A method for preparing limbal stem cells and mesenchymal stem cells through co-culture

Abstract

This invention relates to the field of tissue engineering, and particularly to a method for preparing limbal stem cells and mesenchymal stem cells through co-culture. This invention innovatively constructs a co-culture system for limbal stem cells and mesenchymal stem cells. This system significantly improves the expansion efficiency of limbal stem cells, increasing the number of cells by more than 500-fold within 14-18 days, and allowing for 1-2 additional passages, effectively overcoming the proliferation limit of single-culture. Simultaneously, the co-culture microenvironment significantly downregulates the expression of immune rejection-related genes such as HLA-B and HLA-DPB1, reducing cellular immunogenicity and helping to reduce rejection reactions after clinical transplantation. Notably, although the expression of stem cell genes such as TP63 and ITGB4, and corneal-specific protein genes such as KRT12 and KRT3, is slightly downregulated, the cells still retain the expression of limbal stem cell-specific genes, proliferation and differentiation capabilities, and the potential to differentiate into mature corneal epithelial cells. This strategy, which achieves large-scale in vitro pharmaceutical-grade culture while maintaining stem cell characteristics, better meets the needs of clinical transplantation.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering, and in particular to a method for preparing limbal stem cells and mesenchymal stem cells through co-culture. Background Technology

[0002] A normal cornea is avascular, transparent, and richly nerve-rich, forming the outermost layer of the eye's refractive media and possessing the strongest refractive power. The corneal epithelial cell layer, located on the corneal surface, maintains a dynamic balance through continuous shedding and renewal, thereby preserving corneal transparency, refractive power, and the integrity of the ocular surface, ultimately ensuring intact visual function. This continuous renewal of corneal epithelial cells originates from the proliferation, differentiation, and migration of limbal stem cells (LSCs) within the basal layer of the limbus in the Vogt's palisade structure region. LSCs provide a guarantee for ocular surface stability.

[0003] Limbal stem cell deficiency (LSCD) is an ocular surface disease caused by a reduction in the number or functional impairment of limbal stem cells, leading to an imbalance in corneal epithelial homeostasis. When limbal stem cells are damaged or lost, corneal conjunctivalization can occur, and in severe cases, vision loss can result. Unilateral LSCD can be treated with limbal stem cell transplantation from the healthy eye. Bilateral LSCD requires allogeneic limbal stem cell transplantation. In recent years, in vitro limbal stem cell culture technology has brought new hope to these patients.

[0004] Currently, most methods for culturing corneal stem cells, both domestically and internationally, rely on in vitro expansion. The main drawbacks of this method include limited in vitro proliferative lifespan, low expansion efficiency, and high immunogenicity. Cells cultured in isolation show obvious signs of senescence after the P3 generation, and by around day 30, they may completely lose their adhesion and proliferative abilities, only expanding to about 100 times their original cell volume. Simultaneously, the expression of rejection-related genes such as HLA-B and HLA-DPB1 is significantly upregulated in cells cultured in isolation, leading to a higher risk of rejection after transplantation. Furthermore, this technology places stringent requirements on the timeliness of donor corneas (≤24 hours of harvesting), pathogen screening, and storage conditions. The procedure requires frequent medium changes every 2-3 days, strict control of passage to 70-80% confluence, and the cryopreservation and thawing process easily causes cell damage, ultimately limiting the total number of cells available for clinical treatment.

[0005] Therefore, this invention innovatively constructs a co-culture system for limbal stem cells and mesenchymal stem cells. This system significantly improves the expansion efficiency of limbal stem cells, with cell numbers increasing more than 500-fold within 14-18 days, and allowing for 1-2 additional passages, effectively overcoming the proliferation limit of single-culture. Simultaneously, the co-culture microenvironment significantly downregulates the expression of immune rejection-related genes such as HLA-B and HLA-DPB1, reducing cellular immunogenicity and helping to reduce rejection reactions after clinical transplantation. Notably, although the expression of stem cell genes such as TP63 and ITGB4, and corneal-specific protein genes such as KRT12 and KRT3, is slightly downregulated (possibly related to interference from residual mesenchymal stem cells), the cells still retain limbal stem cell-specific gene expression, proliferation and differentiation capacity, and the potential to differentiate into mature corneal epithelial cells. This strategy of achieving large-scale, pharmaceutical-grade in vitro culture while maintaining stem cell characteristics better meets the needs of clinical transplantation. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for preparing limbal stem cells and mesenchymal stem cells through co-culture, aiming to overcome the proliferation limit of single-culture, achieve large-scale in vitro pharmaceutical-grade culture, and effectively reduce the immunogenicity of cultured cells, thereby reducing rejection reactions after clinical transplantation, and thus providing a sufficient and safe source of transplanted cells for patients with limbal stem cell deficiency.

[0007] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a method for preparing limbal stem cells and mesenchymal stem cells through co-culture, comprising the following steps: S1. Donor cornea acquisition, transportation, and donor screening; S2. Sterilization and cutting of limbal tissue: The cornea was rinsed multiple times at room temperature with PBS containing antibiotics (penicillin 100U / mL + streptomycin 100μg / mL + gentamicin 50μg / mL); the limbal tissue was stored in physiological saline at 4℃ and aseptically cut into small tissue pieces; S3. Isolation and preparation of limbal stem cells: (1) Dispase II low-temperature digestion: Dispase II solution was used to digest small tissue pieces at low temperature for 12 hours; (2) Washing and centrifugation: After rinsing the tissue with PBS, centrifuge and discard the supernatant to obtain the centrifuged tissue; (3) Secondary digestion with trypsin: After digestion with 0.5 mg / mL trypsin and centrifugation, the tissue obtained was centrifuged for 2-3 min, then washed with PBS, centrifuged at 500 rpm for 5 min and the supernatant was discarded. The trypsin digestion, washing and centrifugation steps were repeated once to prepare a single cell suspension. (4) Inoculation and culture: Resuspend cells in serum-free limbal stem cell culture medium, inoculate them into culture plates, and culture at 37°C with 5% CO2 saturated humidity. Change half or all of the medium every 2-3 days. S4. Isolation and preparation of mesenchymal stem cells: Using the same tissue isolation, enzyme digestion, centrifugation and seeding process as steps S1 to S3, mesenchymal stem cells are cultured alone at 37°C and 5% CO2 saturated humidity using mesenchymal stem cell culture medium (in practice, mesenchymal stem cells can also be commercially available, for example, purchased from the Chinese Academy of Sciences Cell Bank). S5. Co-culture of two types of stem cells: When the confluence of limbal stem cells reaches 30% to 50%, trypsin digestion for 5 to 10 minutes is performed to remove the stem cells from the culture wall. After washing with PBS, centrifugation at 1000 rpm for 5 minutes is performed to discard the supernatant. After counting, the cells are seeded into culture flasks at a density of 2000 cells / cm² and co-cultured with mesenchymal stem cells. S6. Cell passage and expansion: Change the medium completely every 48-72 hours; if the cells do not adhere after 3 days of inoculation, add 1-2 mL of fresh serum-free limbal stem cell culture medium while retaining the original culture medium; when the cell confluence reaches 90%, harvest, count, and passage to obtain corneal-oriented cells; S7. Cell cryopreservation: Harvested oriented corneal cells are resuspended in cryopreservation solution and stored in an ultra-low temperature freezer at -80°C.

[0008] Furthermore, the serum-free limbal stem cell culture medium in step S3 consists of the following components: phenol red-free and animal-free DMEM / F-12 basal medium at a volume ratio of 1:1, supplemented with 1% rHSR recombinant human serum albumin, 0.01 mg / mL insulin-transferrin-selenoethanolamine (ITS-X) (enkilife catalog number CMD0017, https: / / www.enkilife.cn / ), 20 ng / mL epidermal growth factor (EGF), 2.5 ng / mL FGF-2 fibroblast growth factor (FGF-2), 5 ng / mL PDGF-BB platelet-derived growth factor-BB, 0.1 mM calcium ions, and 0.1 mM 2-mercaptoethanol, with a final calcium ion concentration of <0.2 mM.

[0009] Furthermore, in step S3, the concentration of Dispase II is 1 U / mL.

[0010] Furthermore, in step S3, the trypsin concentration is 0.5 mg / mL.

[0011] Furthermore, the mesenchymal stem cell culture medium in step S4 consists of the following components: phenol red-free and animal-free DMEM / F-12 basal medium at a volume ratio of 1:1, supplemented with 0.1% rHSR recombinant human serum albumin, ITS-X1×(insulin-transferrin-selenoethanolamine) 0.01 mg / mL, 20 ng / mL basic fibroblast growth factor, 5 ng / mL EGF epidermal growth factor, 5 ng / mL PDGF-BB, and 0.1 mM 3-mercaptoethanol.

[0012] Furthermore, in step S7, the cryopreservation solution is 90% + 10% (volume fraction) dimethyl sulfoxide in serum-free limbal stem cell culture medium.

[0013] Furthermore, the study aims to achieve stable expression of TP63 and ITGB4 stem cell genes and KRT12 and KRT3 corneal-specific protein genes in corneal cells, thereby preserving the stem cell proliferation and differentiation capacity and the potential to differentiate into mature corneal epithelial cells.

[0014] Secondly, the present invention provides the use of co-cultured cells obtained according to the preparation method in the preparation of transplantation preparations for treating limbal stem cell deficiency.

[0015] Compared with the prior art, the advantages of the present invention are as follows: This invention innovatively constructs a co-culture system for limbal stem cells and mesenchymal stem cells. This system significantly improves the expansion efficiency of limbal stem cells, with cell numbers increasing more than 500-fold within 14-18 days, and allowing for 1-2 additional passages, effectively overcoming the proliferation limit of single-culture. Simultaneously, the co-culture microenvironment significantly downregulates the expression of immune rejection-related genes such as HLA-B and HLA-DPB1, reducing cellular immunogenicity and helping to reduce rejection reactions after clinical transplantation. Notably, although the expression of stem cell genes such as TP63 and ITGB4, and corneal-specific protein genes such as KRT12 and KRT3, is slightly downregulated (possibly related to interference from residual mesenchymal stem cells), the cells still retain limbal stem cell-specific gene expression, proliferation and differentiation capacity, and the potential to differentiate into mature corneal epithelial cells. This strategy of achieving large-scale, pharmaceutical-grade in vitro culture while maintaining stem cell characteristics better meets the needs of clinical transplantation. Attached Figure Description

[0016] The present invention will now be further described with reference to the accompanying drawings.

[0017] Figure 1 Images showing the deformation of limbal stem cells and mesenchymal stem cells co-cultured according to the present invention.

[0018] Figure 2 This is a statistical chart of the transcriptome sequencing results for the detection of stem genes in this invention. Detailed Implementation

[0019] The invention will now be further described with reference to the accompanying drawings.

[0020] Example 1

[0021] The method for preparing limbal stem cells according to the present invention includes the following steps: 01. Acquisition and Transportation of Donor Corneas: Human donor corneas (donor source 1: autologous limbal stem cells obtained (can be from the contralateral eye); donor source 2: allogeneic limbal cornea (can be corneal donation or voluntary donation from siblings. Both comply with human ethics) are placed in a humidified box containing physiological saline and transported at 4°C for ≤24 hours. 02. Donor Screening: Pathogen testing is performed on the donor cornea to ensure that the results of HBV, HCV, HIV, syphilis, and fungal tests are all negative, and that the donor has no history of ocular surface tumors or active inflammation. 03. Tissue sterilization and preservation: The cornea was transferred to a culture dish using sterile forceps and rinsed three times with PBS containing antibiotics (penicillin 100U / mL + streptomycin 100μg / mL + gentamicin 50μg / mL) at room temperature for 5 minutes each time; the isolated limbal tissue was placed in 20mL of physiological saline and preserved at 4°C. 04. Cutting of limbal stromal strips: Using a sterile scalpel, cut the limbal stromal strips into 1mm³ pieces and transfer them into 15mL centrifuge tubes; 05. Dispase II digestion: The cut matrix strips were digested with 10 mL of Dispase II (concentration of 1 U / mL) at low temperature for 12 h using an enzymatic digestion method. 06. PBS washing and centrifugation: Wash the digested tissue with PBS, collect it into a 15 mL centrifuge tube, centrifuge at 2000 rpm for 5 min, and discard the supernatant; repeat this step once. 07. Trypsin digestion: The tissue was digested with trypsin (concentration of 0.5 mg / mL) for 2 to 3 minutes. 08. Preparation of single-cell suspension: Wash the digested cells with PBS, collect them into 15mL centrifuge tubes, centrifuge at 500rpm for 5min, and discard the supernatant; repeat the trypsin digestion and washing centrifugation steps once. 09. Cell resuspension and seeding: Resuspend cells in 2 mL of serum-free limbal stem cell culture medium and seed them in 6-well cell culture plates. Incubate at 37°C in a 5% CO2 saturated humidity incubator. Change half or all of the medium every 2 to 3 days depending on the growth. The serum-free limbal stem cell culture medium consists of: DMEM / F-12 (DMEM to Ham's F-12 ratio is 1:1, phenol red, no animal source), supplemented with rHSR (recombinant human serum albumin) 1%, insulin-transferrin-selenoethanolamine 0.01 mg / mL, EGF (epidermal growth factor) 20 ng / mL, FGF-2 (fibroblast growth factor 2) 5 ng / mL, PDGF-BB (platelet-derived growth factor-BB) 5 ng / mL, calcium ions 0.1 mM (final physiological concentration <0.2 mM), and 2-mercaptoethanol 0.1 mM; DMEM / F-12 is a classic basal culture medium formulation formed by mixing DMEM and Ham's F-12 in a 1:1 ratio, which combines the advantages of both media: DMEM (Dulbecco's Modified Eagle's Medium): High concentration of glucose, amino acids and vitamins, supporting rapid proliferation of adherent cells; Ham's F-12: Rich in trace elements, inorganic salts and nucleotide precursors, making it more suitable for low-serum / serum-free culture systems.

[0022] 10. Preparation of mesenchymal stem cells: Mesenchymal stem cells were isolated and cultured using the same methods as 01-09. The mesenchymal stem cell culture was conducted in a 37°C, 5% CO2 saturated humidity incubator. The mesenchymal stem cell culture medium consists of: DMEM / F-12 (1:1, phenol red, animal-free), supplemented with rHSR 0.1%, ITS-X 1× (insulin-transferrin-selenoethanolamine 0.01 mg / mL), basic fibroblast growth factor bFGF 20 ng / mL, EGF 5 ng / mL, PDGF-BB 5 ng / mL, and 3-mercaptoethanol 0.1 mM. 11. Co-culture of limbal stem cells and mesenchymal stem cells: When limbal stem cells reach 30%–50% confluence, trypsin is used to digest cell adhesion proteins (such as integrins and cadherins) for 5–10 minutes to detach adherent cells. The digested cells are washed with PBS and collected into 15 mL centrifuge tubes. The tubes are centrifuged at 1000 rpm for 5 minutes, and the supernatant is discarded. After counting, the cells are seeded in culture flasks at a density of 2000 cells / cm² and co-cultured with mesenchymal stem cells. 12. Cell passage and expansion: Complete medium replacement every 48-72 hours; if no cells adhere by day 3, add 1-2 mL of fresh culture medium without discarding the old medium; when cell confluence reaches 90%, harvest, count, and passage; (In this example, cell confluence refers to the percentage of the surface area of ​​the culture medium bottom covered by adherent cells in the culture dish / flask, which is a quantitative indicator and does not refer to the fusion of two cell types) 13. Cell cryopreservation: The harvested cells were cryopreserved using 10% (volume fraction) dimethyl sulfoxide (DMSO) cryopreservation solution in an ultra-low temperature freezer at -80°C.

[0023] Example 2

[0024] Cell formation and morphology Cultured until Day 7, early limbal stem cells were observed under a microscope.

[0025] On Day 14, the limbal stem cells were digested with 1 ml of trypsin, yielding 1.32 × 10⁻⁶ cells. 5 Mesenchymal stem cells and limbal stem cells were co-cultured, and the culture flasks containing limbal stem cells cultured alone and limbal stem cells co-cultured with mesenchymal stem cells were observed under a microscope.

[0026] After culturing for 18 days, microscopic observation showed that the confluence reached approximately 90%, at which point the stem cells were harvested, counted, and passaged. The number of individually cultured limbal stem cells harvested in P1 was 2.54 × 10⁻⁶. 6 The number of cells obtained after co-culturing limbal stem cells and mesenchymal stem cells (MSCs) and fusion was 1.03 × 10⁻⁶. 7 .

[0027] By Day 23, cell confluence was observed under a microscope to be approximately 90%, at which point the cells were harvested and counted. The number of individually cultured limbal stem cells harvested was 5.42 × 10⁻⁶. 7 The number of cells co-cultured with limbal stem cells and mesenchymal stem cells was 2.56 × 10⁻⁶. 8 A portion of the cells were sampled and sequenced, while the remaining cells were passaged. The number of limbal stem cells passaged was 1×102 7 The co-culture of limbal stem cells and mesenchymal stem cells resulted in a limbal mass of 1×10⁻⁶ cells. 7 .

[0028] By Day 29, cell confluence was observed under a microscope to be approximately 90%, at which point the cells were harvested and counted. The number of individually cultured limbal stem cells harvested at P3 was 5.23 × 10⁻⁶. 7 The number of cells co-cultured with limbal stem cells and mesenchymal stem cells was 8.43 × 10⁻⁶. 7Some cells were cryopreserved, while others were passaged. The number of limbal stem cells passaged was 1×10⁻⁶. 7 The co-culture of limbal stem cells and mesenchymal stem cells resulted in a limbal mass of 1×10⁻⁶ cells. 7 .

[0029] On day 30, under a microscope, many floating cells were observed in the culture medium of limbal stem cells cultured alone. These cells exhibited altered adherence and ceased proliferation, and were discarded. A small number of floating cells were observed in the co-cultured cells of limbal stem cells and mesenchymal stem cells, and cell proliferation slowed down.

[0030] By Day 34, cell confluence was observed under a microscope to be approximately 90%, at which point the cells were harvested and counted. A total of 3.23 × 10⁻⁶ cells (i.e., cells oriented towards corneal growth) co-cultured with limbal stem cells and mesenchymal stem cells were harvested. 7 .

[0031] Example 3

[0032] Transcriptome sequencing and performance testing were performed on the limbal stem cells cultured alone and the cells co-cultured with limbal stem cells and mesenchymal stem cells (corneal cells) prepared in Example 1. The results are as follows: 01. Cell Expansion Efficiency Test By day 14 of culture, the number of corneal limbal stem cells harvested from isolated culture was 1.32 × 10⁻⁶. 5 The number of cells harvested from the co-culture of limbal stem cells and mesenchymal stem cells was 1.03 × 10⁻⁶. 7 .

[0033] By day 23 (P2 generation), the number of corneal limbal stem cells harvested from culture alone was 5.42 × 10⁻⁶. 7 The number of cells harvested from the co-culture of limbal stem cells and mesenchymal stem cells was 2.56 × 10⁻⁶. 8 .

[0034] By day 29 (passage P3), the number of corneal limbal stem cells harvested from culture alone was 5.23 × 10⁻⁶. 7 The number of cells harvested from the co-culture of limbal stem cells and mesenchymal stem cells was 8.43 × 10⁻⁶. 7 .

[0035] By day 34, the number of cells harvested from the co-culture of limbal stem cells and mesenchymal stem cells was 3.23 × 10⁻⁶. 7 .

[0036] The formula for calculating the cell expansion fold is: Fold expansion = Initial number of cells inoculated / Number of cells harvested Calculations show that the co-culture system of limbal stem cells and mesenchymal stem cells can increase the number of cells by more than 500 times within 14 to 18 days, while limbal stem cells cultured alone can only increase to about 100 times the original number of cells.

[0037] 02, Stem gene testing The transcriptome sequencing results are shown in Table 1 and Figure 2 : Table 1. Analysis of gene expression differences between the LSC group and the MSC_LSC group. .

[0038] The results show: (1) TP63, ITGB4 and other limbal stem cell stem genes were expressed in both culture systems, indicating that the cells obtained from the culture were identified as limbal stem cells. (2) The expression of stem genes such as TP63, ITGB4, and KRT14 in cells co-cultured with limbal stem cells and mesenchymal stem cells was downregulated compared with that in cells cultured alone, indicating that limbal stem cells cultured alone have stronger stemness. (3) The expression of corneal-specific protein genes such as KRT12, KRT3, and CLDN7 was downregulated in cells co-cultured with limbal stem cells and mesenchymal stem cells, indicating that co-culture reduced the specificity of corneal cells.

[0039] 03, Immunogenicity testing Transcriptome sequencing results showed that the expression of immune rejection-related genes such as HLA-B and HLA-DPB1 in cells co-cultured with limbal stem cells and mesenchymal stem cells was significantly downregulated compared with that of limbal stem cells cultured alone, indicating that co-culture can effectively reduce the immunogenicity of limbal stem cells and improve their anti-rejection ability.

[0040] Example 4: Detection of Cell Functional Characteristics: In Vitro Experiments Objective: To evaluate the differences in stemness maintenance and differentiation capacity of recombinant corneal epithelial flaps constructed by two systems: limbal stem cell culture alone and limbal stem cell co-culture with mesenchymal stem cells, and to clarify the impact of the co-culture system on cell functional characteristics.

[0041] Methods: Two groups of cells (one group consisted of limbal stem cells cultured alone, and the other group consisted of limbal stem cells co-cultured with mesenchymal stem cells) were seeded onto de-epithelialized human amniotic membrane carriers and cultured for 14 days under air-liquid interface conditions to construct recombinant corneal epithelial sheets. Immunofluorescence staining was used to detect the expression intensity and localization distribution of the limbal stem cell marker TP63 (nuclear positive) and the corneal differentiation marker KRT12 (cytoplasmic positive). ImageJ software was used to quantitatively analyze the proportion of positive cells.

[0042] Results: In the monoculture group, the proportion of TP63-positive cells remained at 68.3%±4.7%, mainly located in the basal layer; while in the co-culture group, the proportion of TP63-positive cells decreased to 41.6%±5.2% (P<<0.01), and the distribution of positive cells was more diffuse. Regarding corneal differentiation, in the monoculture group, the proportion of KRT12-positive cells was 12.5%±3.1%, mainly distributed in the superficial layer; while in the co-culture group, the proportion of KRT12-positive cells increased to 34.8%±4.5% (P<<0.01), and the positive signal distribution expanded to multiple cell layers above the basal layer.

[0043] Conclusion: Co-culture reduced the stemness maintenance capacity of limbal stem cells, but promoted their differentiation and maturation into corneal epithelial cells, suggesting that the co-culture system is more suitable for scenarios that require rapid acquisition of differentiated corneal epithelial cells.

[0044] Example 5: Effect of co-culture system on the therapeutic effect of transplantation of rabbit limbal stem cell defect model Objective: To investigate the effect of a co-culture system of limbal stem cells and mesenchymal stem cells on the therapeutic effect of transplantation in a rabbit limbal stem cell defect model, and to verify the clinical efficacy advantage of co-culture in reducing immunogenicity.

[0045] Methods: A bilateral limbal stem cell defect model was established in New Zealand white rabbits (alkali burn method, 8 mm in diameter). Cell slices from the left eye (co-cultured group) and the right eye (cultured alone group) were randomly selected as self-controls (n=8). Clinical scores were assessed on postoperative days 7, 14, and 28, and histological examination was performed on day 28. Clinical observation indicators included corneal transparency score (0-4 points) and the percentage of neovascularization area.

[0046] Results: 28 days post-surgery, the corneal transparency score in the culture-only group was 2.8±0.5, and the neovascularization area was 18.6%±3.4%. In the co-culture group, the corneal transparency score significantly improved to 1.4±0.3 (P<<0.01), and the neovascularization area decreased to 7.2%±2.1% (P<<0.01). Histological examination showed that in the culture-only group, TP63-positive cells were aggregated in the corneal epithelium (positive rate 15.3%±2.8%), but accompanied by a large number of KRT12-positive cells (positive rate 52.6%±5.3%) and scattered inflammatory cell infiltration. Although the proportion of TP63-positive cells in the co-culture group was slightly lower (11.7%±2.1%), the corneal epithelial structure was more regular, the KRT12-positive cells were evenly distributed (positive rate 61.4%±4.7%), and the inflammatory cell infiltration was significantly reduced.

[0047] Conclusion: The co-culture group significantly improved corneal transparency and anti-angiogenesis after corneal transplantation by reducing immunogenicity (consistent with the downregulation of HLA genes in the transcriptome), while maintaining effective corneal epithelial barrier function.

Claims

1. A method for preparing limbal stem cells and mesenchymal stem cells through co-culture, characterized in that, Includes the following steps: S1. Donor cornea acquisition, transportation, and donor screening; S2. Sterilization and cutting of limbal tissue: The cornea was rinsed multiple times at room temperature with PBS containing antibiotics; the limbal tissue was stored in physiological saline at 4°C and aseptically cut into small tissue pieces; S3. Isolation and preparation of limbal stem cells: (1) Dispase II low-temperature digestion: Dispase II solution was used to digest small tissue pieces at low temperature for 12 hours; (2) Washing and centrifugation: After rinsing the tissue with PBS, centrifuge and discard the supernatant to obtain the centrifuged tissue; (3) Secondary digestion with trypsin: After digestion with 0.5 mg / mL trypsin and centrifugation, the tissue obtained was centrifuged for 2-3 min, then washed with PBS, centrifuged at 500 rpm for 5 min and the supernatant was discarded. The trypsin digestion, washing and centrifugation steps were repeated once to prepare a single cell suspension. (4) Inoculation and culture: Resuspend cells in serum-free limbal stem cell culture medium, inoculate them into culture plates, and culture at 37°C with 5% CO2 saturated humidity. Change half or all of the medium every 2-3 days. S4. Mesenchymal stem cell culture: Mesenchymal stem cells were cultured alone using mesenchymal stem cell culture medium at 37°C and 5% CO2 saturated humidity. S5. Co-culture of two types of stem cells: When the confluence of limbal stem cells reaches 30% to 50%, trypsin digestion for 5 to 10 minutes is performed to remove the stem cells from the culture wall. After washing with PBS, centrifugation at 1000 rpm for 5 minutes is performed to discard the supernatant. After counting, the cells are seeded into culture flasks at a density of 2000 cells / cm² and co-cultured with mesenchymal stem cells. S6. Cell passage and expansion: Change the medium completely every 48-72 hours; if the cells do not adhere after 3 days of inoculation, add 1-2 mL of serum-free limbal stem cell culture medium while retaining the original culture medium; when the cell confluence reaches 90%, harvest, count, and passage to obtain corneal-oriented cells; S7. Cell cryopreservation: Harvested oriented corneal cells are resuspended in cryopreservation solution and stored in an ultra-low temperature freezer at -80°C.

2. The method for preparing limbal stem cells and mesenchymal stem cells co-cultured according to claim 1, characterized in that, The serum-free limbal stem cell culture medium in step S3 consists of the following components: phenol red-free and animal-free DMEM / F-12 basal medium at a volume ratio of 1:1, supplemented with 1% rHSR recombinant human serum albumin, 0.01 mg / mL insulin-transferrin-selenoethanolamine, 20 ng / mL EGF epidermal growth factor, 2.5 ng / mL FGF-2 fibroblast growth factor, 5 ng / mL PDGF-BB platelet-derived growth factor-BB, 0.1 mM calcium ions, and 0.1 mM 2-mercaptoethanol, with a final calcium ion concentration of <0.2 mM.

3. The method for preparing limbal stem cells and mesenchymal stem cells co-cultured according to claim 1, characterized in that, In step S3, the concentration of Dispase II is 1 U / mL.

4. The method for preparing limbal stem cells and mesenchymal stem cells co-cultured according to claim 1, characterized in that, In step S3, the trypsin concentration is 0.5 mg / mL.

5. The method for preparing limbal stem cells and mesenchymal stem cells co-cultured according to claim 1, characterized in that, The mesenchymal stem cell culture medium components described in step S4 are as follows: phenol red-free and animal-free DMEM / F-12 basal medium at a volume ratio of 1:1, supplemented with 0.1% rHSR recombinant human serum albumin, ITS-X1×(insulin-transferrin-selenoethanolamine) 0.01 mg / mL, 20 ng / mL basic fibroblast growth factor, 5 ng / mL EGF epidermal growth factor, 5 ng / mL PDGF-BB, and 0.1 mM 3-mercaptoethanol.

6. The method for preparing limbal stem cells and mesenchymal stem cells co-cultured according to claim 1, characterized in that, In step S7, the cryopreservation solution is 10% dimethyl sulfoxide and 90% serum-free limbal stem cell culture medium.

7. The method for preparing limbal stem cells and mesenchymal stem cells co-cultured according to claim 1, characterized in that, The goal is to promote stable expression of TP63, ITGB4 stem cell genes and KRT12, KRT3 corneal-specific protein genes in corneal cells, thereby preserving the stem cell proliferation and differentiation capacity and the potential to differentiate into mature corneal epithelial cells.

8. The use of co-cultured cells obtained by the preparation method according to any one of claims 1 to 7 in the preparation of transplantation preparations for treating limbal stem cell deficiency.

Images