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In-vitro separation and purification method for tree-shrew corneal endothelial cells

A technology of corneal endothelial and purification methods, applied in cell dissociation methods, biochemical equipment and methods, animal cells, etc., can solve the problems of unsatisfactory purity of CECs, difficult separation of tree shrew corneal endothelial cells, etc., and avoid growth and proliferation interference, good cell morphology, and improved purification efficiency

Active Publication Date: 2016-12-21
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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Problems solved by technology

[0005] In order to solve the problem that the existing tree shrew corneal endothelial cells are difficult to be successfully separated and the purity of the isolated CECs is not ideal, the present invention provides an in vitro separation and purification method for tree shrew corneal endothelial cells, which can extract tree shrew CECs primary cells Very good separation, greatly improving purification efficiency

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  • In-vitro separation and purification method for tree-shrew corneal endothelial cells
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  • In-vitro separation and purification method for tree-shrew corneal endothelial cells

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Embodiment Construction

[0023] The present invention will be described in further detail below in conjunction with the accompanying drawings and embodiments.

[0024] (1) Isolation of tree shrew corneal endothelial primary cells:

[0025] Tear off the elastic layer of dead adult tree shrews completely, add 3mL of digestion solution, place on a shaker at 37°C for 2h, centrifuge at 1500rpm for 10min, wash with culture medium for 3 times, resuspend the cells, and centrifuge at 1500r / min 10min, discard the supernatant, add 5mL of culture medium, pipette and mix well, then move to 25cm 2 In the culture flask, mix the precipitate by pipetting, and incubate at 37°C, 5% CO 2 Cultivate under certain conditions for 25-30 days to the P5 generation, change the culture medium every other day, and obtain culture samples; observe the cell morphology under the microscope every two days and take pictures for records, see figure 1 ;

[0026] Described digestive juice is the DMEM low-sugar medium containing collage...

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Abstract

The invention discloses an in-vitro separation and purification method for tree-shrew corneal endothelial cells. The in-vitro separation and purification method includes the steps that tree-shrew corneal endothelial cells are separated, purified and subjected to passage cultivation through CECs, and corneal endothelial cells are obtained. The separated corneal endothelial cells have high cell activities, purification of the corneal endothelial cells can be achieved in the passed P3 generation is passed, the purified cells have typical endothelial cell morphology, and are identified through NSE immunofluorescence, and the dyeing rate is high. The purification efficiency of the method is greatly improved, the cost is low, using is convenient, and the large quantity of tree-shrew corneal endothelial cells can be obtained. In-vitro cultivation of the tree-shrew corneal endothelial cells is achieved, and the void that a specific corneal-endothelial-primary-cell in-vitro cultivation technology for a tree-shrew species does not exist ate present is filled; the material taking and purification methods of primary cells are easy to operate, the cost is low, and in-vitro cultivation tree-shrew corneal endothelial cells can be kept the good cell states in the long-term passage cultivation process.

Description

technical field [0001] The invention relates to a method for separating and purifying tree shrew corneal endothelial cells in vitro. Background technique [0002] Corneal Endothelial Cells (CECs) are a source of endothelial cells in the innermost layer of the cornea. Its fluid barrier and Na+-K+ ion pump functions are crucial to maintaining corneal transparency. Various damage factors cause corneal Decompensation of endothelial function will result in corneal edema and opacity. Penetrating keratoplasty is an effective method to replace diseased or damaged endothelial cells to restore corneal transparency, but the shortage of donor sources and the occurrence of postoperative immune rejection limit the operation. In recent years, with the development of in vitro cell culture technology, in vitro culture of corneal endothelial cells for endothelial transplantation has become a new development trend, and high-quality seed cells are an important factor affecting the success of e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12Q1/02
CPCC12N5/0621C12N2509/00G01N33/5005
Inventor 苗雨润宋庆凯罕园园匡德宣代解杰孙晓梅仝品芬陆彩霞王文广李娜
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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