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Culture substratum for human pluripotent stem cells, and use thereof

A technology for human pluripotent stem cells and culture substrates, which is applied in the field of culture substrates for human pluripotent stem cells, can solve the problems of difficult technology and low yield, and achieve the effects of low cost, rapid expansion, and high safety

Active Publication Date: 2012-07-18
OSAKA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, since the molecular weight of the three subunit chains constituting laminin is as high as 200,000 to 400,000 Da, there are problems as follows: the technology of directly expressing the heterotrimeric molecule formed by the combination of the above subunit chains in the form of recombinant protein It is not easy in itself, and the yield is also small

Method used

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  • Culture substratum for human pluripotent stem cells, and use thereof
  • Culture substratum for human pluripotent stem cells, and use thereof
  • Culture substratum for human pluripotent stem cells, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0165] [Example 1: Comparison of concentration-dependent adhesion efficiency of human ES cells in various extracellular matrices]

[0166] Dilute rhLM511E8, rhLM332E8, rhLM511, rhLM332, rhLM111, matriogel, fibronectin, and vitronectin with Dulbecco's PBS (Wako Pure Chemical #045-29795) or DMEM-F12 (SIGMA #D6241), in a 96-well microplate (BD Bioscience #351172) was added to each extracellular matrix solution to achieve a final concentration of 0-25 μg / cm 2 range, stand at room temperature for 2 hours for coating.

[0167] Add TrypLE Select (Invitrogen #12563011) to mTeSR1 cultured cells (P1), and treat at 37°C for 3 minutes to disperse the cells into single cells. After counting the number of cells, 5×10 4 Density of cells / well Cells were seeded into microwell plates. Six hours after cell seeding, the supernatant was removed, the wells were washed once with DMEM-F12, and the cells were fixed by adding 10% neutral buffered formalin for 10 minutes. After replacing with 100% e...

Embodiment 2

[0169] [Example 2: Study of Adhesion Efficiency of Single Human ES Cells Dependent on Seeding Density in Culture Vessels Coated with rhLM511E8]

[0170] In the subculture of human ES cells, the viability of the cells is extremely low when they are dispersed into single cells using trypsin / EDTA, etc., so they are generally subcultured while maintaining the colony shape. Therefore, the adhesion efficiency of single human ES cells when rhLM511E8 was applied was compared with that of single human ES cells when matrigel was applied.

[0171] Dilute rhLM511E8 with Dulbecco’s PBS to a final concentration of 1.5 μg / cm 2 . Dilute matriogel with DMEM-F12 to a final concentration of 25 μg / cm 2 . Each dilution was added to a 48-well multiwell plate (BD Bioscience #351178) and allowed to stand at room temperature for 2 hours for coating.

[0172] Add TrypLE Select to mTeSR1 cultured cells (P1), and treat at 37°C for 3 minutes to disperse the cells into single cells. In a 48-well multi...

Embodiment 3

[0174] [Example 3: Morphological observation and surface antigen analysis of human ES cells in culture vessels coated with rhLM511E8 or rhLM332E8]

[0175] Dilute rhLM511E8 or rhLM332E8 with Dulbecco’s PBS to a final concentration of 1.5 μg / cm 2 . Dilute matriogel with DMEM-F12 to a final concentration of 25 μg / cm 2 . Each dilution was added to a 35 mm i-Grip cell culture dish (BD Falcon #353001), and allowed to stand at room temperature for 2 hours for coating.

[0176] Add TrypLE Select to mTeSR1 cultured cells (P1), and treat at 37°C for 3 minutes to disperse the cells into single cells. In a 35mm easy-to-grip cell culture dish coated with rhLM511E8, rhLM332E8 or matriogel, 5×10 4 cells / cm 2 seeded at a cell density. Observation was carried out every day from the beginning of cell inoculation, and the morphological changes were photographed and recorded.

[0177] Five days after cell inoculation, for surface antigen analysis, the cells were treated with TrypLESelect ...

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Abstract

Disclosed are: a culture substratum which can retain and culture human pluripotent stem cells under a feeder-free culture environment while maintaining the pluripotency of the stem cells; and a method for culturing human pluripotent stem cells using the culture substratum. Human pluripotent stem cells which are dispersed in the form of single cells are seeded onto a culture substratum at a density of 4*104 to 10*104 cells / cm<2>, wherein the culture substratum is one intended to be used for culturing human pluripotent stem cells and has been coated with an E8 fragment of human laminin [alpha]5[beta]1[gamma]1 or an E8 fragment of human laminin [alpha]3[beta]3[gamma]2 preferably at a concentration of 0.5 to 25 [mu]g / cm<2>. In this manner, human pluripotent stem cells can be expanded rapidly while maintaining the pluripotency of the stem cells.

Description

technical field [0001] The present invention relates to a culture substrate for human pluripotent stem cells and uses thereof, specifically, to a culture substrate for human pluripotent stem cells, a method for culturing human pluripotent stem cells using the culture substrate, and a method for rapidly expanding human pluripotent stem cells And a method for isolating human pluripotent stem cell clones derived from a single cell. Background technique [0002] The application of human pluripotent stem cells such as human ES cells and human iPS cells in regenerative medicine is attracting worldwide attention. However, in order to culture and maintain human pluripotent stem cells in a state of maintaining pluripotency (pluripotency), generally, a common method is to use mouse or human-derived fibroblasts as feeder cells for co-cultivation, and The use of feeder cells has been a huge constraint in the clinical application of human pluripotent stem cells. In order to solve this ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/00C12N5/07
CPCC12N2533/52C12N5/0696C12N5/0606
Inventor 关口清俊二木杉子溪口征雅林麻利亚中辻宪夫宫崎隆道川濑荣八郎末盛博文
Owner OSAKA UNIV
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