Clinical-grade serum-free medium for adherent culture of human neural stem cells

A serum-free medium and adherent culture technology, applied in the field of stem cell culture, to achieve the effects of protecting cells, improving utilization, and avoiding virus infection

Inactive Publication Date: 2015-04-29
广州吉帝生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problems existing in the existing serum-free neural stem cell culture medium, and to provide a serum-free culture medium that can be used for adherent culture of human neural stem cells

Method used

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  • Clinical-grade serum-free medium for adherent culture of human neural stem cells
  • Clinical-grade serum-free medium for adherent culture of human neural stem cells
  • Clinical-grade serum-free medium for adherent culture of human neural stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Comparison of neural stem cell adherence and suspension culture to clinical transformation.

[0042] The key to the clinical application of neural stem cells lies in the large-scale preparation of stem cells and the use of stem cells that meet the requirements for clinical use. According to the current literature reports, the proliferation of adherent cultured neural stem cells is faster and the adherent cells are easier to prepare in large scale.

[0043] The adherent culture cells (P3) and suspension culture cells (P3) were digested with Accutase enzyme, and the collected cells were frozen in liquid nitrogen with the same freezing medium and the same density for three months. Inoculated into coated culture plates to observe cell morphology. Results The survival rate of suspension cells was only about 60% after cryopreservation, while the survival rate of adherent cultured cells was over 90%. One day after the cells recovered and adhered to the wall, their...

Embodiment 2

[0044] Embodiment 2: Screening of each component in the serum-free medium supplement

[0045] By consulting all kinds of information, various additives that may be beneficial to cell proliferation are screened out, including the following, adenosine, transferrin, progesterone, putrescine, sodium selenite, apotransferrin, HSA, LIF, B-ME, VA, VC, Ve, Ve acetate, estradiol, thyroxine, ethanolamine, lipids, TGF beta, hydrocortisone, catalase, superoxide dismutase, glutathione , Triiodothyronine, L-carnitine, galactose, etc. Among them, the first five are N2 components. The present invention prepares them according to the concentration of each component in N2 as a basic additive, and then adds other various cytokines, BFGF, and EGF at appropriate concentrations therein, and cultivates NSCs from P0 to P3. Cell proliferation speed, cell morphology and cell proliferation ability after subculture were used as evaluation indicators, and the effects of each factor on cell proliferation ...

Embodiment 3

[0056] Example 3 The serum-free stem cell culture of the present invention that can be used in clinical culture of human neural stem cells, after quality inspection, all indicators are up to standard, see Table 4:

[0057] Table 4: Detection items and results of human neural stem cell serum-free medium of the present invention

[0058]

[0059]

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PUM

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Abstract

The invention discloses a clinical-grade serum-free medium for adherent culture of human neural stem cells. The medium disclosed by the invention comprises a basic medium, basic nutrition additives, plant-based human serum albumins, saccharides, lipid, hormones, antioxidants and related substances for promoting metabolism; the basic medium is prepared from commercial DMEM / F12 and a commercial neurobasal medium according to a ratio of 1:1; the basic nutrition additives comprise insulin, holo-transferrin, apo-transferrin, putrescine, progesterone and sodium selenite; the hormones comprise biotin, corticosterone, lipoic acid, Ve and Ve acetic ester; the antioxidants comprise human-derived catalase, human-derived superoxide dismutase, glutathione and Vc; the related substances for promoting metabolism comprise carnitine, T3 and ethanol amine. The medium can improve a cell expansion speed by two to three times, well keeps stem properties of the neural stem cells, keeps the cell differentiation potential of the neural stem cells and eliminates potential animal-origin endotoxin and viral pollution.

Description

technical field [0001] The invention relates to the field of stem cell culture, and relates to a clinical-grade serum-free medium for adherent culture of human neural stem cells and a corresponding method for culturing neural stem cells. Background technique [0002] Neural stem cells (NSC) are a kind of multipotent stem cells capable of self-replication, which can differentiate into neurons, astrocytes and oligodendrocytes under certain conditions. Because of its potential to treat a variety of nervous system diseases, nearly 900 neural stem cell therapy projects have been registered on the international clinical trial registration website, mainly for cerebral palsy, sequelae of stroke (cerebral infarction, cerebral hemorrhage), and spinal cord injury , Motor neuron disease, cerebellar atrophy, mutual aid scrutiny, Parkinson's disease, optic atrophy, Alzheimer's disease and other diseases. With the rapid development of neural stem cell therapy technology, it is very import...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797
Inventor 钟慧霖赖良学邹清雁陈文明唐时幸
Owner 广州吉帝生物科技有限公司
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