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Primary hepatocyte cryopreservation solution, hepatocyte cryopreservation method and hepatocyte resuscitation method

A technology of primary hepatocytes and cryopreservation solution, applied in the field of primary hepatocyte cryopreservation solution, can solve the problems of poor effect of cryopreservation of liver cells, difficult for cells to adhere to the wall, and low cell viability, so as to protect the liver cell membrane and enhance the vitality. The effect of high adherence ability and cell viability

Active Publication Date: 2019-11-19
周明
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This cryopreservation solution has excellent effect on cryopreservation of cancer cells, but the cryopreservation effect on primary hepatocytes is poor, and there are cases where the cell viability is low and the cells are difficult to adhere to the wall
Therefore, this cryopreservation solution and resuscitation method are not suitable for cryopreservation and resuscitation of primary hepatocytes
[0005]The second one is CryoStorCS10 cryopreservation solution, the manufacturer is BioLifeSolutions, the specific ingredients are unknown, because the manufacturer is not disclosed, but the price is expensive, the current market price is 3500 RMB / 100mL
Therefore, this cryopreservation solution has serious limitations

Method used

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  • Primary hepatocyte cryopreservation solution, hepatocyte cryopreservation method and hepatocyte resuscitation method
  • Primary hepatocyte cryopreservation solution, hepatocyte cryopreservation method and hepatocyte resuscitation method
  • Primary hepatocyte cryopreservation solution, hepatocyte cryopreservation method and hepatocyte resuscitation method

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Embodiment 1

[0082]The primary hepatocyte cryopreservation solution of this embodiment is composed of the following components in volume percentage: 45% solution A, 10% fetal bovine serum, 10% dimethyl sulfoxide and 35% water, wherein, The solution A is composed of the following components: 100mg / mL D-glucose, 130mg / mL hydroxyethyl starch, 70mg / mL lactobionic acid, 60mg / mL polyvinylpyrrolidone, 30mg / mL pentahydrate D-raffinose, 15mg / mL potassium hydroxide, 6mg / mL potassium dihydrogen phosphate, 4mg / mL magnesium sulfate heptahydrate, 2mg / mL adenosine, 3mg / mL reduced glutathione, 0.2mg / mL allopurinol, 7×10 -3 mg / mL tauroursodeoxycholic acid and 1×10 -3 mg / mL- diammonium glycyrrhizinate, adjust the pH value to 7.4, and store at 4°C.

[0083] A method for cryopreserving hepatocytes, comprising: adding the above-mentioned primary hepatocyte cryopreservation solution to hepatocytes that have undergone conventional treatment, mixing evenly, and then cryopreserving. Specifically: add the above-m...

Embodiment 2

[0096] The primary hepatocyte cryopreservation solution of the present embodiment is composed of the following components in volume percentage: 45% solution A, 45% fetal calf serum and 10% dimethyl sulfoxide, wherein the solution A consists of The following components are composed: 120mg / mL D-glucose, 111mg / mL hydroxyethyl starch, 79.6mg / mL lactobionic acid, 51mg / mL polyvinylpyrrolidone, 39.6mg / mL pentahydrate D-raffinose, 12.5mg / mL mL potassium hydroxide, 7.56mg / mL potassium dihydrogen phosphate, 2.73mg / mL magnesium sulfate heptahydrate, 2.98mg / mL adenosine, 2mg / mL reduced glutathione, 0.3mg / mL allopurinol, 6× 10 -3 mg / mL tauroursodeoxycholic acid and 1 mg / mL diammonium glycyrrhizinate, adjust the pH value to 7.4, and store at 4°C.

[0097] A method for cryopreserving hepatocytes, comprising: adding the above-mentioned primary hepatocyte cryopreservation solution to hepatocytes that have undergone conventional treatment, mixing evenly, and then cryopreserving. Specifically:...

Embodiment 3

[0110] The primary hepatocyte cryopreservation solution of this embodiment is composed of the following components in volume percentage: 45% of solution A, 25% of human serum albumin, 10% of dimethyl sulfoxide and 20% of water, wherein , the solution A is composed of the following components: 140mg / mL D-glucose, 90mg / mL hydroxyethyl starch, 90mg / mL lactobionic acid, 40mg / mL polyvinylpyrrolidone, 50mg / mL pentahydrate D-raffinose , 10mg / mL potassium hydroxide, 9mg / mL potassium dihydrogen phosphate, 2mg / mL magnesium sulfate heptahydrate, 4mg / mL adenosine, 1mg / mL reduced glutathione, 0.4mg / mL allopurinol, 5× 10 -3 mg / mL tauroursodeoxycholic acid and 10 mg / mL diammonium glycyrrhizinate. Human serum albumin can be purchased commercially, such as purchased from Shenzhen Weiguang Biological Products Co., Ltd., National Drug Approval S20033032, wherein the protein concentration is 20%. When used in this embodiment, use ultrapure water (ddH 2 O) Dilute to 3v / v%. Adjust the pH to 7.4 ...

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Abstract

The invention discloses a primary hepatocyte cryopreservation solution, a hepatocyte cryopreservation method and a hepatocyte recovery method, and belongs to the field of primary hepatocyte cryopreservation and recovery. The primary hepatocyte cryopreservation solution comprises, by volume, 45% of a solution A, 10-45% of fetal bovine serum, 10% of dimethyl sulfoxide, and 0-35% of water, and the solution A comprises D-glucose, hydroxyethyl starch, lactobionic acid, polyvinylpyrrolidone, D-raffinose pentahydrate, potassium hydroxide, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, adenosine, reduced glutathione, allopurinol, tauroursodeoxycholic acid, and diammonium glycyrrhizinate. The invention also discloses the hepatocyte cryopreservation method and the hepatocyte recovery method. The primary hepatocyte cryopreservation solution can reduce the damages of the primary hepatocytes in the cryopreservation process and improve the vitality and adherence efficiency of the primary hepatocytes after thawing, and can be widely used in basic research and new drug development for livers.

Description

technical field [0001] The invention relates to a primary hepatocyte cryopreservation solution, a hepatocyte cryopreservation method and a hepatocyte resuscitation method, belonging to the technical field of hepatocyte cryopreservation and resuscitation. Background technique [0002] Primary hepatocytes refer to hepatocytes cultured immediately after removal from the liver tissue, which can be widely used in basic research and drug development, including liver research, hepatitis virus research, drug metabolism and toxicity research. However, the long-term storage of isolated primary hepatocytes is difficult, the individual differences are large, and the limitations of transportation limit their wide application. Cryopreservation can largely solve the limitations of long-term preservation, large individual differences, and transportation difficulties. [0003] At present, there are two methods for cryopreservation and resuscitation of primary hepatocyte cryopreservation sol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/071
CPCA01N1/0221A01N1/0226C12N5/067C12N2500/32C12N2501/11C12N2501/39C12N2501/998Y02A50/30
Inventor 周明
Owner 周明
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