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Application of DMSO-free cryopreservation solution in organ and tissue cryopreservation

A cryopreservation solution and cryopreservation technology, which is applied in the application field of cryopreservation solution in the cryopreservation of organs and tissues, and can solve problems such as adverse side effects, strong cytotoxicity, and cytotoxic side effects

Pending Publication Date: 2020-10-20
PEKING UNIV THIRD HOSPITAL +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, existing cryopreservation reagents cannot effectively control the growth of ice crystals during rewarming, which can damage cells
Dimethyl sulfoxide (DMSO) is a commonly used cosolvent and permeable cell cryopreservation agent for in vitro cell culture. However, DMSO has adverse side effects in clinical patient trials; at the same time, DMSO also has strong cytotoxicity, and different types of Cells have different sensitivities to DMSO concentration, resulting in toxic and side effects of cryopreservation reagents with DMSO as the main protective agent component on cells, and its application is limited
The high concentration (≥15%) of DMSO commonly used in the current vitrification method seriously affects the survival rate and even the (offspring) safety and functional expression of cryopreserved subjects after recovery
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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  • Application of DMSO-free cryopreservation solution in organ and tissue cryopreservation
  • Application of DMSO-free cryopreservation solution in organ and tissue cryopreservation
  • Application of DMSO-free cryopreservation solution in organ and tissue cryopreservation

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preparation example Construction

[0100] The preparation method of the present invention will be further described in detail in conjunction with specific examples below. It should be understood that the following examples are only for illustrating and explaining the present invention, and should not be construed as limiting the protection scope of the present invention. All technologies realized based on the above contents of the present invention are covered within the scope of protection intended by the present invention.

[0101] The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents and materials used in the following examples can be obtained from commercial sources unless otherwise specified.

[0102]The PVA used in the embodiment of the present invention has a syndiotacticity of 50%-55%, a molecular weight of 13-23kDa, and a degree of hydrolysis of 98%.

[0103] In the embodiment of the present invention, poly-L-proline with a degree of ...

Embodiment 1

[0105] Example 1. Cryopreservation of Mouse Oocytes and Embryos

[0106] 1. Preparation of cryopreservation solution: prepare cryopreservation solution according to the following formula

[0107] Cryopreservation solution A: Each 100ml contains the following components:

[0108] substance Dosage Poly-L-proline (g) 1.5 PVA (g) 2.0 Ethylene glycol (mL) 10 Sucrose (mol L -1 )

0.5 DPBS (mL) margin

[0109] Heat 2.0g of PVA in a water bath at 80°C and dissolve it in 25mL of DPBS with magnetic stirring, adjust the pH to 7.0, which is solution 1; ultrasonically dissolve 1.5g of poly-L-proline in another 20mL of DPBS, Regulating pH is 7.0, is solution 2; The sucrose of 17g (0.05mol) (the final concentration of sucrose in cryopreservation solution is 0.5mol L -1 ) was ultrasonically dissolved in 25mL of DPBS. After the sucrose was completely dissolved, 10mL of ethylene glycol was added in turn to form solution 3. After solution 1, so...

Embodiment 2

[0146] Embodiment 2: Cryopreservation of human umbilical cord mesenchymal stem cells

[0147] 1. Preparation of cryopreservation solution: prepare cryopreservation solution according to the following formula

[0148] Cryopreservation solution E: total volume 100mL, containing ethylene glycol 10mL, serum 20mL, sucrose 17g (0.5mol L -1 ), poly-L-arginine (polymerization degree is 8) 4.0g, PVA 1.0g, DPBS balance.

[0149] Cryopreservation solution F: total volume 100mL, containing ethylene glycol 25mL, serum 20mL, sucrose 17g (0.5mol L -1 ), L-Arg 16g, L-Thr 8g, DPBS balance.

[0150] Cryopreservation solution G: total volume 100mL, containing 10mL ethylene glycol, 20mL serum, 17g sucrose (0.5mol L -1 ), PVA 2.0g, balance of DPBS.

[0151] Cryopreservation solution H: total volume 100mL, containing ethylene glycol 10mL, serum 20mL, sucrose 17g (0.5mol L -1 ), TR28g, DPBS margin.

[0152] Cryopreservation solution I: total volume 100mL, containing ethylene glycol 10mL, sucro...

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Abstract

The invention discloses an application of a DMSO-free cryopreservation solution in organ and / or tissue cryopreservation. Every 100 mL of the cryopreservation solution contains 0.01-50.0 g of a bionicice control material, 5.0-30 mL of polyol, 1-30 g of water-soluble sugar, 0-30 mL of serum and the balance of a buffer solution, wherein the bionic ice control material is selected from PVA and / or anamino acid bionic ice control material. The cryopreservation solution and a freezing equilibrium solution do not contain DMSO, and have high preservation efficiency when used for cryopreservation of ovarian tissues or organs. The DMSO-free and serum-free cryopreservation solution further solves the problems of poor stability, introduction of parasitic biological pollutants and the like caused by serum contained in commercialized cryopreservation solutions commonly used in clinic at present. The cryopreservation solution disclosed by the invention is simple in composition, convenient in raw material source and low in cost, and can be widely applied to cryopreservation of various organs or tissues.

Description

[0001] This application requires the patent application number 201910281978.2 submitted to the State Intellectual Property Office of China on April 9, 2019, and the prior application with the title of invention "A DMSO-free cryopreservation solution and its preparation method", and the patent application number 201910281986.7 , the priority of the earlier application entitled "A Peptide Compound and a Cryopreservation Solution Containing the Compound". The entire contents of the above two prior applications are incorporated by reference in this application. technical field [0002] The invention belongs to the technical field of biomedical materials, and in particular relates to the application of a DMSO-free cryopreservation solution in organ and tissue cryopreservation. Background technique [0003] Since the advent of cryopreservation technology, it has become one of the indispensable research methods in the field of natural science and has been widely used. With the imp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K5/062C07K5/068C07K5/078C07K5/083A01N1/02
CPCC07K5/0606C07K5/06095C07K5/06165C07K5/0806C07K5/081A01N1/0221A01N1/0205
Inventor 严杰乔杰闫丽盈李蓉王健君金晟琳
Owner PEKING UNIV THIRD HOSPITAL
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