Stem cell culture medium and method

A stem cell culture, stem cell technology, applied in tissue culture, biochemical equipment and methods, microorganisms, etc., can solve problems such as death, and achieve the effect of improving survival rate

Inactive Publication Date: 2009-10-21
RIKEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, even if human ES cells are not completely dissociated into single cells (such as cultured from small cell clumps), the problem remains that they still die in large numbers (Frisch et al., Curr. Opin. Cell Biol. 13, 555 -562(2001))
However, there is no report on the culture of stem cells such as ES cells with media containing ROCK inhibitors

Method used

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  • Stem cell culture medium and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1. Using ROCK Inhibitor Y-27632 to Improve Human Embryonic Stem Cell Cloning Efficiency

[0093] experiment method

[0094] The human embryonic stem cells used in the experiments described here refer to the embryonic stem cells (KhES-1, KhES-2 and KhES-3) obtained from human blastocysts, which were obtained from the Institute for Frontier Medical Sciences, Kyoto University, Japan. ), its distribution and use (mainly KhES-1) complies with the Japanese government guidelines for human embryonic stem cells. According to the method of Nakatsuji laboratory (Suemori et al., Biochem Biophys Res Commun. 345, 926-32 (2006)), in a plastic dish, in mouse embryonic fibroblasts (inhibited with mitomycin) composed of Undifferentiated human embryonic stem cells were cultured under feeder cell layer conditions. More specifically, the medium used contained KSR (Invitrogen / Gibco-BRL company) and the final concentration was 20%, 1X NEAA (non-essential amino acid, Invitrogen / Gibco...

Embodiment 2

[0101] Example 2. Activation of Rho in dissociated human embryonic stem cells

[0102] experiment method

[0103] The maintenance culture of human embryonic stem cells uses the passaged cells of the small cell mass described in Example 1.

[0104] As described in Example 1, the human embryonic stem cells were dissociated into single cells by trypsin digestion, suspended in the culture medium to maintain culture, and incubated at 37°C. After incubating for 0 minutes, 15 minutes, 30 minutes, 60 minutes, and 120 minutes, the cells were obtained by centrifugation, and then treated with the GTPase Activation Kit (Cytoskeleton, Denver, Colorado) according to the instructions. down method) analysis. Activation of Rho was judged by Western blotting based on an increase in the ratio of activated Rho (GTP-associated Rho) to total Rho. Cell samples were taken in a 10cm culture plate (approximately 1x10 6 cells) are prepared in batches.

[0105] test results

[0106] Significant Rho...

Embodiment 3

[0108] Example 3. Test method for clonogenic efficiency of human embryonic stem cells in maintenance culture under different kinase inhibitors

[0109]The effect of other ROCK inhibitors on maintaining human embryonic stem cells in culture was evaluated by the method described in Example 1. The ROCK inhibitors used were fasudil / HA1077 (10 μM) and H-1152 (200 nM). Inhibitors of other kinases were also used as references. Inhibitors of other kinases used were: cAMP-Rp (1-100 μM) and KT5720 (5-500 nM), both protein kinase A inhibitors; bisindolymaleimide (0.01-5 μM) and staurosporine (1-50nM), both protein kinase C inhibitors; PD98059 (0.5-50μM), a MAPK inhibitor; LY294002 (1-50μM), a PI3K inhibitor and ML-7 (0.3-30 μM), which is an MLCK inhibitor.

[0110] test results

[0111] Cloning efficiency was significantly enhanced with ROCK inhibitors (fasudil / HA1077 and H-1152) compared to no inhibitor, but no improvement was observed with other kinase inhibitors.

[0112] Thus, i...

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Abstract

The present invention discloses a stem cell medium and a culturing method. Stem cells such as embryonic stem cells (ES cells), including human ES cells, are cultured in a medium comprising a ROCK inhibitor, and a stem cell culture medium, optionally serum free, comprises a ROCK inhibitor.

Description

technical field [0001] The invention provides a method for cultivating stem cells such as embryonic stem cells (ES cells), a medium for cultivating the stem cells and applications thereof. Background technique [0002] ES cells provide an efficient candidate cell source for cell transplantation to treat central nervous diseases such as Parkinson's disease and diabetes. [0003] In ES cell research, mouse ES cells are commonly used at present, but considering clinical application, it is necessary to conduct research and development of human ES cells. However, human ES cells are more prone to death in cell culture than mouse ES cells. [0004] For example, in maintenance cultures of human ES cell subcultures, cell pellets are separated from feeder cells or culture medium by enzymatic treatment or mechanical dissociation by pipetting small cell pellets and immediately suspended cultured and then inoculated into new medium. However, compared with common cell lines and mouse E...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/02C12N5/22
Inventor 笹井芳树渡边毅一
Owner RIKEN
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