A kind of fat mesenchymal stem cell cryopreservation liquid and cryopreservation method thereof

A technique of stromal cells and cryopreservation method, applied in the field of adipose-derived mesenchymal stem cell cryopreservation solution, can solve the problems of cryopreserved cells damage, low survival rate, limited promotion and application, etc., and achieves prevention of degeneration and inactivation and high cell survival rate. , to ensure the effect of cell viability

Active Publication Date: 2021-10-08
GUANGDONG CELL BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, DMSO is toxic and will cause damage to frozen cells, while exogenous animal serum may contaminate cells, long-term use will affect cell viability, and subsequent use will affect the promotion of clinical treatment of cells
Even if the existing adipose-derived mesenchymal stem cell cryopreservation solution does not contain fetal calf serum or DMSO, the cryopreservation effect is not ideal, and there are problems such as poor cell recovery rate and low survival rate after long-term storage, which greatly limits its clinical application. Promoted application of

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  • A kind of fat mesenchymal stem cell cryopreservation liquid and cryopreservation method thereof
  • A kind of fat mesenchymal stem cell cryopreservation liquid and cryopreservation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] A fat-derived mesenchymal stem cell cryopreservation solution, which consists of the following raw materials: DMEM / F12 medium, ethylene glycol monomethyl ether 55 μg / mL, acetylglucosamine 3.0 mg / mL, bilobalide 28 ng / mL, pyrophosphate Sodium 40ng / mL, alfalfa saponin 85μg / mL, lipoic acid 105μg / mL.

[0024] A cryopreservation method for adipose-derived mesenchymal stem cells, comprising the following steps:

[0025] (1) Add ethylene glycol monomethyl ether and acetylglucosamine to the DMEM / F12 medium, and then add the adipose-derived mesenchymal stem cells in the logarithmic growth phase to be frozen, and the cell density is 1.5× per ml medium 10 6 , pre-cooled at 4°C for 10 minutes;

[0026] (2) Add bilobalactone, sodium pyrophosphate, alfalfa saponin and lipoic acid to the precooled medium in step (1), and prefreeze at -30°C for 1.5h;

[0027] (3) The adipose-derived mesenchymal stem cell mixture pre-frozen in step (2) was cooled down to -85°C at a rate of 3°C / min, fr...

Embodiment 2

[0029] A fat-derived mesenchymal stem cell cryopreservation solution, which consists of the following raw materials: DMEM / F12 medium, ethylene glycol monomethyl ether 50 μg / mL, acetylglucosamine 1.5 mg / mL, bilobalide 25 ng / mL, pyrophosphate Sodium 35ng / mL, alfalfa saponin 80μg / mL, lipoic acid 100μg / mL.

[0030] A cryopreservation method for adipose-derived mesenchymal stem cells, comprising the following steps:

[0031] (1) Add ethylene glycol monomethyl ether and acetylglucosamine to the DMEM / F12 medium, then add the adipose-derived mesenchymal stem cells in the logarithmic growth phase to be frozen, and the cell density is 1.0× per ml medium 10 6 , pre-cooled at 6°C for 5 minutes;

[0032] (2) adding bilobalactone, sodium pyrophosphate, alfalfa saponin and lipoic acid to the precooled medium in step (1), and prefreezing at -20°C for 2 hours;

[0033] (3) The adipose-derived mesenchymal stem cell mixture pre-frozen in step (2) was cooled to -85°C at a rate of 2°C / min, froz...

Embodiment 3

[0035] A fat-derived mesenchymal stem cell cryopreservation solution, which consists of the following raw materials: DMEM / F12 medium, ethylene glycol monomethyl ether 60 μg / mL, acetylglucosamine 4.0 mg / mL, bilobalide 30 ng / mL, pyrophosphate Sodium 45ng / mL, alfalfa saponin 90μg / mL, lipoic acid 110μg / mL.

[0036] A cryopreservation method for adipose-derived mesenchymal stem cells, comprising the following steps:

[0037] (1) Add ethylene glycol monomethyl ether and acetylglucosamine to the DMEM / F12 medium, then add the adipose-derived mesenchymal stem cells in the logarithmic growth phase to be frozen, and the cell density is 2.0× per ml medium 10 6 1, 10min in a precooling place at 4°C;

[0038] (2) adding bilobalactone, sodium pyrophosphate, alfalfa saponin and lipoic acid to the precooled medium in step (1), and prefreezing at -40°C for 1 hour;

[0039] (3) Cool down the adipose-derived mesenchymal stem cell mixture pre-frozen in step (2) to -85°C at a rate of 4°C / min, fr...

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Abstract

The invention discloses a fat mesenchymal stem cell cryopreservation solution, which is composed of the following raw materials: DMEM / F12 medium, 50-60 μg / mL of ethylene glycol monomethyl ether, 1.5-4.0 mg / mL of acetylglucosamine, ginkgo Lactone 25-30ng / mL, sodium pyrophosphate 35-45ng / mL, alfalfa saponin 80-90μg / mL, lipoic acid 100-110μg / mL. The invention provides a fat-derived mesenchymal stem cell cryopreservation solution, which does not add animal serum and DMSO, and has a high cell survival rate after resuscitation. Adding ethylene glycol monomethyl ether and acetyl glucosamine to the cryopreservation solution of the present invention has no damage to cells, improves cell permeability, maintains stable osmotic pressure inside and outside cells, and helps cells maintain activity. The combination of bilobalide and sodium pyrophosphate is added to prevent the denaturation and inactivation of cell protein molecules, and help cells quickly recover their activity after long-term freezing. The cryopreservation solution of the present invention is also added with alfalfa saponin and lipoic acid to remove free radicals generated during the cryopreservation process and has an anti-oxidation effect. The invention also provides a cryopreservation method of adipose-derived mesenchymal stem cells, which is simple and easy to operate.

Description

technical field [0001] The invention relates to the field of stem cells, in particular to a cryopreservation liquid for adipose-derived mesenchymal stem cells and a cryopreservation method thereof. Background technique [0002] Adipose Mesenchymal Stem Cells (ADSC) are adult pluripotent stem cells that exist in adipose tissue, and can differentiate into various tissue cells such as bone, cartilage, cardiac muscle, smooth muscle, nerve and endothelium under certain conditions. At present, it has been found that adipose-derived mesenchymal stem cells can treat more than 80 diseases including the human nervous system, immune system, digestive system, sports system and other systems. Adipose-derived mesenchymal stem cells are an important regenerative medicine resource in disease treatment and human anti-aging The application field has very important use value. [0003] The adipose-derived mesenchymal stem cell cryopreservation solution used in the prior art mostly contains fet...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 史辛艺王泰华梁杰马超群
Owner GUANGDONG CELL BIOTECHNOLOGY CO LTD
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