Mesenchymal stem cell cryopreservation liquid, cryopreservation method, preservation kit and thawing method

A technology for stem cells and preservation reagents, applied in the field of cell biology, can solve the problems of high DMSO content, easy to be unevenly heated, and high cytotoxicity, and achieve the effect of reasonable cell density, protection of cell activity, and maintenance of cell activity.

Active Publication Date: 2020-04-17
GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, animal serum is usually used in this cell cryopreservation solution, which will produce exogenous contamination from xenobiotic sources, high DMSO content, and high cytotoxicity. This cell cryopreservation solution cannot be used clinically
Moreover, the general cell recovery process is prone to uneven heating, secondary crystallization, and greater damage to cells.

Method used

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  • Mesenchymal stem cell cryopreservation liquid, cryopreservation method, preservation kit and thawing method
  • Mesenchymal stem cell cryopreservation liquid, cryopreservation method, preservation kit and thawing method
  • Mesenchymal stem cell cryopreservation liquid, cryopreservation method, preservation kit and thawing method

Examples

Experimental program
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Effect test

Embodiment 1

[0044] A method for cryopreserving mesenchymal stem cells, comprising the following steps:

[0045] Digestion: Take out the mesenchymal stem cells with a confluence of 80%, wash them once with normal saline, add an appropriate amount of 0.25% trypsin to digest them, add selective medium to stop the digestion after the cells become round, centrifuge at 300g for 5min, and retain the precipitate. Add the mesenchymal stem cell cryopreservation solution, resuspend the cell solution, and control the cell concentration to 5×10 6 pc / mL; Among them, the mesenchymal stem cell cryopreservation medium uses the mesenchymal stem cell selective medium as the base fluid, and each 100mL mesenchymal stem cell cryopreservation medium includes the following components: 30mL mesenchymal stem cell culture supernatant , 5mL glycerol, 5g PVP, 0.5g PVA, 0.5g trehalose, 1g HA nanoparticles, 1mg all-trans retinoic acid, 10mL serum replacement; the mesenchymal stem cell culture supernatant was obtained b...

Embodiment 2

[0051] A mesenchymal stem cell cryopreservation method, which differs from the cryopreservation method in Example 1 in that each 100mL mesenchymal stem cell cryopreservation solution includes the following components: 30mL mesenchymal stem cell culture supernatant, 5mL glycerol, 5g PVP, 0.5g PVA, 0.5g trehalose, 2g HA nanoparticles, 1mg all-trans retinoic acid, 10mL serum replacement.

[0052] The recovery method of the cryopreserved cells is the same as the recovery method in Example 1.

Embodiment 3

[0054] A cryopreservation method for mesenchymal stem cells, which is the same as the cryopreservation method in Example 1.

[0055] The resuscitation method for cryopreserved cells is different from the resuscitation method in Example 1 in that each 100 mL of resuscitation solution includes the following components: 0.3 g of glucose, 1 mg of all-trans retinoic acid, and 1 mg of ascorbic acid.

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Abstract

The invention provides a mesenchymal stem cell cryopreservation liquid, a cryopreservation method, a preservation kit and a thawing method, and relates to the technical field of cell biology. The mesenchymal stem cell cryopreservation liquid comprises the following components with the following working concentrations: 25-35 ml / 100ml of a mesenchymal stem cell culture supernatant; 4-6 ml / 100ml of glycerol, 4-6 ml / 100ml of polyvinylpyrrolidone, 0.5-1.5 g / 100ml of polyvinyl alcohol, 0.5-2.5 g / 100mL of trehalose, 1-2.5 g / 100mL of hydroxyapatite nanoparticles, 0.5-1.5 mg / 100mL of all-trans retinoicacid and 8-12 ml / 100ml of a serum substitute; the mesenchymal stem cell culture supernatant is prepared by the following method: culturing mesenchymal stem cells at the convergence degree of 78-82% for 22-26 hours, filtering, and retaining a filtrate, thereby obtaining the mesenchymal stem cell culture supernatant. The cryopreservation liquid can effectively reduce damage to cells in cryopreservation and resuscitation processes and improve the activity of the resuscitated cells.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a mesenchymal stem cell cryopreservation solution, a cryopreservation method, a preservation kit and a recovery method. Background technique [0002] Cryoprotectant refers to a substance that can protect cells from frostbite in an extremely low temperature environment, also known as cell cryopreservation solution. Adding cryoprotectants to cell suspensions can protect cells from solution damage and ice crystal damage. The cryoprotectant combines with the water molecules in the solution to cause a hydration reaction, weakens the crystallization of water, increases the viscosity of the solution to reduce the formation of ice crystals, and at the same time maintains a certain molar concentration inside and outside the cells to reduce the concentration of unfrozen solutions inside and outside the cells. The concentration of electrolytes in the medium protects cells from damage ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/0775
CPCA01N1/0221A01N1/0226C12N5/0662
Inventor 孙灿兴李静静赵蓝刘小翠江嘉豪
Owner GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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