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A cryopreservation solution and method for a large number of cryopreserved mesenchymal stem cells

A quality stem cell and cryopreservation technology, applied in the field of stem cells, can solve the problems of affecting cell viability, cumbersome operations, waste of time and manpower, etc., and achieve the effect of shortening the time required for recovery, high survival rate, and reducing impact.

Active Publication Date: 2021-01-01
JILIN TUO HUA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing mesenchymal stem cells are all cryopreserved in 1ml or 1.5ml cryopreservation tubes. For multiple patients, dozens of tubes need to be resuscitated at one time. The operation is extremely cumbersome, wastes time and manpower, and affects the survival rate of cells.
[0003] In addition, dimethyl sulfoxide (DMSO) is mostly added to the cryopreservation solution in the prior art. DMSO is a osmotic protective agent that can lower the freezing point of cells and reduce the formation of ice crystals, thereby reducing the damage to cells caused by cryopreservation; however , DMSO has cytotoxicity. At deep low temperature, the cytotoxicity of DMSO is inhibited, but the toxicity to cells is very serious during resuscitation. Therefore, operators are required to act quickly when resuscitating cells and wash off dimethyl sulfoxide as soon as possible; obviously , for a large number of mesenchymal stem cells cryopreserved in cryopreservation tubes, even if the operator is skilled, it is easy to affect the survival rate of the cells

Method used

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  • A cryopreservation solution and method for a large number of cryopreserved mesenchymal stem cells
  • A cryopreservation solution and method for a large number of cryopreserved mesenchymal stem cells
  • A cryopreservation solution and method for a large number of cryopreserved mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0052]Prepare a 300ml cryopreservation solution for freezing and storing a large number of mesenchymal stem cells, add propylene glycol, azone, albumin and low-molecular dextran to the α-MEM basal medium; among them, the concentration of propylene glycol is 1.5mol / L, and azone The concentration of albumin is 2ml / 100ml, the concentration of albumin is 10mg / ml, and the concentration of low molecular dextran is 8ml / 100ml.

[0053] The method for freezing a large number of mesenchymal stem cells using the above-mentioned freezing solution comprises the following steps:

[0054] (1) Prepare the above freezing solution, mix it evenly, divide it into 30ml / tube, and store it at 4°C for later use;

[0055] (2) Collect 30 flasks of mesenchymal stem cells in T175 culture flasks with a cell fusion of more than 85%, add digestive enzymes to digest the cells, dilute and neutralize with normal saline, centrifuge at 1500rpm for 5 minutes, and discard the supernatant;

[0056] (3) Use 1 tube ...

Embodiment 2

[0071] Prepare a 300ml cryopreservation solution for freezing and storing a large number of mesenchymal stem cells, add propylene glycol, azone, albumin and low-molecular dextran to the α-MEM basal medium; among them, the concentration of propylene glycol is 1.2mol / L, and azone The concentration of albumin is 0.5ml / 100ml, the concentration of albumin is 12mg / ml, and the concentration of low molecular dextran is 8.5ml / 100ml.

[0072] The method for freezing a large number of mesenchymal stem cells using the above-mentioned freezing solution comprises the following steps:

[0073] (1) Prepare the above freezing solution, mix it evenly, divide it into 35ml / tube, and store it at 4°C for later use;

[0074] (2) Collect 35 flasks of mesenchymal stem cells in T175 culture flasks with a cell fusion of more than 85%, add digestive enzymes to digest the cells, dilute and neutralize with normal saline, centrifuge at 1500rpm for 5 minutes, and discard the supernatant;

[0075] (3) Use 1 ...

Embodiment 3

[0081] Prepare a 300ml cryopreservation solution for freezing and storing a large number of mesenchymal stem cells, add propylene glycol, azone, albumin and low-molecular dextran to the α-MEM basal medium; among them, the concentration of propylene glycol is 1.8mol / L, and azone The concentration of albumin is 4ml / 100ml; the concentration of albumin is 8mg / ml, and the concentration of low molecular dextran is 7.5ml / 100ml.

[0082] The method for freezing a large number of mesenchymal stem cells using the above-mentioned freezing solution comprises the following steps:

[0083] (1) Prepare the above-mentioned freezing solution, mix it evenly, divide it into 25ml / tube, and store it at 4°C for later use;

[0084] (2) Collect 25 flasks of mesenchymal stem cells in T175 culture flasks with a cell fusion of more than 85%, add digestive enzymes to digest the cells, dilute and neutralize with normal saline, centrifuge at 1500rpm for 5 minutes, and discard the supernatant;

[0085] (3)...

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Abstract

The invention discloses cryopreservation liquid for cryopreserving large quantities of mesenchymal stem cells. The cryopreservation liquid comprises alpha-MEM (minimum Eagle media), propylene glycol,azone, albumin and low-molecular dextran. The concentration of the propylene glycol is 1.2-1.8 mol / L, and the concentration of the azone is 0.5-4.0 ml / 100 ml; the concentration of the albumin is 8-12mg / ml, and the concentration of the low-molecular dextran is 7.5-8.5 ml / 100 ml. The invention further provides a method for cryopreserving the mesenchymal stem cells by the aid of cryopreservation bags with the volumes of 50 ml. The cryopreservation liquid and the method have the advantages that various components are combined with one another at specific ratios, accordingly, the formed cryopreservation liquid is little in cell injury when used for cryopreserving the large quantities of mesenchymal stem cells, and the cryopreserved cells are stable in quality, short in recovery time and high in survival rate; the cell cryopreservation quality can be guaranteed by the aid of the method, the required recovery time can be shortened to a great extent, recovery processes can be simplified, andinfluence of excessively long recovery time on the mesenchymal stem cells can be lowered.

Description

technical field [0001] The invention relates to the field of stem cells, and more specifically relates to a cryopreservation solution and method for a large number of mesenchymal stem cells. Background technique [0002] At present, the clinical transformation of mesenchymal stem cells is becoming more and more widely used, and the requirements for the number of mesenchymal stem cells in clinical applications are also getting higher and higher. However, the existing mesenchymal stem cells are all cryopreserved in 1ml or 1.5ml cryopreservation tubes. For multiple patients, dozens of tubes need to be resuscitated at one time. The operation is extremely cumbersome, wastes time and manpower, and affects the survival rate of cells. . [0003] In addition, dimethyl sulfoxide (DMSO) is mostly added to the cryopreservation solution in the prior art. DMSO is a osmotic protective agent that can lower the freezing point of cells and reduce the formation of ice crystals, thereby reduci...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 毕薇薇
Owner JILIN TUO HUA BIOTECH
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