Cell aggregation promoting agent
A technology of accelerators and cells, which is applied in the direction of cell culture active agents, embryonic cells, animal cells, etc., can solve the problems of difficulty in expanding the scale, the number of cells depends on the culture area, and the difficulty in supplying regenerative medical cells, etc., to achieve high yield and live cells. high rate effect
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[0178]
[0179] Another aspect of the present invention is a method for producing cell aggregates, including the step of suspending culturing cells in a medium containing an SRF inhibitor, or an SRF inhibitor and a ROCK inhibitor.
[0180] According to this method, moderately sized cell aggregates can be prepared in high yield. In particular, when the cells are stem cells, cell aggregates of an appropriate size can be produced in high yield while maintaining the undifferentiated nature of the stem cells.
[0181] The specific embodiments of SRF inhibitor, ROCK inhibitor, culture medium and cells are as above.
[0182] For the concentration of the SRF inhibitor in the medium in the above-mentioned process, for example, the SRF inhibitor is CCG-1423 (Cayman Chemical, CAS No.285986-881, C 18 h 14 CIF 6 N 2 o 3, molecular weight = 454.8), the lower limit of the concentration of the inhibitor is not particularly limited as long as it can promote cell aggregation, but it is p...
Embodiment 1
[0208]
[0209] As human iPS cells, TkDN4-M strain (Institute of Medical Sciences, The University of Tokyo) was used. Inoculate human iPS cells on culture dishes coated with Vitronectin (Thermo Fisher Scientific), using Essential 8 as a medium TM (Thermo Fisher Scientific) maintained culture. Accutase (Thermo Fisher Scientific) was used as a cell detachment agent during passage. In addition, Y-27632 (Wako Pure Chemical Industries, Ltd.) was added to the medium at a concentration of 10 μM only at the time of cell inoculation. Media changes were performed daily. Human iPS cells up to passage 50 were used in the experiment.
Embodiment 2
[0210]
[0211] The effect of promoting cell aggregation by adding an SRF inhibitor was investigated.
[0212] (method)
[0213] The human iPS cells cultured according to the procedure of Example 1 were treated with Accutase for 3 minutes to 5 minutes, detached and dispersed into single cells. The cells were suspended in Essential 8 containing BSA (Wako Pure Chemical Industries, Ltd.) at a final concentration of 5 mg / mL and Y-27632 (Wako Pure Chemical Industries, Ltd.) at a final concentration of 2.5 μM.TM A part of the culture medium was stained with trypan blue, and the number of cells was checked. Prepared to contain 2 x 10 per 1 mL 5 cells. Separately adjust the cell aggregation promoter so that the final concentration of CCG-1423 (Cayman, 10010350) is 4.55mg / mL (10mM), add the above-mentioned adjusted cell aggregation promoter to the above-mentioned cell suspension, so that the final concentration of CCG-1423 The concentration was 10 μM, and then, 1.3 mL / well was ino...
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