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Adipose tissue-derived stromal 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: 2020-12-15
GUANGDONG CELL BIOTECHNOLOGY CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Method used

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  • Adipose tissue-derived stromal cell cryopreservation liquid and cryopreservation method thereof
  • Adipose tissue-derived stromal cell cryopreservation liquid and cryopreservation method thereof

Examples

Experimental program
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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 provides an adipose tissue-derived stromal cell cryopreservation liquid. The adipose tissue-derived stromal cell cryopreservation liquid is prepared from the following raw materials: a DMEM / F12 culture medium, 50 to 60 [mu] g / mL of ethylene glycol monomethyl ether, 1.5 to 4.0 mg / mL of acetylchitosamine, 25 to 30 ng / mL of bilobalide, 35 to 45 ng / mL of sodium pyrophosphate, 80 to 90 [mu] g / mL of alfalfa saponin and 100 to 110 [mu] g / mL of lipoic acid. According to the adipose tissue-derived stromal cell cryopreservation liquid provided by the invention, animal-derived serum and DMSO are not added, and the survival rate of resuscitated cells is high. The ethylene glycol monomethyl ether and the acetylchitosamine are added into the cryopreservation liquid, so that cells are not damaged, the permeability of the cells is improved, the osmotic pressure inside and outside the cells is maintained to be stable, and the activity of the cells is maintained. The bilobalide and the sodium pyrophosphate are added and compounded for use, so that denaturation and inactivation of cell protein molecules are prevented, and rapid activity recovery of cells after long-time cryopreservationis facilitated. Alfalfa saponin and lipoic acid are also added into the cryopreservation liquid, free radicals generated in the cryopreservation process are removed, and the antioxidation effect is achieved. The invention further provides a cryopreservation method of the adipose tissue-derived stromal cells, and the method is simple and convenient 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 Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 梁杰马超群
Owner GUANGDONG CELL BIOTECHNOLOGY CO LTD
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