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Freezing liquid for human stem cells and freezing storage method

A technology of stem cells and freezing liquid, applied in the field of cell tissue engineering, can solve the problems of easy damage, reduction, and differentiation potential of stem cells due to oxidative damage, and achieve the effects of avoiding oxidative damage, high cell recovery rate, and maintaining the vitality of stem cells.

Inactive Publication Date: 2017-09-22
山东翰康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both DMSO and glycerin can effectively help cells resist the harmful effects of freezing, but stem cells frozen in a cryogenic solution containing large doses of DMSO and glycerin are not suitable for the human body, they are easily damaged, and DMSO is likely to have toxic side effects
In addition, when using the ordinary freezing solution FBS / DMSO / glycerol mixture, the activity of cells after recovery is often poor, and the process of recovery may easily lead to oxidative damage and reduced differentiation potential of stem cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Preparation of freezing liquid X1

[0029] Take 25ml DMSO, 85ml glycerin, 225ml fetal bovine serum, add PBS buffer to make 1000ml basic freezing solution, then add 20mg salidroside, 60g hydroxyethyl modified starch 450Kd, 45g carboxylated poly-L-lysine amino acid, 50g sucrose, 80g trehalose, 7g antifreeze glycoprotein, and 7g glycoprotein to make 1000ml of human stem cell freezing liquid mother solution, and then divide into 10 parts of 100ml for freezing.

[0030] Cryopreservation of human stem cells 1:

[0031] According to the total amount of stem cells to be frozen, calculate the amount of cryopreservation solution X1 required, put the cells into an appropriate amount of freezing solution X1, freeze at -20°C for 1 hour, and continue to freeze at -80°C for 12 hours, and then transferred to liquid nitrogen for long-term storage.

Embodiment 2

[0033] Preparation of freezing liquid X2

[0034] Take 20ml DMSO, 100ml glycerin, 200ml fetal bovine serum, add HEPES buffer to make 1000ml basic freezing solution, then add 20mg salidroside, 100g hydroxyethyl modified starch 450Kd, 10g carboxylated poly-L-lysine Amino acid, 100g sucrose, 150g trehalose, 5g antifreeze protein Ⅰ, 5g glycoprotein, make 1000ml of human stem cell freezing liquid mother liquid, and then divide into 10 parts of 100ml for freezing.

[0035] Cryopreservation method of human stem cells 2

[0036] According to the total amount of stem cells to be frozen, calculate the amount of freezing solution X2 required, put the cells into an appropriate amount of freezing solution X2, freeze at -20°C for 1 hour, and continue to freeze at -80°C for 12 hours, and then transferred to liquid nitrogen for long-term storage.

Embodiment 3

[0038] Preparation of freezing liquid X3

[0039] Take 50ml DMSO, 60ml glycerin, 300ml fetal bovine serum, and then add PBS buffer to make 1000ml basic freezing solution, then add 50mg salidroside, 50g hydroxyethyl modified starch 450Kd, 50g carboxylated poly-L-lysine Amino acid, 50g sucrose, 20g trehalose, 20g antifreeze protein Ⅳ, 20g glycoprotein, make 1000ml of human stem cell freezing liquid mother liquid, and then pack into 10 parts of 100ml for freezing.

[0040] Cryopreservation method of human stem cells 3

[0041] According to the total amount of stem cells to be frozen, calculate the amount of cryopreservation solution X3 required, put the cells into an appropriate amount of freezing solution X3, freeze at -20°C for 1 hour, and continue to freeze at -80°C for 12 hours, and then transferred to liquid nitrogen for long-term storage.

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PUM

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Abstract

The invention relates to a freezing liquid for human stem cells and a freezing storage method. Every 1000 ml of the freezing liquid comprises the following components: 20-50 mg of salidroside, 50-100 g of hydroxyethyl modified starch 450 Kd, 10-50 g of carboxylated poly-L-lysine, 50-100 g of sucrose, 20-150 g of trehalose, 5-20 g of antifreeze protein, 5-20 g of glycoprotein, 20-50 ml of DMSO, 60-100 ml of glycerin, and 200-300 ml of fetal calf serum, with the balance being a buffer solution. The freezing liquid for human stem cells is low in dimethyl sulfoxide content, has no side or toxic effect on frozen cells, can effectively keep the activity of stem cells, prevents oxidative injury, and is high in cell thawing rate.

Description

technical field [0001] The invention belongs to the field of cell tissue engineering, in particular to a freezing liquid and a freezing storage method of human stem cells. Background technique [0002] Over the past decade, pluripotent stem cells have become a useful tool in many fields to help people explore developmental biology, study disease pathology, and develop cell therapies. [0003] No matter what the purpose of stem cells is, cryopreservation is inseparable. Cryopreservation or freezing can preserve precious stem cell resources, but freezing stem cells is not as simple as freezing differentiated cells. It requires appropriate freezing reagents and protocols, otherwise the preserved stem cells will lose their value if they undergo random differentiation. [0004] The usual freezing scheme is to first use protease to dissociate the cells, then centrifuge, and finally resuspend in the medium containing the freezing solution, which includes dimethyl sulfoxide (DMSO),...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0226
Inventor 郭长生赵振峰
Owner 山东翰康生物科技有限公司
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