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A type of corneal epithelial cells, tissue engineering corneal epithelium, preparation and application

A technology of corneal epithelial cells and epithelial cells, applied in the field of tissue engineered corneal epithelium and its preparation and application, and corneal epithelial cells, which can solve the problem of the small number of adult stem cells, the difficulty of separating, differentiated and purified cell lines, and the lack of specific molecular markers, etc. problem, to achieve the effect of weakening western blot, reducing immune rejection, and convenient material collection

Active Publication Date: 2020-03-06
LUOYANG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the extremely small number of adult stem cells in tissues and the lack of specific molecular markers, it is difficult to separate, differentiate and purify to establish cell lines (Watt FM, et al. 2010, Phil. Trans. R. Soc. B; 365: 155 -163)

Method used

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  • A type of corneal epithelial cells, tissue engineering corneal epithelium, preparation and application
  • A type of corneal epithelial cells, tissue engineering corneal epithelium, preparation and application
  • A type of corneal epithelial cells, tissue engineering corneal epithelium, preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] (1) Isolation and expansion of sheep skin epidermal cells

[0059] ① Aseptically take the ear skin of Boer goats (raised in the test field), place it in normal saline containing penicillin, rinse with PBS containing penicillin, and remove blood stains and hair;

[0060] ②Separate the skin from the cartilage under a dissecting microscope, and cut the skin into 0.5×1cm 2 The skin slices of the same size were evenly placed in a glass petri dish, added with digestion solution containing 0.25wt% Dispase enzyme and 0.02wt% EDTA, overnight at 4°C, and the epidermis was separated the next day; In the EDTA digestion solution, treat at 37°C for 6 minutes, stop digestion with serum-containing medium (M199+15% fetal bovine serum), centrifuge to obtain cells, and use serum-free medium (F12+1.2%BSA+25ng / mL EGF+ 2.5μg / mL insulin + 10ng / mL IGF-1 + 0.4μg / mL hydrocortisone + 100U / mL penicillin + 100μg / mL streptomycin) to culture epidermal cells, when the cell proliferation reaches 75% c...

Embodiment 2

[0071] (1) Separation and expansion of skin epidermal cells

[0072] ① Aseptically take the ear skin of Boer goats (raised in the test field), place it in normal saline containing penicillin, rinse with PBS containing penicillin, and remove blood stains and hair;

[0073] ②Separate the skin from the cartilage under a dissecting microscope, and cut the skin into 0.3×0.8cm 2 The skin slices of the same size were evenly placed in a glass petri dish, added with digestion solution containing 0.25wt% Dispase enzyme and 0.02wt% EDTA, overnight at 4°C, and the epidermis was separated the next day; In the EDTA digestion solution, treat at 37°C for 5 minutes, stop digestion with serum-containing medium (M199+15% fetal bovine serum), centrifuge to obtain cells, and use serum-free medium (F12+1.0%BSA+20ng / mL EGF+ 2.5μg / mL insulin+15ng / mL IGF-1+0.6μg / mL hydrocortisone+100U / mL penicillin+100μg / mL streptomycin) to culture epidermal cells, when the cell proliferation reached 75% confluence, ...

Embodiment 3

[0084] (1) Separation and expansion of skin epidermal cells

[0085] ① Aseptically take the ear skin of Boer goats (raised in the test field), place it in normal saline containing penicillin, rinse with PBS containing penicillin, and remove blood stains and hair;

[0086] ②Separate the skin from the cartilage under a dissecting microscope, and cut the skin into 0.4×1.2cm 2 The skin slices of the same size were evenly placed in a glass petri dish, added with digestion solution containing 0.25wt% Dispase enzyme and 0.02wt% EDTA, overnight at 4°C, and the epidermis was separated the next day; In the EDTA digestion solution, treat at 37°C for 8 minutes, stop digestion with serum-containing medium (M199+15% fetal bovine serum), centrifuge to obtain cells, and use serum-free medium (F12+1.1%BSA+23ng / mL EGF+ 2.5μg / mL insulin+12ng / mL IGF-1+0.5μg / mL hydrocortisone+100U / mL penicillin+100μg / mL streptomycin) to culture epidermal cells, when the cell proliferation reached 75% confluence, ...

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Abstract

The invention belongs to the technical field of tissue engineering, and in particular relates to a method and application for constructing tissue-engineered corneal epithelium using corneal epithelial cells as seed cells. The invention provides a two-step method to induce skin-derived epidermal cells to transdifferentiate into corneal epithelial cells, and use the induced corneal epithelial cells as seed cells to replace limbal stem cells to construct tissue-engineered corneal epithelium. The engineered corneal epithelium has the characteristics of good transparency, good elasticity, and strong operability. It can be used to transplant and repair the ocular surface of humans or animals with severe vision loss or blindness caused by corneal limbal stem cell defects. It is used for blindness due to corneal limbal stem cell defects. Both autograft and allograft in animal models can achieve good results.

Description

technical field [0001] The invention belongs to the technical field of tissue engineering, and in particular relates to a kind of corneal epithelial cells, tissue engineered corneal epithelium and its preparation and application. Background technique [0002] According to survey statistics, corneal blindness accounts for about 15.38% of patients with binocular blindness in my country, and has become the second blinding eye disease. There are about 4 million corneal blind patients (including monocular patients) in my country every year, and more than 75% of these patients can achieve the goal of vision recovery through corneal transplantation. Due to the serious shortage of corneal donors, there are only about 4,000 cases of surgery in my country every year, accounting for only 2‰ of patients. [0003] Constructing tissue-engineered corneas that can be used for human transplantation to meet the needs of patients has become an urgent medical problem that needs to be solved. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071C12N5/079A61L27/36A61L27/38
CPCA61L27/3604A61L27/3687A61L27/3813A61L2430/16A61L2430/40C12N5/0621C12N5/0625C12N5/0697C12N2533/92
Inventor 杨学义郭燕杰马西亚张沛
Owner LUOYANG NORMAL UNIV
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