Method for constructing tissue-engineered cornea based on human amniotic epithelial stem cells and application thereof

Abstract

The application provides a method for constructing a tissue-engineered cornea based on human amniotic epithelial stem cells and application thereof, and belongs to the technical field of biotechnology. The method comprises the following steps: 1) culturing human amniotic epithelial stem cells in a corneal epithelial cell induction and differentiation culture medium to obtain human corneal epithelial cell-like cells; culturing the human amniotic epithelial stem cells in a corneal stromal cell induction and differentiation culture medium to obtain corneal stromal cells; 2) inoculating the human corneal epithelial cell-like cells and the corneal stromal cells obtained in step 1) on the surface and inside of a decellularized corneal stromal scaffold respectively, and co-culturing for 5 days to construct a tissue-engineered cornea. The application successfully constructs a tissue-engineered cornea by inoculating two kinds of corneal cells derived from human amniotic epithelial stem cells on a human decellularized corneal stromal at the same time for the first time. In addition, the obtained corneal cells can be inoculated on various types of biomaterials such as hydrogels, thereby providing a wider application possibility for developing various types of artificial cornea products.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method and application for constructing tissue-engineered corneas based on human amniotic epithelial stem cells. Background Technology

[0002] The cornea, as the outermost structure of the eyeball, is characterized by high light transmittance and avascularity, serving as a crucial refractive medium and an important barrier protecting the internal structures of the eye. From the outside in, the cornea is mainly composed of the corneal epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Several known corneal diseases, such as limbal stem cell deficiency, corneal burns, or keratoconus, cause visual impairment by damaging the corneal structure, eventually progressing to corneal blindness.

[0003] Benefiting from the rapid development of novel biomaterials research in the field of regenerative medicine, researchers are attempting to construct tissue-engineered corneas to alleviate the severe shortage of corneal donors. Tissue-engineered corneas mainly consist of two components: biomaterials and seed cells. Various biomaterials, such as decellularized corneas and various types of hydrogels, have been proven to have good biocompatibility and are suitable as biological cell scaffolds. In contrast, the insufficient source of seed cells is the main obstacle to constructing tissue-engineered corneas. Researchers generally use stem cells directly as seed cells, or cells induced from stem cell differentiation as seed cells. Commonly used stem cells include human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). However, for these two types of cells, the ethical issues and potential tumorigenicity of hESCs, as well as the genetic instability of iPSCs, cannot be ignored, severely limiting the clinical translation and application of these two types of stem cells. Summary of the Invention

[0004] To overcome the limitations of stem cells and the shortcomings of differentiation methods mentioned above, this invention provides a method and application for constructing tissue-engineered corneas based on human amniotic epithelial stem cells. For the first time, two types of corneal cells derived from human amniotic epithelial stem cells (hAESCs) are simultaneously seeded into the human decellularized corneal stroma to construct tissue-engineered corneas and alleviate the current shortage of corneal donors.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for constructing tissue-engineered corneas based on human amniotic epithelial stem cells, comprising:

[0007] 1) Human amniotic epithelial stem cells were cultured in corneal epithelial cell induction differentiation medium to obtain human corneal epithelial cell-like cells; human amniotic epithelial stem cells were cultured in corneal stromal cell induction differentiation medium to obtain corneal stromal cells;

[0008] 2) The human corneal epithelial cell-like cells and corneal stromal cells obtained in step 1) were seeded on the surface and inside of the decellularized corneal stromal scaffold, respectively, and cultured for 5 days to construct a tissue-engineered cornea.

[0009] Preferably, in step 1), human amniotic epithelial stem cells are added to corneal epithelial cell induction differentiation medium and cultured for 5-20 days, with the medium being changed every two days to obtain human corneal epithelial cell-like cells; the corneal epithelial cell induction differentiation medium is CnT-PR-D epithelial cell differentiation medium containing 5-50 ng / mL keratinocyte growth factor (KGF), 0.1-5 μM retinoic acid and 1× double antibiotic.

[0010] Preferably, in step 1), the human amniotic epithelial stem cells are P0-P2 generation human amniotic epithelial stem cells.

[0011] Preferably, in step 1), human amniotic epithelial stem cells are cultured in corneal stromal cell induction differentiation medium for 3-12 days to obtain corneal stromal cells; the corneal stromal cell induction differentiation medium is DMEM / F12 medium containing 1% insulin-transferrin-selenium (ITS), 2mM L-glutamine, 1mM non-essential amino acids, 1mM sodium pyruvate, 0.05-2mM ascorbic acid 2-phosphate, 5-20ng / mL basic fibroblast growth factor, 0.1-10μM retinoic acid and 1× double antibiotic.

[0012] Preferably, in step 2), the decellularized corneal stromal scaffold is prepared by the following method: collecting the waste corneal-scleral limbus tissue remaining after human corneal transplantation, cutting off the sclera part with a blade, and treating it with 1.5M hypertonic sodium chloride solution for decellularization to obtain the decellularized corneal stromal scaffold.

[0013] Preferably, in step 2), the method for seeding human corneal epithelial cell-like cells onto the surface of a decellularized corneal stromal scaffold is as follows: Take the human corneal epithelial cell-like cells obtained in step 1), digest them, and prepare them into a concentration of 1×10⁻⁶. 6 A single-cell suspension of 200 μL per cell was dropped onto the surface of a decellularized corneal stromal scaffold. After standing, human corneal epithelial-like cells naturally settled and adhered to the surface of the decellularized cornea.

[0014] Preferably, in step 2), the method for seeding corneal stromal cells into the interior of a decellularized corneal stromal scaffold is as follows: the corneal stromal cells are digested and prepared into a concentration of 1×10⁻⁶. 6 A single-cell suspension of 200 μL per cell was injected into the interior of a decellularized corneal stromal scaffold using an insulin needle.

[0015] Preferably, in step 2), the culture medium used for co-culture is a 1:1 mixture of corneal epithelial cell induction differentiation medium and corneal stromal cell induction differentiation medium, and the culture environment is a cell culture incubator containing 5.5% CO2 at 37°C, with the medium changed once every 2 days.

[0016] The present invention also provides a tissue-engineered cornea, which is constructed by any of the methods described above.

[0017] The present invention further provides the application of the above-described tissue-engineered cornea in the preparation of products for treating and / or improving corneal blindness. Corneal blindness includes all types of visual impairment caused by damage to the corneal structure, such as infectious keratitis, traumatic corneal injury, corneal dystrophy, corneal degeneration, congenital corneal abnormalities, autoimmune disease-related keratitis, and limbal stem cell deficiency.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] (1) This invention employs an in vitro differentiation system for human corneal epithelial cells that is free of animal serum, has a clearly defined chemical composition, and does not rely on the microenvironment of animal corneal tissue. The system consists of CnT-PR-D epithelial cell differentiation medium containing keratinocyte growth factor and retinoic acid. Human amniotic epithelial stem cells derived from discarded placenta are induced to differentiate into human corneal epithelial cell-like cells, achieving the reuse of waste biological resources. The technology is highly reproducible, the system is stable, and it is closer to clinical translation and application. The induced human corneal epithelial cell-like cells, in addition to expressing classic corneal epithelial markers, also possess good barrier function, successfully obtaining the cell phenotype and function of human corneal epithelium. It is worth mentioning that previous corneal cell differentiation systems typically required the microenvironment provided by animal corneal tissue to achieve cell differentiation. This invention overcomes this technical limitation, obtaining differentiated corneal cells without seeding hAESCs into corneal tissue. The differentiated corneal cells can be seeded onto various types of biomaterials such as corneal stromal scaffolds or hydrogels, thus providing a wider range of application possibilities for developing various novel artificial corneal products.

[0020] (2) Considering that some corneal donors and discarded corneal transplant scraps lose cell viability due to long-term or improper preservation, these tissues can be used as cell scaffolds for tissue-engineered corneas after decellularization. This invention is the first to simultaneously seed two types of corneal cells derived from hAESCs into the decellularized corneal stroma, constructing tissue-engineered corneas through an innovative in vitro co-culture system. This can replace corneal donors for the treatment of corneal blindness, providing a new approach to alleviating the shortage of corneal donors and showing promise for clinical translation. In addition, the differentiated corneal cells can be seeded into various types of biomaterials such as hydrogels, providing a wider range of application possibilities for the development of various novel artificial corneal products. Attached Figure Description

[0021] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration:

[0022] Figure 1 Morphology and expression of corneal epithelial cell-like cells (hAESC-CECs) derived from hAESC. (A) Cell morphology of hAESC-CECs after 10 and 20 days of differentiation. (B) Cell morphology of human corneal epithelial cell line (hCECs). (C) Expression of corneal epithelial cell-specific markers by hAESC-CECs at different differentiation time points. (D) Expression of eye development-related genes and corneal epithelial cell-specific markers by hAESCs, hAESC-CECs, and hCECs, where hAESC-CECs are cells obtained after 10 days of differentiation. (E, F) Immunofluorescence detection of corneal epithelial cell-specific marker expression by hAESC-CECs and hCECs.

[0023] Figure 2 Retinoic acid enhances the barrier function of hAESC-CECs. (A) Schematic diagram of the corneal epithelial cell induction protocol optimized with retinoic acid. The CK differentiation protocol is CnT-PR-D epithelial cell differentiation medium with 20 ng / mL keratinocyte growth factor added; the RA differentiation protocol is the CK differentiation protocol with 1 μM retinoic acid added. The expression of (B) corneal epithelial-associated mucin and (C) corneal epithelial-specific markers by hAESC-CECs in different differentiation systems, where the NC group refers to the hAESCs group. (D, E) Rose Bengal staining results of hAESC-CECs in different differentiation systems, the larger the white area, the stronger the epithelial barrier function. (F) hAESC-CECs induced by the RA differentiation protocol express tight junction protein ZO-1; (G) hAESC-CECs induced by the RA differentiation protocol express corneal epithelial-specific markers, where the NC group refers to the hAESCs group.

[0024] Figure 3 (A) Schematic diagram of hAESC-CECs seeding on the decellularized corneal stroma. (B) Staining of the cytoskeleton (F-actin) in the decellularized cornea and the cornea seeded with hAESC-CECs. (C) Expression of corneal epithelial-specific markers after seeding the decellularized cornea with hAESC-CECs. (D, E) Staining of basement membrane components human type IV collagen (COL IV) and laminin in the decellularized cornea and after seeding the decellularized cornea with hAESC-CECs. Short white arrows indicate discontinuous or uneven portions of the basement membrane on the surface of the decellularized cornea.

[0025] Figure 4 : Construction of tissue-engineered corneas based on corneal cells derived from hAESCs. (A) Schematic diagram of constructing tissue-engineered corneas using corneal epithelial cells and corneal stromal cells derived from hAESCs. (B) HE staining and DAPI staining results of tissue-engineered corneas. (C) Expression of corneal epithelial cell markers and corneal stromal cell markers in tissue-engineered corneas. Detailed Implementation

[0026] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. Experimental conditions and methods not specifically specified in the embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0027] Example

[0028] I. Culture of Human Amniotic Epithelial Stem Cells (hAESCs)

[0029] 1. Preparation of culture medium: Add 15% (v / v) KnockOut serum substitute (KSR), 1× non-essential amino acids, 1× L-glutamine, 1× sodium pyruvate, 1× penicillin-streptomycin mixed solution, and 10 ng / mL epidermal growth factor (EGF) to DMEM / F12 medium (all reagents were purchased from ThermoFisher, USA).

[0030] 2. Culture of hAESCs: Resuspend hAESCs in the above culture medium to a concentration of 5 × 10⁻⁶. 4 Cells / mL were inoculated into culture dishes and placed in a 37°C, 5.5% CO2 cell culture incubator. The culture medium was changed every two days.

[0031] II. Induction of Differentiation of Human Corneal Epithelial Cell-like Cells (hAESC-CECs)

[0032] 1. Preparation of culture medium for inducing hAESCs to differentiate into human corneal epithelial cells: Add keratinocyte growth factor (KGF) (purchased from Peprotech, catalog number 100-19) and 1× penicillin-streptomycin mixed solution (purchased from Gibco, catalog number 15140-122) to CnT-PR-D epithelial cell differentiation medium (CnT-Prime 2D Diff, Epithelial Culture Medium, purchased from CELLnTEC, Switzerland) to a final concentration of 20 ng / mL to obtain the corneal epithelial cell differentiation induction medium.

[0033] 2. Method for inducing hAESCs to differentiate into human corneal epithelial cells: Take P0-P2 generation hAESCs and differentiate them at a rate of 5 × 10⁻⁶ cells / year. 5 Cells were seeded at a rate of 100 cells / well in 12-well plates. After 24 hours, the supernatant was discarded, and 1 mL of corneal epithelial cell differentiation induction medium was added to each well. The plates were then incubated at 37°C in a cell culture incubator containing 5.5% CO2. The medium was changed every other day, and the plates were cultured for a total of 10 days.

[0034] 3. Record cell morphological changes regularly using an optical microscope.

[0035] 4. The expression of corneal epithelial-specific markers was detected using quantitative real-time PCR and immunofluorescence. The KRT3 (catalog number ab77869) and KRT12 (catalog number ab185627) antibodies used were purchased from Abcam.

[0036] Figure 1 The results in Figures A and B show that after induced differentiation, the cell morphology of hAESC-CECs is similar to that of hCECs.

[0037] Figure 1 The results in Figures C and D show that the expression of eye development-related genes (OTX2 and SIX6) and corneal epithelial cell-specific markers (KRT3 and KRT12) is upregulated in hAESC-CECs.

[0038] Figure 1 The results in Figures E and F show that the expression levels of corneal epithelial cell-specific markers in hAESC-CECs are close to those in hCECs.

[0039] 5. Based on the aforementioned induction protocol, the effect of adding 1 μM retinoic acid (purchased from Sigma, catalog number R2625) on the corneal epithelial barrier function between hAESC-CECs cells was investigated; the expression of corneal epithelial mucins MUC1, MUC4, and MUC16 was detected using quantitative real-time PCR; and the barrier function between hAESC-CECs cells was assessed by rose benzene staining and tight junction protein ZO-1 staining (antibody purchased from Abcam, catalog number ab221547). The rose benzene staining procedure for hAESC-CECs cells is as follows:

[0040] 1) Preparation of Tiger Red Staining Solution: Dissolve 10 mg of tiger red dye in 10 mL of PBS solution free of Ca2+ and Mg2+ to prepare a 0.1% staining solution.

[0041] 2) Discard the culture supernatant of hAESC-CECs, wash 3 times with 1×PBS buffer, add an appropriate amount of red cinnabar staining solution for staining for 5 minutes.

[0042] 3) Observe and photograph the staining process under an optical microscope.

[0043] Figure 2 The results of the AC diagram showed that, based on the aforementioned induction protocol, the expression levels of mucins MUC1, MUC4 and MUC16 in hAESC-CECs were significantly upregulated after treatment with 1 μM retinoic acid, without affecting the expression levels of corneal epithelial-specific markers.

[0044] Figure 2 The results in Figures D and E show that the red retinoic acid staining experiment indicates that after treatment with 1 μM retinoic acid, a stronger barrier function was formed between hAESC-CECs (the larger the area not stained by red retinoic acid, the better the barrier function between cells).

[0045] Figure 2 The results of the middle F figure show that tight junctions formed between hAESC-CECs cells.

[0046] Figure 2 The results from the G-plot showed that hAESC-CECs cells could still maintain the expression of corneal epithelial-specific markers after treatment with 1 μM retinoic acid.

[0047] 3. Inoculating hAESC-CECs onto human decellularized corneal tissue

[0048] 1. Collect the remaining waste corneal-limbal tissue after human corneal transplantation, cut off the sclera with a blade, and decellularize it with 1.5M hypertonic sodium chloride solution to prepare corneal stromal scaffold.

[0049] 2. Preparation of cell culture medium: Add a mixture of KGF, 1 μM retinoic acid and 1× penicillin-streptomycin to CnT-PR-D medium at a final concentration of 20 ng / mL.

[0050] 3. Prepare a single-cell suspension of hAESC-CECs with a concentration of 1×10⁻⁶. 6 Cells / 200 μL. The cell suspension was dropped onto the surface of the corneal stromal scaffold and allowed to settle and adhere naturally. The scaffold was then incubated at 37°C in a cell culture incubator containing 5.5% CO2 for 5 days, with the medium changed every 2 days.

[0051] 4. Frozen sections of corneal tissue

[0052] 1) Tissue pretreatment: The corneal tissue obtained in step 3 was initially rinsed with 1×PBS buffer.

[0053] 2) Fixation: Immerse the rinsed corneal tissue in 4% paraformaldehyde (PFA) fixative and fix it at 4°C for 12-16 hours.

[0054] 3) Gradient dehydration: The fixed tissue was sequentially placed in 10%, 20%, and 30% sucrose solutions for gradient dehydration treatment, specifically: 10% sucrose solution for 2 hours, 20% sucrose solution for 2 hours, and 30% sucrose solution for 12-16 hours. The entire process was carried out at a low temperature of 4°C.

[0055] 4) Low-temperature embedding: The dehydrated tissue was embedded using a special embedding agent for frozen sections, and immediately transferred to an ultra-low temperature environment of -80℃ for preservation after embedding.

[0056] 5) Section preparation: The sectioning operation was carried out using a cryostat pre-cooled to -20℃, which specifically included: initially setting the thickness to 50μm for pre-sectioning, and adjusting it to 7μm for formal sectioning.

[0057] 6) Section preservation: Store the obtained tissue sections at -80℃ for later use.

[0058] 5. Fluorescent staining of frozen sections

[0059] 1) Sample pretreatment: Take out the frozen tissue sections stored at -80℃ and equilibrate them at room temperature for 30 minutes.

[0060] 2) Buffer washing: The slides were washed three times with 1×PBS buffer for 5 minutes each time.

[0061] 3) Tissue permeabilization treatment: Add 1×PBS buffer containing 0.25% (v / v) Triton X-100 to the surface of the tissue section and incubate at room temperature for 5 minutes to enhance membrane permeability.

[0062] 4) Washing after permeabilization: After removing the permeabilization solution, wash three times with 1×PBS buffer, 5 minutes each time.

[0063] 5) Blocking: Cover the tissue area with blocking solution (formulation: 850 μL PBS + 100 μL 10% BSA + 50 μL HBS) and block at room temperature for 1 hour to reduce background staining.

[0064] 6) Primary antibody incubation: Draw a circle around the tissue with an oil-based pen, add the primary antibody working solution, and incubate at 4°C for 12-16 hours.

[0065] The KRT3 (catalog number ab77869, mouse), KRT12 (catalog number ab185627, rabbit), PAX6 (catalog number ab195045, rabbit), P63 (catalog number ab124762, rabbit), COL IV (catalog number ab236640, rabbit) and Laminin (catalog number ab11575, rabbit) antibodies used here were all purchased from Abcam; the F-actin antibody (catalog number 40734ES75) was purchased from Yeasen.

[0066] 7) Primary antibody washing: After removing the primary antibody, wash three times with 1×PBS buffer for 5 minutes each time.

[0067] 8) Fluorescent secondary antibody labeling: Dilute the fluorescently labeled secondary antibodies, including Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) (ab150080) (both purchased from Abcam), to a concentration of 1:500 with 1×PBS buffer; incubate at room temperature for 1 hour in the dark.

[0068] 9) Secondary antibody washing: After removing the secondary antibody, wash three times with 1×PBS buffer in the dark, 10 minutes each time.

[0069] 10) Nuclear staining: Dilute DAPI staining solution at a ratio of 1:300 and stain at room temperature for 10 minutes.

[0070] 11) Final wash: After removing the staining solution, wash three times with 1×PBS buffer, 5 minutes each time.

[0071] 12) Mounting and observation: The slides were mounted with anti-fluorescence quenching mounting medium and images were immediately acquired under a fluorescence microscope.

[0072] Figure 3The results in Figures A and B show that after 5 days of inoculation and culture, hAESC-CECs can form stratified epithelium on the decellularized corneal surface, which is structurally similar to the natural corneal epithelium.

[0073] Figure 3 The results in Figure C show that hAESC-CECs seeded on decellularized corneas maintain the expression of corneal epithelial cell-specific markers.

[0074] Figure 3 The results in Figures D and E show that hAESC-CECs can express and secrete components of the corneal subepithelial basement membrane, such as COL IV and laminin, making the basement membrane of decellularized cornea more intact.

[0075] This demonstrates that hAESC-CECs can integrate well with human decellularized cornea and exhibit the natural corneal epithelial phenotype.

[0076] IV. Tissue-engineered corneal construction

[0077] 1. Induction of corneal stromal cell differentiation from hAESCs: Corneal stromal cell differentiation induction medium was prepared by adding 1mM L-ascorbic acid 2-phosphate, 10 ng / mL basic fibroblast growth factor, and 1 μM retinoic acid to DMEM / F12 medium containing 1% insulin-transferrin-selenium (ITS), 2 mM L-glutamine, 1 mM non-essential amino acids, 1 mM sodium pyruvate, and 1× penicillin-streptomycin mixture (purchased from Gibco, catalog number: 15140-122). hAESCs were then cultured in this medium for 5 days to obtain hAESC-derived corneal stromal cells for later use.

[0078] 2. hAESC-CECs were seeded onto the surface of a decellularized corneal stromal scaffold. The specific seeding method was as follows: differentiated hAESC-CECs cells were digested and prepared into a single-cell suspension with a concentration of 1×10⁻⁶. 6 hAESC-CECs cells / 200μL. The cell suspension was dropped onto the surface of the corneal stromal scaffold, and after standing, the hAESC-CECs cells naturally settled and adhered to the decellularized corneal surface.

[0079] 3. HAESCs-derived corneal stromal cells were seeded into a decellularized corneal stromal scaffold. The specific seeding method was as follows: Corneal stromal cells were digested and prepared into a single-cell suspension. 200 μL of a suspension containing 1×10⁻⁶ cells was injected using an insulin needle. 5 A suspension of corneal stromal cells was injected into the decellularized corneal stromal scaffold.

[0080] 4. Culture conditions for constructing tissue-engineered corneas: The culture medium used for co-culture was a 1:1 mixture of corneal epithelial cell induction differentiation medium and corneal stromal cell induction differentiation medium. The culture environment was a cell culture incubator at 37°C with 5.5% CO2. The medium was changed every 2 days, and the culture was carried out for a total of 5 days.

[0081] Figure 4 Figure A in the middle is a schematic diagram of the process of constructing tissue-engineered corneas based on corneal epithelial and stromal cells derived from hAESCs.

[0082] Figure 4 Images B and C show that hAESC-CECs form stratified epithelium on the corneal stromal surface, while corneal stromal cells derived from hAESCs migrate and disperse among stromal fibers. Furthermore, both cell types stably express corneal cell-specific markers. The KRT3 antibody used here was purchased from Abcam; the Keratocan (KERA) antibody was purchased from Bioss.

[0083] In summary, we successfully constructed a tissue-engineered cornea using two types of corneal cells derived from hAESCs and decellularized corneal stroma.

[0084] This invention marks the first successful construction of a tissue-engineered cornea by simultaneously seeding two types of corneal cell derived from hAESCs into the human decellularized corneal stroma. The hAESC-CECs induced from human amniotic epithelial stem cells exhibit morphological similarities to hCECs and highly express classic corneal epithelial cell markers. Furthermore, they can form tight junctions, a crucial condition for the corneal epithelium's barrier function. When differentiated hAESC-CECs were seeded onto decellularized corneas and cultured, tissue section staining revealed that the differentiated cells integrated well into the decellularized cornea and maintained the expression of cell markers. Simultaneously, hAESC-CECs can secrete components of the corneal epithelial basement membrane, another important function of corneal epithelial cells.

[0085] Obviously, the above embodiments of the present invention are merely examples to illustrate the present invention more clearly, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all implementation methods here. Any obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A method for constructing a tissue-engineered cornea based on human amniotic epithelial stem cells, characterized by, Comprise: 1) culturing human amniotic epithelial stem cells in corneal epithelial cell induction differentiation medium for 5-20 days, replacing the medium every two days, to obtain human corneal epithelial cell-like cells; culturing human amniotic epithelial stem cells in corneal stromal cell induction differentiation medium for 3-12 days to obtain corneal stromal cells; the corneal epithelial cell induction differentiation medium uses CnT-PR-D epithelial cell differentiation medium containing 5-50 ng / mL keratinocyte growth factor, 1 μM retinoic acid and 1× double-antigen; the corneal stromal cell induction differentiation medium uses DMEM / F12 medium containing 1% insulin-transferrin-selenium, 2 mM L-glutamine, 1 mM non-essential amino acids, 1 mM sodium pyruvate, 0.05-2 mM ascorbic acid-2-phosphate, 5-20 ng / mL basic fibroblast growth factor, 1 μM retinoic acid and 1× double-antigen; 2) seeding the human corneal epithelial cell-like cells and corneal stromal cells obtained in step 1) on the surface and inside of the acellular corneal stromal scaffold respectively, co-culturing for 5 days to construct a tissue-engineered cornea, the method for seeding the human corneal epithelial cell-like cells on the surface of the acellular corneal stromal scaffold is as follows: taking the human corneal epithelial cell-like cells obtained in step 1), preparing a single-cell suspension with a concentration of 1×10 6 cells / 200 μL after digestion, and dropping the single-cell suspension on the surface of the acellular corneal stromal scaffold, and then allowing the human corneal epithelial cell-like cells to naturally settle and adhere to the surface of the acellular cornea; the method for seeding the corneal stromal cells inside the acellular corneal stromal scaffold is as follows: preparing a single-cell suspension with a concentration of 1×10 6 cells / 200 μL after digestion, and injecting the single-cell suspension into the inside of the acellular corneal stromal scaffold using an insulin needle; the co-culturing medium is a 1:1 mixture of the corneal epithelial cell differentiation culture medium and the corneal stromal cell differentiation culture medium, and the culture environment is a cell culture box with 5.5% CO2 at 37℃, and the medium is replaced once every 2 days during the culture.

2. The method of claim 1, wherein, In step 1), the human amniotic epithelial stem cells are P0-P2 generation human amniotic epithelial stem cells.

3. The method of claim 1, wherein, In step 2), the decellularized corneal stromal scaffold is prepared by the following method: collecting the residual waste corneo-scleral limbal tissue after human corneal transplantation, cutting off the sclera part with a blade, and decellularizing with 1.5 M high-osmotic sodium chloride solution to prepare a decellularized corneal stromal scaffold.

Images