Primary culture method of limbal stem cells
A corneal limbal stem cell and primary culture technology, applied in the field of stem cell culture, can solve the problems of limbal insufficiency, limbal stem cells prone to differentiation, loss of limbal stem cells, etc., achieving no risk of disease, high safety, Guaranteed effect of stability
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[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...
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