Vitrification freezing liquid and freezing method for ova or cleavage stage embryos

A technology of vitrification and freezing, which is applied in the field of vitrification and freezing of eggs or cleavage-stage embryos, and can solve embryo damage, long-term (pre-protection solution needs to be used for 10-15 minutes, and protection solution needs to be used for 3-3 minutes). 5min, poor freezing protection effect and time-consuming problems, to achieve the effect of short operation time and reduced damage

Active Publication Date: 2020-06-05
佛山辅康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high dosage of DMSO is more cytotoxic and harmful to embryos
Moreover, the existing cryoprotective solutions for eggs and cleavage stage embryos genera

Method used

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  • Vitrification freezing liquid and freezing method for ova or cleavage stage embryos
  • Vitrification freezing liquid and freezing method for ova or cleavage stage embryos
  • Vitrification freezing liquid and freezing method for ova or cleavage stage embryos

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The preparation method of embodiment 1 vitrification liquid:

[0046] (1) Preparation of base solution: Take 2ml of SSS (Serum Sustitute Supplement) and dissolve it with 8ml of M199 culture medium (with HEPES).

[0047] (2) Preparation of pre-protection solution: Take 2ml of SSS, 0.75ml of ethylene glycol, 0.375ml of propylene glycol, and 0.375ml of dimethyl sulfoxide, and dissolve them in 6.5ml of M199 culture medium (with HEPES).

[0048](3) Preparation of protective solution: Dissolve 1.891g trehalose (molecular weight: 378.3) with 4ml of M199 culture medium (containing HEPES, American sigma company), then add 2ml SSS, 1.5ml ethylene glycol, 0.75ml propylene glycol, 0.75ml Dimethyl sulfoxide, use M199 culture medium (with HEPES) to make up to 10ml.

Embodiment 2

[0050] Preparation method of vitrification liquid:

[0051] (1) Preparation of base solution: Take 2ml of SSS (Serum Sustitute Supplement) and dissolve it with 8ml of M199 culture medium (with HEPES).

[0052] (2) Preparation of pre-protection solution: Take 2ml of SSS, 0.75ml of ethylene glycol, 0.375ml of propylene glycol, and 0.375ml of dimethyl sulfoxide, and dissolve them in 6.5ml of M199 culture medium (with HEPES).

[0053] (3) Preparation of protection solution: Dissolve 1.891g trehalose in 3.75ml of M199 culture medium (with HEPES), then add 2ml of SSS, 1.5ml of ethylene glycol, 0.75ml of propylene glycol, 1ml of dimethyl sulfoxide, and use M199 culture medium (with HEPES) to 10ml.

Embodiment 3

[0054] The preparation method of embodiment 3 vitrification liquid:

[0055] (1) Preparation of base solution: Take 2ml of SSS (Serum Sustitute Supplement) and dissolve it with 8ml of M199 culture medium (with HEPES).

[0056] (2) Preparation of pre-protection solution: Take 2ml of SSS, 0.75ml of ethylene glycol, 0.375ml of propylene glycol, and 1ml of dimethyl sulfoxide, and dissolve them in 5.875ml of M199 culture medium (with HEPES).

[0057] (3) Preparation of protective solution: dissolve 1.891g trehalose with 4ml of M199 culture medium (with HEPES), then add 2ml of SSS, 1.25ml of ethylene glycol, 1ml of propylene glycol, and 0.75ml of dimethyl sulfoxide, and use M199 culture medium (with HEPES) to dissolve 1.891g of trehalose. with HEPES) to 10ml.

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Abstract

The invention relates to a vitrification freezing liquid and a freezing method for ova or cleavage stage embryos. The vitrification freezing liquid comprises a base fluid, a pre-protection fluid and aprotection fluid, wherein the basic liquid comprises an M199 culture solution and a serum substitute; the pre-protection fluid comprises an M199 culture solution, a serum substitute, ethylene glycol,dimethyl sulfoxide and propylene glycol; the protection fluid comprises an M199 culture solution, a serum substitute, ethylene glycol, dimethyl sulfoxide, propylene glycol and trehalose; and the molar concentration of trehalose in the protection fluid is 0.3 to 1.0 mol/L. On the basis of reducing the addition amount of DMSO, the stability of ova or cleavage stage embryonic membranes can be improved by the vitrification freezing liquid, the osmotic injury is reduced, and the survival rate of ova or cleavage stage embryos is increased.

Description

technical field [0001] The invention relates to animal embryo engineering, in particular to a vitrification liquid and a freezing method for eggs or cleavage-stage embryos. Background technique [0002] Vitrification technology is to cool the cells and their protective agent solution at a fast enough cooling rate, supercool to the "glass transition temperature" to be solidified into a complete glass state, and store it at a low temperature in this glass state, avoiding The formation of ice crystals can reduce the freezing damage of ice crystals to cells and achieve the effect of cryoprotection. [0003] In recent years, the vitrification technology of blastocyst stage embryos has made significant progress, and the survival rate after freezing and thawing has been greatly improved. Compared with cleavage stage embryos, blastocyst stage embryos have better developmental potential and resistance to freezing damage, and have higher survival rate and pregnancy rate after freezin...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 胡威姜永存曾玉洁赵衡斌陈烨周星宇吴秀芝吴文林
Owner 佛山辅康生物科技有限公司
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