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Ovary vitrification cryopreservation method under intervention of luteinizing hormone

A technology of luteinizing hormone and vitrification cryopreservation, which is applied in the preservation, application, animal husbandry and other directions of human or animal body, can solve the problems of high cost, etc., and achieve the effect of long validity period, optimal reproductive function and endocrine function.

Inactive Publication Date: 2016-12-07
NINGXIA MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Now there are methods of cryopreservation of ovaries using melatonin, vascular endothelial growth factor, erythropoietin and other drugs to intervene to improve the survival rate of ovarian follicles after cryopreservation, but most of them are used in combination and the cost is high

Method used

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  • Ovary vitrification cryopreservation method under intervention of luteinizing hormone
  • Ovary vitrification cryopreservation method under intervention of luteinizing hormone
  • Ovary vitrification cryopreservation method under intervention of luteinizing hormone

Examples

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Effect test

Embodiment 1

[0053] The adult mouse ovary vitrification cryopreservation method under the intervention of luteinizing hormone comprises the following steps:

[0054] Preculture of isolated ovaries:

[0055] Soak the isolated ovaries in culture medium at 37°C, 5% CO 2 Cultivate in a constant temperature incubator under the conditions for 240 minutes to obtain pre-cultured isolated ovaries; the culture solution is mixed according to the ratio of 12% bovine serum albumin 5ml and base solution 45ml, and then add luteinizing hormone to 0.60 IU / ml. The base solution was prepared according to 5 g of DMEM powder, 5.3 g of F12, 0.1 g of streptomycin sulfate, 2.38 g of 4-hydroxyethylpiperazineethanesulfonic acid, 2.38 g of NaHCO3, 10 ml of dimethyl sulfoxide, and 150 g of penicillin Mix in the proportion of IU, then use 5.6% NaHCO3 solution to adjust the pH value to 7.1-7.4, and finally add 1000ml triple distilled water.

[0056] Pre-infiltration balance of isolated ovaries:

[0057] At room temp...

Embodiment 2

[0074] The adult mouse ovary vitrification cryopreservation method under the intervention of luteinizing hormone comprises the following steps:

[0075] Preculture of isolated ovaries:

[0076] Soak the isolated ovary in the culture medium, place it in a constant temperature incubator under the condition of 37°C and 5% CO2 for 240 minutes, and obtain the isolated ovary after pre-cultivation; The ratio of 45ml of base solution was mixed, and then luteinizing hormone was added to 0.30 IU / ml; the base solution was prepared according to 5 g of DMEM powder, 5.3 g of F12, 0.1 g of streptomycin sulfate, and 4-hydroxyethylpiperazine ethyl sulfonate Mix 2.38 g NaHCO3, 2.38 g NaHCO3, 10 ml dimethyl sulfoxide, and 150 IU penicillin, then use 5.6% NaHCO3 solution to adjust the pH value to 7.1-7.4, and finally add 1000 ml triple-distilled water.

[0077] Pre-infiltration balance of isolated ovaries:

[0078] At room temperature, transfer the pre-cultured isolated ovary to a pre-osmotic b...

Embodiment 3

[0095] The adult mouse ovary vitrification cryopreservation method under the intervention of luteinizing hormone comprises the following steps:

[0096] Preculture of isolated ovaries:

[0097] Soak the isolated ovaries in culture medium at 37°C, 5% CO 2 Cultivate in a constant temperature incubator under the conditions for 240 minutes to obtain pre-cultured isolated ovaries; the culture solution is mixed according to the ratio of 12% bovine serum albumin 5ml and base solution 45ml, and then add luteinizing hormone to 0.15 IU / ml. The base solution was prepared according to 5 g of DMEM powder, 5.3 g of F12, 0.1 g of streptomycin sulfate, 2.38 g of 4-hydroxyethylpiperazineethanesulfonic acid, 2.38 g of NaHCO3, 10 ml of dimethyl sulfoxide, and 150 g of penicillin Mix in the proportion of IU, then use 5.6% NaHCO3 solution to adjust the pH value to 7.1-7.4, and finally add 1000ml triple distilled water.

[0098] Pre-infiltration balance of isolated ovaries:

[0099] At room temp...

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Abstract

The invention discloses an ovary vitrification cryopreservation method under intervention of luteinizing hormone. The ovary vitrification cryopreservation method comprises the following steps: soaking an in-vitro ovary into a culture solution, putting into a constant-temperature incubator with 5 percent of CO2 at the temperature of 37 DEG C for culture, then transferring into a permeable cryoprotective agent-containing ethylene glycol pre-equilibrium solution for soaking, after then, transferring into a high-concentration cryoprotective agent-containing vitrified cryopreservation fluid for continuous permeable equilibrium, and finally transferring into liquid nitrogen for soaking for at least 24 hours. A thawing process comprises the steps of taking out the cryopreserved ovary from the liquid nitrogen, placing the ovary into 0.50 mol / L, 0.25 mol / L and 0.125 mol / L of thawing solutions which are preheated at the temperature of 37 DEG C and has the sucrose concentration gradually decreased in sequence for 10 min, then transferring the ovary into the culture solution, and putting into the constant-temperature incubator with 5 percent of CO2 at the temperature of 37 DEG C for culture; the culture solution, the pre-equilibrium solution, the vitrified cryopreservation fluid and the thawing solution contain the luteinizing hormone, so that the survival rate of the cryopreserved ovary can be increased, and the reproductive function and the endocrine function can be retained to a relatively large extent.

Description

technical field [0001] The invention relates to vitrification cryopreservation technology, in particular to a vitrification cryopreservation method of ovaries under the intervention of luteinizing hormone. Background technique [0002] The in vitro culture and cryopreservation of isolated ovarian tissue can provide convenient conditions for obtaining female reproductive stem cells and all levels of follicles from ovarian tissue, so that the development of these techniques is not limited by time and place. At the same time, isolated ovarian tissue is convenient for studying the development mechanism of egg cells, combined with in vitro fertilization or parthenogenetic activation techniques to obtain a large number of embryonic stem cells for scientific research, and also helps to establish egg banks to provide reproductive insurance for women. [0003] Currently isolated ovarian tissue is mainly cultured directly after isolation, and then used for research, but due to field c...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/021A01N1/0221
Inventor 王燕蓉裴秀英常青陈杰
Owner NINGXIA MEDICAL UNIV
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