Method for producing brain organoids

A manufacturing method and organoid technology, applied in the field of brain organoid manufacturing, can solve problems such as limited utilization of brain tissue and inability to understand human cerebral cortex in detail.

Pending Publication Date: 2021-06-18
JSR CORPORATIOON +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Unlike the generation of the cerebral cortex in mice, for which a great deal of information is available, the generation of the human cerebral cortex cannot be understood in detail due to the limited availability of fetal brain tissue

Method used

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  • Method for producing brain organoids
  • Method for producing brain organoids
  • Method for producing brain organoids

Examples

Experimental program
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Effect test

experiment example 1

[0176] According to "Nakagawa M., et al., A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells stem cells), Scientific Reports (Scientific Reports), 4, 3594, 2014", human iPS cells (PCiPS771 strain, Lot. A01QM28, manufactured by ReproCELL) were cultured without a feeder layer. As the feeder-free medium, StemFit AK02N (manufactured by Ajinomoto Co., Ltd.) or iMatrix-511 (manufactured by Nippi Co., Ltd.) was used in a feeder-free scaffold.

[0177] As a specific expansion culture operation, first, use phosphate buffered saline (hereinafter abbreviated as "PBS (Phosphate Buffered Saline)") to reach 60% to 80% confluence (60% to 80% of the culture area is covered with cells) Human iPS cells (PCiPS771 strain, Lot.A01QM28, manufactured by ReproCELL) were washed and dispersed into single cells using TrypLE Select (manufactured by Thermo Fisher Scientific). Then, the human iPS cells dispersed into single cells were seeded on a plasti...

experiment example 2

[0180] 80% confluent human iPS cells (PCiPS771 strain, Lot.A01QM28, manufactured by ReproCELL) were treated for 2 hours in the presence of Y27632 (ROCK inhibitor, 10 μM) to obtain human iPS cells.

[0181] The human iPS cells after the 2-hour treatment were subjected to single-cell treatment using a cell dispersion solution (product name "TrypLE Select", manufactured by Thermo Fisher Scientific Corporation) by pipetting. Human iPS cells made into single cells were placed in 100 μL of aggregation medium at 37° C. in a container with 5 vol% CO 2 Suspension culture was carried out in the presence of non-cell adherent 96-well culture plate (product name "PrimeSurface 96V bottom plate", manufactured by Sumitomo Bakelite (BakeLite) Co., Ltd.) 2 × 10 4 indivual.

[0182]As the medium for aggregation, StemFit AK02N (manufactured by Ajinomoto Co.) supplemented with non-essential amino acids (Non-essential Amino Acids) (manufactured by Thermo Fisher Scientific, diluted 200 times), peni...

experiment example 3

[0187] On the 7th day after the start of the suspension culture in Experimental Example 2, 230 μL of the aggregation medium was removed from each well. Next, the first medium was added in units of 150 μL / well, and mixed on an ice bath so that the extracellular matrix and other components were uniformly dispersed, at 37° C. and 5 vol% CO in the container. 2 Suspension culture was carried out without agitation in the presence of .

[0188] As the first medium, various kinds of Dulbecco's Modified Eagle Medium: NutrientMixture F-12 (manufactured by Thermo Fisher Scientific) added with 1×N2 Supplement (Supplement) (manufactured by Thermo Fisher Scientific, diluted concentration 200 times), heparin sodium salt (Heparin Sodium Salt) (manufactured by Sigma, final concentration 10 μg / mL), non-essential amino acids (Non-essential Amino Acids) (manufactured by Thermo Fisher Scientific, diluted 200 times) , Penicillin / Streptomycin (manufactured by Nacalai Tesque, diluted 100 times), glu...

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Abstract

A method for producing brain organoids comprising a step of culturing a neuroectoderm marker-positive cell aggregate in a culture medium that contains extracellular matrix at a concentration of more than 10 volume%.

Description

technical field [0001] The invention relates to a method for manufacturing brain organoids (Brain Organoids). More specifically, the present invention relates to a method for producing a brain organoid, a brain organoid, a kit for evaluating a drug effect of a test substance, a method for evaluating a drug effect of a test substance, and a method for evaluating the drug effect of a nervous system cell or nervous tissue. Therapeutic drugs for diseases caused by diseases and pharmaceutical compositions for diseases caused by disorders of nervous system cells or nerve tissues. This application claims priority based on Japanese Patent Application No. 2018-214930 filed in Japan on November 15, 2018, and the content thereof is incorporated herein by reference. Background technique [0002] The mammalian cerebral cortex has a multi-layered structure (layers I-VI), which is slowly formed from the formation of the fetal cerebral cortex. The cerebral cortex grows from the neuroepith...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079A61K35/30A61P25/00C12Q1/02
CPCA61K35/30A61P25/00G01N33/5082C12N5/0697G01N2800/28C12N2506/45C12N2501/727C12N2533/90C12N5/0619C12N2501/15C12N2513/00C12N2501/415C12N5/0618
Inventor 平峯勇人石川充冈野栄之
Owner JSR CORPORATIOON
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