Cryopreservation and resuscitation method of neural stem cells

A neural stem cell cryopreservation technology, applied in the field of neural stem cell cryopreservation and recovery, can solve the problems of high price, unstable quality, complex serum components, etc.

Active Publication Date: 2011-11-02
SHANGHAI ANGECON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, there are two types of cryopreservation solutions used for neural stem cell cryopreservation: serum-containing and serum-free. Due to the disadvantages of complex components, unstable quality, and high price of serum, serum-free cryopreservation and resuscitation technology has become a development trend.

Method used

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  • Cryopreservation and resuscitation method of neural stem cells
  • Cryopreservation and resuscitation method of neural stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Obtain 1.0×10^6 cells of neural stem cells from the cortex of mouse brain tissue, add a certain volume of cryopreservation solution, and make the final concentration of the cell suspension 1.0×10^6 cells / ml, and place the cell suspension at -20°C For 30 minutes, place at -70°C for 12-16 hours, and then transfer to liquid nitrogen for freezing.

[0025] After 1 week, take out the frozen cells in liquid nitrogen and incubate in a 40°C water bath for 2 minutes; add 10 times the volume of 37°C preheated resuscitation solution to resuspend, centrifuge to remove the supernatant; add 5 times the volume of DMEM for culture The solution was resuspended, and the supernatant was removed by centrifugation again; the washing step was repeated; then 1 volume of Hibernation medium (purchased from Invitrogen Company) was added to resuspend, and counted by an automatic cell counter to obtain 9.6×10 cells / ml , the recovery rate was 96% (see figure 1 a).

[0026] In the present embodime...

Embodiment 2

[0055] Obtain 1.0×10^6 cells of neural stem cells from the cortex of mouse brain tissue, add a certain volume of cryopreservation solution, and make the final concentration of the cell suspension 1.0×10^6 cells / ml, and place the cell suspension at -20°C For 30 minutes, place at -70°C for 12-16 hours, and then transfer to liquid nitrogen for freezing.

[0056] After 1 week, take out the frozen cells in liquid nitrogen and incubate in a 40°C water bath for 2 minutes; add 10 times the volume of 37°C preheated resuscitation solution to resuspend, centrifuge to remove the supernatant; add 5 times the volume of DMEM for culture The liquid was resuspended, centrifuged again to remove the supernatant; the previous step was repeated and washed again with DMEM; 1 volume of Hibernation medium (purchased from Invitrogen) was added to resuspend, and counted by an automatic cell counter to obtain 9.4×10^5 cells / ml, the recovery rate was 94% (see figure 2 a).

[0057] In the present embo...

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Abstract

The invention provides a cryopreservation method and a resuscitation method of neural stem cells (NSCs), and provides a cryopreserved NSC solution and a resuscitated NSC solution with cell survival rates higher than 90%. The solutions are prepared with the methods provided by the invention. Also, the invention provides a cryopreservation medium and a resuscitation medium used in the NSC cryopreservation method and resuscitation method.

Description

technical field [0001] The invention relates to the field of biological products, in particular to a method for cryopreservation and recovery of neural stem cells. In the present invention, by adding two cytokines, EGF and bFGF, the cryopreservation and recovery rate of neural stem cells is greatly improved, from about 40% in the prior art to more than 90%. The present invention successfully freezes and recovers neural stem cells, It is of great significance for clinical elective stem cell transplantation and establishment of "stem cell bank". Background technique [0002] Neural stem cell (Neural Stem Cell, NSC) is a kind of stem cell existing in embryonic or adult brain, spinal cord and other tissues. It is a kind of mother cell with division potential and self-renewal ability. Various types of cells in nervous tissue such as cells, astrocytes, and oligodendrocytes can also be transdifferentiated into blood cells and skeletal muscle cells. In all nervous tissues such as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/0797
CPCA01N1/0221A01N1/0226
Inventor 金宜强姚静杨立敏
Owner SHANGHAI ANGECON BIOTECH
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