Method for constructing human corneal epithelial cell system

A construction method and epithelial cell technology, which is applied in the field of construction of human corneal epithelial cell lines, can solve the problems of adult cells that are difficult to divide, proliferate and adhere to the wall, and achieve the effect of reducing costs

Active Publication Date: 2011-08-31
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

[0004] At present, relevant studies on the construction of human corneal endothelial cell lines using human corneal endothelial cells have been carried out. The establishment of human corneal epithelial tissue still has the problem that adult cells are difficult to divide, prolifera

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023]Take 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 20% Qing Damycin, soaked for 35 minutes, and disinfected in the ultra-clean workbench; the following operations were carried out in the ultra-clean workbench, and all supplies were sterile; the cornea was taken out from the gentamicin solution with ophthalmic forceps, and the cornea Put the concave side up in a glass culture dish, add 5ml of 0.2% trypsin to the culture dish to digest for 2 minutes, remove the trypsin solution, use ophthalmic forceps to tear off the corneal epithelium with Descemet's layer, and use ophthalmic scissors to cut off the corneal epithelium Cut the corneal epithelium with Bowman's membrane into 8 pieces on average at the center point, put the corneal epithelium face down into the bottom of the 24-well culture plate with ophthalmic forceps, add DMEM containing 20%...

Embodiment 2

[0025] 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 70% qing Damycin, soaked for 15 minutes, and then sterilized in an ultra-clean workbench; the following operations are all in a sterile state; use ophthalmic forceps to take out the cornea from the gentamicin solution, and place the cornea with the concave surface facing upwards on a glass petri dish Add 10 ml of 0.5% trypsin to the petri dish to digest for 1 minute; remove the trypsin solution, tear off the Bowman's membrane with ophthalmic forceps, and cut the corneal epithelium with the Bowman's membrane into 8 Use ophthalmic tweezers to attach the corneal epithelium face down to the bottom of the well of a 24-well culture plate, add 0.3 ml of DMEM / F12 culture solution containing 20% ​​fetal bovine serum to the culture well pasted into the corneal epithelium; place the culture plate P...

Embodiment 3

[0027] 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; suck out the normal saline, add 20ml of 50% qing Damycin, soaked for 20 minutes, disinfected in an ultra-clean workbench; the following operations are all sterile; use ophthalmic forceps to take out the cornea from the gentamicin solution, and place the concave surface of the cornea upwards in a glass petri dish , add 7.5 ml of 0.35% trypsin to the petri dish to digest for 1.5 minutes; remove the trypsin solution, tear off the Bowman's membrane with ophthalmic forceps, and cut the corneal epithelium with Bowman's membrane into 8 pieces on average along the center of the cornea with ophthalmic scissors , use ophthalmic forceps to put the corneal epithelium face down into the well bottom of the 24-well culture plate; add 0.2 ml of DMEM / F12 culture solution containing 20% ​​fetal bovine serum to the culture well pasted into the corneal epi...

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PUM

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Abstract

The invention relates to a method for constructing a human corneal epithelial cell system. The construction method comprises the following steps of: disinfecting cornea, digesting the cornea, taking off corneal epithelium with a front elastic layer, cutting the corneal epithelium into small tissue blocks, flatly attaching the tissue blocks to the bottom of culture holes downwards, adding culture solution into a carbon dioxide culture box, and performing plate-attaching culture at the temperature of 37 DEG C, changing the culture solution during culturing, performing subculture when the cells grow into a single layer, and presently, completing the construction of the human corneal epithelial cell system above 60 generations, wherein the culture solution contains 20 percent of fetal calf serum, 0.002 to 0.004 percent of human epidermic cell growth factor, 0.001 to 0.002 percent of human alkali fibroblast growth factor, 0.04 to 0.1 percent of carboxymethyl chito-oligosaccharide, 0.025 to0.1 percent of DMEM/F12 culture solution of chondroitin sulfate, and 0.008 to 0.01 percent of IV type collagen. In the construction method, the non-transfection human corneal epithelial cell system is successfully constructed by utilizing the human corneal epithelial tissues, and the constructed cell system can realize continuous transfer of culture, and a great number of human corneal epithelialcells are provided.

Description

technical field [0001] The invention belongs to the technical field of corneal cell culture, and in particular relates to a method for constructing a human corneal epithelial cell line. Background technique [0002] The corneal epithelial layer (epithelial layer), as the first barrier of the cornea, is a structure about 50-60 μm thick composed of 5-7 layers of squamous cells, wing cells and columnar cells. It not only supports and stabilizes the tear film, Moreover, it can also prevent the loss of corneal moisture and the invasion of external pathogens. Among the three types of cells in the corneal epithelium, columnar cells are the only cells capable of dividing, and can differentiate upward to produce wing cells and squamous cells, and downward to produce basement membrane. The source of columnar cell renewal and regeneration is located in the cornea Limbal stem cells in the limbal basal layer. Current studies have shown that many severe ocular surface diseases such as s...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12R1/91
Inventor 樊廷俊赵君杨洪收徐彬孙爱
Owner 青岛彩晖生物科技有限公司
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