Neural stem cell culture amplification method and used culture medium

A neural stem cell and culture medium technology is applied to the medium for culturing and expanding neural stem cells, and the field of culturing and expanding neural stem cells can solve the problems of the application of neural stem cells, the high price of bioreactors, and the low expansion efficiency, etc. The effect of scientific research and industrial production

Inactive Publication Date: 2012-12-26
SHANGHAI ANGECON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Neural stem cells can be cultured in two ways: static culture and dynamic culture. The former is cultured under static conditions in an incubator with cell culture flasks, and the expansion efficiency is low under serum-free conditions ("Research Progress in Mammalian Neural Stem Cell Expansion Technology" , Dong Liang, Qi Hanshi, Biotechnology, 2005, 15(4)); the latter uses a cell culture bioreactor to cultivate under dynamic conditions such as rotation, and has a high amplification efficiency, but the bioreactor is expensive, and In some cases, for the convenience of experiments or due to the requirements of experimental conditions, neural stem cells are not suitable for cultivation and expansion in bioreactors
[0007] In addition, the current culture of neural stem cells requires the addition of antibiotics, which may affect the further application of neural stem cells

Method used

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  • Neural stem cell culture amplification method and used culture medium
  • Neural stem cell culture amplification method and used culture medium
  • Neural stem cell culture amplification method and used culture medium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] The composition of I culture medium:

[0060] The composition of culture medium A is: DMEM / F12 solution, containing B27 additive (1×), N2 additive (1×), L-glutamine (2mM), sodium pyruvate (1mM), NAC (1mM), bFGF (20ng / ml), EGF (20ng / ml) and LIF (10ng / ml).

[0061] The nutritional supplement solution is composed of: DMEM / F12 solution, containing B27 additive (1×), N2 additive (1×), L-glutamine (2mM), sodium pyruvate (1mM), NAC (1mM), bFGF (100ng / ml), EGF (100ng / ml) and LIF (10ng / ml).

[0062] The above reagents NAC were purchased from SIGMA Company, and other reagents were purchased from Invitrogen Company.

[0063] II culture steps

[0064] Resuscitated human fetal neural stem cell cryopreservation product (Cyagen company, product number HUXNF-01001) 1.0 × 10 6 cells (packaged in frozen tubes, transported on dry ice) obtained 8.2×10 5 cells. at 1.0×10 5 Cells / ml density was inoculated into 2 T25cm cells containing 4ml culture solution A 2 cultured in a square f...

Embodiment 2

[0067] The composition of I culture medium:

[0068] The composition of culture medium A is: DMEM / F12 solution, containing B27 additive (1×), N2 additive (1×), L-glutamine (2mM), sodium pyruvate (1mM), NAC (1mM), bFGF (20ng / ml), EGF (20ng / ml) and LIF (5ng / ml).

[0069] The nutritional supplement solution is composed of: DMEM / F12 solution, containing B27 additive (1×), N2 additive (1×), L-glutamine (2mM), sodium pyruvate (1mM), NAC (1mM), bFGF (100ng / ml), EGF (100ng / ml) and LIF (5ng / ml).

[0070] The above reagents NAC were purchased from SIGMA Company, and other reagents were purchased from Invitrogen Company.

[0071] II culture steps

[0072] The mononuclear cell layer was separated by density gradient centrifugation from human umbilical cord blood (Shanghai Blood Center Cord Blood Bank), and then adhered to the wall to obtain mesenchymal stem cells, which were induced to differentiate into neural stem cells (by adding growth factors EGF and bFGF each induced by 20ng / m...

Embodiment 3

[0074] The composition of I culture medium:

[0075] The composition of culture medium A is: DMEM solution, containing B27 additive (1×), N2 additive (1×), L-glutamine (2mM), sodium pyruvate (1mM), NAC (1mM), bFGF (20ng / ml ), EGF (20ng / ml) and LIF (10ng / ml).

[0076] The nutritional supplement solution is composed of: DMEM solution containing B27 additive (1×), N2 additive (1×), L-glutamine (2mM), sodium pyruvate (1mM), NAC (1mM), bFGF (100ng / ml ), EGF (100ng / ml) and LIF (10ng / ml).

[0077] The above reagents NAC were purchased from SIGMA Company, and other reagents were purchased from Invitrogen Company.

[0078] II culture steps

[0079] A density of 3.4 × 10 was obtained from the ICR mouse brain tissue cortex 7 P3 generation neurosphere suspension of 10 cells (references: Yin Guocai, Jia Zuo, Qu Suqing, etc., the culture of neural stem cells in neonatal rat cerebral cortex and its cell replacement in compatriot rats, Chinese Journal of Perinatal Medicine, July 2005, No....

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Abstract

The invention discloses a neural stem cell culture amplification method, which comprises the following steps of culturing different sources of neural stem cells with a culture solution, adding a nutritional supplement solution, replacing a fresh culture solution, selecting a certain size of neurospheres for passage and the like. A formula of a relative culture solution, the nutritional supplement solution and other compositions is also provided. The method obviously improves the proliferation efficiency of neural stem cells under static culture, no antibiotics are used, and the cost is lower. The method is suitable for industrial production.

Description

technical field [0001] The present invention relates to the field of cell culture expansion. The invention particularly relates to a medium for culturing and expanding neural stem cells and a method for cultivating and expanding neural stem cells using the medium. Background technique [0002] Neural stem cells (Neural Stem Cells, NSCs) are a kind of stem cells that exist in nervous tissues such as embryonic and adult brains and spinal cords. Various types of cells in nervous tissue such as neurons, astrocytes, and oligodendrocytes can also be transdifferentiated into blood cells and skeletal muscle cells. In all nervous tissues such as the brain and spinal cord, different types of neural stem cells produce different types of progeny cells, and the distribution is also different. The current science and technology can cultivate and expand neural stem cells in vitro, which can be used in life science research, drug screening test, clinical application research and other fie...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0797
Inventor 金宜强刘军杨立敏
Owner SHANGHAI ANGECON BIOTECH
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