Human retinal pigment epithelial cell separation and cryopreservation method

A technique for retinal pigment and epithelial cells, which is applied in the field of cryopreservation and separation of human retinal pigment epithelial cells, can solve problems such as being unsuitable for processing retinal pigment epithelial cells, and achieves shortening the time for releasing the adhesion, reducing cell damage, and steps easy effect

Pending Publication Date: 2017-02-22
EYECURE THERAPEUTICS INC JIANGSU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The purpose of the present invention is to solve the problem that existing digestive enzymes are not suitable for processing retinal pigment epithelial cells, and to provide a method for separating and freezing human retinal pigment epithelial cells

Method used

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  • Human retinal pigment epithelial cell separation and cryopreservation method
  • Human retinal pigment epithelial cell separation and cryopreservation method
  • Human retinal pigment epithelial cell separation and cryopreservation method

Examples

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Embodiment 1

[0029] A method for separating and freezing human retinal pigment epithelial cells, comprising the steps of:

[0030] (1) Clearing the culture medium: take the retinal pigment epithelial cells out of the carbon dioxide incubator, and suck out the culture medium.

[0031] (2) Washing: Add 5ml of DPBS (CTS) into the culture bottle, shake gently, and remove the liquid after complete washing.

[0032] (3) Digestion: Add 2ml of retinal pigment epithelial cell digestion solution to the culture bottle, mix well and put it in a carbon dioxide incubator for 10 minutes.

[0033] (4) Preparation: Take one 15ml centrifuge tube and add 3ml of retinal pigment epithelial cell culture medium for later use;

[0034] (5) Termination: Take out the RPE cells after the time is over, add 2ml RPE cell culture medium to terminate the reaction, pipette the cells with a manual pipette, and collect the cells into a prepared 15ml centrifuge tube;

[0035] (6) Observation: put the discarded culture bott...

Embodiment 2

[0041] A method for separating and freezing human retinal pigment epithelial cells, comprising the steps of:

[0042] (1) Clearing the culture medium: take the retinal pigment epithelial cells out of the carbon dioxide incubator, and absorb the culture medium;

[0043] (2) Washing: Add 10ml of DPBS (CTS) to the culture bottle, shake gently, and remove the liquid after complete washing;

[0044] (3) Digestion: Add 3ml of retinal pigment epithelial cell digestion solution to the culture bottle, mix well and put it in a carbon dioxide incubator for 15 minutes;

[0045] (4) Preparation: Take one 15ml centrifuge tube and add 5ml of retinal pigment epithelial cell culture medium for later use;

[0046] (5) Termination: Take out the RPE cells after the time is over, add 3ml RPE cell culture medium to terminate the reaction, pipette the cells with a manual pipette, and collect the cells into a prepared 15ml centrifuge tube;

[0047] (6) Observation: put the discarded culture bottle ...

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Abstract

The invention relates to a human retinal pigment epithelial cell separation and cryopreservation method which includes the steps: firstly, cleaning and washing retinal pigment epithelial cells by nutrient solution; secondly, mixing Dispase II separase and DPBS buffer solution according to the ratio of 1:(40-50)w/v to prepare Dispase II separase solution; thirdly, mixing the Dispase II separase solution and Tryple select solution according to the volume ratio of 1:(2-3) to prepare digestive fluid for digestive separation; finally, performing cryopreservation after counting and centrifuging. The separation method is simple, the steps are easily controlled, the digestive fluid prepared by mixing the Dispase II separase solution with the Tryple select solution is used for performing digestive separation for the retinal pigment epithelial cells, adherence removing time of the retinal pigment epithelial cells can be shortened, cell integrity is kept, and cell injury is greatly reduced.

Description

technical field [0001] The invention relates to a method for separating and freezing human retinal pigment epithelial cells, belonging to the technical field of biological materials. Background technique [0002] Retinal degenerative diseases are the main cause of irreversible blindness, including age-related macular degeneration, retinitis pigmentosa, and Stargardt disease. Among them, there are about 30 to 50 million patients with age-related macular degeneration worldwide, and about one million patients with retinitis pigmentosa. At present, there are more than 20 million patients with age-related macular degeneration in my country, and it is expected to double in 2050. Therefore, studying human retinal pigment epithelial cells and finding a targeted treatment plan is one of the key topics in the current biomedical research. In the digestion process of human retinal pigment epithelial cells, the effect of traditional trypsin is not good: the digestion ability of retinal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/079A01N1/02
CPCC12N5/0625A01N1/0221C12N5/0621C12N2500/30C12N2500/32C12N2500/84C12N2501/39C12N2501/395
Inventor 谢磊李宁范国平方攀峰秦承学陆梦华
Owner EYECURE THERAPEUTICS INC JIANGSU
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