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A cryopreservation method and recovery method without ice crystals in a supercooled state

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

Active Publication Date: 2022-04-29
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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  • A cryopreservation method and recovery method without ice crystals in a supercooled state
  • A cryopreservation method and recovery method without ice crystals in a supercooled state
  • A cryopreservation method and recovery method without ice crystals in a supercooled state

Examples

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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 a cryogenic preservation method and a recovery method without ice crystals in a supercooled state, and relates to the technical field of cryogenic medicine. The method for cryopreservation without ice crystals in a supercooled state includes: adding an oil substance to a cryoprotective solution in which cells or tissues are suspended, so that the oil substance completely covers the surface of the cryoprotectant solution to form a sample, and the sample is cryopreserved. The application can inhibit the freezing of cryoprotective solution at ‑20℃~0℃, and has obvious effects of inhibiting the formation of ice crystals and preventing the growth of ice crystals, so that the use of cytotoxic cryoprotective solutions such as DMSO can be avoided in tissue cell banks, etc. It is more conducive to the construction of a safe and non-toxic biological sample bank. This application can perform cryopreservation of tissue cells for a longer time (>2 days) at lower temperatures, reducing ischemia-reperfusion injury, and the survival rate of tissue cells after resuscitation is higher, and can maintain the original biological properties.

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 Patents(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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