Culture method and application of porcine corneal limbus epithelial stem cells

Abstract

The invention belongs to the technical field of cytobiology and regenerative medicine, and particularly relates to a porcine-derived corneal limbus epithelial stem cell culture method and application, during culture, pretreatment is firstly carried out, then a culture dish is coated, cells are cultured and passaged after centrifugation, dryness identification is carried out through cell immunofluorescence color, and the porcine-derived corneal limbus epithelial stem cells are obtained. The stem cell engineering vector can be applied to damage repair of corneal diseases including corneal limbal stem cell deficiency, during repair, the stem cell engineering vector is compounded with a biological material to form the stem cell engineering vector, the stem cell engineering vector is implanted into a damaged part, the separation and culture method is simple in step, the cell obtaining amount is large, and the stem cell engineering vector is suitable for being separated from eyeballs of a large animal-pig; the corneal limbus epithelial stem cells cultured in vitro can form cell clusters called stem cell islands, the obtained corneal limbus epithelial stem cells have phenotypes of BMI1 + and delta Np63 alpha +, and CK14 + and ABCG < 2 + >, and a new way can be provided for cell transplantation based on experimental research and clinical research.

Description

Technical field:

[0001] The present invention belongs to the technical field of cell biology and regenerative medicine, and specifically relates to a method and application of culturing porcine limbal epithelial stem cells to repair limbal stem cell deficiency

[0002] (LSCD) and other corneal diseases. Background technology:

[0003] Limbal stem cell deficiency (LSCD) is a complex disease that may lead to vision loss or even blindness. There are many methods for treating corneal blindness, for example, Chinese patents disclose.

[0004] Chinese Patent No. 202211138120.9 discloses a flexible piezoelectric corneal contact lens for repairing corneal damage, which comprises, from top to bottom: a first outer packaging film, a first inner packaging film, a piezoelectric sensing layer, an electrode pair, a second inner packaging film, and a second outer packaging film; the flexible piezoelectric corneal contact lens also includes a rectifier, which is used to electrically connect the piezoelectric sensing layer and the electrode pair; the piezoelectric sensing layer is used to convert the energy generated during blinking into a pulse voltage signal, and the rectifier is used to convert the piezoelectric sensing layer into a pulse voltage signal. The pulse voltage signal collected by the layer is converted into direct current and output to the electrode pair. The electrode pair is used to convert the direct current signal into an external enhanced electric field to enhance the endogenous electric field; the piezoelectric sensing layer is a piezoelectric electret film with a 12-pointed star-shaped snowflake structure. The piezoelectric electret film consists of three layers, namely a top electrode layer, an intermediate organic material layer and a bottom electrode layer, which constitute a flat plate capacitor; the pattern of the electrode pair is two concentric circles with a connecting line between the two concentric circles, and the circumference lines and connecting lines of the two circles are both serpentine lines. Chinese Patent 202010496058.5 discloses an eye drop suitable for limbal stem cell deficiency, comprising the following components: human adipose stem cell exosomes 1-40 mg / L, excipients: sodium hyaluronate 0.5-2 g / L, vitamin B6 0.5-3 g / L, benzalkonium chloride 0.05-0.3 g / L, the balance is medical saline, the pH is 6.5-7.5, and the preparation method comprises the following steps: (1) selecting 3-5 generations of human adipose stem cells, culturing the cells to 70%-80% fusion using stem cell culture medium, replacing it with stem cell serum-free culture medium, and continuing to culture the stem cells for 36h-48h, collecting the culture supernatant, extracting human adipose stem cell exosomes, and resuspending the exosomes to obtain a human adipose stem cell exosome solution; (2) mixing the human adipose stem cell exosome solution prepared in step (1) with other components according to the concentration of the exosome component of the eye drops of claim 1, and adjusting the pH to 6.5-7.5, the eye drops are prepared, in step (1), the exosomes are extracted by ultraspeed gradient centrifugation, and the specific centrifugal force is selected as 300×g, 20min, and the supernatant is retained; 10000×g, 30min, and the supernatant is retained; 100000×g, 1h, the supernatant is removed, and the precipitate is human adipose stem cell exosomes, and in step (2), according to the components of the eye drops of claim 1, sodium hyaluronate is dissolved in medical saline, and then vitamin B6 and benzalkonium chloride are dissolved in turn, the pH is adjusted to 6.5-7.5, and then the human adipose stem cell exosome solution prepared in step (1) is added at a corresponding concentration, and the eye drops are prepared by mixing evenly. However, the therapeutic effects of the above schemes are not ideal.Actual research has found that human donor corneal transplantation, which relies on healthy donor corneas, exquisite surgical techniques and advanced medical equipment, is an effective method for treating corneal blindness. However, the severe shortage of donor corneas limits the popularity of this treatment method, as one donor cornea can only meet the needs of about 70 patients. In addition, allogeneic transplants have the risk of immune rejection and infection, especially in patients with inflammation and severe corneal lesions.

[0005] Studies have shown that LSCD requires specialized stem cell therapy to restore eye health and vision, and cannot rely solely on traditional donor corneal transplants. The limbus contains a group of self-renewing stem cells, called limbal epithelial stem / progenitor cells (LSC), which are responsible for maintaining the integrity of the corneal surface and the continuous renewal of the corneal epithelium. Under steady state, LSC maintains a slow cell cycle, but after corneal injury, limbal homeostasis is disturbed by stimulating factors and LSC begins to proliferate. LSC can proliferate through symmetric division and asymmetric division. In asymmetric division, LSC divides to produce a daughter stem cell to replenish the stem cell pool, as well as a progenitor cell that has not yet differentiated but has limited proliferation potential. Under normal physiological conditions, LSC divides and proliferates, then migrates to the central cornea and produces terminally differentiated cells, which eventually fall off the corneal surface due to biological functions such as blinking. LSCs are supported and regulated by the limbal niche, which is a highly controlled microenvironment of the human eye. When LSCs are lost or the limbal niche is destroyed, resulting in a decrease in the number of LSCs or loss of function, it may cause limbal barrier destruction, delayed corneal epithelial wound healing, corneal neovascularization, inflammatory infiltration and scar formation. Based on this, the key to treating LSCD is to restore corneal health and function by supplementing limbal epithelial stem cells to rebuild the limbal niche.

[0006] At present, the study of LSC is limited by the lack of specific markers. Although people have been looking for specific markers that can directly identify LSC, since these markers have been found to be expressed in central corneal epithelial cells and conjunctiva in recent years, no one or a group of absolutely specific marker molecules for LSC has been found so far. This limitation means that it is impossible to accurately identify corneal epithelial stem cells through existing markers, which poses a challenge to research fields such as the isolation, culture and transplantation of limbal stem cells. Currently, the identification of LSC is through the combined labeling of multiple relatively specific markers distributed in different cell locations. Summary of the invention:

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and to develop a porcine limbal epithelial stem cell culture method and application to repair cells of corneal diseases such as limbal stem cell deficiency (LSCD).

[0008] In order to achieve the above-mentioned purpose, the specific process of a method for culturing porcine limbal epithelial stem cells according to the present invention is as follows:

[0009] (1) Pretreatment of tissue

[0010] First, in a biosafety cabinet, the pig eyeball tissue was immersed in a pre-cooled PBS solution (phosphate buffered saline) containing 10% antibiotics and pH = 7.4, and rinsed three times, each time for 3 to 5 minutes;

[0011] Then, cut the cornea, remove the iris attached to the cornea, remove the limbal ring, place it in a dispase II solution with a concentration of 1.2-2U / ml, and digest it in a refrigerator at a temperature of 4°C for 14-16 hours;

[0012] Finally, the epithelial cell layer of the limbal ring was separated and transferred to an incubator at 37°C and digested with trypsin for 5 min;

[0013] Among them, dispase II is a neutral protease that can act precisely on the basement membrane area, destroy the corneal elastic layer, and achieve rapid, effective and gentle separation of the epithelium and cornea, while maintaining the integrity of epithelial cells. It is highly stable to the interference of temperature, pH value and serum components, and has little effect on the epithelial cells themselves, thus separating the epithelial cells from the matrix without damaging the epithelial cells.

[0014] (2) Coated culture dish

[0015] First, laminin (recombinant LN521, BIOLAMINA) was diluted to 5ug / ml laminin coating solution with 1× DPBS (Dubrich's phosphate buffered saline) containing Ca++ and Mg++;

[0016] Then, add 2 ml of laminin coating solution to the culture dish to be coated;

[0017] Finally, seal the culture dish and incubate overnight at 2-8°C;

[0018] Among them, the coating concentration of LN521 coated culture dish is 0.5-1ug / cm 2 ;

[0019] (3) Initial centrifugation

[0020] First, the suspension was dispersed by pipetting until no obvious cell pieces were left, and further digestion was stopped by adding complete stem cell culture medium, followed by centrifugation at 1000 g for 5 min;

[0021] Then, discard the supernatant, add 1 ml of stem cell complete medium, pipette and mix, filter the cell suspension, and adjust the cell density to 1×106 / ml;

[0022] Finally, the cells were inoculated into a culture dish (LN521 coated) with complete stem cell culture medium and incubated in a humidified atmosphere at 37.0°C and 5% CO2 saturation;

[0023] (4) Cultivate cells and pass them on to future generations

[0024] When the cell density reaches 70-80%, preheat the stem cell complete medium, 0.25% trypsin and PBS solution to 37°C, remove the old stem cell complete medium, rinse the cells twice with PBS solution, and add trypsin for digestion;

[0025] After the cells shrink and disperse, add stem cell complete medium to terminate digestion, transfer the cells to an Eppendorf tube, centrifuge at 1000g for 5 minutes, discard the supernatant, resuspend the cells with stem cell complete medium, take 1 / 2 and add to a new culture dish, mix well and place in a CO2 incubator for culture. The obtained cells are the first generation cells (P1);

[0026] The cells obtained by subculture are the second generation cells (P2). Similarly, the inoculation concentration of subcultured cells is 0.5×10 4 / cm 2 -1.0×10 4 / cm 2 ;

[0027] The culture conditions of the CO2 incubator are: temperature of 37°C, CO2 saturation of 5%, and humidity of 95%.

[0028] The antibiotics involved in the present invention are antibiotics containing 100U / mL of penicillin and 100μg / mL of streptomycin, referred to as double antibiotics;

[0029] Complete stem cell culture medium contains 5% hematoxylin and eosin Cell culture supplements, Dakota) and 1% antibiotics in MSCBM (human mesenchymal stem cell basal medium, Dakota).

[0030] The present invention relates to a porcine limbal epithelial stem cell, which is identified by cell immunofluorescence. The specific process of the identification is as follows:

[0031] First, place the cell slides in a six-well plate, add 1 ml of 5ug / ml laminin coating solution to each well, seal the plate, and incubate overnight at 2-8°C.

[0032] Then, the first-generation cells and the second-generation cells were subjected to standard digestion treatment, and the obtained cell suspension was transferred to a well plate.

[0033] Place the well plate in a humidified environment at 37.0°C and 5% CO2 saturation for 12-24 hours to allow the cell coverage to reach 60-70%;

[0034] Finally, four relatively specific antibodies were selected: BMI1 (CST, 6964T) and ABCG2 (Santa Cruz Animal Health, sc-18841), ΔNp63α (CST, 13109T) and CK14 (Wuhan Mitsubishi, 60320-1-IG), and paired in pairs for double immunofluorescence staining to identify stem cell markers;

[0035] Among them, BMI1 is highly expressed in the basal cells of the limbal epithelium and is a specific biomarker molecule for identifying LSCs; ABCG2 is a relatively specific marker for limbal stem cells; keratin K14 often exists as a dimer with other keratins and is expressed in the basal cells of the limbal epithelium, serving as a positive marker molecule for LSCs; ΔNp63α is located in the limbus and is not expressed in corneal epithelial cells, and is a relatively specific marker currently widely used; DAPI is used to mark cell nuclei.

[0036] The porcine limbal epithelial stem cells involved in the present invention are used for repairing damage to corneal diseases including limbal stem cell deficiency, and the repair method is simple and easy: they are combined with biomaterials to form a stem cell engineering carrier and implanted into the damaged part for regeneration and repair.

[0037] Compared with the prior art, the present invention is suitable for isolation from the eyeball of a large animal, a pig. The isolation and culture method has simple steps and a large amount of cells can be obtained. The limbal epithelial stem cells cultured in vitro can form cell clusters, called stem cell islands. The obtained limbal epithelial stem cells have the phenotypes of BMI1+ and ΔNp63α+, as well as CK14+ and ABCG2+, and can be applied to experimental research and clinical research, providing a new approach for cell transplantation. Description of the drawings:

[0038] Figure 1 The present invention is a schematic diagram of separating pig cornea and pig eyeball in a biological safety cabinet.

[0039] Figure 2 It is a schematic diagram of the corneal limbal ring obtained after peeling off the central cornea under a stereo microscope involved in the present invention.

[0040] Figure 3 This is a schematic diagram of the growth status of the P0 generation cells involved in the present invention after being cultured in a CO2 incubator, wherein A is the first day and B is the third day.

[0041] Figure 4This is a schematic diagram of the growth status of the P1 generation cells involved in the present invention after being cultured in a CO2 incubator, wherein A is the 3rd day and B is the 7th day.

[0042] Figure 5 The schematic diagram of the results of double immunofluorescence staining for labeling LSC-specific marker molecules involved in the present invention, wherein the upper left figure is a schematic diagram of cell immunofluorescence staining of cytokeratin 14 (CK14); the upper right figure is a schematic diagram of cell immunofluorescence staining of the ΔNp63α subtype in the ΔNp63 (ΔNp63α, β and γ) lacking the N-terminal domain in the cell cycle regulatory factor p63 family; the lower left figure is a schematic diagram of DAPI nuclear staining labeling; the lower right figure is a schematic diagram of the co-localization of CK14, ΔNp63α and DAPI.

[0043] Figure 6 It is a schematic diagram of the results of double immunofluorescence staining to label LSC-specific marker molecules involved in the present invention, wherein the upper left figure is a schematic diagram of cell immunofluorescence staining of the polycomb gene BMI1; the upper right figure is a schematic diagram of cell immunofluorescence of ATP-binding cassette transporter family member ABCG2; the lower left figure is a schematic diagram of DAPI nuclear staining labeling; the lower right figure is a schematic diagram of the co-localization of BMI1, ABCG2 and DAPI. Specific implementation method:

[0044] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0045] Embodiment 1:

[0046] The specific process of a method for culturing porcine limbal epithelial stem cells involved in this embodiment is as follows:

[0047] (1) Fresh intact porcine eyeball tissue obtained from a local slaughterhouse was immediately placed in D-Hank's buffer at 4°C and transported to a sterile laboratory within 1-2 hours;

[0048] (2) Place the pig eyeball tissue in a biosafety cabinet and wash the blood stains with pre-cooled PBS solution containing 10% double antibody. Repeat the washing three times, each time for 3-5 minutes;

[0049] (3) Fix the eyeball of the pig and use ophthalmic scissors sterilized in an autoclave to cut the complete cornea along the limbus of the cornea and sclera. Figure 1 As shown, the iris was placed in a sterile PBS solution or normal saline containing 10% double antibody, and the iris attached to the cornea was removed by ophthalmic forceps;

[0050] The cornea is composed of five layers. After removing the anterior and posterior layers of the Descemet's membrane and the endothelium, the epithelial cell layer and the stroma layer constitute the main part of the cornea. The corneal stroma layer accounts for 90% of the total thickness of the cornea, while the corneal epithelium is relatively thin, accounting for only 10% of the total thickness of the cornea.

[0051] (4) Under a stereo microscope, use ophthalmic scissors to make a small cut at the corneal scleral edge of the cornea, and then peel off the complete central cornea along the grayish-white area inside the corneal scleral edge to obtain the following Figure 2 Limbal ring shown;

[0052] Dispase II solution was prepared using Hepe buffered saline (50 mM Hepes / KOH pH 7.4, 150 mM NaCl) and diluted to 1.2-2 U / ml with complete stem cell culture medium;

[0053] Immerse the limbal ring in the diluted dispase II solution and digest it in a refrigerator at 4°C for 14-6 hours;

[0054] (5) Place the corneal limbal ring with the front side facing up in a culture dish containing complete stem cell culture medium. With the assistance of a stereomicroscope, use ophthalmic forceps to separate the epithelial cell layer of the corneal limbal ring (presenting a transparent film-like structure with strong refractive properties), place it in an incubator at a temperature of 37° C., and digest it with 0.25% trypsin for 5 minutes.

[0055] (6) Thaw laminin (LN521) in a refrigerator at 4°C in advance, and dilute it with 1× DPBS containing Ca++ and Mg++ to make a 5 ug / ml laminin coating solution (LN521 solution);

[0056] Depending on the size of the culture dish, add 1-3 ml of LN521 solution (0.5-1 ug / cm 2 ), seal the culture dish to prevent evaporation of the LN521 solution and external contamination, and incubate overnight at a temperature of 2-8°C to ensure that the laminin evenly covers the surface of the culture dish. At the same time, avoid drying the surface of the culture dish to prevent inactivation of the laminin;

[0057] When ready to use, aspirate the excess LN521 solution in the culture dish and add 1 ml of stem cell complete medium in advance to prevent the surface of the culture dish from drying out;

[0058] (7) Use a pipette to gently pipette the cell suspension after trypsin digestion until no cell sheets are visible to the naked eye;

[0059] Complete stem cell medium was added to terminate digestion, and the mixture was centrifuged at 1000 g for 5 min;

[0060] Discard the supernatant, add 1-2 ml of stem cell complete medium, and mix well by pipetting;

[0061] Filter the cell suspension through a 200-mesh cell strainer and adjust the cell density to 1 × 10 6 / ml, inoculated into a culture dish pre-coated with LN521 solution (containing 1ml of stem cell complete medium), and incubated at a temperature of 37.0°C, a CO2 saturation of 5%, and a humidity of 95%;

[0062] On the second day after inoculation, closely observe the cell adhesion status and replace with fresh stem cell complete medium every 2-3 days;

[0063] (8) On the third day after culture, the cells reached 85-90% fusion. Figure 3 As shown in B, 1 ml of 0.25% trypsin was used to digest the cells attached to the bottom of the culture dish for 5 min. When the cells retracted and dispersed under a microscope, an equal amount of stem cell complete medium was immediately added to terminate the digestion. The cells were transferred to an EP tube and centrifuged at 1000 g for 5 min.

[0064] Discard the supernatant, add stem cell complete medium to resuspend the cells, take 1 / 2 and add it to a new culture dish, shake gently to mix;

[0065] Place in a CO2 incubator and culture. After 12-24 hours, observe the cell status under an inverted microscope. If bacterial contamination is observed, discard it. If there is no contamination, continue to culture.

[0066] Continue to culture. After 2-5 days, it is observed that the cells gradually form stem cell islands, which are clustered and evenly distributed. After 6-10 days, it is observed that the cell clusters gradually extend to the surroundings, the cell state gradually relaxes, and the cell clusters gradually form cell sheets. Then the cell sheets fuse into a complete cell layer, which is paved with pebbles. After the cells reach 80-90% fusion, they are digested and centrifuged with trypsin, and resuspended to obtain the first generation of cells (P1), which are transferred to another new culture dish. In this way, the third generation of cells (P3) are obtained by subculturing, and they still maintain a strong proliferation ability.

[0067] The cells of the P0 generation Figure 3 As shown, Figure 3 A is the growth status on the first day, Figure 3 B is the growth state on the 3rd day, showing a tendency to form cell clusters; the cells of the P1 generation are as follows Figure 4 As shown, Figure 4 A is the growth state on the third day, forming evenly distributed stem cell islands. Figure 4 B is the growth state on the 7th day, with vigorous proliferation and the formation of a cell layer that looks like paving pebbles.

[0068] Embodiment 2:

[0069] The specific process flow for phenotypic identification of porcine limbal epithelial stem cells involved in this embodiment is as follows:

[0070] (1) Place the cell slide in a six-well plate, add 1 ml of 5 ug / ml LN521 solution to each well, seal the plate, and incubate overnight at 2-8°C;

[0071] (2) Take P1 cells and P2 cells and inoculate them onto cell slides coated with LN521 solution;

[0072] (3) When the cell density reaches 60-70%, remove the excess stem cell complete medium in the well plate, add 1-2 ml of 4% paraformaldehyde, seal the well plate, let it stand at room temperature for 20 minutes, and wash it three times with PBS solution, each time for 3 minutes;

[0073] (4) Permeabilize with 0.2% Triton X-100 for 5 min at room temperature and wash with PBS solution three times, 3 min each time;

[0074] (5) After blocking for 30 minutes, specific primary antibodies (BMI1, ΔNp63α, CK14, ABCG2) were added and incubated overnight at 4°C;

[0075] (6) Add fluorescently labeled secondary antibody and incubate for 1 hour in dark conditions;

[0076] (7) Use DAPI for nuclear staining and let stand at room temperature for 10 minutes;

[0077] (8) Add 20 μL of anti-fluorescence quencher to seal the slide, observe under a fluorescence microscope, and collect images.

Claims

1. A method for culturing porcine limbal epithelial stem cells, characterized in that: The process is as follows: (1) Pretreatment of tissue First, in a biosafety cabinet, the porcine eyeball tissue was immersed in a pre-cooled PBS solution containing 10% antibiotics and pH = 7.4 and rinsed; Then, cut the cornea, remove the iris attached to the cornea, remove the limbal ring, place it in a dispase II solution with a concentration of 1.2-2U / ml, and digest it in a refrigerator at a temperature of 4°C for 14-16 hours; Finally, the epithelial cell layer of the limbal ring was separated and transferred to an incubator at 37°C and digested with trypsin for 5 min; (2) Coated culture dish First, laminin was diluted into 5ug / ml laminin coating solution with 1× DPBS containing Ca++ and Mg++; Then, add 2 ml of laminin coating solution to the culture dish to be coated; Finally, seal the culture dish and incubate overnight at 2-8°C; (3) Initial centrifugation First, the cells were dispersed by pipetting until there were no obvious cell pieces in the suspension, and then digestion was terminated by centrifugation at 1000 g for 5 minutes; Then, discard the supernatant, add 1 ml of stem cell complete medium, pipette and mix well, and adjust the cell density to 1×10 6 / ml; Finally, the cells were inoculated into a culture dish with complete stem cell culture medium and incubated in a humidified atmosphere at 37.0°C and 5% CO2 saturation; (4) Cultivate cells and pass them on to future generations When the cell density reaches 70-80%, preheat the stem cell complete medium, 0.25% trypsin and PBS solution to 37°C, remove the old stem cell complete medium, rinse the cells with PBS solution, and add trypsin for digestion; After the cells shrink and disperse, stop digestion and transfer the cells to an EP tube. Centrifuge at 1000g for 5 minutes, discard the supernatant, resuspend the cells, take 1 / 2 and add to a new culture dish. Mix well and place in a CO2 incubator for culture. The obtained cells are the first generation cells. The cells obtained by subculture are the second generation cells. Similarly, the inoculation concentration of subculture cells is 0.5×10 4 / cm 2 -1.0×10 4 / cm 2 .

2. The method for culturing porcine limbal epithelial stem cells according to claim 1, characterized in that: The coating concentration of the LN521 coated culture dish involved in step (2) is 0.5-1ug / cm 2 The culture conditions of the CO2 incubator involved in step (4) are: temperature of 37°C, CO2 saturation of 5%, and humidity of 95%.

3. The method for culturing porcine limbal epithelial stem cells according to claim 1, characterized in that: The antibiotics were antibiotics containing 100 U / mL of penicillin and 100 μg / mL of streptomycin, referred to as double antibiotics; the complete stem cell culture medium was MSCBM containing 5% heparin and 1% antibiotics.

4. The method for culturing porcine limbal epithelial stem cells according to claim 1, characterized in that: The specific process is as follows: (1) Place the porcine eyeball tissue obtained from the slaughterhouse in D-Hank's buffer at 4°C and transport it to a sterile laboratory within 1-2 hours; (2) Place the pig eyeball tissue in a biosafety cabinet and wash the blood stains with pre-cooled PBS solution containing 10% double antibody. Repeat the washing three times, each time for 3-5 minutes; (3) Fix the eyeball of the pig eye tissue, cut the complete cornea along the corneoscleral limbus using ophthalmic scissors sterilized in an autoclave in advance, place it in a sterile PBS solution or physiological saline containing 10% double antibody, and remove the iris attached to the cornea using ophthalmic forceps; (4) Under a stereo microscope, use ophthalmic scissors to make a small cut at the corneal scleral edge of the cornea, and then peel off the complete central cornea along the grayish-white area on the inner side of the corneal scleral edge; Prepare dispase II solution using Hepe buffered saline and dilute to 1.2-2 U / ml with complete stem cell culture medium; Immerse the limbal ring in the diluted dispase II solution and digest it in a refrigerator at 4°C for 14-6 hours; (5) placing the corneal limbal ring with the front side facing up in a culture dish containing complete stem cell culture medium, using ophthalmic forceps to separate the epithelial cell layer of the corneal limbal ring with the assistance of a stereomicroscope, placing it in an incubator at a temperature of 37° C., and digesting it with 0.25% trypsin for 5 minutes; (6) Thaw the laminin in a refrigerator at 4°C in advance and dilute it with 1× DPBS containing Ca++ and Mg++ to make a 5 ug / ml laminin coating solution; Depending on the size of the culture dish, add 1-3 ml of LN521 solution to the culture dish to be coated, seal the dish, and incubate overnight at 2-8°C; When ready to use, aspirate the excess LN521 solution in the culture dish and add 1 ml of stem cell complete medium in advance; (7) Use a pipette to gently pipette the cell suspension after trypsin digestion until no cell sheets are visible to the naked eye; Complete stem cell medium was added to terminate digestion, and the mixture was centrifuged at 1000 g for 5 min; Discard the supernatant, add 1-2 ml of stem cell complete medium, and mix by pipetting; Filter the cell suspension through a 200-mesh cell strainer and adjust the cell density to 1 × 10 6 / ml, inoculated into a culture dish pre-coated with LN521 solution, and incubated at 37.0°C, 5% CO2 saturation, and 95% humidity; Replace the stem cell complete medium every 2-3 days; (8) On the third day after culture, when the cells reached 85-90% fusion, 1 ml of 0.25% trypsin was used to digest the cells attached to the bottom of the culture dish for 5 min. When the cells began to shrink and disperse under a microscope, an equal amount of stem cell complete medium was added to terminate the digestion. The cells were transferred to an EP tube and centrifuged at 1000 g for 5 min. Discard the supernatant, add stem cell complete medium to resuspend the cells, take 1 / 2 and add it to a new culture dish, mix well; Place in a CO2 incubator and culture. After 12-24 hours, observe the cell status under an inverted microscope. If bacterial contamination is observed, discard it. If there is no contamination, continue to culture. Continue to culture. After 2-5 days, the cells are observed to gradually form stem cell islands, which are clustered and evenly distributed. After 6-10 days, the cell clusters are observed to gradually extend to the surroundings, the cell state gradually relaxes, and the cell clusters gradually form cell sheets. Then the cell sheets fuse into a complete cell layer, which is paved with pebbles. After the cells reach 80-90% fusion, they are digested and centrifuged with trypsin, resuspended to obtain the first generation of cells, and transferred to another new culture dish. The second generation of cells is obtained by subculture in this way.

5. The method for culturing porcine limbal epithelial stem cells according to any one of claims 1 to 4, characterized in that: Porcine limbal epithelial stem cells are used in the repair of corneal diseases including limbal stem cell deficiency.

6. The method for culturing porcine limbal epithelial stem cells according to claim 5, characterized in that: During repair, porcine corneal limbal epithelial stem cells are combined with biomaterials to form a stem cell engineering carrier and implanted into the damaged area.

7. The method for culturing porcine limbal epithelial stem cells according to any one of claims 1 to 4, characterized in that: The porcine corneal limbal epithelial stem cells were identified by cell immunofluorescence. The identification process is as follows: First, place the cell slides in a six-well plate, add 1 ml of 5ug / ml laminin coating solution to each well, seal the plate, and incubate overnight at 2-8°C. Then, the first-generation cells and the second-generation cells were subjected to standard digestion treatment, and the obtained cell suspension was transferred to a well plate. Place the well plate in a humidified environment at 37.0°C and 5% CO2 saturation for 12-24 hours to allow the cell coverage to reach 60-70%; Finally, four relatively specific antibodies were selected: BMI1 and ABCG2, ΔNp63α and CK14, and paired in pairs for double immunofluorescence staining to identify stem cell markers.

8. The method for culturing porcine limbal epithelial stem cells according to claim 7, characterized in that: The specific process of phenotypic identification is as follows: (1) Place the cell slide in a six-well plate, add 1 ml of 5 ug / ml LN521 solution to each well, seal the plate, and incubate overnight at 2-8°C; (2) Take P1 cells and P2 cells and inoculate them onto cell slides coated with LN521 solution; (3) When the cell density reaches 60-70%, aspirate the excess stem cell complete medium in the well plate, add 1-2 ml of 4% paraformaldehyde, seal the well plate, let it stand at room temperature for 20 minutes, and wash with PBS solution; (4) Permeabilize with 0.2% Triton X-100 for 5 min at room temperature and wash with PBS solution; (5) After blocking for 30 minutes, add specific primary antibody and incubate overnight at 4°C; (6) Add fluorescently labeled secondary antibody and incubate for 1 hour in dark conditions; (7) Use DAPI for nuclear staining and let stand at room temperature for 10 minutes; (8) Add 20 μL of anti-fluorescence quencher to seal the slide, observe under a fluorescence microscope, and collect images.

Images