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Method for producing cell mass including pituitary tissue, and cell mass thereof

A technology of cell aggregates and pituitary glands, applied in the direction of bone/connective tissue cells, biochemical equipment and methods, artificially induced pluripotent cells, etc., can solve problems such as failure to achieve reproducible structures

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

However, the prepared cells are cultured two-dimensionally, and cannot reproduce the structure of the complex pituitary tissue of an organism that is important for functioning

Method used

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  • Method for producing cell mass including pituitary tissue, and cell mass thereof
  • Method for producing cell mass including pituitary tissue, and cell mass thereof
  • Method for producing cell mass including pituitary tissue, and cell mass thereof

Examples

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Effect test

preparation example Construction

[0141] 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.

[0142] 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.

[0143] The "mammal" in the present invention includes r...

Embodiment 1

[0368] Example 1: Cell clumps containing pituitary tissue made from human ES cells

[0369] 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.

[0370] As a specific maintenance culture operation, first, subconfluent human ES cells (KhES-1 strain) were washed with PBS, treated with enzymes using Accumax (manufactured by Innovative Cell Technologies), and then StemFit medium was added and removed from the culture dish using a cell scraper. The cells are peeled off the surface and dispersed into single cells by pipetting. After that, the above-mentioned human ES cells dispersed as single cells were seeded on plastic culture dishes coated with laminin 511-E8, and in the presence of Y27632 (ROCK inhibitor, manufactured by Wako Pure Chemical Indust...

Embodiment 2

[0376] Example 2: Maturation by Long-Term Culture of Cell Aggregates Made from Human ES Cells Containing Pituitary Tissue

[0377] 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.

[0378] As a specific maintenance culture operation, it was carried out in the same manner as in Example 1.

[0379] Thereafter, suspension culture using a 96-well plate was carried out under the same conditions as in Example 1.

[0380] On the 28th day after the start of the suspension culture, the cell aggregates were transferred to a 10 cm suspension culture plate (manufactured by Sumitomo BakeLite Co., Ltd.) in the form of 48 cell aggregates per plate, and the suspension culture was carried out until the 84th day by culturing in a serum-free medium . For the...

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Abstract

The present invention addresses the problem of providing a technique for efficiently producing a cell mass including pituitary tissue from pluripotent stem cells. A method for producing a cell mass including pituitary tissue, the method including the following steps (1) and (2): (1) a first step for suspension-culturing pluripotent stem cells in the presence of a Wnt signaling pathway inhibitor, and forming an aggregate of cells; and (2) a second step for suspension-culturing the aggregate obtained in the first step in the presence of a BMP signaling pathway agonist and a sonic hedgehog signaling pathway agonist, and obtaining a cell mass including pituitary tissue.

Description

technical field [0001] The present invention relates to a method for preparing a cell mass containing pituitary tissue and the cell mass. The present invention further relates to a cell mass comprising nervous system cells or nerve tissue, and pituitary tissue, and mesenchymal cells in the cell mass. Background technique [0002] The pituitary gland is an endocrine organ located in the head, and produces various pituitary hormones important for the maintenance and growth of living bodies, such as adrenocorticotropic hormone (ACTH) and growth hormone. In the case of pituitary dysfunction due to diseases such as pituitary hypoplasia, hypopituitarism, and pituitary adenoma, serious conditions such as developmental disorders, genital-related abnormalities, and abnormalities of the adrenal gland and thyroid gland occur. Generally speaking, damaged pituitary tissue seldom regenerates naturally to restore function. [0003] In Patent Document 1 and Non-Patent Documents 1 and 2, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735A61K35/30A61P5/06A61P5/10A61P43/00C12N5/079C12N5/10
CPCA61P5/06A61P5/10C12N2506/02C12N2513/00C12N2501/415C12N2501/41C12N2501/155C12N5/0616C12N2501/727C12N2506/45C12N2500/99C12N5/0618C12N5/0697C12N5/0652C12N2533/52C12N2501/15C12N5/0621A61K35/30A61P43/00C12N2501/113C12N2501/115C12N2501/42C12N5/0018C12N5/10
Inventor 中野德重
Owner SUMITOMO CHEM CO LTD
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