Primary culture method of limbal stem cells

Abstract

The invention relates to a primary culture method of limbal stem cells. It is characterized in that the limbal tissue treated with gentamycin solution is cut into pieces, then digested and filtered in the tissue digestive fluid, and the limbal tissue cell pellet obtained after the filtrate is centrifuged is temporarily frozen, and then the remaining filtrated The tissue block (i.e. filter residue) was resuspended in corneal limbal stem cell trophoblast culture medium, inoculated on the cell culture plate pretreated with coating solution for culture to obtain limbal fibroblasts, and then treated with mitomycin C solution to grow to Limbal stem cell feeder layer was made from limbal fibroblasts in the logarithmic phase. Finally, the short-term frozen tissue cells were recovered, resuspended in the primary culture medium of limbal stem cells, and seeded on the cells covered with limbal stem cell feeder layer. Primary cultures were carried out in culture plates. The limbal stem cells have high safety and good stability, and fully meet the high requirements of clinical treatment.

Description

technical field

[0001] The invention belongs to the technical field of stem cell culture, and in particular relates to a method for primary culture of limbal stem cells. Background technique

[0002] Studies have found that limbal stem cells are located in the unique wavy structure "Vogt fence" in the limbal basal layer, and are the source of corneal epithelial cell renewal. The continuous renewal of limbal stem cells can maintain the continuous horizontal centripetal and vertical upward movement of limbal epithelial cells, thereby ensuring the integrity of the corneal epithelial layer structure and normal function. Moreover, the proliferative pressure of limbal stem cells can inhibit the ingrowth of conjunctival epithelial cells and prevent the invasion of conjunctival blood vessels in the limbus. When various injury factors, such as chemical burn, mechanical injury, pathogenic microorganism infection, etc., will lead to the loss of corneal limbal stem cells or affect thei...

Examples

preparation example Construction

[0034] 5. Preparation of limbal stem cell trophoblast: resuspend the remaining tissue block (i.e., filter residue) after filtering with a 200-mesh cell sieve in corneal limbal stem cell trophoblast culture medium, and spread evenly on the cells pretreated with coating solution. In the culture plate, after the cells migrated out and covered the monolayer, conventional cell passage was carried out to obtain limbal fibroblasts, and then the limbal fibroblasts were inoculated in the cell culture plate pretreated by the coating solution. Grow to the logarithmic growth phase, suck out the culture medium, add mitomycin C solution, incubate at 37°C in the dark for 2 hours, discard the mitomycin C solution, rinse with sterile PBS balanced salt solution, and use it as the limbus stem cell trophoblast;

[0035] 6. Subsequent primary culture of limbal stem cells: routinely resuscitate the above-mentioned short-term frozen tissue cells, centrifuge at 500 rpm for 10 min, remove the supernat...

Embodiment 1

[0037] First wash the limbal tissue with sterile 0.9% (w / v) saline until there are no impurities, then take 10 ml DMEM / F12 (1:1) medium, add 10 mg gentamicin (potency 1000 U / mg ), was completely dissolved and sterilized by filtration with a 0.22 μm syringe filter, and then diluted to 100 ml with DMEM / F12 (1:1) medium to prepare a gentamicin solution. Then the cleaned limbal tissue was immersed in gentamicin solution, treated at 37°C for 10 min, then rinsed with sterile PBS balanced salt solution for 3 times, and rinsed with DMEM / F12 (1:1) medium for 3 times ;

[0038] Then take 10 ml of DMEM / F12 (1:1) medium, add 24 mg of Dispase II enzyme (enzyme activity 10 U / mg), completely dissolve it and filter it with a 0.22 μm syringe filter, then add 5 ml of fetal bovine Serum was finally diluted to 100 ml with DMEM / F12 (1:1) medium, and prepared as tissue digestion solution. Put the limbal tissue in 500 μl tissue digestion solution, and cut the tissue into a size of about 1 mm with ...

Embodiment 2

[0045] First wash the limbal tissue with sterile 0.9% (w / v) saline until there are no impurities, then take 10 ml DMEM / F12 (1:1) medium, add 10 mg gentamicin (potency 1000 U / mg ), was completely dissolved and sterilized by filtration with a 0.22 μm syringe filter, and then diluted to 100 ml with DMEM / F12 (1:1) medium to prepare a gentamicin solution. Then the cleaned limbal tissue was immersed in gentamicin solution, treated at 37°C for 10 min, then rinsed with sterile PBS balanced salt solution for 3 times, and rinsed with DMEM / F12 (1:1) medium for 3 times ;

[0046] Then take 10 ml of DMEM / F12 (1:1) medium, add 48 mg of Dispase II enzyme (enzyme activity 10 U / mg), completely dissolve, filter and sterilize with a 0.22 μm syringe filter, and then add 5 ml of fetal bovine Serum was finally diluted to 100 ml with DMEM / F12 (1:1) medium, and prepared as tissue digestion solution. Put the limbal tissue in 500 μl tissue digestion solution, and cut the tissue into a size of about 0...

technical field

[0001] The invention belongs to the technical field of stem cell culture, and in particular relates to a method for primary culture of limbal stem cells. Background technique

[0002] Studies have found that limbal stem cells are located in the unique wavy structure "Vogt fence" in the limbal basal layer, and are the source of corneal epithelial cell renewal. The continuous renewal of limbal stem cells can maintain the continuous horizontal centripetal and vertical upward movement of limbal epithelial cells, thereby ensuring the integrity of the corneal epithelial layer structure and normal function. Moreover, the proliferative pressure of limbal stem cells can inhibit the ingrowth of conjunctival epithelial cells and prevent the invasion of conjunctival blood vessels in the limbus. When various injury factors, such as chemical burn, mechanical injury, pathogenic microorganism infection, etc., will lead to the loss of corneal limbal stem cells or affect thei...

Examples

preparation example Construction

[0034] 5. Preparation of limbal stem cell trophoblast: resuspend the remaining tissue block (i.e., filter residue) after filtering with a 200-mesh cell sieve in corneal limbal stem cell trophoblast culture medium, and spread evenly on the cells pretreated with coating solution. In the culture plate, after the cells migrated out and covered the monolayer, conventional cell passage was carried out to obtain limbal fibroblasts, and then the limbal fibroblasts were inoculated in the cell culture plate pretreated by the coating solution. Grow to the logarithmic growth phase, suck out the culture medium, add mitomycin C solution, incubate at 37°C in the dark for 2 hours, discard the mitomycin C solution, rinse with sterile PBS balanced salt solution, and use it as the limbus stem cell trophoblast;

[0035] 6. Subsequent primary culture of limbal stem cells: routinely resuscitate the above-mentioned short-term frozen tissue cells, centrifuge at 500 rpm for 10 min, remove the supernat...

Embodiment 1

[0037] First wash the limbal tissue with sterile 0.9% (w / v) saline until there are no impurities, then take 10 ml DMEM / F12 (1:1) medium, add 10 mg gentamicin (potency 1000 U / mg ), was completely dissolved and sterilized by filtration with a 0.22 μm syringe filter, and then diluted to 100 ml with DMEM / F12 (1:1) medium to prepare a gentamicin solution. Then the cleaned limbal tissue was immersed in gentamicin solution, treated at 37°C for 10 min, then rinsed with sterile PBS balanced salt solution for 3 times, and rinsed with DMEM / F12 (1:1) medium for 3 times ;

[0038] Then take 10 ml of DMEM / F12 (1:1) medium, add 24 mg of Dispase II enzyme (enzyme activity 10 U / mg), completely dissolve it and filter it with a 0.22 μm syringe filter, then add 5 ml of fetal bovine Serum was finally diluted to 100 ml with DMEM / F12 (1:1) medium, and prepared as tissue digestion solution. Put the limbal tissue in 500 μl tissue digestion solution, and cut the tissue into a size of about 1 mm with ...

Embodiment 2

[0045] First wash the limbal tissue with sterile 0.9% (w / v) saline until there are no impurities, then take 10 ml DMEM / F12 (1:1) medium, add 10 mg gentamicin (potency 1000 U / mg ), was completely dissolved and sterilized by filtration with a 0.22 μm syringe filter, and then diluted to 100 ml with DMEM / F12 (1:1) medium to prepare a gentamicin solution. Then the cleaned limbal tissue was immersed in gentamicin solution, treated at 37°C for 10 min, then rinsed with sterile PBS balanced salt solution for 3 times, and rinsed with DMEM / F12 (1:1) medium for 3 times ;

[0046] Then take 10 ml of DMEM / F12 (1:1) medium, add 48 mg of Dispase II enzyme (enzyme activity 10 U / mg), completely dissolve, filter and sterilize with a 0.22 μm syringe filter, and then add 5 ml of fetal bovine Serum was finally diluted to 100 ml with DMEM / F12 (1:1) medium, and prepared as tissue digestion solution. Put the limbal tissue in 500 μl tissue digestion solution, and cut the tissue into a size of about 0...