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Corneal limbal stem cell culture medium, and culture method

A technology of corneal limbal stem cells and culture medium, which is applied in the field of corneal limbal stem cell culture medium and its cultivation, can solve the problems of affecting the light and visual sensitivity of corneal limbal stem cells, the inability to stably separate and cultivate corneal limbal stem cells, and opacity. Good sex and proliferation ability, improved purity and stability, and reduced damage

Active Publication Date: 2017-07-04
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although the existing methods of culturing limbal stem cells can obtain epithelial cells, because NIH 373 cells are exogenous non-human cells, it is easy to cause immune reactions and the spread of mouse-derived pathogens; human amnion is difficult to obtain and consumes a lot of money. Need to further test whether it contains HIV, hepatitis virus, etc., and it is opaque and has a certain thickness. It is easy to integrate with the corneal stroma and has a certain impact on the thickness and structure of the cornea.
The above factors will affect the light sensitivity and visual sensitivity of the cultured limbal stem cells after transplantation, and the existing methods can only be used for one-time use of limbal stem cells, and cannot be used for stable isolation and culture of limbal stem cells

Method used

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  • Corneal limbal stem cell culture medium, and culture method
  • Corneal limbal stem cell culture medium, and culture method
  • Corneal limbal stem cell culture medium, and culture method

Examples

Experimental program
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Embodiment 1

[0039] Limbal stem cell culture medium: 220mL DMEM, 220mL DMEM / F12, 50mL FBS, 5mL 100× double antibody (100IU penicillin, 100ug / ml streptomycin), 1mL human recombinant EGF stock solution, 1mL insulin stock solution, 1mL 3,3 ´, 5-Triiodo-L-Thyronine stock solution, 1mL hydrocortisone stock solution, 1mL cholera toxin stock solution, stored at 4°C, rewarmed at 37°C before use.

[0040] Under the operating microscope, use tissue forceps and corneal scissors to scissor the human corneal limbal tissue, wash it twice with PBS containing double antibody (penicillin-streptomycin, 1×), 5 min each time; Tissue shredded.

[0041] According to the volume of the tissue block, every 1cm 3 Add 5ml 0.2 wt% IV collagenase solution to the tissue block, gently shake and digest at 37°C for 2h, remove the IV collagenase solution, add 5mL 0.25% trypsin solution, suspend the cells evenly, filter through a 100μm mesh sieve, and place at 37 After further digestion at ℃ for 15 minutes, DMEM containin...

Embodiment 2

[0045] The experimental method is the same as in Example 1, the only difference is that the limbal stem cell culture medium used in this example consists of: 204.5mL DMEM, 204.5mL DMEM / F12, 75mL FBS, 5mL 100× double antibody (100IU penicillin, 100ug / ml streptomycin), 2mL human recombinant EGF stock solution, 2mL insulin stock solution, 2.5mL 3,3´,5-Triiodo-L-Thyronine stock solution, 2.5mL hydrocortisone stock solution, 2mL cholera toxin stock solution.

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Abstract

The invention discloses a corneal limbal stem cell culture medium, and a culture method thereof. The corneal limbal stem cell culture medium comprises 5ml of 100Xdouble-antibody, 10 to 20ng / ml of human recombined EGF, 5 to 10<mu>g / ml of insulin, 1 to 5*10<9>M<3-> iodothyronine, 0.2 to 1<mu>g / ml of hydrocortisone, 10 to 20ng / ml of cholera toxin, 10 to 15% of fetal calf serum, and the balance DMEM and / or DMEM / F12. According to the culture method, I-type collagen is taken as a substrate substance of growth of corneal limbal stem cells to coat materials such as culture plates so as to increase the purity and stability of corneal limbal stem cells. The corneal limbal stem cell culture medium is used for separating corneal limbal stem cells, P63, Pax6 antibody positive rate ranges from 96 to 100%. The corneal limbal stem cell culture method is free of trophoderm and carrier, and a stable cell resource is provided for study on corneal limbal stem cell specificity mechanism and transplantation therapy.

Description

technical field [0001] The invention relates to the technical field of stem cell culture, and more specifically, to a limbal stem cell culture medium and a culture method thereof. Background technique [0002] Limbal stem cells are the junction of the cornea, conjunctiva and sclera, and the distinguishing mark from the cornea is the termination of Bowman's membrane; the distinguishing mark from the conjunctiva is that there are no goblet cells, and the limbal stem cells are about 1-2mm wide, here only There are epithelial layer and stromal layer, and the epithelial cell layer contains 10 layers of cells, arranged irregularly, the cells are small cylindrical, and the nucleus is deeply stained. The deep stromal cells are a layer of small cylindrical or cuboidal cells with oval nuclei parallel to the surface and formed in the basal papilla, forming a special "palisade"-like epithelial structure, which contains pigment and rich vascular network, And closely associated with the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797
CPCC12N5/0623C12N2500/30C12N2500/32C12N2500/84C12N2501/01C12N2501/11C12N2501/33C12N2509/00C12N2533/54
Inventor 欧阳宏
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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