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Vitrification refrigerating liquid and refrigerating method thereof

A technology of vitrification and freezing liquid, which is applied in the field of assisted reproduction, and can solve problems such as reduction, spindle disorder, freezing efficiency, and cytoskeleton damage.

Inactive Publication Date: 2018-07-06
成都艾伟孚生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cytoskeleton damage caused by freezing (microtubule depolymerization leads to spindle disorder) is considered to be one of the important reasons for the reduction of freezing efficiency.

Method used

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  • Vitrification refrigerating liquid and refrigerating method thereof
  • Vitrification refrigerating liquid and refrigerating method thereof
  • Vitrification refrigerating liquid and refrigerating method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The preparation of embodiment 1 vitrification liquid

[0047] 1. Prepare frozen base solution (pretreatment solution):

[0048] 1) M199 culture medium as solvent, NaHCO 35~10mmol / L, HEPES 10~14mmol / L, HEPES-Na 10~15mmol / L, penicillin 0.065~0.077g / L, streptomycin 0.05~0.09g / L, fetal bovine serum (volume content) 10%~ 20%, paclitaxel 0.1~1.5μmol / L;

[0049] 2) Mix well, filter through a 0.22 μm disposable filter, and set aside.

[0050] 2. Freezing solution 1: add 6.5% (v / v) ethylene glycol and 7% (v / v) DMSO to the freezing base solution.

[0051] 3. Freezing solution 2: Add 18% (v / v) ethylene glycol + 18% (v / v) DMSO and 0.75 mol / L sucrose to the freezing base solution.

Embodiment 2

[0052] The way of adding paclitaxel in the vitrification liquid of embodiment 2

[0053] 1. Grouping

[0054] Vitrification solution without paclitaxel was used as the control group, paclitaxel was added only to the pretreatment solution as the experimental group 1, and paclitaxel was added to the pretreatment solution, freezing solution 1 and freezing solution 2 as the experimental group 2.

[0055] 2. Experimental method

[0056] Three groups of vitrification solutions are used for freezing mature oocytes, and the OPS (openpulled straw) method is used for vitrification:

[0057] 1) Equilibrate the basic freezing liquid, freezing liquid 1, and freezing liquid 2 at room temperature for 25 minutes;

[0058] 2) Move the oocytes to the basic freezing solution for 5-8 minutes;

[0059] 3) After the treatment, move the oocytes into freezing solution 1 to balance for 20-40 seconds;

[0060] 4) Transfer to freezing solution 2, suck the oocytes into the OPS tube within 25s, and qu...

Embodiment 3

[0066] The added concentration of paclitaxel in the vitrification liquid of embodiment 3

[0067] 1. Grouping

[0068] The vitrification solution without paclitaxel was used as the control group, and the vitrification solution with three different concentrations of paclitaxel was used as the experimental group.

[0069] 2. Experimental method

[0070] Four groups of vitrification fluids are used for freezing mouse mature oocytes: adopt OPS (open pulled straw) method for vitrification, first move the oocytes to the freezing base fluid for 25 minutes, and then transfer them to the freezing fluid 1 to balance 30s, then transfer to vitrification solution II, within 25s, inhale oocytes and a small amount of freezing solution into OPS and put them into liquid nitrogen for cryopreservation.

[0071] 3. Experimental results

[0072] Record the survival rate of mouse mature oocytes after vitrification, the development rate of in vitro fertilized embryos, cleavage rate and mulberry b...

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Abstract

The invention belongs to the field of assisted reproduction and particularly relates to vitrification refrigerating liquid. According to the vitrification refrigerating liquid provided by the invention, taxol is added to serve as a cryoprotectant and the vitrification refrigerating liquid obtained with reference to the taxol adding mode is treated, so that the development rate of in vitro fertilized blastocyst after the oocyte is thawed can be increased. The taxol serving as an additive in the refrigerating liquid can achieve the effects of reducing freezing injury of the mature oocytes and improving the development capability of the embryo after fertilization. The optimal concentration of the adding mode is obtained by researching the adding concentration of the taxol. The vitrification refrigerating liquid provided by the invention can greatly increase recovery rate after the frozen mature oocytes are thawed and the development rate of the blastula and achieves the protective effecton the cell ultrastructure.

Description

technical field [0001] The invention belongs to the field of assisted reproduction, and in particular relates to a vitrification liquid added with paclitaxel. Background technique [0002] Cryopreservation of human gametes and embryos has become an essential part of artificial reproduction. In the past two decades, the development of cryopreservation technology can also reflect the development of artificial reproductive technology. [0003] Due to the large size and high water content of oocytes, intracellular ice crystals are easily formed during the freezing process and cause mechanical damage to the cells. Therefore, the key to oocyte preservation is to reduce the formation of intracellular ice crystals during freezing. During the freezing process, the mechanical damage caused by the formation of ice crystals in the cells and the osmotic damage caused by the high-concentration protective solution to the cells cause the rupture and hardening of the zona pellucida of the o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 余裕炉左晓玲刘洪君严飞戴甄
Owner 成都艾伟孚生物科技有限公司
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