Limbal stem cell primary culturing method

Abstract

The invention relates to a limbal stem cell primary culturing method. The limbal stem cell primary culturing method is characterized by comprising shredding limbal tissues processed with gentamicin solution, then digesting and filtering the shredded tissues in tissue digestion solution, centrifuging the filter liquor to obtain limbal tissue cell precipitates for temporary frozen storage, resuspending filtered tissue blocks, namely filter residues, through limbal stem cell trophoderm culture solution and inoculating the mixture onto cell culture plates preprocessed with coating liquid to obtainlimbal fibroblasts, processing the limbal fibroblasts during logarithmic growth through mitomycin C solution to obtain a limbal stem cell trophoderm, recovering and resuspending the temporarily frozen tissue cells through limbal stem cell primary culture solution, and inoculating the resuspended tissue cells onto the cell culture plates spread with the limbal stem cell trophoderm to obtain limbalstem cells. The cultured limbal stem cells are high in safety and stability and well meets the high requirements of clinical treatment.

Description

Technical field

[0001] The invention belongs to the technical field of stem cell culture, and specifically 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 basal layer of the limbus, and they are the source of corneal epithelial cell renewal. The continuous renewal of limbal stem cells can maintain the continuous horizontal and vertical upward movement of limbal epithelial cells, thereby ensuring the integrity and normal function of the corneal epithelial layer. Moreover, the proliferation pressure of limbal stem cells can inhibit the growth of conjunctival epithelial cells and prevent the invasion of conjunctival blood vessels in the limbus. When various damage factors, such as chemical burns, mechanical damage, pathogenic microbial infections, etc., will cause the loss of limbal stem cells or affect their survival microenvironment an...

Examples

preparation example Construction

[0034] 5. Preparation of limbal stem cell trophoblast: Resuspend the remaining tissue mass (ie filter residue) after filtering with a 200-mesh cell sieve in limbal stem cell trophoblast culture medium, and spread it evenly on the cells pretreated with the coating solution In the culture plate, after the cells have migrated out and grown into a monolayer, perform conventional cell passage to obtain limbal fibroblasts, and then inoculate limbal fibroblasts into the cell culture plate pretreated with the coating solution. It grows to the logarithmic growth phase, aspirate the culture solution, add mitomycin C solution, incubate at 37°C for 2 h in the dark, discard the mitomycin C solution, and rinse with sterile PBS balanced salt solution as the limbus Stem cell trophoblast;

[0035] 6. The subsequent primary culture of limbal stem cells: the above-mentioned transiently frozen tissue cells are subjected to routine recovery, centrifuged at 500 rpm for 10 minutes, and the supernatant ...

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 (titer 1000 U / mg ), after complete dissolution, filter and sterilize with a 0.22 μm syringe filter, and then dilute 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 3 times, and DMEM / F12 (1:1) medium rinsed 3 times ;

[0038] Then take 10 ml DMEM / F12 (1:1) medium, add 24 mg Dispase II enzyme (enzyme activity 10 U / mg), completely dissolve it, filter and sterilize it with a 0.22 μm syringe filter, and then add 5 ml fetal cattle Serum, finally dilute to 100 ml with DMEM / F12 (1:1) medium, and prepare tissue digestion solution. Place the limbal tissue in 500 μl tissue digestion solution, and cut the tissue into a size of about 1 mm with ophthalmic scissors 3 Ad...

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 (titer 1000 U / mg ), after complete dissolution, filter and sterilize with a 0.22 μm syringe filter, and then dilute 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 3 times, and DMEM / F12 (1:1) medium rinsed 3 times ;

[0046] Then take 10 ml DMEM / F12 (1:1) medium, add 48 mg Dispase II enzyme (enzyme activity 10 U / mg), completely dissolve it, filter and sterilize with a 0.22 μm syringe filter, and then add 5 ml fetal cattle Serum, finally dilute to 100 ml with DMEM / F12 (1:1) medium, and prepare tissue digestion solution. Place the limbal tissue in 500 μl tissue digestion solution, and cut the tissue into a size of about 0.5 mm with ophthalmic scissors 3 Add...

Technical field

[0001] The invention belongs to the technical field of stem cell culture, and specifically 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 basal layer of the limbus, and they are the source of corneal epithelial cell renewal. The continuous renewal of limbal stem cells can maintain the continuous horizontal and vertical upward movement of limbal epithelial cells, thereby ensuring the integrity and normal function of the corneal epithelial layer. Moreover, the proliferation pressure of limbal stem cells can inhibit the growth of conjunctival epithelial cells and prevent the invasion of conjunctival blood vessels in the limbus. When various damage factors, such as chemical burns, mechanical damage, pathogenic microbial infections, etc., will cause the loss of limbal stem cells or affect their survival microenvironment an...

Examples

preparation example Construction

[0034] 5. Preparation of limbal stem cell trophoblast: Resuspend the remaining tissue mass (ie filter residue) after filtering with a 200-mesh cell sieve in limbal stem cell trophoblast culture medium, and spread it evenly on the cells pretreated with the coating solution In the culture plate, after the cells have migrated out and grown into a monolayer, perform conventional cell passage to obtain limbal fibroblasts, and then inoculate limbal fibroblasts into the cell culture plate pretreated with the coating solution. It grows to the logarithmic growth phase, aspirate the culture solution, add mitomycin C solution, incubate at 37°C for 2 h in the dark, discard the mitomycin C solution, and rinse with sterile PBS balanced salt solution as the limbus Stem cell trophoblast;

[0035] 6. The subsequent primary culture of limbal stem cells: the above-mentioned transiently frozen tissue cells are subjected to routine recovery, centrifuged at 500 rpm for 10 minutes, and the supernatant ...

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 (titer 1000 U / mg ), after complete dissolution, filter and sterilize with a 0.22 μm syringe filter, and then dilute 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 3 times, and DMEM / F12 (1:1) medium rinsed 3 times ;

[0038] Then take 10 ml DMEM / F12 (1:1) medium, add 24 mg Dispase II enzyme (enzyme activity 10 U / mg), completely dissolve it, filter and sterilize it with a 0.22 μm syringe filter, and then add 5 ml fetal cattle Serum, finally dilute to 100 ml with DMEM / F12 (1:1) medium, and prepare tissue digestion solution. Place the limbal tissue in 500 μl tissue digestion solution, and cut the tissue into a size of about 1 mm with ophthalmic scissors 3 Ad...

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 (titer 1000 U / mg ), after complete dissolution, filter and sterilize with a 0.22 μm syringe filter, and then dilute 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 3 times, and DMEM / F12 (1:1) medium rinsed 3 times ;

[0046] Then take 10 ml DMEM / F12 (1:1) medium, add 48 mg Dispase II enzyme (enzyme activity 10 U / mg), completely dissolve it, filter and sterilize with a 0.22 μm syringe filter, and then add 5 ml fetal cattle Serum, finally dilute to 100 ml with DMEM / F12 (1:1) medium, and prepare tissue digestion solution. Place the limbal tissue in 500 μl tissue digestion solution, and cut the tissue into a size of about 0.5 mm with ophthalmic scissors 3 Add...