Method for preparing in-vitro oocyte maturation culture solution

A technology for in vitro maturation culture and oocyte, which is applied in the preparation of oocyte in vitro maturation medium and in the field of culture medium preparation, which can solve the problems of low embryonic development ability and poor oocyte developmental potential, and improve embryonic development. Potential, improve the effect of embryo stoppage problem

Pending Publication Date: 2018-12-07
FOURTH MILITARY MEDICAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a preparation method of oocyte in vitro maturation culture medium, and the present invention improves the problem that oocyte development potential after IVM is poor, and embryo development ability is relatively low after parthenogenetic activation

Method used

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  • Method for preparing in-vitro oocyte maturation culture solution

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Embodiment 1

[0021] A method for preparing oocyte maturation culture medium in vitro, the specific steps are as follows:

[0022] Step 1) preparing basic IVM medium;

[0023] Step 2) adding lipoproteins to the basic IVM medium to obtain: culture medium for in vitro maturation of oocytes.

Embodiment 2

[0025] A method for preparing oocyte maturation culture medium in vitro, the specific steps are as follows:

[0026] Step 1) preparing basic IVM medium;

[0027] Step a) using commercial tissue culture medium TCM199 as the basic cell culture medium;

[0028] Step b) Replace commercially available 0.075-0.10IU / ml FSH and 0.5-0.8IU / ml HCG, 0.1-0.2μg / ml 17β-estradiol, and 10%-20% by volume of commercially available human Serum was added to commercial tissue culture medium TCM199;

[0029] Step c) filter the commercialized tissue culture fluid TCM199 added in step b) with a 0.22 μm filter;

[0030] Step d) After filtering, low-temperature storage is carried out in an environment with a temperature of 4° C. to obtain the basic IVM medium. The basic IVM medium was used within 2 weeks.

[0031] Step 2) Add 20ug / ml lipoprotein to the basic IVM medium to obtain: culture medium for in vitro maturation of oocytes.

[0032] Before culturing the oocytes in the oocyte in vitro maturati...

Embodiment 3

[0035] This embodiment is the experimental example that embodiment 2 is done, and experimental process is as follows:

[0036] Select female mice aged 6 to 8 weeks, inject 10IU PMSG intraperitoneally for superovulation treatment, kill the mice after 46-48 hours, take out the ovaries and put them in M2 culture medium, and use a needle under a stereomicroscope Crush the follicles, select GV stage oocytes, and culture them in the following groups. Control group: basic IVM medium; experimental group: add 20ug / ml lipoprotein to the basic IVM medium, and add 10-15 oocytes to each culture drop Oocytes were cultured under the above conditions, and the occurrence rate of MII was counted after 24 hours of culture, and the MII eggs were selected and placed in a pre-balanced solution containing 20mMSrCl 2 parthenogenetic activation in ksom at 37°C, 5% CO 2 Wash 5 times with the KSOM culture solution pre-balanced for more than 4 hours under certain conditions, and then transfer the eggs i...

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Abstract

The invention relates to a method for preparing an in-vitro oocyte maturation culture solution. The preparation method is characterized by comprising the following steps: 1) preparing a basal IVM (InVitro Maturation) culture medium; and 2) adding lipoprotein into the basal IVM culture medium, thereby obtaining the in-vitro oocyte maturation culture solution. According to the method disclosed by the invention, the problems that the developmental potential of oocyte is poor after IVM and developmental potential of embryos is relatively low after parthenogenetic activation can be solved, the embryo can continuously develop to blastula after the lipoprotein is added, the developmental potential of the embryos is improved by the IVM culture solution added with the lipoprotein, and the embryo damage problem is effectively improved.

Description

technical field [0001] The invention relates to a preparation method of a culture solution, in particular to a preparation method of an oocyte maturation culture solution in vitro. Belongs to the field of medicine. Background technique [0002] In vitro maturation of oocytes (in vitro maturation, IVM) refers to the process of culturing and developing oocytes in GV or M I phases to the second meiotic metaphase (M II phase) in vitro. IVM can be used as a technology, combined with in vitro fertilization (IVF) technology can be effectively applied in the field of reproductive medicine to treat infertility. [0003] As an important part of assisted reproductive technology, oocyte in vitro maturation technology can not only improve the utilization efficiency of oocytes, reduce the occurrence of ovarian hyperstimulation syndrome, but also provide more options for egg donation and female fertility preservation. In recent years, with the rapid development of reproductive medicine, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2500/35C12N2501/31
Inventor 李博陈书强芦洁
Owner FOURTH MILITARY MEDICAL UNIVERSITY
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