Method for preparing carrier bracket of tissue engineering artificial corneal endothelium by using fresh amniotic membrane

A technology of artificial cornea and tissue engineering, applied in prosthetics, medical science, etc., can solve the problem of limiting the clinical application of large-scale in vitro reconstruction of tissue engineering artificial corneal endothelium, unable to meet the large number of clinical transplantation needs of patients with corneal endothelial blindness, seed cell Tumor or limited quantity, etc., to achieve the effect of easy attachment and growth, low cost, and strong mechanical properties

Inactive Publication Date: 2010-09-29
青岛宇明生物技术有限公司
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Problems solved by technology

Scholars at home and abroad have used immortalized human corneal endothelial cells transfected with oncogenes, primary cultured human corneal endothelial cells, or human corneal endothelial cells cultured to the 4th or 5th passage in vitro as seed cells, respectively using the Descemet's membrane , de-epithelial amniotic membrane, collagen-chitosan blend membrane, polyhydroxyethyl acrylate, acellular porcine corneal stroma and N-(1-methylethyl)-2-acrylamide homopolymer as carriers Scaffolds successfully reconstructed human corneal endothelial tissue analogues in vitro in shape, transparency and tissue structure similar to normal corneal endothelium. Gene

Method used

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preparation example Construction

[0019] 4. Preparation of de-epithelialized amniotic membrane: Scrape the epithelial surface of the amniotic membrane lightly with a cell scraper to remove all residual epithelial cells, rinse with sterile D-Hanks solution for 3-5 times to obtain de-epithelialized amniotic membrane.

[0020] 5. Dry pasting of epithelial amniotic membrane slices: Use a punch or ophthalmic scissors to punch or cut the epithelial amniotic membrane into discs with a diameter of 15.6 mm, and lay them flat in the wells of a 24-well culture plate with the epithelial side facing upwards. Put in 5% CO 2 Dry paste in an incubator at 37°C for 15 to 25 hours.

[0021] 6. Coating modification of epithelial amniotic membrane slices: under sterile conditions, gently add 1 ml / well special coating solution for epithelial amniotic membrane removal to the wells of a 24-well culture plate with amnion attached, and culture The plate is placed in a 37°C incubator for coating treatment for 15-25 hours, and the coati...

Embodiment 1

[0023] Get 5 million units of tobramycin sulfate injection, add 500 milliliters of 0.9% normal saline, and mix to obtain a 1:1000 tobramycin sulfate disinfectant. Use ophthalmic tweezers to tear off the fibroblast layer and sponge layer of fresh amniotic membrane preserved at low temperature, rinse it with 0.9% normal saline, put it in the above-mentioned disinfectant solution, soak it in the sterile operating table for 20 minutes, and then use sterile D- Hanks solution rinse for 3 minutes.

[0024] Take 25 ml of 0.6% trypsin, add 25 ml of 0.04% EDTA solution, and mix well to obtain 0.3% trypsin-0.02% EDTA digestion solution. Spread the sterilized filter paper on a sterile glass plate, add the above digestive solution dropwise to fully wet the filter paper, spread the rinsed amnion on the filter paper with the epithelial side facing down, and place it in a 37°C incubator for digestion 30 minutes, that is, the inverted digestion method. Then, gently scrape the epithelial surf...

Embodiment 2

[0027] Get 5 million units of tobramycin sulfate injection, add 500 milliliters of 0.9% normal saline, and mix to obtain a 1:1000 tobramycin sulfate disinfectant. Use ophthalmic tweezers to tear off the fibroblast layer and sponge layer of the fresh amniotic membrane preserved at low temperature, rinse it with 0.9% normal saline, put it in the above-mentioned disinfectant solution, soak it in the sterile operating table for 25 minutes, and then use sterile D- Hanks solution rinse for 4 minutes.

[0028]Take 25 ml of 0.4% trypsin, add 25 ml of 0.02% EDTA solution, and mix well to obtain 0.2% trypsin-0.01% EDTA digestion solution. Spread the sterilized filter paper on a sterile glass plate, add the above digestive solution dropwise to fully wet the filter paper, spread the rinsed amnion on the filter paper with the epithelial side facing down, and place it in a 37°C incubator for digestion 50 minutes, that is, the inverted digestion method. Then, gently scrape the epithelial s...

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Abstract

The invention relates to a method for preparing a carrier bracket of tissue engineering artificial corneal endothelium by using a fresh amniotic membrane. The method comprises the following steps: soaking and disinfecting the fresh amniotic membrane by tobramycin sulfate injection according to the mass ratio of 1:1000; after reverse digestion using trypsin-EDTA digestive juice, lightly scraping the epithelial surface of the amniotic membrane by a cell scraper to completely remove residual epithelial cells to obtain the denuded amniotic membrane which is tiled in culture plate holes for fixingand dry-posting; coating by special coating liquid for the denuded amniotic membrane; and sucking and then drying the coating liquid to obtain the carrier bracket of the tissue engineering artificialcorneal endothelium. The method has scientific and reasonable process, the prepared carrier bracket can be mass produced to meet the heavy demand of scale reconstruction of the tissue engineering artificial corneal endothelium and create conditions for sight rehabilitation of corneal endothelium blindness through clinical corneal transplantation, and the preparation method of the carrier bracket has low cost in in-vitro reconstruction and clinical treatment of the tissue engineering artificial corneal endothelium.

Description

technical field [0001] The invention relates to a method for preparing a tissue engineering artificial corneal endothelial carrier support by using fresh amniotic membrane, which belongs to the tissue engineering artificial corneal endothelial technology. Background technique [0002] The human corneal endothelium plays a vital role in maintaining corneal thickness, transparency and providing nutrients for the cornea. When the cornea is infected or traumatized by surgery, the corneal endothelial cells will be reduced to varying degrees. Once the density of corneal endothelial cells is lower than the critical density for forming a complete corneal endothelial layer and maintaining the physiological function of the corneal endothelium, the corneal endothelium will appear irreversible lesions. That is, corneal endothelial blindness. According to incomplete statistics, there are more than 800,000 patients with corneal endothelial blindness in my country at present, and most of ...

Claims

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Application Information

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IPC IPC(8): A61L27/36A61L27/24A61L27/22
Inventor 樊廷俊赵君马西亚丛日山杨洪收于昊泽
Owner 青岛宇明生物技术有限公司
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