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Test method for induced differentiating human embryo stem cells to retina pigment epithelial cells

A human embryonic stem cell, retinal pigment technology, applied in embryonic cells, animal cells, vertebrate cells and other directions, can solve the problems of low, until 1998, Thomson in the United States was first established and other problems, to achieve rich sources, economical, high success rate, Overall good effect

Inactive Publication Date: 2012-05-23
薛志刚 +1
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Problems solved by technology

Due to the low success rate of human ES cell line establishment, it is known that Thomson in the United States first established human ES cell line in 1998, so there are still few studies on the directed differentiation of human ES cells.

Method used

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Embodiment Construction

[0016] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, so as to define the protection scope of the present invention more clearly.

[0017] A test method for inducing differentiation of human embryonic stem cells into retinal pigment epithelial cells in vitro, the specific steps are: A) 0.25% trypsin and 0.1mg / mL collagenase iv preparation in 1mM CaCl 2 and 20% KSR in PBS solution to digest ES clones to make ES cell clusters of 5-10 cells. Digested and suspended in serum-free medium containing WNT inhibitor DKK1 (100 ng / mL) and NODAL inhibitor LEFTY-A (500 ng / mL); B) Short-term incubation of ES clones in gelatin-coated dishes to remove Feeder cells; C) ES cell clusters were cultured in non-adherent bacterial culture dishes at a concentration of 6.7×102 cell clusters per millil...

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Abstract

The invention provides a test method for induced differentiating human embryo stem cells to retina pigment epithelial cells, which comprises the following steps that: A) 0.25% pancreatic enzyme and 0.1mg / mL collagenase iv are mixed into phosphate buffered saline (PBS) solution containing 1mM calcium chloride (CaCl2) and 20% KSR, the solution is used for digesting embryo stem (ES) clones, so the ES becomes an ES cell group with 5 to 10 cells; B) the ES clone is incubated in a culture dish which is wrapped by gelatin in a short time so as to remove the feeder layer cells; and C) the ES cell group is cultured in a continuous differential culture medium with the concentration of 6.7*102 cell groups per liters in a non-adhesive bacteria culture dish. The test method has the advantages such as simple and convenient method, good repeatability, high successful rate, good omnisexuality, rich and economical source and the like.

Description

technical field [0001] The present invention relates to a technology of directed differentiation of embryonic stem cells, in particular to a test method for inducing differentiation of human embryonic stem cells into retinal pigment epithelial cells in vitro. Background technique [0002] Human embryonic stem cells (human Embnyonic Stem Cells, ES cells) are totipotent hepatocytes and have the potential to differentiate into various human tissue cells of the three germ layers. How to induce ES cells to differentiate into a single, specific cell is the focus of current research, which will bring new hope for tissue engineering and organ replacement therapy. At present, ES cells of uncut animals (mice, rats, guinea pigs, etc.) can be induced in vitro to differentiate into various types of cells such as neurons, hematopoietic cells, phenotype cells, chondrocytes, cardiomyocytes, and vascular skin cells. Due to the low success rate of establishing human ES cell lines, it is know...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
Inventor 薛志刚范国平
Owner 薛志刚
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