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Vitrification liquid and freezing method of ovum or cleavage stage embryo

A vitrification and freezing method technology, applied in the field of vitrification and freezing of eggs or cleavage stage embryos, can solve the problem of long (pre-protection solution needs to be used for 10-15min, protection solution needs to be used for 3-5min, freezing The protection effect is poor and time-consuming, and the cytotoxicity of DMSO is high, so as to reduce the osmotic damage, shorten the operation time, and improve the stability.

Active Publication Date: 2022-03-25
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 generally have defects such as poor cryoprotective effect and long time-consuming (10-15 minutes for pre-protection solution and 3-5 minutes for protection solution).

Method used

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  • Vitrification liquid and freezing method of ovum or cleavage stage embryo
  • Vitrification liquid and freezing method of ovum or cleavage stage embryo
  • Vitrification liquid and freezing method of ovum or cleavage stage embryo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

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

[0048](3) Preparation of protective solution: dissolve 1.891g of 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, make up to 10 ml with M199 medium (with HEPES).

Embodiment 2

[0050] The preparation method of vitrification freezing liquid:

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

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

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

Embodiment 3

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

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

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

[0057] (3) Preparation of protective solution: dissolve 1.891g of 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 present invention relates to a vitrification solution and a freezing method for eggs or cleavage stage embryos. The vitrification solution includes base solution, pre-protection solution and protection solution; the base solution includes M199 culture solution and serum substitute; the pre-protection solution includes M199 culture solution, serum substitute, ethylene glycol, dimethyl sulfoxide Sulfone and propylene glycol; the protection solution includes M199 culture solution, serum substitute, ethylene glycol, dimethyl sulfoxide, propylene glycol and trehalose; the molar concentration of trehalose in the protection solution is 0.3-1.0mol / L . The vitrification liquid of the invention can improve the stability of the membrane of the egg or the cleavage stage embryo, reduce the osmotic damage and improve the survival rate of the egg or the cleavage stage embryo on the basis of reducing the added amount of DMSO.

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 freezing technology is to solidify the cells and their protective agent solutions into a complete glass state at a sufficiently fast cooling rate, supercooling to the "glass transition temperature", and store them at low temperature in this glass state, avoiding the need for The formation of ice crystals, thereby reducing the freezing damage of ice crystals to cells, achieves the effect of cryoprotection. [0003] In recent years, the vitrification technology of blastocyst stage embryos has made remarkable 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 thawing injury, and have higher survival and pregnancy r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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