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Reconstruction method of tissue engineering human corneal epithelium

A corneal epithelium and tissue engineering technology, applied in the field of tissue-engineered human corneal epithelium reconstruction, can solve problems such as limited experimental research, inability to carry out tissue-engineered human corneal epithelium large-scale in vitro reconstruction, limited source of seed cells, etc., and achieves low cost. Effect

Inactive Publication Date: 2011-08-31
OCEAN UNIV OF CHINA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These research results have opened up the way for the in vitro reconstruction of tissue engineered human corneal epithelium, but because the seed cells used are all cells induced to differentiate, the source of seed cells is limited, or because they still have the characteristics of the original type of cells, tissue engineering cannot be carried out Large-scale in vitro reconstruction of human corneal epithelium, or in vitro reconstruction of tissue engineered human corneal epithelium cannot be used clinically due to unstable structure and function, and is still limited to experimental research

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024]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

[0031] Human corneal epithelial cells were taken, suspended in 20% calf serum-DMEM / F12 culture medium, inoculated into a culture flask with a bottom area of ​​75 square centimeters, and cultured at 37°C for 80 hours. Take 80 ml of conventionally prepared DMEM / F12 culture solution, add 8 mg of type IV collagen, filter and sterilize with a 0.22 micron microporous membrane after completely dissolving, add 10 ml of calf serum, and add conventionally prepared DMEM / F12 The culture fluid to 100 milliliters is the special culture fluid for corneal epithelial reconstruction of the present invention. For the culture bottle after the expansion culture, use a glass dropper to suck out the culture solution, add 0.25% trypsin solution to digest for 1.5 minutes, add the old culture solution sucked out before to stop the digestion, centrifuge at 1500 rpm for 10 minutes, obtain the cell pellet, and use Suspend 3 ml of the above-mentioned special culture medium evenly to make a suspension of hu...

Embodiment 3

[0034] Human corneal epithelial monoclonal cells were taken, suspended in 20% calf serum-DMEM / F12 culture medium, inoculated into a culture flask with a bottom area of ​​75 cm2, and placed at 37°C for expansion and culture for 72 hours. Take 80 ml of conventionally prepared DMEM / F12 culture solution, add 6 mg of type IV collagen, filter and sterilize with a 0.22 micron microporous membrane after completely dissolving, add 10 ml of calf serum, and add conventionally prepared DMEM / F12 The culture fluid to 100 milliliters is the special culture fluid for corneal epithelial reconstruction of the present invention. For the culture bottle after the amplification culture, use a glass dropper to suck out the culture solution, add 0.25% trypsin solution to digest for 1 minute, add the old culture solution sucked out before to stop the digestion, centrifuge at 1000 rpm for 15 minutes, obtain the cell pellet, and use Suspend 3 ml of the above-mentioned special culture medium evenly to ma...

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Abstract

The invention relates to a reconstruction method of tissue engineering human corneal epithelium. The method comprises the following steps of: adopting a DMEM / F12 culture medium containing 20% calf serum to carry out the in vitro culture on human corneal epithelium cells to a logarithmic growth phase, and adopting trypsin and a trypsin-EDTA digestion method to obtain a digestive amniotic carrier bracket of which the epithelium is completely removed; and after the digestive amniotic carrier bracket of which the epithelium is removed is flatly laid in a plug-in Petri dish and is firmly and pasted in a drying way, inoculating the human corneal epithelium cells at the logarithmic growth phase suspended in the DMEM / F12 culture medium containing IV type collagen and 20% calf serum to the plug-inPetri dish flatly laid with the digestive amniotic carrier bracket of which the epithelium is removed, and carrying out the in vitro reconstruction on the tissue engineering human corneal epithelium by a gas-liquid interface culture method. The invention has scientific and reasonable process, the reconstructed tissue engineering human corneal epithelium can be used for mass production, a lot of demands of vast blind patients with corneal epithelium diseases for the tissue engineering human corneal epithelium in clinical corneal transplantation treatment can be met, and the costs of the in vitro reconstruction and clinical treatment of the tissue engineering human corneal epithelium are low.

Description

technical field [0001] The invention belongs to the technical field of corneal epithelium construction, and in particular relates to a method for reconstructing tissue-engineered human corneal epithelium, that is, a method for reconstructing tissue-engineered human corneal epithelium by using human corneal epithelial cells and de-epithelialized amniotic membranes. Background technique [0002] The human corneal epithelium is composed of multiple layers of epithelial cells, which is the first barrier of the cornea, which can prevent the loss of corneal water and the invasion of external pathogens, and prevent the liquid and electrolytes in tears from entering the stroma, so that the cornea is in a transparent state; in addition, The microvilli on the surface of the corneal epithelium interact with the tear film to provide an optical interface for the anterior refractive surface of the transparent cornea (Xie Lixin, 2000). Because the corneal epithelium is in direct contact wi...

Claims

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Application Information

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IPC IPC(8): A61L27/38C12N5/071
Inventor 樊廷俊徐彬杨洪收孙爱王宝泉
Owner OCEAN UNIV OF CHINA
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