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Neural stem cell induced differentiation culture medium and induced differentiation method

A technology of neural stem cells and induction medium, applied in nervous system cells, biochemical equipment and methods, animal cells, etc., can solve the problems of undisclosed applications in the field of nerve regeneration, and achieve huge economic and social effects

Pending Publication Date: 2022-07-12
IREGENE THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the development direction of this molecule is mainly based on drug development and treatment of lung cancer, liver cancer and glioblastoma (Pharmaceutics.2020May 18; 12(5):459), and does not disclose any application in the field of nerve regeneration

Method used

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  • Neural stem cell induced differentiation culture medium and induced differentiation method
  • Neural stem cell induced differentiation culture medium and induced differentiation method
  • Neural stem cell induced differentiation culture medium and induced differentiation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1: Preparation of Neural Stem Cell Induction Medium

[0059] The formula of neural induction basal medium (hereinafter referred to as NouvNeu001): Duchenne's modified Eagle / F12 medium (DMEM / F12), minimum essential medium non-essential amino acids (MEM non-essential amino acids, Minimum EssentialMedium Non-Essential Amino Acids, Thermo Fisher, product number: 11140076), sodium chloride (SodiumChloride, 0.5g / L), sodium selenite (Sodium selenite, 13.6μg / L), insulin (22μg / ml), recombinant human transferrin (100ng / ml) .

[0060] The neural stem cell induction medium used in the present invention is prepared by adding 55nM-24μM LY2157299 (Selleck, S2230) to NouvNeu001 basal medium, and the final concentration is preferably 300nM, 500nM, 700nM, 900nM, 1μM, 2.5μM, 5μM , 7.5 μM, 10 μM, 12.5 μM, 15 μM, 17.5 μM, 20 μM or 22 μM, most preferably 12.5 μM. The above-mentioned medium is referred to as the LY induction medium for short, and the experimental group using this me...

Embodiment 2

[0062] Example 2: Induction and identification of neural stem cells

[0063] 2.1 Chemical induction of neural stem cells:

[0064] Human pluripotent stem cells include embryonic pluripotent stem cells, such as the H9 cell line and human induced pluripotent stem cells. Wherein, the human induced pluripotent stem cells used in the present invention are carried out according to the "reprogramming medium and the method for culturing reprogramming induced pluripotent stem cells" (the method in the patent ZL201910050800.7), from CD34 + Cell reprogramming was obtained.

[0065] Human pluripotent stem cells were used to coat T25 cell culture flasks with Matrigel (STEMCELL Technologies), and then placed in a 37°C incubator for more than one hour after plating. Follow 1×10 6 Cells were seeded in T25 flasks for expansion and passage.

[0066] For neural induction, use 50μg / ml polylysine (SIGMA, Cat. No.: P6407) to coat a 6-well culture plate, place the plate in a 37°C incubator and i...

Embodiment 3

[0081] Example 3: Identification of Differentiation Function of Neural Stem Cells

[0082] The neural stem cells obtained by the LY induction method in Example 2 were subjected to directed differentiation in NouvNeu001 basal medium. For neuronal differentiation, use 50μg / ml polylysine (SIGMA ALDRICH, Cat. No.: P6407) to coat a 6-well culture plate, place the plate in a 37°C incubator and incubate for more than 3 hours until cells are seeded.

[0083] 3.1 Pain receptor neurons:

[0084] The neural stem cells obtained in Example 2 were 1 × 10 5 The ratio of each bottle was inoculated into a T25 culture flask, and 3 μM CHIR99021 (Selleck, Cat. No.: S2924), 10 μM SU5402 (Tocris, Cat. No.: 3300 / 1), 10 μM DAPT (Selleck, Cat. No.: S2215) were added to NouvNeu001 basal medium, Fresh medium was replaced every 3 days until day 21. Culture conditions are 37°C, 5% CO 2 . The experimental results show that the neural stem cells obtained in Example 2 have axonal structure after differe...

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Abstract

The invention provides a new application of a TGF-beta small-molecule inhibitor in the field of nerve regeneration. The TGF-beta small-molecule inhibitor can be used for in-vitro regeneration and directional differentiation of various nerve cells and brain-like organs. By adding the culture medium into a group of basic culture media with clear chemical components, the pluripotent stem cells can be induced and converted into adult cells derived from various neural stem cells, and the number of nerve cells obtained through induction is greatly increased. The induction system provided by the invention expands the brand-new function of a single small molecule in the field of ectoderm cell induced differentiation, and avoids the use of B27 equivalent serum at the same time, thereby completely avoiding the potential danger caused by the existence of animal-derived components in the cell culture process, and greatly expanding the clinical prospects of transplantation of various nerve cells.

Description

technical field [0001] The invention belongs to the field of biology, and particularly relates to a method for culturing ectodermal cells, a culture medium, and their application in nerve injury diseases. Background technique [0002] The ectoderm is the outermost layer formed during embryonic development. With the beginning of organogenesis, ectoderm cells gradually differentiate into important systems such as the brain, spinal cord, and sensory organs. The nervous system is an important system responsible for functions such as thinking, emotion, perception, and movement. Compared with diseases such as tumors, there are currently fewer drugs for neurological diseases and the development cycle is long. One of the most important reasons is the particularity of various primary cells in the ectodermal lineage, such as the non-regeneration of primary neurons. Sexuality has contributed to the scarcity of in vitro drug screening platforms for nervous system drugs. [0003] In ad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12N5/0793C12Q1/02A61K35/30A61P25/00
CPCC12N5/0623C12N5/0619G01N33/5058A61K35/30A61P25/00C12N2500/25C12N2500/32C12N2500/12C12N2500/90C12N2501/155C12N2506/45C12N2506/08C12N2533/52C12N2500/30C12N2503/02C12N2501/405G01N2500/10A61P25/08G01N33/5088G01N33/5082C12N5/0618C12N5/0697C12N2501/15C12N2513/00
Inventor 魏君蔡萌周佳侯梦莹云轩
Owner IREGENE THERAPEUTICS LTD
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