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Cryopreservation liquid, preparation method thereof and application of cryopreservation liquid in human normal hepatocytes

A technology for cryopreservation liquid and liver cells, which can be used in applications, preservation of human or animal bodies, animal husbandry, etc. It can solve the problem of severe cell damage, poor cell state, lack of ability to control ice crystal nucleation and growth, and inhibit ice recrystallization and other problems, to achieve the effects of reducing production costs, reducing the growth rate of ice crystals, and inhibiting the recrystallization process of ice crystals

Active Publication Date: 2022-03-18
SONGSHAN LAKE MATERIALS LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, DMSO is cytotoxic, and the higher the concentration, the greater the harm to the cells, and the state of the cells after freezing is not good; secondly, the freezing solution containing DMSO will freeze again and recrystallize ice crystals during the rewarming process. Traditional cryopreservatives do not have the effect of controlling the nucleation and growth of ice crystals and inhibiting ice recrystallization, so the damage to cells caused by ice crystals during rewarming cannot be avoided

Method used

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  • Cryopreservation liquid, preparation method thereof and application of cryopreservation liquid in human normal hepatocytes
  • Cryopreservation liquid, preparation method thereof and application of cryopreservation liquid in human normal hepatocytes
  • Cryopreservation liquid, preparation method thereof and application of cryopreservation liquid in human normal hepatocytes

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Embodiment 1

[0032] Embodiment 1: This embodiment is illustrated by taking the cryopreservation and recovery of normal human liver cells as an example, but it is not used to limit the scope of the present invention.

[0033] (1) Preparation of cryopreservative: Take 90 mL of water for injection and put it into a beaker placed in a water bath at 4°C, put in a stirrer, and adjust the rotation speed to 500 rpm; add 0.1 g of L-glutamic acid, 0.1 g of sodium alginate, and three 3 g of hydroxymethylaminomethane, stirred for 10 minutes; followed by 5 mL of standard PBS buffer solution, 0.3 g of sodium dihydrogen phosphate and 0.7 g of disodium hydrogen phosphate, and stirred for 30 minutes.

[0034] (2) Cryopreservation of normal human liver cells:

[0035] (S1) Digest the cells in a 10cm culture dish (or T75 culture flask) and grow to a density of 80%-90%, add 2mL of trypsin for digestion, 4mL of complete medium (containing 10% FBS) to stop digestion, collect cell suspension;

[0036] (S2) Cen...

Embodiment 2

[0052] Embodiment 2: This embodiment is basically the same as Embodiment 1, the only difference is that the cell density during freezing is 10 4 cells / mL, 10 5 cells / mL, 10 6 cells / mL, 10 7 cells / mL. The L-O2 cells were cryopreserved according to the cryopreservation solution and the cryopreservation method provided in Example 1. After being frozen in liquid nitrogen for 10 days, the cells were recovered and the cell viability was detected. The results are shown in Table 1.

[0053] Table 1

[0054]

[0055] The results show that the results show that the cell viability of the different cell concentration groups of the test group is within 10 4 cells / m1-10 7 Within the range of cells / mL cell concentration, there was no significant difference.

Embodiment 3

[0056] Embodiment 3: This embodiment is basically the same as Embodiment 1, the only difference is that the cryopreservation agent includes the following components in mass percentage: 0.003% of L-glutamic acid, 0.002% of sodium alginate, trihydroxy 0.04% of methyl aminomethane, 0.2% of sodium dihydrogen phosphate, 0.5% of disodium hydrogen phosphate, 3% of PBS buffer solution, and the balance is water for injection.

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Abstract

The invention discloses a human normal hepatocyte freezing medium as well as a preparation method and application thereof in human normal hepatocytes. The freezing medium comprises the following components: L-glutamic acid, sodium alginate, trihydroxymethyl aminomethane, sodium dihydrogen phosphate, disodium hydrogen phosphate, a PBS buffer solution and water for injection. According to the formula of the cryopreservation agent, ice control materials such as L-glutamic acid, sodium alginate and tris (hydroxymethyl) aminomethane serve as main components, and damage of ice crystals to L-02 cells in the cryopreservation process can be effectively reduced. Compared with a traditional cryopreservation agent, the cryopreservation agent does not contain toxic molecules such as dimethyl sulfoxide and ethylene glycol and expensive components such as serum, and after recovery, the survival rate, the multiplication capacity and the cell morphology of L-02 cells are superior to those of an existing DMSO-containing cryopreservation protective agent. The components of the cryopreservation liquid are green and non-toxic, convenient to obtain and low in cost, and the cryopreservation liquid is a reliable human normal hepatocyte cryopreservation means.

Description

technical field [0001] The invention relates to the technical field of cryopreservation, in particular to a human normal hepatocyte cryopreservation solution, a preparation method thereof and an application in human normal hepatocytes. Background technique [0002] Both hepatocyte transplantation and hepatocyte-related medical research require a large number of hepatocytes. L-02 cells are a hepatic cell line established by domestic scholars in the 1980s. It has the advantages of easy in vitro culture and convenient source, and has been widely used in medical research on hepatocytes. [0003] In the scientific research and practical application of L-02 cells, it is often necessary to freeze the cells to meet the needs of long-distance transportation or long-term storage. Uncontrollable freezing during cryopreservation can cause severe mechanical damage to cells, which directly leads to direct cell inactivation. Therefore, cryopreservation solution is a protective agent that...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/021Y02A50/30
Inventor 刘红吕健勇赵立山卢思彤
Owner SONGSHAN LAKE MATERIALS LAB
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