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Constructing method for human corneal endothelium cell system

A construction method and technology of endothelial cells, applied in the field of construction of human corneal endothelial cell lines, can solve the problems that the cell lines are far apart, cannot be constructed with artificial corneas and clinical corneal transplantation

Active Publication Date: 2006-03-22
青岛彩晖生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Later, scholars explored the in vitro culture conditions of different mammalian corneal endothelial cells, and found that some growth factors have a certain mitogenic effect on corneal endothelial cells, but they are still far from the establishment of cell lines.
In the 1990s, many scholars used simian red herpes virus, lymphoma virus, simian red herpes virus T antigen and different oncogenes to conduct transfection research on corneal endothelial cells of humans, rabbits and mice, and obtained passageable cells. The cultured corneal endothelial cells even obtained several immortal cell lines, but because of the potential tumorigenicity of the cells carrying tumor virus genes or oncogenes, they cannot be used for the construction of artificial corneas and clinical corneal transplantation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017]Take out 2 complete human corneas from the eye bank, put them into a 100ml glass beaker, add 10ml of 0.9% normal saline, wash for 5 minutes; after sucking out the normal saline, add 10ml of 1 / 6000 of Soak in mercuric chloride solution for 10 minutes for the first disinfection; then, suck out the mercuric chloride solution, then add 10 ml of gentamicin with a concentration of 20%, soak for 15 minutes, and carry out secondary disinfection in an ultra-clean workbench; All operations were carried out in a clean workbench, and all supplies were sterile; the cornea was taken out of the gentamicin solution with ophthalmic forceps, and the concave surface of the cornea was placed on a glass petri dish, and 0.2% Digest 0.5 ml of trypsin for 3 minutes; remove the trypsin solution, cut each cornea into 4 pieces on average along the center of the cornea with ophthalmic scissors, and stick the endothelial side of the cornea into the bottom of the well of the 24-well culture plate with...

Embodiment 2

[0019] Take 2 complete human corneas from the eye bank, put them into a 100ml glass beaker, add 30ml of 0.9% normal saline, wash for 9 minutes; after sucking out the normal saline, add 30ml of 1 / 2000 Soak in mercuric chloride solution for 20 minutes for the first disinfection; then, suck out the mercuric chloride solution, then add 30 ml of gentamicin with a concentration of 70%, soak for 35 minutes, and carry out secondary disinfection in an ultra-clean workbench; The operations were all in a sterile state; the cornea was taken out from the gentamicin solution with ophthalmic forceps, and the concave surface of the cornea was placed on a glass petri dish, and 1 ml of 0.5% trypsin was added to the concave area to digest for 6 minutes; remove Use trypsin solution, use ophthalmic scissors to cut each cornea into 4 pieces on average along the center of the cornea, use ophthalmic tweezers to paste the corneal endothelium face down into the bottom of the well of the 24-well culture ...

Embodiment 3

[0021] Take 2 complete human corneas from the eye bank, put them into a 100ml glass beaker, add 20ml of 0.9% normal saline, wash for 7 minutes; after sucking out the normal saline, add 20ml of 1 / 4000 Soak in mercuric chloride solution for 15 minutes for the first disinfection; then, suck out the mercuric chloride solution, then add 20 ml of gentamicin with a concentration of 50%, soak for 20 minutes, and carry out secondary disinfection in an ultra-clean workbench; The operations are all sterile; take out the cornea from the gentamicin solution with ophthalmic forceps, put the concave surface of the cornea upwards on a glass petri dish, add 0.7 ml of 0.4% trypsin to the concave area to digest for 5 minutes; remove the pancreatic Protease solution, use ophthalmic scissors to cut each cornea into 4 pieces on average along the center of the cornea, use ophthalmic tweezers to paste the corneal endothelium face down into the bottom of the well of the 24-well culture plate, and stick...

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PUM

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Abstract

The construction of human corneal endothelium cell line with corneal endothelium as material includes the steps of: the first adherent culture of human corneal endothelium cell inside DMEM / F12 culture liquid with ox embryo serum in 20 wt%, chondroitin sulfate oxidizing and degrading matter, epidermal cell growth factor, basic fibroblast growth factor and ox eye auxin to obtain pure corneal endothelium cell; and the subsequent secondary culture in trypsinization process. The technological process is scientific and reasonable, and up to now, passage cell of 106-th generation has been obtained. The human corneal endothelium cell line of the present invention has no any virus or cancer genetic transfection, has no any tumorigenicity, and is expected to be used in direct artificial cornea production and clinical application.

Description

technical field [0001] The invention relates to a construction method for establishing a corneal endothelial cell line using human cornea. Background technique [0002] The corneal endothelial cell layer of higher animals is formed by a single layer of hexagonal dendritic cells, but this layer of cells loses the ability to divide after adulthood, and only relies on the expansion and migration of adjacent cells to fill the defect area when it is slightly damaged. Once the density of corneal endothelial cells is lower than the critical density for maintaining the physiological function of endothelial cells, irreversible lesions will appear in the cornea. At present, there are nearly one million patients with corneal endothelial blindness in my country. Although most of them can be cured by corneal transplantation, due to the extremely limited number of donated corneas, only 2,000 to 2,500 corneal transplants are completed each year in the country. Most patients cannot see agai...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N5/071
Inventor 樊廷俊付永锋丛日山汤志宏孙文杰于秋涛王晶赵君
Owner 青岛彩晖生物科技有限公司
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