Limbus corneae stem cell serum-free medium and culture method thereof

A serum-free medium and corneal limbal stem cell technology, applied in the field of stem cell culture, can solve the problems of unsatisfactory stem cell proliferation rate, stem cell purity and quantity, unsatisfactory culture effect, etc., achieve rapid and stable cell source, improve purity and stability , the effect of improving cell quality

Active Publication Date: 2019-05-14
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Serum-free medium has the characteristics of clear components, but the existing serum-free medium has problems such as unsatisfactory culture effect, unsatisfactory proliferation speed of stem cells, and unsatisfactory purity and quantity of stem cells after culture.

Method used

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  • Limbus corneae stem cell serum-free medium and culture method thereof
  • Limbus corneae stem cell serum-free medium and culture method thereof
  • Limbus corneae stem cell serum-free medium and culture method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] 1. A serum-free culture medium for limbal stem cells

[0066] A serum-free medium for limbal stem cells, consisting of the following components:

[0067] 216 mL Ham's F12, 216 mL DMEM, 5 mL 100× penicillin-streptomycin double antibody solution, 1 mL human recombinant EGF stock solution, 500 μL insulin stock solution, 500 μL vitamin C stock solution, 500 μL 3,3', 5-triiodo-L-thyronine stock solution, 500 μL hydrocortisone stock solution, 1 μL forskolin stock solution, 0.5 μL manganese sulfate monohydrate, 0.5 μL sodium selenite, 500 μL silicon metasilicate sodium phosphate, 0.5 μL ammonium metavanadate, 0.5 μL nickel chloride hexahydrate, 0.5 μL stannous chloride dihydrate, 0.5 μL ethanolamine, 500 μL phosphoethanolamine, 0.5 μL ammonium molybdate tetrahydrate, 2.7 g 4-hydroxyethyl piperazine ethanesulfonic acid, 10 g bovine serum albumin, 5 mL lipid concentrate and 50 mL serum replacement.

[0068] The concentration of each added component was 10 ng / mL human recombina...

Embodiment 2

[0081] Example 2 Medium formula

[0082] The experimental method is the same as in Example 1, the only difference is that the concentration of each added component in the serum-free medium of limbal stem cells used in this example is:

[0083] 10 ng / mL human recombinant EGF, 10 μg / mL insulin, 5×10 -9 M 3-iodothyronine, 0.2 μg / mL hydrocortisone, 2×10 -5 M Forskolin, 0.5×10 -9 M manganese sulfate monohydrate, 5×10 -7 M sodium selenite, 0.1×10 -3 M sodium metasilicate, 8×10 -6 M ammonium metavanadate, 3×10 -10 M nickel chloride hexahydrate, 8×10 -10 M stannous chloride dihydrate, 3×10 -7 M ethanolamine, 8×10 -6 M Phosphoethanolamine, 1×10 -9 M ammonium molybdate tetrahydrate, 8 g / L 4-hydroxyethylpiperazineethanesulfonic acid, 20 μg / mL vitamin C, 3% bovine serum albumin, 0.5% lipid concentrate, and 15% serum replacement.

Embodiment 3

[0084] Embodiment 3 culture medium formula

[0085] The experimental method is the same as in Example 1, the only difference is that the concentration of each added component in the serum-free medium of limbal stem cells used in this example is:

[0086] 20 ng / mL human recombinant EGF, 5 μg / mL insulin, 1×10 -9 M 3-iodothyronine, 1 μg / mL hydrocortisone, 0.5×10 -5 M Forskolin, 2×10 -9 M manganese sulfate monohydrate, 10×10 -7 M sodium selenite, 1×10 -3 M sodium metasilicate, 3×10 -6 M ammonium metavanadate, 8×10 -10 M Nickel chloride hexahydrate, 3×10 -10 M stannous chloride dihydrate, 8×10 -7 M ethanolamine, 3×10 -6 M Phosphoethanolamine, 6×10 -9 M ammonium molybdate tetrahydrate, 2 g / L 4-hydroxyethylpiperazineethanesulfonic acid, 50 μg / mL vitamin C, 1% bovine serum albumin, 2% lipid concentrate, and 5% serum replacement.

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Abstract

The invention discloses a limbus corneae stem cell serum-free medium and a culture method thereof. The serum-free medium comprises a basic culture medium and additives, wherein the additives include human recombinant EGF, insulin, 3,3',5-triiodo-L-thyronine, hydrocortisone, forskolin, manganese sulfate monohydrate, sodium selenite, sodium metasilicate, ammonium metavanadate, nickel chloride hexahydrate, stannous chloride dihydrate, ethanol amine, phosphorylethanolamine, ammonium molybdate tetrahydrate, 4-hydroxyethylpiperazine ethane sulfonic acid, vitamin C, bovine serum albumin, lipid concentrate and serum substitution. The serum-free medium contains no fetal calf serum or any animal source ingredient, can supply sufficient nutrients and good environment needed by cell growth proliferation and can effectively replace the effect of serum, cells grow well, the cell purity and stability are improved, rapid and stable cell sources are provided for limbus corneae stem cell specificity mechanism research and transplantation therapy, and the clinic application prospect is wide.

Description

technical field [0001] The invention belongs to the technical field of stem cell culture. More specifically, it relates to a serum-free culture medium for limbal stem cells and a culture method thereof. Background technique [0002] The limbus is 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 it does not contain goblet cells and is about 1 to 2 mm wide, where there are only epithelial layers and stromal layer; its epithelial cell layer contains 10 layers of cells, arranged irregularly, and the cells are small cylindrical, with deeply stained nuclei; the deep stromal cells are a layer of small cylindrical or cuboidal cells, with oval nuclei, and the surface In parallel, papillae form at the base, forming a special "palisade"-like epithelial structure, which contains pigment and rich vascular network, and is closely connected with the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797
CPCC12N5/0623C12N2500/05C12N2500/20C12N2500/36C12N2500/38C12N2500/42C12N2500/46C12N2500/90C12N2501/11C12N2501/33C12N2501/39C12N2500/30
Inventor 欧阳宏朱进
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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