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Isolated culture method as well as freeze storage and recovery method of mesenchymal stem cell

A technology for the isolation and culture of mesenchymal stem cells, applied to the isolation and culture of mesenchymal stem cells, cryopreservation, and recovery, to achieve good cell quality

Pending Publication Date: 2018-07-24
SHENZHEN ZHIBO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] To carry out clinical trial treatment, a large number of cells need to be prepared, and the cell preparation technology for stem cell therapy is diverse, complex and specific

Method used

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  • Isolated culture method as well as freeze storage and recovery method of mesenchymal stem cell
  • Isolated culture method as well as freeze storage and recovery method of mesenchymal stem cell
  • Isolated culture method as well as freeze storage and recovery method of mesenchymal stem cell

Examples

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Embodiment 1

[0037] Such as figure 1 As shown, a method for isolating and culturing mesenchymal stem cells, comprising:

[0038] Step S1: obtaining primary cells of mesenchymal stem cells;

[0039] Step S2: Culture and expansion of primary mesenchymal stem cells: After culturing primary mesenchymal stem cells for 5-10 days, replace with serum-free DMEM nutrient solution until the cells are 65-75% confluent, remove the culture supernatant, Then add the first digestion solution, digest for 0.5-2min, add the removed culture supernatant to neutralize the cells after contraction, then centrifuge, add complete medium to resuspend, inoculate according to 1 pass 1-1.5, and culture for 4- After 5 days, when the cells are 65-75% confluent, subculture at a ratio of 1:6-8; and

[0040] Step S3: Isolation of mesenchymal stem cells: collecting cells in the P2 or P3 logarithmic growth phase and the degree of cell fusion not exceeding 80% in the culture and expansion of the primary cells of mesenchymal ...

Embodiment 2

[0069] A method for cryopreservation and recovery of mesenchymal stem cells, wherein the mesenchymal stem cells obtained by the method for isolating and culturing mesenchymal stem cells described in Example 1 are cryopreserved and recovered, comprising:

[0070] Step T1: cryopreservation of mesenchymal stem cells: resuspend the mesenchymal stem cells with cell freezing solution, then place them in a container filled with isopropanol, store them overnight at -90 to -70°C, and then transfer them to liquid nitrogen tank; and

[0071] Step T2: Recovery of mesenchymal stem cells: Take frozen mesenchymal stem cells and put them into a 40-45°C water bath to melt immediately, then add complete medium and mix well, discard the supernatant after centrifugation, and replace with complete medium After resuspension, store at 35-40°C, 5% CO 2 And cultured under the condition of saturated humidity, wait until 65-75% of the cells adhere to the wall.

[0072]In step T1, preferably, the cell ...

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Abstract

The invention relates to the technical field of biology, and provides an isolated culture method as well as freeze storage and recovery method of mesenchymal stem cells. The isolated culture method ofthe mesenchymal stem cell comprises the following steps: acquiring a primary cell of the mesenchymal stem cell; performing the culture amplification for the primary cell of the mesenchymal stem cell;and isolating the mesenchymal stem cell. By adopting the isolated culture method of the mesenchymal stem cell, the mesenchymal stem cell with good cell quality, safety and biological effect can be obtained. For the mesenchymal stem cell obtained by the isolated culture method of the mesenchymal stem cell, the invention also provides a freeze storage and recovery method of the mesenchymal stem cell, so that the mesenchymal stem cell can be well stored and recovered.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating and culturing mesenchymal stem cells and a method for freezing and resuscitating them. Background technique [0002] Stem cells are a class of cells with different differentiation potentials that self-renew in an undifferentiated state. Stem cell therapy refers to the process of using human autologous or allogeneic stem cells to treat diseases after in vitro manipulation (or implantation) into the human body. This in vitro operation includes the isolation, purification, expansion, cryopreservation and recovery of stem cells after cryopreservation. At present, clinical application studies of various stem cell types have been carried out at home and abroad, and the main diseases involved include bone and joint diseases, liver cirrhosis, graft-host rejection (GVHD), spinal cord injury, degenerative nervous system diseases, and diabetes. Mesenchymal stem cells (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A01N1/02
CPCA01N1/0221C12N5/0663C12N5/0667C12N5/0668C12N2509/00C12N5/0665
Inventor 杨涛隋昳
Owner SHENZHEN ZHIBO BIOTECH CO LTD
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