Method for constructing human corneal epithelial cell system

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...

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, proliferate and adhere to the wall in vitro

In addition, there are also studies on the construction of human corneal epithelial cell lines using corneal limbal tissue, but so far, there is no report on the construction of human corneal epithelial cell lines using human corneal epithelial tissue