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Cryoprotectant and preparation method thereof

A cryopreservation solution and cryopreservation technology, applied in the field of biomedical materials, can solve the problems of cytotoxic side effects, high toxicity of reagents, and no ice crystal growth.

Active Publication Date: 2020-10-20
北京玳衲芈珂生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, existing cryopreservation reagents cannot effectively control the growth of ice crystals during rewarming, which can damage cells
The high-concentration (≥15%) organic solvents used in the current vitrification method, such as: DMSO, lead to toxic side effects of cryopreservation reagents on cells, seriously affecting the survival rate and even (offspring) safety of cryopreservation objects after recovery and the functional expression
In summary, the cryopreservation reagents currently used do not have the ability to effectively control the growth of ice crystals during the rewarming process, and there is a problem of high reagent toxicity

Method used

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  • Cryoprotectant and preparation method thereof
  • Cryoprotectant and preparation method thereof
  • Cryoprotectant and preparation method thereof

Examples

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preparation example Construction

[0074] The preparation method of the present invention will be further described in detail below in conjunction with specific examples. It should be understood that the following examples are only illustrative and explanation of the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the foregoing contents of the present invention are covered by the scope of the present invention.

[0075] The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, materials, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0076] The PVA used in the embodiment of the present invention has a syndiotacticity of 50%-55%, a molecular weight of 13-23 kDa, and a degree of hydrolysis of 98%.

[0077] In the embodiment of the present invention, poly-L-proline has a degree of polymerization of 8 o...

Embodiment 1

[0079] Example 1: Cryopreservation of oocytes and embryos

[0080] 1. Preparation of cryopreservation solution: prepare cryopreservation solution according to the following formula

[0081] Cryopreservation Solution A:

[0082] Each 100mL contains the following components:

[0083] substance content PVA(g)2.0 Ethylene glycol (mL)10 DMSO(mL)10 Sucrose (mol L -1 )

0.5 Fetal Bovine Serum (mL)20 DPBS(mL) margin

[0084] Heat 2.0 g of PVA in a water bath at 80°C and magnetically stir and dissolve it in 25 mL of DPBS. After all PVA is dissolved and cooled to room temperature, the pH is adjusted to 7.0, which is solution 1; 17 g (0.05 mol) of sucrose (sucrose is frozen The final concentration in the preservation solution is 0.5mol L -1 ) Dissolve in 25mL of DPBS by ultrasonic, add 10mL of ethylene glycol and 10mL of DMSO after all the sucrose is dissolved to form solution 2. After solution 1 and solution 2 return to room temperature, mix the two solutions evenly and adjust the pH v...

Embodiment 2

[0128] Example 2: Freeze storage of stem cells

[0129] Cryopreservation Solution C1:

[0130] Each 100ml contains the following components:

[0131] substance Dosage PVA(g)1.0 Ethylene glycol (mL)10 Sucrose (mol L -1 )

0.5 Fetal Bovine Serum (mL)20 DPBS(ml) margin

[0132] Cryopreservation Solution D:

[0133] Each 100ml contains the following components:

[0134] substance Dosage PVA(g)2.0 Ethylene glycol (mL)10 Sucrose (mol L -1 )

0.5 DPBS(mL) margin

[0135] Cryopreservation Solution F:

[0136] Each 100ml contains the following components:

[0137]

[0138]

[0139] The preparation method of the cryopreservation solution is the same as in Example 1.

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Abstract

The invention discloses a cryoprotectant and a preparation method thereof. The cryoprotectant contains PVA, ethylene glycol and sucrose. PVA is used as a main cryoprotectant component of the cryoprotectant; and the cryoprotectant is low in DMSO content and low in toxicity, has a same or even higher cell survival rate and functional expression stability as a commercial cryoprotectant (containing 15% of DMSO) when being used for cryopreservation of cells or tissues, especially oocytes, and has higher preservation efficiency. The cryoprotectant is simple in composition and can be widely applied to cryopreservation of various cells, tissues and organs; the raw materials are easily available, and the cost is low.

Description

[0001] This application claims the priority of the prior application whose patent application number is 201910282416.X, and the invention title is "a PVA-based cryopreservation solution and its preparation method" filed with the State Intellectual Property Office of China on April 9, 2019. The full text of this previous application is incorporated into this application by reference. Technical field [0002] The invention belongs to the technical field of biomedical materials, and specifically relates to a PVA-based cryopreservation liquid and a preparation method thereof. Background technique [0003] Cryopreservation refers to the preservation of cells, tissues or organs in ultra-low temperature to slow down or stop cell metabolism and division. Once they return to normal physiological temperature, they can continue to develop. Since its inception, this technology has become one of the indispensable research methods in the natural sciences and has been widely adopted. In recent ...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 王健君金晟琳严杰乔杰闫丽盈李蓉
Owner 北京玳衲芈珂生物科技有限公司
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