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Method for evaluating cell differentiation state

A cell and state technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve problems such as cell preciousness

Active Publication Date: 2019-08-30
SYSMEX CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cells prepared in regenerative medicine are very precious, and it is not preferable to consume some of the cells for destructive testing in order to evaluate the quality of differentiated cells as in Non-Patent Document 1.

Method used

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  • Method for evaluating cell differentiation state
  • Method for evaluating cell differentiation state
  • Method for evaluating cell differentiation state

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Comparison of measured values ​​of miR302 in the culture supernatant of iPS cells and the culture supernatant of cancer cells

[0059] (1) Preparation of measurement samples

[0060] 75cm inside which was coated with cell culture substrate iMatrix-511 (manufactured by Nippi Corporation) 2 15 mL of cell culture medium Essential 8 (manufactured by Gibco) was added to the flask (manufactured by Corning Incorporated). Add iPS cells (10 5 cells / 1.5mL), adherent culture for 1 day. After culturing, the supernatant was recovered.

[0061] Similarly for colorectal cancer cell line HCT116(10 5 cells / 1.5mL) for 1-day adherent culture. The culture solution was centrifuged at 1500 g for 10 minutes, and the supernatant was recovered.

[0062] 50 μL of the recovered supernatant and 50 μL of a negative control (Essential 8 not used for cell culture without cells) were used as measurement samples.

[0063] (2) Determination of miR302

[0064] Total RNA was recovered f...

Embodiment 2

[0067] Example 2: Comparison of measured miRNA values ​​of miR302 / 367 cluster in iPS cell culture supernatant, vascular endothelial cell (iPS cell-derived) culture supernatant, and vascular endothelial cell (cell line) culture supernatant

[0068] (1) Preparation of measurement samples

[0069] For the culture supernatant of iPS cells, the same procedure as in Example 1 was carried out. 75cm inside which was coated with cell culture substrate fibronectin (manufactured by Invitorogen Corporation) 2 15 mL of a cell culture medium VascuLife VEGF Medium Complete Kit (manufactured by Kurabo) was added to the flask (manufactured by Corning Incorporated). To this was added vascular endothelial cells differentiated from iPS cells iCell (trademark) Endotherial Cells (manufactured by Cellular Dynamics International) (10 5 cells / 1.5mL), adherent culture for 1 day. After the cultivation, the culture solution was centrifuged at 1500 g for 10 minutes, and the supernatant was recovered. ...

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Abstract

To provide a method whereby pluripotent stem cells can be detected at a high sensitivity without disrupting the cells after inducing differentiation. The present invention provides a method which comprises measuring miRNA in miR302 / 367 cluster in a liquid phase fraction of a cell culture liquid during and / or after inducing differentiation of pluripotent stem cells and evaluating the differentiation state of the cells in the cell culture liquid on the basis of the measurement value of miRNA. The present invention also provides a method for detecting pluripotent stem cells, said method comprising a step for measuring miRNA in miR302 / 367 cluster in a liquid phase fraction of a cell culture liquid during and / or after inducing differentiation of pluripotent stem cells and a step for detecting the pluripotent stem cells in the cell culture liquid on the basis of the measurement value of miRNA.

Description

technical field [0001] The present invention relates to methods of assessing the differentiation state of cells. Background technique [0002] In regenerative medicine, a procedure of differentiating pluripotent stem cells into desired cells is performed. However, not all pluripotent stem cells are necessarily differentiated even through the differentiation-inducing operation, and pluripotent stem cells may remain. If cell transplantation is performed in a state where pluripotent stem cells remain, tumors may be generated from the transplanted cells. Therefore, it is necessary to evaluate the differentiation state of the cells in the culture medium. [0003] As a technique for detecting hiPSCs (human induced pluripotent stem cells; human iPS cells) after a differentiation-inducing operation, the method described in Non-Patent Document 1 is known. Non-Patent Document 1 describes a method in which some cells are harvested after the differentiation induction operation, RNA i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12N15/09
CPCC12N15/113C12Q1/6881C12Q2600/178C12N2506/45
Inventor 相原祐希
Owner SYSMEX CORP