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Limbal stem cell culture medium and culture method thereof

A corneal limbal stem cell and culture medium technology, which is applied to the corneal limbal stem cell culture medium and its culture field, can solve problems such as affecting the limbal stem cells' sensitivity to light and vision, unable to stably separate and culture the limbal stem cells, and opacity, etc. Good properties and proliferation, improved purity and stability, and reduced damage

Active Publication Date: 2019-01-01
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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  • Limbal stem cell culture medium and culture method thereof
  • Limbal stem cell culture medium and culture method thereof
  • Limbal stem cell culture medium and culture method thereof

Examples

Experimental program
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Effect test

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, 1mL3,3′ , 5-Trioodo-L-Thyronine stock solution, 1 mL hydrocortisone stock solution, 1 mL cholera toxin stock solution, stored at 4°C, and rewarmed at 37°C before use.

[0040] Under the operating microscope, use tissue tweezers and corneal scissors to cut the human limbal tissue and rinse it twice with PBS containing double antibody (penicillin-streptomycin, 1×) in a sterile environment for 5 min each time; Tissue shredded.

[0041] According to the volume of the tissue block, every 1cm 3 Add 5ml of 0.2wt% IV collagenase solution to the tissue block, and after gently shaking at 37°C for 2 hours, remove the IV collagenase solution, add 5mL of 0.25% trypsin solution, suspend the cells evenly, filter through a 100 μm mesh sieve, and place them in 37 The digestion was ...

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), 2 mL human recombinant EGF stock, 2 mL insulin stock, 2.5 mL 3,3′,5-Triodo-L-Thyronine stock, 2.5 mL hydrocortisone stock, 2 mL cholera toxin stock.

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Abstract

The invention discloses a limbal stem cell culture medium and a culture method thereof. The culture medium comprises: 5mL 100× double antibody, 10-20ng / ml human recombinant EGF, 5-10ug / ml insulin, 1-5x10‑9M3 ‑Iodothyronine, 0.2~1ug / ml hydrocortisone, 10~20ng / ml cholera toxin, 10~15% fetal bovine serum, the balance is DMEM and / or DMEM / F12; type I collagen is used as The base material for the growth of limbal stem cells is coated with culture plates and other materials to improve the purity and stability of limbal stem cells. The culture medium provided by the invention is used to separate limbal stem cells, and the positive rate of p63 and Pax6 antibodies in the cells is 96%. ~100%. A trophoblast-free and carrier-free culture method of limbal stem cells was established, which provided a stable source of cells for research on the specific mechanism of limbal stem cells and transplantation therapy.

Description

technical field [0001] The invention relates to the technical field of stem cell culture, and more particularly, to a corneal limbal stem cell culture medium and a culture method thereof. Background technique [0002] The corneal limbal stem cells are the junction of the cornea, the conjunctiva and the sclera, and the identification mark with the cornea is the termination of Bowman's membrane; the identification mark with the conjunctiva is that it does not contain goblet cells, and the limbal stem cells are about 1-2 mm wide. There are epithelial layer and stromal layer. The epithelial cell layer contains 10 layers of cells, which are irregularly arranged, and the cells are small cylindrical with hyperchromatic nuclei. Its 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 to form a special "palete"-like epithelial structure, which contains pigments and a rich vascular network, an...

Claims

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Application Information

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Patent Type & Authority Patents(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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