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Production method for cell mass including neural cells/tissue and non-neural epithelial tissue, and cell mass from same

A cell clump and epithelial tissue technology, applied in the field of cell clumps, can solve the problem of high cost of recombinant proteins

Pending Publication Date: 2020-07-07
SUMITOMO CHEM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, since the recombinant protein (BMP4) is expensive, a method of preparing a cell mass including nervous system cells or neural tissue and non-neuroepithelial tissue without using the recombinant protein at a high concentration for a long time is strongly desired.

Method used

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  • Production method for cell mass including neural cells/tissue and non-neural epithelial tissue, and cell mass from same
  • Production method for cell mass including neural cells/tissue and non-neural epithelial tissue, and cell mass from same
  • Production method for cell mass including neural cells/tissue and non-neural epithelial tissue, and cell mass from same

Examples

Experimental program
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Effect test

preparation example Construction

[0115] Preparation of targeting vectors for homologous recombination of target genes and effective screening of homologous recombinants can be carried out according to the methods described in: Gene Targeting, A Practical Approach (gene targeting, practical methods), IRL Press at Oxford University Press (1993); Biomanual Series 8, Gene Targeting, Making of Mutant Mouse using ES cell (gene targeting, using ES cells to produce mutant mice), YODOSHA CO., LTD. (1995); etc. As the targeting vector, either of a replacement type or an insertion type can be used. As the screening method, methods such as positive selection, promoter selection, negative selection, polyadenylic acid (polyA) selection and the like can be used.

[0116] Examples of methods for selecting desired homologous recombinants from selected cell lines include DNA hybridization method (Southern hybridization method) for genomic DNA, PCR method, and the like.

[0117] The "mammal" in the present invention includes r...

Embodiment approach

[0291] One embodiment of the implementation method of the compound irritation test of the present invention is the implementation method of the compound irritation test, and the method includes the following steps (A) to (D).

[0292] (A) The step of contacting the "cell aggregate including nervous system cells or nerve tissue and non-neuroepithelial tissue" or "non-neuroepithelial tissue sheet" with the substance to be tested;

[0293] (B) A step of staining the "cell mass containing nervous system cells or nerve tissue and non-neuroepithelial tissue" or "non-neuroepithelial tissue sheet" that has been in contact with the test substance with a dye;

[0294] (C) The step of extracting the dye from the stained "cell mass containing nervous system cells or nerve tissue and non-neuroepithelial tissue" or "non-neuroepithelial tissue slice";

[0295] (D) A step of quantifying the amount of the extracted dye and evaluating the irritation of the compound to be evaluated.

[0296] ...

Embodiment 1

[0333] Example 1: Cell aggregates made from human ES cells comprising nerve tissue and non-neuroepithelial tissue

[0334] Human ES cells (KhES-1 strain, obtained from Kyoto University) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium was used as the feeder-free medium, and laminin 511-E8 was used for the feeder-free scaffold. As a specific maintenance culture operation, it was carried out in the same manner as in Comparative Example 1. Thereafter, under the same conditions as in Comparative Example 1, suspension culture using a 96-well plate was started. Then, on the second day after the start of the suspension culture, a serum-free medium containing IWP-2, SB-431542, and BMP4 containing no Y27632 was added at 100 μl per well. As for BMP4, 3 nM was added to the supplemented media so that the final concentration in the wells was 1.5 nM. Afterwards, on the 6th, 10th, 13th, 17th, 21st, and 24th ...

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Abstract

The present invention addresses the problem of providing a means of efficiently producing, from pluripotent stem cells, a cell mass that includes neural cells / tissue and non-neural epithelial tissue.Provided is a production method for a cell mass that includes 1) neural cells or neural tissue, and 2) non-neural epithelial tissue, said method comprising steps (1) and (2): (1) a first step in whichpluripotent stem cells are suspension-cultured in the presence of a Wnt-signaling pathway inhibitor, and a cell aggregation is formed; and (2) a second step in which the aggregate obtained in the first step is suspension-cultured in the presence of a BMP-signaling pathway activator, and a cell mass that includes 1) neural cells / tissue, and 2) non-neural epithelial tissue, is obtained.

Description

technical field [0001] The present invention relates to a method for preparing a cell mass comprising nervous system cells or nerve tissue and non-neuroepithelial tissue from pluripotent stem cells and the cell mass. Background technique [0002] In Non-Patent Document 1, it is reported that human corneas were produced by suspension culture of aggregates produced from human iPS cells in the presence of Wnt signal transduction pathway inhibitors and a basement membrane preparation (matrigel) derived from mouse sarcoma organoids. However, from the viewpoint of avoiding the mixing of heterogeneous components and undetermined factors, the preparation of cell aggregates including nervous system cells or neural tissues and non-neuroepithelial tissues without using basement membrane preparations derived from mouse sarcoma has been strongly desired. method. [0003] In Non-Patent Document 2, it is reported that human iPS cells were cultured adherently on a flat surface to produce ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079A61K35/30A61K35/545A61L27/36A61L27/38A61P27/02C12N5/071C12Q1/02
CPCA61P27/02C12N5/0619C12N2501/155C12N2501/415C12N2506/02C12N2506/45C12N2500/90C12N2501/727C12N2513/00C12N2533/52C12N2500/99C12N2501/41C12N5/0621C12N5/062C12N5/0697G01N33/5008C12N5/0618C12Q1/025A61K35/30A61L27/36A61L27/38A61P27/00C12N2501/15C12N2501/42
Inventor 中野德重
Owner SUMITOMO CHEM CO LTD
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