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

A serum-free medium, neural stem cell technology, applied in the field of stem cell culture, to achieve the effect of improving utilization, avoiding potential animal endotoxin virus infection, and increasing cell proliferation rate

Inactive Publication Date: 2018-03-16
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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  • A clinical-grade serum-free medium for adherent culture of human neural stem cells
  • A clinical-grade serum-free medium for adherent culture of human neural stem cells
  • A 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 stem cells used meet the requirements of clinical use. According to current literature reports, the neural stem cells in adherent culture proliferate faster and adherent cells are easier to prepare on a large scale.

[0043] The adherent cultured cells (P3) and suspension cultured cells (P3) were digested with Accutase enzyme, and the cells were collected and stored in liquid nitrogen at the same density with the same cryopreservation solution for three months. Resuscitated the cells for viable cell count and at the same density Inoculate the coated culture plate to observe the cell morphology. Results The survival rate of suspended cells after cryopreservation was only about 60%, while the survival rate of adherent cultured cells was over 90%. One d...

Embodiment 2

[0044] Example 2: Screening of ingredients in serum-free medium additives

[0045] By consulting various materials, various additives that may be beneficial to cell proliferation were screened out including the following, insulin, transferrin, progesterone, putrescine, sodium selenite, apotransferrin, HSA, LIF, B-ME, VA, VC, Ve, Ve acetate, estradiol, thyroxine, ethanolamine, lipids, TGFβ, hydrocortisone, catalase, superoxide dismutase, glutathione , Triiodothyronine, L-carnitine, galactose, etc. Among them, the first five are N2 components. The present invention formulates them according to the concentration of each component in N2 as a basic addition, and then adds appropriate concentrations of other various cytokines, BFGF, and EGF to cultivate NSC from P0 to P3. Cell proliferation speed, cell morphology and cell proliferation ability after passage are used as evaluation indicators to comprehensively evaluate the effects of various factors on cell proliferation and differenti...

Embodiment 3

[0056] Example 3 The serum-free stem cell culture of the present invention that can be used for culturing human neural stem cells in the clinic has been tested for quality and all indicators are up to standard, as shown in Table 4:

[0057] Table 4: Test items and results of serum-free medium for neural stem cells of the present inventors

[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 adherently culturing human neural stem cells and a corresponding method for culturing neural stem cells. Background technique [0002] Neural stem cells (NSC) are a type of self-replicating pluripotent stem cells that can differentiate into neurons, astrocytes and oligodendrocytes under certain conditions. Because of its potential to treat a variety of neurological diseases, there are currently nearly 900 neural stem cell therapy items registered on the international clinical trial registration website, mainly used for cerebral palsy, sequelae of stroke (cerebral infarction, cerebral hemorrhage), and spinal cord injury , Motor neuron disease, cerebellar atrophy, co-examination, Parkinson's disease, optic atrophy, Alzheimer's and other diseases. With the rapid development of neural stem cell therapy technology, it is important to develop a culture ...

Claims

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Application Information

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Patent Type & Authority Patents(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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