Ice-crystal-free low-temperature preservation method in supercooled state and ice-crystal-free low-temperature recovery method in supercooled state

A low-temperature preservation and state-of-the-art technology, applied in the field of low-temperature medicine, can solve the problems of affecting cell differentiation, damaging cells, and uneven heat diffusion, and achieves the effects of large density and cell mass, inhibition of ice crystal formation, and high survival rate

Active Publication Date: 2022-01-11
GUANGDONG UNISUN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Vitrification technology requires rapid cooling. If the amount of preserved cells is too large, the density is too high, or the tissue volume is large, the thermal stress caused by uneven diffusion of heat during the cooling process can damage the cells. Therefore, vitrification is only suitable for a small amount of cells. , low-density cells and small-volume tissues
In addition, vitrification requires a high concentration of cryoprotectant, the most commonly used is dimethyl sulfoxide (DMSO), which itself has cytotoxicity and can affect cell differentiation

Method used

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  • Ice-crystal-free low-temperature preservation method in supercooled state and ice-crystal-free low-temperature recovery method in supercooled state
  • Ice-crystal-free low-temperature preservation method in supercooled state and ice-crystal-free low-temperature recovery method in supercooled state
  • Ice-crystal-free low-temperature preservation method in supercooled state and ice-crystal-free low-temperature recovery method in supercooled state

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The present embodiment provides a method for low-temperature storage without ice crystals in a supercooled state, which includes the following steps:

[0050] S1. Select UW solution as cryoprotectant solution.

[0051] S2. Acquiring human cells and tissues.

[0052] S3. Suspend human cells and tissues with cryoprotectant solution pre-cooled to 4°C, pour into a 15ml centrifuge tube, and make the cell density reach 1×10 6 / ml.

[0053] S4. Inject an appropriate amount of light chain mineral oil. It can be seen that the light chain mineral oil floats on the liquid surface of the cryoprotectant solution and gradually completely covers the liquid surface to form a sample. Air bubbles should be avoided during the operation.

[0054] S5. Place the sample at a temperature pre-cooled to 4°C for temperature balance for 15 minutes, and then quickly place it at a temperature pre-cooled to -16°C for 24 hours in a supercooled state.

Embodiment 2

[0056] The present embodiment provides a method for low-temperature storage without ice crystals in a supercooled state, which includes the following steps:

[0057] S1. Select DMEM medium containing 10% fetal bovine serum as cryoprotectant solution.

[0058] S2. Acquiring human cells and tissues.

[0059] S3. Suspend human cells and tissues with cryoprotectant solution, pour them into a 15ml centrifuge tube, and make the cell density reach 5×10 6 / ml.

[0060] S4. Inject an appropriate amount of olive oil. It can be seen that the olive oil floats on the liquid surface of the cryoprotectant liquid and gradually completely covers the liquid surface to form a sample. Air bubbles should be avoided during the operation.

[0061] S5. Place the sample at a temperature pre-cooled to 4°C for temperature balance for 20 minutes, and then quickly place it at a temperature pre-cooled to -16°C for cryopreservation in a supercooled state.

[0062] S6. Place the sample at a temperature of...

Embodiment 3

[0064] This example provides a cryopreservation method without ice crystals in a supercooled state, which is basically the same as Example 1, the difference is that the cryoprotectant solution in this application includes UW solution, 5% PEG and 0.2mol / L 3-OMG .

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Abstract

The invention discloses an ice-crystal-free low-temperature preservation method in a supercooled state and an ice-crystal-free low-temperature recovery method in the supercooled state, and relates to the technical field of low-temperature medicine. The ice-crystal-free low-temperature preservation method in the supercooled state comprises the following steps: adding an oil substance into a cryoprotectant in which cells or tissues are suspended, allowing the oil substance to completely cover the surface of the cryoprotectant to form a sample, and carrying out low-temperature preservation on the sample. According to the invention, the cryoprotectant can be inhibited from freezing at a temperature of -20 DEG C to 0 DEG C, and the obvious effects of inhibiting ice crystal formation and preventing ice crystal growth are obtained, so a tissue cell bank and the like can be prevented from using cryoprotectants with cytotoxicity, such as DMSO (dimethylsulfoxide), and a safe and non-toxic biological sample bank is more favorably constructed. Compared with the prior art, the methods of the invention have the advantages that tissue cell cryopreservation can be performed for a longer time (longer than 2 days) at a lower temperature, ischemia-reperfusion injury is relieved, the survival rate of tissue cells after resuscitation is higher, and original biological characteristics can be kept.

Description

technical field [0001] The invention relates to the technical field of cryogenic medicine, in particular to a cryopreservation method and recovery method without ice crystals in a supercooled state. Background technique [0002] There are more than one million new severe hepatitis patients in my country every year, and less than 1% of them are cured through liver transplantation. It is urgent to meet the treatment needs of these patients clinically. Bioartificial liver has been confirmed by clinical practice as an effective treatment for organ replacement. In fact, with the extensive use of bioartificial livers in clinical practice, it is necessary to establish a "cell factory" in which a team with rich experience uses special equipment to conduct standardized evaluations of hepatocyte expansion, function, and safety under sterile conditions, and In this way, a bioartificial liver cell bank similar to a blood bank can be established to meet clinical needs. [0003] Cell cr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/021A01N1/0221
Inventor 高毅李阳黄楚颖
Owner GUANGDONG UNISUN BIOTECHNOLOGY CO LTD
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