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Vitrification reagent used for reproduction freezing, kit and use method thereof

A vitrification freezing, cryoprotectant technology, applied in the application, preservation of human or animal body, animal husbandry, etc., can solve problems such as methylation, toxicity, cell damage affecting cell differentiation, avoid recrystallization, and achieve good results. Thermal conductivity, the effect of eliminating toxicity

Active Publication Date: 2018-07-06
江苏康麟医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high concentrations of cryoprotectants are toxic and affect the quality of germ cells and the growth and development of embryos
For example, dimethyl sulfoxide increases the flow of extracellular calcium ions into the cell, causing potential damage to the cell, affecting cell differentiation and DNA methylation
However, if the concentration of ethylene glycol is too high, it will affect the function of cell mitochondria.
As far as the current vitrification effect is concerned, the development rate, clinical pregnancy rate and implantation rate of embryos are difficult to reach a satisfactory level
[0008] (2) Germ cells or embryos undergoing vitrification have been kept in liquid nitrogen. When their activity needs to be restored, they must be rewarmed to ambient temperature. However, during the rewarming process, the vitrified cells and their surrounding cryoprotective solution Easy but extremely easy to re-form ice crystals, causing serious damage to cells
However, the above attempts cannot completely avoid the use of dimethyl sulfoxide and ethylene glycol
CN104839144A Although proline is added to the vitrification liquid, it still needs to add 5-15v / v% dimethyl sulfoxide and ethylene glycol, so it still has a certain toxic effect on cells
However, the existing nanoparticles, such as titanium dioxide nanoparticles, have strong toxicity to cells, affecting the survival rate and development rate of cells.

Method used

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  • Vitrification reagent used for reproduction freezing, kit and use method thereof
  • Vitrification reagent used for reproduction freezing, kit and use method thereof
  • Vitrification reagent used for reproduction freezing, kit and use method thereof

Examples

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Embodiment 1

[0070] A kit for vitrification, said kit comprising balance solution, vitrification solution and thawing solution, wherein the specific formulations of said balance solution, vitrification solution and thawing solution are as follows:

[0071] Equilibrium solution: DPBS buffer solution of 0.5M glucose, 0.2M betaine and 1.5mM HSA, pH=7.2.

[0072] The vitrification solution is composed of independently packaged intracellular vitrification solution and extracellular vitrification solution. Intracellular vitrification solution: liposomes encapsulating glucose, wherein the concentration of glucose is 1M; extracellular vitrification solution: 2M Glucose, 0.25M proline, 0.3M betaine and 1.5mM HSA in DPBS buffer, pH=7.2.

[0073] Thawing solution 1: DPBS buffer solution of 1M glucose, 0.2M betaine and 1.5mM HSA, pH=7.2;

[0074] Thawing solution 2: DPBS buffer solution of 0.5M glucose, 0.2M betaine and 1.5mM HSA, pH=7.2;

[0075] Thawing solution 3: 0.2M betaine and 1.5mM HSA in DP...

Embodiment 2

[0077] A kit for vitrification, said kit comprising balance solution, vitrification solution and thawing solution, wherein the specific formulations of said balance solution, vitrification solution and thawing solution are as follows:

[0078] Equilibrium solution: DPBS buffer solution of 0.25M trehalose, 0.2M betaine and 1.5mM HSA, pH=7.2.

[0079] The vitrification solution is composed of independently packaged intracellular vitrification solution and extracellular vitrification solution. Intracellular vitrification solution: liposome-encapsulated glucose, the concentration of glucose is 0.75M; extracellular vitrification solution: 1M seaweed Sugar, 0.5M proline, 0.3M betaine and 1.5mM HSA in DPBS buffer, pH=7.2.

[0080]Thawing solution 1: DPBS buffer solution of 0.5M glucose, 0.2M betaine and 1.5mM HSA, pH=7.2;

[0081] Thawing solution 2: DPBS buffer solution of 0.2M glucose, 0.2M betaine and 1.5mM HSA, pH=7.2;

[0082] Thawing solution 2: 0.2M betaine and 1.5mM HSA in ...

Embodiment 3

[0084] A kit for vitrification, said kit comprising balance solution, vitrification solution and thawing solution, wherein the specific formulations of said balance solution, vitrification solution and thawing solution are as follows:

[0085] Equilibrium solution: DPBS buffer solution of 0.75M glucose, 0.2M betaine and 1.5mM HSA, pH=7.2.

[0086] The vitrification solution is composed of independently packaged intracellular vitrification solution and extracellular vitrification solution. Intracellular vitrification solution: liposomes encapsulating glucose, wherein the concentration of glucose is 2M; extracellular vitrification solution: 3M Glucose, 0.3M betaine and 1.5mM HSA in DPBS buffer, pH=7.2.

[0087] Thawing solution 1: DPBS buffer solution of 1.5M glucose, 0.2M betaine and 1.5mM HSA, pH=7.2;

[0088] Thawing solution 2: DPBS buffer solution of 0.5M glucose, 0.2M betaine and 1.5mM HSA, pH=7.2;

[0089] Thawing solution 3: DPBS buffer solution of 0.1M glucose, 0.2M b...

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Abstract

The invention relates to a vitrification reagent used for reproduction freezing, a kit and a use method thereof. The vitrification reagent used for reproduction freezing comprises an intracellular vitrification reagent, the intracellular vitrification reagent comprises a transmembrane carrier and a cryoprotectant enveloped in the transmembrane carrier, and the cryoprotectant is selected from a saccharides cryoprotectant, a sugar alcohol cryoprotectant or an amine cryoprotectant; and therefore, by adopting the vitrification reagent, the toxicity of cryoprotectant to cells can be greatly reducedwhile avoiding forming of ice crystals in the cells, the survival rate of the cells is increased, and the normal development of the cells is ensured.

Description

technical field [0001] The invention relates to the field of reproductive freezing, in particular to a vitrification reagent for reproductive freezing, a kit and a use method thereof. Background technique [0002] Reproductive cryopreservation refers to the technology of stopping metabolism of germ cells and embryos at -196°C through special protection measures and cooling procedures, and recovering their metabolic capacity after warming up. With the continuous development of in vitro fertilization-embryo transfer and its derivative technologies, reproductive cryopreservation technology involving germ cells and embryos has become an indispensable part in the field of human assisted reproduction or animal breeding. [0003] Currently, conventional reproductive freezing techniques include slow freezing and vitrification. [0004] Among them, the slow freezing method is the main method for cryopreservation of most mammalian reproduction. In this method, a lower concentration ...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 严杰乔杰李蓉刘平郑晓英黄锦庄新杰李军生林胜利袁一峰
Owner 江苏康麟医疗科技有限公司
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