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In-vitro construction method of tissue engineering posterior lamellar cornea

A tissue engineering and construction method technology, applied in tissue regeneration, tissue culture, biochemical equipment and methods, etc., to achieve the effects of low production cost, good integrity, and easy attachment

Pending Publication Date: 2020-05-08
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention is based on the non-transfected human corneal stromal cell line and human corneal endothelial cell line that our laboratory has successfully established, and screens out human corneal stromal cells and human corneal endothelial cells with normal karyotype and normal structure and function, and successfully Solved the problem of the source of sufficient normal seed cells required for the large-scale construction of lamellar cornea after tissue engineering

Method used

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Examples

Experimental program
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Effect test

preparation example Construction

[0036] 1. Preparation of the carrier bracket: soak the carrier bracket in the carrier bracket rehydration solution for 1-2 hours for rehydration treatment;

[0037] 2. The preparation method of corneal stromal seed cells: take human corneal stromal cells or corneal stromal-like cells induced and differentiated from limbal stem cells, and use DMEM / F12 culture medium containing 15%-20% calf serum at 37°C. Expansion culture, after the cells are covered with a single layer, suck out the culture medium, add 0.25% trypsin solution to digest for 1-2 minutes, add the old culture solution aspirated before to stop the digestion, centrifuge at 1000-1500 rpm for 10-15 minutes, Suspend the cell pellet with 4mL special culture medium A for construction to make a homogeneous corneal stromal cell suspension; after counting the cells, use the above special culture medium A for construction to adjust the cell concentration to 2×10 5 -6×10 5 cells / mL;

[0038] 3. In vitro construction of the c...

Embodiment 1

[0043] Inject human corneal stromal cells into the carrier bracket with Descemet's layer, add culture medium A for construction, and then replace culture medium B for construction to continue culturing to obtain the corneal stroma part; then inoculate human corneal endothelial cells On the Descemet descemet's layer of the corneal stroma part obtained above, and then replace the construction special culture medium D to continue culturing to obtain a tissue-engineered posterior lamellar cornea with a tissue structure and function similar to that of a normal posterior lamellar cornea.

[0044] First take 80 mL of conventionally prepared DMEM / F12 culture solution, add 1 g of tobramycin, and filter it through a 0.22 μm microporous membrane to sterilize after it is completely dissolved. ;

[0045] After soaking the carrier bracket in the carrier bracket rehydration solution for 2 hours for rehydration treatment, it is used as the lamellar cornea carrier bracket after tissue engineer...

Embodiment 2

[0054] Inject human corneal stromal cells into the carrier bracket with Descemet's layer, add culture medium A for construction, and then replace culture medium B for construction to continue culturing to obtain the corneal stroma part; then inoculate human corneal endothelial cells On the Descemet descemet's layer of the corneal stroma part obtained above, and then replace the construction special culture medium D to continue culturing to obtain a tissue-engineered posterior lamellar cornea with a tissue structure and function similar to that of a normal posterior lamellar cornea.

[0055] First take 80 mL of conventionally prepared DMEM / F12 culture solution, add 1 g of tobramycin, and filter it through a 0.22 μm microporous membrane to sterilize after it is completely dissolved. ;

[0056] After soaking the carrier bracket in the carrier bracket rehydration solution for 2 hours for rehydration treatment, it is used as the lamellar cornea carrier bracket after tissue engineer...

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Abstract

The invention relates to an in-vitro construction method of a tissue engineering posterior lamellar cornea. The tissue engineering posterior lamellar cornea includes human corneal stromal cells and human corneal endothelial cells which are adopted as corneal seed cells and an acellular porcine corneal matrix which has a posterior elastic layer and is adopted as a tissue engineering corneal carrierscaffold, wherein the in-vitro construction method of the tissue engineering posterior lamellar cornea is characterized by comprising the steps: inoculating the corneal stromal seed cells to the scaffold firstly so as to construct the stromal layer part of the tissue engineering posterior lamellar cornea, and then inoculating the corneal endothelial seed cells to the posterior elastic layer so asto construct the tissue engineering posterior lamellar cornea which has a tissue structure and functions similar to the tissure structure and functions of a normal posterior lamellar cornea. The international problem is solved that human corneal endothelial cells cannot be proliferated in vitro, and a high-density and tight-connection single-layer endothelial structure with 3400-3600 cells / mm<2>is formed by the corneal endothelial cells; and an injection inoculation method is adopted for inoculation of the corneal stromal cells, not only can the in-vitro culture time be shortened greatly, but also the structure integrity of the carrier scaffold can be maintained; and therefore, needs of high quality and low cost can be met, and mass production and promotion and application are achieved.

Description

technical field [0001] The invention belongs to the construction technology in the field of tissue engineering of regenerative medicine, and in particular relates to an in vitro construction method of lamellar cornea after tissue engineering. Background technique [0002] The cornea is located at the front of the eyeball and is the only immune-pardoned area in the human body. It has no blood vessels and is transparent, providing most of the refractive power for the eyes. From outside to inside, the tissue structure of the cornea is the corneal epithelium, Bowman's membrane, stroma, Bowman's membrane and endothelium. The corneal endothelial layer is composed of a single layer of flat, tightly connected hexagonal endothelial-like cells, which plays a key role in maintaining the transparency and normal thickness of the cornea, and has the function of the cornea-aqueous humor barrier. There are no blood vessels in the cornea, so it can only obtain nutrients / oxygen from the aque...

Claims

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Application Information

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IPC IPC(8): A61L27/38A61L27/50C12N5/079
CPCA61L27/3804A61L27/3839A61L27/3895A61L27/3808A61L27/50C12N5/0621A61L2430/16A61L2430/40C12N2531/00C12N2533/56C12N2501/115C12N2501/11
Inventor 樊廷俊徐彬赵君
Owner OCEAN UNIV OF CHINA
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