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Novel serum-free medium for inducing neural stem cells and application of novel serum-free medium

A serum-free medium and neural stem cell technology, which is applied in the field of biomedicine, can solve the problems of restricting neural stem cells and cannot be used for clinical use, and achieves huge economic and social effects, as well as the effect of solving pollution.

Active Publication Date: 2018-07-24
IREGENE THERAPEUTICS LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] From pluripotent stem cells to neural stem cells, there are currently two most widely used induction methods. The first method aggregates the cells into clusters to form embryoid bodies, and then the cells are further induced to become neural stem cells. The medium used in this method The use of animal-derived growth factors restricts the clinical use of neural stem cells
The second is the SMAD pathway dual inhibition method (Chambers et al., 2009), the neural stem cells obtained by this method can differentiate into other types of nerve cells (Fasano et al., 2010), the principle is to inhibit the BMP and TGFB pathways To simulate the signaling pathways in the early stages of embryonic development, thereby inducing the generation of neural stem cells, this method does not use trophoblast cells, but still requires the use of animal-derived cytokines or protein signaling pathway antagonists, which cannot be used in clinical use

Method used

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  • Novel serum-free medium for inducing neural stem cells and application of novel serum-free medium
  • Novel serum-free medium for inducing neural stem cells and application of novel serum-free medium
  • Novel serum-free medium for inducing neural stem cells and application of novel serum-free medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Human induced pluripotent stem cell culture and expansion of neural stem cells

[0051] 1. Preparation of culture medium

[0052] Table 1 Neural stem cell induction medium

[0053]

[0054]

[0055] Table 2 Neural stem cell proliferation medium

[0056]

[0057] Table 3 Neuronal differentiation medium

[0058]

[0059] After the medium is prepared, mix it evenly, seal it and store it in a refrigerator at 4°C, and use it up within 2 weeks.

[0060] 2. Subculture of human pluripotent stem cells:

[0061] First, 1 μg / mL laminin Laminin (STEMCELL Technologies) was used to coat T25 culture flasks, and then incubated in a 37°C incubator for at least 1 hour after plating; human induced pluripotent stem cells were cultured in TeSRTM-e8 medium, using 0.05 % trypsin / EDTA, incubate at 37 degrees for 5 minutes for digestion; resuspend the digested human induced pluripotent stem cells in TeSRTM-e8 medium, according to 2x10 6 The number of cells per plate ...

Embodiment 2

[0067] Example 2: Effects of Different Types of Recombinant FGF on the Proliferation of Human Neural Stem Cells

[0068] Take the neural stem cells induced and cultured in Example 1, according to 5 × 10 4 The number of cells in each well was inoculated, and three groups of parallel repetitions were set up (the average value of the three groups was calculated as the data), and two kinds of neural stem cell expansion media were used to expand the culture respectively, wherein the first medium included N2G21 basal culture Base and plant-derived human basic growth factor OsrbFGF (Table 2), the second including N2G21 basal medium and containing animal-derived human basic growth factor GDNF. Cell culture conditions were 37°C, 5% carbon dioxide. Samples were taken at 24 hours, 48 ​​hours and 72 hours respectively. Cell proliferation was performed using CyQuant kit (Invitrogen), and data was read using SpectraMax i3Multi-Mode MicroplateReader. The cell proliferation was as follows f...

Embodiment 3

[0070] Example 3: Induction of neural stem cells derived from pluripotent stem cells into cortical neurons in vitro

[0071] The human induced neural stem cells cultured by the medium of the present invention can be induced into cortical neurons.

[0072] 1. Induction of neural stem cells into cortical neurons in vitro

[0073] Will 4x10 4 The induced neural stem cells in Example 1 were inoculated in a 24-well plate coated with D-polylysine, and cultured using the neural differentiation medium of the present invention (see Table 3), and the medium was changed every two days during the culture. once. Cell culture conditions were 37°C, 5% carbon dioxide. After 14 days of continuous culture, cortical neurons began to form (eg figure 2 shown).

[0074] 2. Conclusion

[0075] Differentiation result as figure 2 as shown, figure 2 Middle a is the picture of the first day of cell induction, b is the picture of the 21st day of cell induction, c is the picture of subculture o...

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Abstract

The invention provides a novel serum-free medium for inducing neural stem cells. The medium comprises a neural stem cell-induced medium, a neural stem cell proliferation culture medium and a neuronaldifferentiation medium, wherein the neural stem cell-induced medium consists of an animal basal culture medium N2G21 and neural induction compounds PD98059 and JW55; the neural stem cell proliferationculture medium consists of the animal basal culture medium N2G21 and a growth factor OsrbFGF; the neuronal differentiation medium is an N2G21 basal culture medium. The culture medium adopts a scientific design and a simple formula; compared with the prior art, the novel serum-free medium disclosed by the invention has the advantages that pure chemical molecules replace a protein signal antagonist; a non-animal original growth factor is used to replace a traditional animal original growth factor; neural stem cells which can self update and are differentiated into neuronal cells are obtained byhuman multipotential stem cell culture; in addition, the neural stem cells prepared by using the culture medium have higher clinical safety.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a novel serum-free culture medium for inducing neural stem cells and its application. Background technique [0002] Neurodegenerative diseases are currently common diseases of aging, and the cost of treatment and care is extremely expensive, and there are no specific drugs on the market for effective treatment. Neurodegenerative diseases include amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), Alzheimer's disease (AD) and other diseases. According to the statistics of the World Health Organization, there will be more than 30 million patients with neurodegenerative diseases in my country in 2050, and the expected medical expenses will exceed 1 trillion. At present, drugs are mainly used to supplement or stimulate the insufficient levodopa in the brain, nerve nucleus damage surgery or deep brain stimulation surgery, etc., but none of them can achieve ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797C12N5/0793C12N5/0735C12N5/10
CPCC12N5/0619C12N5/0623C12N2500/90C12N2501/115C12N2501/724C12N2501/999C12N2506/02C12N2506/45C12N2510/00
Inventor 魏君蔡萌
Owner IREGENE THERAPEUTICS LTD
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