Cornea-like epithelioid cell, tissue-engineered corneal epithelium as well as preparation and application

A corneal epithelial cell and epithelial cell technology, applied in tissue engineering corneal epithelium and its preparation and application, corneal epithelial cell-like cell field, can solve the problem of small number of adult stem cells, difficult to separate, differentiate and purify cell lines, lack of specific molecular markers, etc. problems, to achieve the effect of weakening western blotting, reducing immune rejection, and convenient material sampling

Active Publication Date: 2019-03-26
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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  • Cornea-like epithelioid cell, tissue-engineered corneal epithelium as well as preparation and application
  • Cornea-like epithelioid cell, tissue-engineered corneal epithelium as well as preparation and application
  • Cornea-like epithelioid cell, tissue-engineered corneal epithelium as well as 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 / mLEGF+2.5 μg / 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% confluence...

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 / mLEGF+2.5 μg / mL insulin+15ng / mL IGF-1+0.6μg / mL hydrocortisone+100U / mL penicillin+100μg / mL streptomycin) to culture epidermal cells, when the cell proliferation reaches 75% confluence, u...

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 / mLEGF+2.5 μg / mL insulin+12ng / mL IGF-1+0.5μg / mL hydrocortisone+100U / mL penicillin+100μg / mL streptomycin) to culture epidermal cells, when the cell proliferation reaches 75% confluence, u...

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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 by taking cornea-like epithelioid cells as seed cells. The invention provides a two-step method for inducing skin-derived epidermic cells to be transformed and differentiated into the cornea-like epithelioid cells, so that the cornea-like epithelioid cells obtained by induction serving as the seed cells replace the corneal limbal stem cells for constructing the tissue-engineered corneal epithelium. The prepared tissue-engineered corneal epithelium has the characteristics of being excellent in transparency, excellent in elasticity, high in operability and the like, can be applied to transplantation and repair of ocularsurfaces of animals or animals with severe vision losses or blindness due to corneal limbal stem cell deficiency, and can achieve excellent effects when applied to autotransplantation and allotransplantation of blind animal models due to the corneal limbal stem cell deficiency.

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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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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