Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Limbal stem cell primary culturing method

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

Active Publication Date: 2019-03-08
OCEAN UNIV OF CHINA
View PDF4 Cites 4 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the limbal stem cells obtained by the existing method are prone to differentiation, and the properties of the limbal stem cells are lost to a considerable extent, which leads to the risk of limbal insufficiency after transplantation

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

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...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N5/0797
CPCC12N5/0621C12N5/0623C12N2500/25C12N2501/11C12N2501/115C12N2501/2306C12N2501/30C12N2533/52
Inventor 徐彬樊廷俊田成磊郑明月
Owner OCEAN UNIV OF CHINA
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com