Method for efficiently isolating retinal pigmented epithelium (RPE) cells and neural retina progenitor cells (RPC) induced in vitro

A technology for separating bodies and cells, applied in the biological field, can solve the problems of low cell yield, large differences between batches, and long time-consuming, and achieve the effects of high cell purity and yield, simple method and high success rate.

Active Publication Date: 2017-05-03
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, most of these reported methods form a mixed cell population containing pigment clumps in the later stage, and mechanical picking is generally used to separate cells
This method has problems such as time-consuming, large batch-to-batch variability, and low cell yield.

Method used

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  • Method for efficiently isolating retinal pigmented epithelium (RPE) cells and neural retina progenitor cells (RPC) induced in vitro
  • Method for efficiently isolating retinal pigmented epithelium (RPE) cells and neural retina progenitor cells (RPC) induced in vitro
  • Method for efficiently isolating retinal pigmented epithelium (RPE) cells and neural retina progenitor cells (RPC) induced in vitro

Examples

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Embodiment

[0025] This example is a method for efficiently separating retinal pigment epithelial cells and retinal neuron precursor cells induced in vitro.

[0026] In this embodiment, the mixed cell is a mixed cell population containing pigment clumps obtained by differentiation of multiple stem cells, and this method can be used to achieve the purpose of separation.

[0027] In this embodiment, the method for separating cells includes the following steps:

[0028] a. Enzymatically treat the mixed cells with pigment clumps derived from hESCs into single cells, discard the supernatant after centrifugation, add medium to resuspend and inoculate them in a culture dish for suspension culture;

[0029] In this step, the hESCs are the 50th generation H1 cell line; the mixed cells with pigment clumps are obtained after 45 days of differentiation of hESCs by directed induction, as figure 1 A; The enzyme treatment into single cells is to apply 0.25% pancreatin for 20 minutes at 37°C, and pipette into sin...

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Abstract

The invention relates to a method for efficiently isolating retinal pigmented epithelium (RPE) cells and neural retina progenitor cells (RPC) induced in vitro and belongs to the field of biotechnology. The method is simple and can separate and purify a lot of mature RPE cells and RPC from a mixed cell population derived from hESCs. Compared with the prior art, the method is suitable for pigment agglomerate-containing mixed cells derived from hESCs. Compared with the traditional mechanical separation method, the method provided by the invention has the advantages of simple method, good reproducibility, high success rate, high cell purity and high yield and can simultaneously separate RPE cells and RPC.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a method for efficiently separating RPE cells and RPCs induced in vitro. Background technique [0002] Retinal degenerative diseases are the leading cause of blindness in the world. In this disease, the damage and dysfunction of retinal pigment epithelium (RPE) leads to the degeneration of photoreceptor cells, which ultimately affects the vision of the patient until blindness. Due to the non-renewability of RPE and photoreceptor cells, there is a lack of clinically effective treatment measures for these diseases. Compared with other treatment methods, cell therapy has greater prospects for the treatment of retinal degenerative diseases. [0003] Because embryonic stem cells (ESCs) have the ability to proliferate and differentiate into various cells indefinitely, they can be used as an ideal source of transplanted cells. There have been reports confirming the feasibility and safet...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/0793
Inventor 徐国彤李宗义高芙蓉吕立夏
Owner TONGJI UNIV
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