. On day 7, the original medium was discarded, and 2 mL of fresh reprogramming medium was added. From day 8 to day 25, the reprogramming medium was changed daily, and the cells were observed until iPSCs appeared.

[0097] 2. Differentiate patient-derived iPSCs into corneal endothelial cells (CECs).

[0098] On day 0, iPSCs at a cell density of 80% were passaged at a ratio of 1:12 and then cultured in mTeSR medium for 4 days. On day 4, the medium was replaced with a dual Smad inhibitor medium (80% DMEM / F12, 20% KnockOut serum substitute, 1% non-essential amino acids, 1mM L-glutamine, 0.1mM 2-mercaptoethanol, 8ng / mL basic fibroblast growth factor, 500ng / mL recombinant human Noggin protein, 10μM SB431542, 100μg / mL N-acetylglucosamine). On day 6, the culture medium was replaced with corneal medium (80% DMEM / F12, 20% KnockOut serum substitute, 1% non-essential amino acids, 1 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 8 ng / mL basic fibroblast growth factor, 0.1×B27 supplement, 10 ng / mL recombinant human platelet-derived growth factor BB, 10 ng / mL recombinant human DKK-2 protein, 100 μg / mL N-acetylglucosamine). On day 7, the differentiated CECs were transferred to new stromal plates and cultured in corneal medium for 6 days. On day 13, the medium was replaced with CY medium (92% Opti-MEM). TM The cells were cultured in a medium containing 8% fetal bovine serum, 200 mg / L calcium chloride, 0.08% chondroitin sulfate, 50 mg / mL gentamicin, 5 ng / mL epidermal growth factor, 20 μg / mL ascorbic acid, 10 μM rho-associated protein kinase (ROCK) inhibitor Y27632, and 100 μg / mL N-acetylglucosamine for 7 days at 37°C in an incubator containing 5% CO2, with the CY medium replaced every other day. The resulting cells are corneal endothelial cells.

[0099] 3. Verify whether the reprogrammed iPSCs maintain an undifferentiated stem cell state.

[0100] (1) The expression of pluripotency markers OCT4 and Nanog and cell surface proteins TRA-1-81 and SSEA4 was observed by immunofluorescence staining.

[0101] (i) Remove the culture medium from the cultured iPSC cells, wash the cells twice with an appropriate amount of PBS buffer, add 4% paraformaldehyde fixative, and incubate at room temperature for 10 min.

[0102] (ii) Discard the 4% paraformaldehyde fixative, rinse 3 times with PBS buffer for 5 min each time, discard the PBS buffer, add permeation blocking buffer (5% BSA and 0.5% Triton X-100), and incubate at room temperature for 1 h.

[0103] (iii) Discard the permeation blocking solution, add the primary antibody incubation solution, and incubate overnight at 4°C.

[0104] (iv) After the primary antibody incubation is complete, rinse three times with PBS for 10 minutes each time, discard the PBS, add the secondary antibody incubation solution, and incubate at room temperature in the dark for 1 hour.

[0105] (v) After the secondary antibody incubation is complete, rinse three times with PBS for 10 minutes each time, discard the PBS, add DAPI staining solution and stain for 10 minutes, then rinse three times with PBST for 5 minutes each time, and finally observe and photograph with a fluorescence microscope.

[0106] (2) Teratoma experiment:

[0107] 1×10 6 Cells were resuspended in a mixture of DMEM, matrix gel, and collagen (in a 2:1:1 ratio) and injected intramuscularly into immunocompromised nude mice, where teratomas formed within 4 to 6 weeks. The teratomas were then embedded in paraffin, sectioned, and identified by hematoxylin and eosin staining.

[0108] 4. Orthotopic transplantation of corneal endothelial cells for the treatment of corneal endothelial blindness

[0109] (1) Establishing a canine corneal endothelial blindness animal model

[0110] The dog's legs were shaved beforehand to expose the veins. The dog was anesthetized with isoflurane using an anesthesia machine. The dog's left eye was disinfected from the inside out with povidone-iodine. The eye was dilated with an eyelid speculum. A 1.6mm incision was made at the corneal limbus, and viscoelastic was injected. Endothelial hooks were used to remove corneal endothelial cells on the posterior elastic lamina, about 6mm in diameter. The removed corneal endothelial cells were rinsed out with saline solution, and excess water was gently squeezed out.

[0111] (2) Experimental treatment

[0112] The normal group consisted of normal dogs, while the other groups consisted of canine corneal endothelial blindness animal models, treated according to the following groupings:

[0113] Control group: Injected with Opti-MEM TM I. Serum-reduced culture medium;

[0114] Group without adhesive: injected with corneal endothelial cell suspension (containing Y27632 100μM);

[0115] Experimental group (with adhesive): Carboxymethyl chitosan adhesive was applied first, followed by injection of corneal endothelial cell suspension (containing Y27632 100μM).

[0116] After treatment, apply tobramycin and dexamethasone eye ointment, and administer a dexmedetomidine hydrochloride injection (10 μg / mL / kg) intravenously to the puppy's leg. Place the puppy on a fixed board and keep it in a prone position with its eyes facing down.

[0117] Experimental results

[0118] 1. Mononuclear cells extracted from the patient's peripheral blood were reprogrammed into iPSCs.

[0119] First, peripheral blood was collected from patients, and erythroid progenitor cells were enriched and expanded. Then, Oct4, Sox2, Klf4, and N-Myc factors were electroporated. After a period of culture, clonal cell clusters were observed, with a high nucleocytoplasmic ratio and tightly packed central portions (Figure 3A). To verify whether the reprogrammed iPSCs maintained an undifferentiated stem cell state, the expression of pluripotency markers OCT4 and Nanog, and cell surface proteins TRA-1-81 and SSEA4 was observed using immunofluorescence staining. The results showed that the reprogrammed iPSCs expressed OCT4, Nanog, TRA-1-81, and SSEA4 (Figure 3B). To further verify the differentiation capacity of iPSCs, teratoma experiments revealed that iPSCs had the ability to differentiate into ectoderm, mesoderm, and endoderm (Figure 3C). These results demonstrate the successful preparation of iPSCs from patient peripheral blood.

[0120] 2. Differentiate patient-derived iPSCs into corneal endothelial cells (CECs).

[0121] After passaged iPSCs at a 1:12 ratio, the medium was replaced with a double Smad inhibitor medium on day 4 and corneal medium on day 6. Morphological observation on day 13 revealed polygonal cell morphology, and on day 20, the cells were found to be tightly packed, exhibiting hexagonal or polygonal shapes (Figure 4A). During iPSC differentiation into CECs and passage, the addition of 100 μg / mL N-acetylglucosamine promoted cell adhesion and proliferation. Immunofluorescence staining was used to detect the expression of the corneal endothelial cell marker ZO-1 in iPSC-derived CECs, revealing ZO-1 expression at cell boundaries, indicating tight junctions between CECs (Figure 4B). Further passage of CECs with the addition of 100 μg / mL N-acetylglucosamine in the medium resulted in continued ZO-1 expression in the CECs at passage 7 (Figure 4C). These results demonstrate the successful preparation of autologous corneal endothelial seed cells.

[0122] 3. Orthotopic transplantation of corneal endothelial cells for the treatment of corneal endothelial blindness

[0123] As shown in Figure 5, on postoperative day 1, all three groups showed significant corneal edema and opacity. On postoperative day 11, the control group still exhibited corneal edema and opacity; in the group without adhesive, only the corneal periphery began to become transparent, while the central portion remained mostly opaque; in the group with adhesive, most of the cornea had recovered transparency. These results indicate that in the treatment of corneal endothelial blindness, the addition of carboxymethyl chitosan as an adhesive can improve cell adhesion to the Descemet's membrane and contribute to the recovery of corneal endothelial blindness.

[0124] An animal model of corneal endothelial blindness was constructed using dogs. In the experimental group, carboxymethyl chitosan was applied as an adhesive, followed by injection of cell suspension, and the group was kept in a prone position. Slit-lamp microscopy revealed that 30 days after corneal endothelial tearing, the control group still exhibited corneal edema, thickening, and opacity, while most of the cornea in the experimental group had become transparent. At 57 days post-surgery, the control group's cornea remained opaque, while the experimental group's cornea had fully recovered transparency and achieved the same thickness as the normal group (Figure 6A). OCT observation of the cornea showed significant scar hyperplasia at the central corneal endothelial tearing site in the control group at 30 days post-surgery, while a small portion of the central corneal endothelial tearing area in the experimental group also showed scar hyperplasia, but the thickness was significantly reduced compared to the control group. At 57 days post-surgery, the central corneal endothelial tearing area in the experimental group was smooth, with no obvious hyperplastic scarring, and the corneal thickness was consistent with the normal group (Figure 6B). Microscopic observation of the corneal endothelium showed that 30 days after corneal endothelial exfoliation, the corneal endothelial cell density in the experimental group recovered to 3115 cells / mm², consistent with the normal group (Figure 6C). These results demonstrate that the addition of adhesives and in situ cell transplantation therapy can rapidly adhere corneal endothelial cells, forming a monolayer, promoting the recovery of corneal transparency and thickness, and facilitating the restoration of corneal endothelial cell density and functional reconstruction.

[0125] Example 4

[0126] 1. Mononuclear cells extracted from the patient's peripheral blood were reprogrammed into induced pluripotent stem cells (iPSCs).

[0127] First, mononuclear cells are extracted from the patient's peripheral blood. Then, stem cell-related transcription factors (Oct4, Sox2, Klf4, and N-Myc) are introduced into the mononuclear cells using electroporation. After a period of culture and cultivation, the transcription factors begin to reprogram the mononuclear cells, causing them to transform into induced pluripotent stem cells.

[0128] 2. Differentiate patient-derived iPSCs into corneal endothelial cells (CECs).

[0129] On day 0, iPSCs at a cell density of 80% were passaged at a ratio of 1:12 and then cultured in mTeSR medium for 4 days. On day 4, the medium was replaced with a dual Smad inhibitor medium (80% DMEM / F12, 20% KnockOut serum substitute, 1% non-essential amino acids, 1mM L-glutamine, 0.1mM 2-mercaptoethanol, 8ng / mL basic fibroblast growth factor, 500ng / mL recombinant human Noggin protein, 10μM SB431542, 100μg / mL N-acetylglucosamine). On day 6, the culture medium was replaced with corneal medium (80% DMEM / F12, 20% KnockOut serum substitute, 1% non-essential amino acids, 1 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 8 ng / mL basic fibroblast growth factor, 0.1×B27 supplement, 10 ng / mL recombinant human platelet-derived growth factor BB, 10 ng / mL recombinant human DKK-2 protein, 100 μg / mL N-acetylglucosamine). On day 7, the differentiated CECs were transferred to new stromal plates and cultured in corneal medium for 6 days. On day 13, the medium was replaced with CY medium (92% Opti-MEM). TM The cells were cultured for 7 days in a medium containing 8% fetal bovine serum, 200 mg / L calcium chloride, 0.08% chondroitin sulfate, 50 mg / mL gentamicin, 5 ng / mL epidermal growth factor, 20 μg / mL ascorbic acid, 10 μM rho-associated protein kinase (ROCK) inhibitor Y27632, and 100 μg / mL N-acetylglucosamine. The resulting cells were corneal endothelial cells.

[0130] 3. Preparation and aseptic treatment of chitosan curvature membranes and chitosan + gelatin curvature membranes.

[0131] (1) Preparation and sterilization of chitin curvature membranes: 0.5g of purified chitin product was weighed and thoroughly mixed with 10mL of formic acid and 0.04g of potassium chloride to obtain a 5% chitin gel solution. The gel solution was cast onto a stainless steel concave-convex curvature plate using a casting method. The concave plate was air-dried overnight to form a dried membrane. The concave-convex plates were combined and the membrane was further cross-linked at 40℃ for 48h. The concave plate was opened and immersed in physiological saline to separate the membrane from the plate. A 1.2cm diameter membrane was drilled using a trephine drill, washed three times with physiological saline, and then immersed in 75% ethanol. The membrane was then dispensed into sterile bottles, immersed in 75% ethanol, and stored at 4℃ for later use.

[0132] (2) Preparation and aseptic treatment of chitosan + gelatin curvature film:

[0133] (1) Weigh 0.5g of purified chitin product and mix it thoroughly with 10mL of formic acid and 0.05g of potassium acetate to obtain a 5% chitin gel solution. Pour 10mL of the gel solution onto a clean glass plate (10cm*10cm), spread the gel solution evenly, and dry it in a fume hood to form a film. Place the carrier glass plate into a 0.2mol / L NaOH solution to neutralize the acid and remove the film. Wash the film thoroughly in sterile distilled water until neutral, and dry the film on the glass plate to obtain the chitin film.

[0134] (2) Preparation of gelatin solution: Add 500mg gelatin to 8mL water, heat in a water bath to form a gel solution, add 30mg crosslinking agent and 0.5mL anhydrous ethanol solution.

[0135] (3) Take out the prepared chitin membrane, place it on sterile filter paper, absorb the liquid, dry the membrane, and keep it in shape.

[0136] (4) Use tweezers to pick up the membrane and immerse it in the gelatin solution (prepared in step (2)) to make the chitosan membrane flat and smooth.

[0137] (5) Carefully place the membrane into the concave pressing device, adjust it to the center and keep it flat. Operate as quickly as possible to prevent moisture evaporation and cause the membrane to become uneven and curled.

[0138] (6) Pressing, maintaining a temperature of approximately 45°C for 30-60 seconds. (The appropriate conditions should be determined experimentally based on the actual conditions required to form the concave curvature of the pressed diaphragm.)

[0139] (7) Decompress and remove the membrane. If the membrane is too tightly attached to the pressing surface, you can apply water (or 50% ethanol aqueous solution) to the syringe needle and remove the pressed membrane.

[0140] (8) Place the fully formed curved diaphragm under sealed conditions and dry it at 40-45℃ for 24 hours.

[0141] (9) Wash the membrane three times with physiological saline, then soak it in 75% ethanol, then dispense the membrane into sterile bottles, soak it in 75% ethanol and store it at 4°C for later use.

[0142] 4. Chitosan curvature membranes and chitosan + gelatin curvature membranes were loaded with corneal endothelial cells, respectively.

[0143] The prepared curvature mold was placed in a 24-well plate, and then chitin curvature membranes and chitin + gelatin curvature membranes were placed on the molds respectively. Collagen was added and incubated at room temperature for 1 hour. The corneal endothelial cells were digested with trypsin and resuspended in CY medium. 200 μL of cell suspension was placed on each curvature membrane and incubated in a 37°C, 5% CO2 incubator. Cell adhesion was observed on the second day.

[0144] 5. Tissue-engineered corneas for corneal endothelial blindness repair

[0145] (1) Establishing a canine corneal endothelial blindness animal model

[0146] The dog was anesthetized with isoflurane using an anesthesia machine. The left eye was disinfected from the inside out with povidone-iodine, a drape was applied, and the eye was dilated with an eyelid speculum. A 1.6mm incision was made at the corneal limbus, and viscoelastic was injected. Endothelial hooks were used to remove corneal endothelial cells on the posterior elastic lamina, with a diameter of about 6mm. The scraped corneal endothelial cells were then rinsed out with saline.

[0147] (2) Experimental treatment

[0148] Process according to the following groups:

[0149] Control group: Chitosan + gelatin curvature membrane implanted;

[0150] Experimental group: First, apply 10 μL of adhesive, then implant a chitin + gelatin curvature membrane loaded with corneal endothelial cells.

[0151] After the procedure, the surgical incision is sutured, the membrane is unfolded, air bubbles are injected to support the curvature of the membrane and its integration with the posterior elastic layer, and tobramycin-dexamethasone eye ointment is applied.

[0152] Experimental results

[0153] 1. Corneal endothelial cells were loaded onto chitosan curvature films and chitosan + gelatin curvature films, respectively.

[0154] As shown in Figure 7, compared with the chitin curvature membrane, the chitin + gelatin curvature membrane can better load cells and form a dense monolayer of cells. Even after culturing for 7 days, the chitin + gelatin curvature membrane can still stably load corneal endothelial cells, demonstrating good biocompatibility.

[0155] For corneal endothelial cell carriers, the transparency of the membrane is crucial. Chitosan solution was used to prepare a uniform and transparent chitosan + gelatin curvature scaffold material through a casting process and curvature treatment (Figure 8A). Next, cultured corneal endothelial cells were seeded onto the chitosan + gelatin curvature membrane. On day 1 post-seedling, an inverted microscope was used to observe the corneal endothelial cells adhering to the membrane and forming a dense monolayer. This was continued until day 14, when microscopic observation revealed that the corneal endothelial cells remained tightly adhered to the membrane (Figure 8B). These results demonstrate that the chitosan + gelatin curvature membrane has good biocompatibility with corneal endothelial cells.

[0156] 2. Tissue-engineered cornea for the treatment of corneal endothelial blindness

[0157] An animal model of corneal endothelial blindness was constructed using dogs. In the experimental group, carboxymethyl chitosan was applied as an adhesive, followed by the implantation of a chitin-gelatin curvature membrane loaded with corneal endothelial cells. On postoperative day 31, bright-field observation showed that the implanted curvature membrane in the experimental group was stably degrading, and on day 66, the cornea gradually regained transparency (Figure 8, C). OCT observation of the cornea showed that the corneal thickness in the experimental group returned to normal on day 66 (Figure 8, D). These results indicate that tissue-engineered corneas are beneficial for the recovery of corneal endothelial blindness.

[0158] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A tissue engineered cornea, characterized in that, The tissue-engineered cornea comprises: corneal endothelial cells and a curvature membrane; wherein the curvature membrane is a chitin membrane cross-linked with gelatin; and the corneal endothelial cells are loaded on the surface of the curvature membrane.

2. The tissue engineered cornea of claim 1, wherein, The curvature membrane contains 3-7% chitosan, 5-8% gelatin, 0.1-1% potassium acetate, 0.2-0.5% crosslinking agent, 4%-8% ethanol, and has a radius of curvature of 5-9 mm; or... The curvature membrane is prepared by the following method: chitin membrane is impregnated with gelatin and crosslinking agent solution and then dried by concave pressing.

3. The tissue engineered cornea of claim 2, wherein, In the method, the chitin membrane is obtained by uniformly flattening and drying chitin gel solution; Preferably, in the method, the chitosan adhesive solution comprises chitosan and a formic acid solution of potassium acetate; the gelatin + crosslinking agent adhesive solution is an aqueous solution containing gelatin, a crosslinking agent, and ethanol. More preferably, in the method, the concentration of chitin is 3-7%; the concentration of potassium acetate is 0.1-1%; the concentration of gelatin is 5-8%; the concentration of the crosslinking agent is 2-6 mg / mL; the concentration of ethanol is 4%-8%; and the curvature of the concave pressing is 5-9 mm.

4. The tissue-engineered cornea according to any one of claims 1-3, characterized in that, The method for preparing the corneal endothelial cells includes: culturing iPSC cells using a cell culture medium; wherein the cell culture medium comprises corneal endothelial culture medium and N-acetylglucosamine; Preferably, the components of the cell culture medium are selected from any one of the following groups: (1) DMEM / F12, KnockOut serum substitute, non-essential amino acids, L-glutamine, 2-mercaptoethanol, basic fibroblast growth factor, recombinant human Noggin protein, SB431542 and N-acetylglucosamine; (2) DMEM / F12, KnockOut serum substitute, non-essential amino acids, L-glutamine, 2-mercaptoethanol, basic fibroblast growth factor, B27 additive, recombinant human platelet-derived growth factor BB, recombinant human DKK-2 protein and N-acetylglucosamine. (3) The cell culture medium contains Opti-MEM TM I-reduced serum culture medium, fetal bovine serum, calcium chloride, chondroitin sulfate, gentamicin, epidermal growth factor, ascorbic acid, rho-associated protein kinase inhibitor Y27632, and N-acetylglucosamine. More preferably, the concentrations of each component of the cell culture medium are as follows: DMEM / F12, 60-90%; KnockOut serum substitute, 10-30%; Non-essential amino acids, 0.5-1.5%; L-Glutamine, 0.5-1.5 mM; 2-Mercaptoethanol, 0.05-0.15 mM; Basic fibroblast growth factor, 6-10 ng / mL; Recombinant human Noggin protein, 300-700 ng / mL; SB431542, 5-15μM; N-acetylglucosamine, 50-400 μg / mL; Additive B27, 0.08-1.5×; Recombinant human platelet-derived growth factor BB, 5-15 ng / mL; Recombinant human DKK-2 protein, 5-15 ng / mL; Opti-MEM TM I serum-free medium, 80-95% Fetal bovine serum, 6-10%; Calcium chloride, 100-300 mg / L; Chondroitin sulfate, 0.05-0.2%; Gentamicin, 30-70 mg / mL; Epidermal growth factor, 3-7 ng / mL; Ascorbic acid, 10-40 μg / mL; rho-related protein kinase inhibitor Y27632, 5-15 μM.

5. The tissue engineered cornea of claim 4, wherein the collagen matrix is derived from a human cornea. The method for preparing the corneal endothelial cells includes the following steps: (i) The passaged iPSC cells were cultured in mTeSR medium for 3-6 days; (ii) Replace with the cell culture medium as described in claim 4, and culture for 1-3 days; (iii) Replace with the cell culture medium as described in claim 4, and culture for 1-3 days; (iv) Transfer the cells to a new substrate gel plate and culture them for 4-8 days in the cell culture medium as described in claim 4, in addition to (2) of the cell culture medium. (v) Replace with the cell culture medium as described in claim 4, and culture for 5-9 days; Preferably, the culture conditions are 37°C and 5% CO2.

6. A method of preparing a tissue engineered cornea according to any one of claims 1 to 5, characterized in that, The corneal endothelial cell suspension was placed on the curved membrane and cultured at 37°C with 5% CO2.

7. A curvature membrane characterized by, The curvature membrane is the curvature membrane in the tissue-engineered cornea as described in any one of claims 1-3.

8. A method of making a curved membrane as claimed in claim 7, characterised in that, The method includes the method in tissue-engineered cornea as described in claim 2 or 3.

9. A method of inducing differentiation of iPSCs into corneal endothelial cells, characterized by, The method includes the preparation method of tissue-engineered cornea as described in claim 4 or 5.

10. A corneal endothelial cell, characterized by, The corneal endothelial cells are prepared by the preparation method described in claim 4 or 5 for tissue-engineered corneas.

11. A composition characterized in that, The composition comprises the corneal endothelial cells as described in claim 10 and pharmaceutically acceptable excipients; Preferably, the composition further comprises chitosan, such as carboxymethyl chitosan.

12. A cell culture medium, characterized in that, The cell culture medium is the cell culture medium used in the tissue-engineered cornea as described in claim 4.

13. The use of one or more of the following in the preparation of a medicament for treating blindness: the tissue-engineered cornea as described in any one of claims 1-5, the curvature membrane as described in claim 7, the corneal endothelial cells as described in claim 10, and the composition as described in claim 11; Preferably, the blindness is corneal endothelial blindness.

14. A method for transplanting corneal endothelial cells, characterized by, The transplantation method includes: applying an adhesive to the surface of the posterior elastic layer of the eye of a subject in need, and then transplanting the tissue-engineered cornea as described in any one of claims 1-5 and / or the corneal endothelial cells as described in claim 10; Preferably, the adhesive comprises chitosan, such as carboxymethyl chitosan; the corneal endothelial cells are differentiated from autologous iPSCs.

15. A tissue-engineered cornea as described in any one of claims 1-5, a curvature membrane as described in claim 7, corneal endothelial cells as described in claim 10, or a composition as described in claim 11 for treating blindness; Preferably, the blindness is corneal endothelial blindness.

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