Serum-free cultured corneal limbus stromal stem cells and method for in-vitro inducing balling and induced differentiation

一种无血清培养、基质细胞的技术，应用在角膜缘基质干细胞体外诱导成球和体外诱导分化的培养领域，能够解决供体来源不足、术后排斥和各种并发症、临床治疗效果不理想等问题，达到批次可控性强、易于推广和市场化、质量稳定的效果

Active Publication Date: 2019-12-13
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are a series of problems in corneal transplantation, such as insufficient donor sources, postoperative rejection and various complications, etc., and the clinical treatment effect is not satisfactory.

Method used

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  • Serum-free cultured corneal limbus stromal stem cells and method for in-vitro inducing balling and induced differentiation
  • Serum-free cultured corneal limbus stromal stem cells and method for in-vitro inducing balling and induced differentiation
  • Serum-free cultured corneal limbus stromal stem cells and method for in-vitro inducing balling and induced differentiation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 A culture medium for limbal stromal stem cells and its culture method

[0077] 1. Culture medium formula

[0078] A culture medium for limbal stromal stem cells, consisting of the following components:

[0079] Each 500 mL medium contains: 5 mL of 10× penicillin-streptomycin double antibody solution, 500 µL of gentamicin, 5 mL of ITS, 1 mL of human recombinant EGF, 1 mL of L-ascorbic acid-2-phosphate ester, 1 mL of cholera toxin, and 288 mL of low-sugar DMEM, with the balance being MCDB-201.

[0080] Among them, the concentration of each component in the medium is: 100 IU of penicillin, 100 µg / mL of streptomycin, 50 µg / mL of gentamicin, ITS, 20 ng / mL of human recombinant EGF, 0.1 mM L-ascorbic acid-2-phosphate (ascorbicacid-2-phosphate), 1×10 -8 M of dexamethasone and 100 ng / mL of cholera toxin.

[0081] 2. A serum-free method for culturing limbal stromal stem cells in vitro, comprising the steps of:

[0082]S11. After the limbal tissue was cleaned with D...

Embodiment 2

[0088] Example 2 A culture medium for limbal stromal stem cells forming spheres and its culture method

[0089] 1. Culture medium formula

[0090] A sphere-forming medium for limbal stromal stem cells, consisting of the following components:

[0091] Each 500 mL medium contains: 5 mL of 10× penicillin-streptomycin double antibody solution, 500 µL of gentamicin, 5 mL of ITS, 1 mL of human recombinant EGF, 5 mL of L-glutamine and 5 mL of B27, the balance was Advanced DMEM.

[0092] Among them, the concentration of each component in the medium is: 100 IU penicillin, 100 µg / mL streptomycin, 50 µg / mL gentamicin, 20 ng / mL human recombinant EGF, 2 mM L-Glutamine, 2% B27 and ITS.

[0093] 2. A culture method for inducing limbal stromal stem cells into spheres in vitro, comprising the steps of:

[0094] S21. When the limbal stromal stem cells P1-P3 generation cells cultured in step S14 in the above example 1 grow to 80%-90% confluence, discard the liquid, wash it once with PBS, and...

Embodiment 3

[0101] Example 3 A culture medium of limbal stromal cells and its culture method

[0102] 1. Culture medium formula

[0103] A limbal stromal cell culture medium, consisting of the following components:

[0104] Each 500 mL medium contains: 5 mL of 10× penicillin-streptomycin double antibody solution, 500 µL of gentamicin, 5 mL of ITS, 1 mL of L-ascorbic acid-2-phosphate and 1 mL of human Recombinant FGF2, the balance is low-sugar DMEM.

[0105] Among them, the concentration of each component in the medium is: 100 IU of penicillin, 100 µg / mL of streptomycin, 50 µg / mL of gentamicin, ITS, 1 mM of L-ascorbic acid-2-phosphate ester (ascorbic acid-2-phosphate) and 100ng / mL of human recombinant FGF2.

[0106] 2. A culture method for inducing limbal stromal stem cells to differentiate into corneal stromal cells in vitro, comprising the steps of:

[0107] S31. When the limbal stromal stem cells P2-P4 cultured in the above step S14 grow to 80%-90% confluence, discard the liquid, wa...

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Abstract

The invention discloses serum-free cultured corneal limbus stromal stem cells and a method for inducing differentiation and stem cell balling of the corneal limbus stromal stem cells. The invention further provides a culture medium combination for inducing corneal limbus stromal stem cells to be pelletized or differentiated into corneal limbus stromal cells in vitro. The serum-free culture mediumcombination used in the invention can provide sufficient nutrition and a good environment required by cell growth and proliferation, can stably carry out in-vitro amplification culture on corneal limbus stromal stem cells, and can ensure that the dryness and specificity of the corneal limbus stromal stem cells are still maintained after amplification culture. Meanwhile, a system for inducing and differentiating into corneal limbus stromal cells is successfully constructed, can be used for experimental research of the corneal limbus stromal stem cells, cell treatment of corneal lesions and transplantation of corneal injuries, and the serum-free cultured corneal limbus stromal stem cells has wide scientific benefits and social and economic benefits.

Description

technical field [0001] The invention belongs to the technical field of cell engineering. More specifically, it relates to a serum-free culture method for limbal stromal stem cells, and a culture method for inducing sphere formation and in vitro differentiation of limbal stromal stem cells in vitro. Background technique [0002] The cornea is a layer of transparent tissue that exists at the front of the eyeball, and has a certain radius of curvature in space. Physiologically, the cornea is divided into a central transparent corneal region and a peripheral limbal region. The central transparent corneal area is divided into five layers, from the outside to the inside are the corneal epithelium, Bowman's membrane, stroma, Bowman's membrane and endothelial cell layer. Among them, the stroma accounts for 90% of the thickness of the cornea and is mainly composed of type I and type V collagen, adhesives and corneal stromal cells. Corneal stromal cells are developmentally derived ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12N5/079
CPCC12N5/0621C12N5/0623C12N2500/90C12N2501/11C12N2501/01C12N2500/38C12N2501/855C12N2501/115C12N2500/25C12N2500/42C12N2503/02C12N5/0018C12N2501/999C12N2501/998
Inventor 欧阳宏朱丽琼
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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