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Chemical induction method of cortex neurons, and culture medium

A neuron and culture medium technology, applied in the field of neuron induction, to achieve the effects of high purity and yield advantages, clear chemical composition, and avoid potential dangers

Pending Publication Date: 2021-03-26
IREGENE THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In view of this, the purpose of the present invention is to provide a method and culture medium for chemically inducing cortical neurons, which solves the potential dangers caused by the presence of animal-derived components in the cell culture process in the prior art, and is thus suitable for use in drugs for nervous system diseases in vitro screening for neurodegenerative diseases

Method used

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  • Chemical induction method of cortex neurons, and culture medium
  • Chemical induction method of cortex neurons, and culture medium
  • Chemical induction method of cortex neurons, and culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of human-derived nerve cell induction basal medium NouvNeu-002 and cortical neuron induction medium NouvNeu-C

[0038] Configure the basal medium for human neural cell induction according to the following formula, hereinafter referred to as NouvNeu-002:

[0039] Duchenne's modified Eagle's medium (DMEM medium), vitamin E (1ug / ml), vitamin B12 (1.2uM), vitamin C (L-ascorbic acid, 64mg / L), progesterone (Progesterone, 6.3ng / ml ), putrescine (Putrescine, 23ug / ml), sodium bicarbonate (NaHCO 3 , 1.2g / L), sodium chloride (Sodium Chloride, 0.5g / L), sodium selenite (13.6μg / L), D(+)-galactose (D(+)-galactose, 12.5ug / ml ), LY2157299 (12.5uM), JW55 (5uM), insulin (Insulin, 20mg / L), recombinant human transferrin (100ng / ml).

[0040] Add 1-20 μM Crenigacestat (the optimal concentration is 7.5uM) to NouvNeu-002 cell culture medium. Form the cortical neuron induction medium NouvNeu-C.

[0041] For the control experiment, refer to the article by Jue Wang et al....

Embodiment 2

[0042] Example 2 Induction and culture of human neural stem cells

[0043] Human pluripotent stem cells include embryogenic pluripotent stem cells, such as H9 cell lines and human induced pluripotent stem cells. Among them, human induced pluripotent stem cells are obtained from CD34+ cell reprogramming according to "reprogramming medium and culture method of reprogramming induced pluripotent stem cells" (ZL201910050800.7).

[0044] Human pluripotent stem cells include embryogenic pluripotent stem cells, such as H9 cell lines and human induced pluripotent stem cells. Among them, human induced pluripotent stem cells are obtained from CD34+ cells by reprogramming according to "reprogramming medium and culture method of reprogramming induced pluripotent stem cells" (Patent Publication No. CN109628383A).

[0045] Human pluripotent stem cells were coated with Matrigel (STEMCELL Technologies) in T25 cell culture flasks, and placed in a 37°C incubator for more than one hour after pla...

Embodiment 3

[0054] Example 3 Induction of human cortical neuron cells

[0055] For the induction culture of human cortical neuron cells, poly-L-ornithine solution (Sigma) was used to coat the first layer of cell culture flasks, and after plating, they were incubated in a 37°C incubator for more than 16 hours. The second layer was coated with laminin (Biolaminin 521LN), and placed in a 37°C incubator for more than 2 hours after plating. According to 4*10 4 piece / cm 2 Seed the human neural stem cells in culture flasks or well plates for passage. Cultured in NouvNeu-C containing 1 / 5 / 10 / 20 μM Crenigacestat at 37°C, 5% CO 2 Induction culture was carried out in the cell culture incubator for the concentration screening of Crenigacestat. For the control group, the same batch of cells was cultured in medium containing 10 μM DAPT.

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Abstract

The invention relates to the technical field of neuron induction, and particularly discloses a chemical induction method of cortex neurons, and a culture medium. The culture medium is a chemical induction system which does not use animal serums and does not contain animal origin ingredients. The culture medium comprises a serum-free basic culture medium and a secretase inhibitor. The induction method disclosed by the invention has clear chemical ingredients and high differentiation efficiency. In addition, an obtained humanized cortex neuron has the characteristics of low immunogenicity, shortdifferentiation time and stable electrophysiology activity. Compared with an internationally common serum and micromolecule combination induction method at present, the method disclosed by the invention completely avoids potential hazards brought by existence of the animal origin ingredients in a cell culture process, is especially suitable for in vitro screening of nervous system disease medicines and treatment of neurodegenerative diseases, and has great economic effects and social effects.

Description

technical field [0001] The invention belongs to the technical field of neuron induction, and in particular relates to a method and culture medium for chemically inducing cortical neurons. Background technique [0002] The ectoderm is the outermost layer formed during embryonic development. As organogenesis begins, 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 thinking, emotion, perception, movement and other functions. Ectodermal cells are related to the occurrence of various degenerative diseases. For example, neurodegenerative diseases are caused by the aging and death of neurons. It is a common aging disease at present. The treatment and nursing costs of this disease are extremely expensive, and there is no Specific drugs can be effective treatment. Neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/079C12N5/0797
CPCC12N5/0619C12N2500/90C12N2500/38C12N2500/12C12N2500/25C12N2500/36C12N2500/34C12N2500/46C12N2501/392C12N2501/15C12N2533/52C12N2533/50C12N2533/90Y02A50/30
Inventor 魏君蔡萌云轩
Owner IREGENE THERAPEUTICS LTD
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