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Novel bovine oocyte in vitro maturation culture solution

A technology for in vitro maturation and culture of oocytes, applied in tissue culture, microorganisms, biochemical equipment and methods, etc., can solve the problems of high oocyte quality requirements, low in vitro fertilization rate and blastocyst formation rate, etc.

Active Publication Date: 2009-12-02
北京科润维德生物技术有限责任公司
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0002] Since the 1930s, many scientific researchers have done a lot of work in the field of bovine oocyte growth and development in vitro. Mother cell culture conditions have been studied extensively and deeply, and great progress has been made. At present, the bovine oocyte maturation in vitro (invitro matrue, IVM) culture technology system has been established, but due to the oocyte maturation mechanism, maturation regulation There are still many problems that have not yet been clarified and some problems need to be solved urgently, so that the current method has high requirements for oocyte quality, low in vitro fertilization rate and blastocyst formation rate, etc.

Method used

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Embodiment 1

[0016] 1. Oocyte in vitro maturation culture method (the supernatant of amniotic membrane epithelial cell culture fluid is used as IVM medium supplement)

[0017] 1.1 Amniotic membrane epithelial cell culture supernatant-in vitro maturation (AECS-IVM) supplement and medium preparation

[0018] 1.1.1 About 1mm 3 Cut bovine or human amniotic membrane into pieces, shake in D-hank's containing 0.125% Trypsins (pancreatin) for 30 minutes at 37°C, and rotate at 400-600rpm;

[0019] 1.1.2 Filter the suspension with a cell sieve to collect amniotic epithelial cells;

[0020] 12.1.3 According to the conventional concentration (the number of cells is 10 5 ~10 6 each / bottle) to inoculate amnion epithelial cells in 75cm 2 Culture bottle;

[0021] 1.1.4 Culture amnion epithelial cells in vitro, and take the culture supernatant within 5-7 days;

[0022] 1.1.5 Centrifuge to remove cells and cell debris, 4°C, 1500-2500rpm, 15-30 minutes;

[0023] 1.1.6 Take the supernatant, remove the ...

Embodiment 2

[0046] 1. In vitro maturation culture method of oocytes (amnion epithelial cells as IVM medium supplement)

[0047] 1.1 Culture amnion epithelial cells as IVM trophoblasts

[0048] 1.1.1 About 1mm 3 The amniotic membrane was cut into pieces, and shaken at 37°C for 30min with 0.125% Trypsins D-hank’s at 400-600rpm

[0049] 1.1.2 Filter the suspension with a cell sieve to collect amniotic epithelial cells

[0050] 1.1.3 Inoculate amnion epithelial cells at 75cm according to conventional concentration 2 Culture flask

[0051] 1.1.4 Culture bovine or human amniotic epithelial cells in vitro, pour out the supernatant, and add 100ml IVM medium (M199+10%FBS+10μg / ml FSH+0.1μg / ml LH+1μg / ml E 2 ), as an amniotic epithelial cell-in vitro maturation (AEC-IVM) culture system

[0052] 1.2 In vitro culture of immature bovine eggs

[0053] 1.2.1 Routine collection and collection of bovine oocytes;

[0054] 1.2.2 Bovine oocytes were fully balanced in AEC-IVM medium for 2 hours, washed t...

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Abstract

The invention relates to a novel bovine oocyte in vitro maturation (IVM) culture solution, belonging to the field of embryo biotechnolory. In the invention, the routine IVM culture solution is added with amniotic epithelia or the supernatant of the culture solution thereof to establish a novel bovine immature oocyte IVM culture system, so as to improve the quality and maturation rate of the IVM bovine oocyte, thus solving the problem of low fertilization rate and low formation rate of blastula in the existing culture method.

Description

technical field [0001] The invention relates to the field of embryo engineering, in particular to bovine oocyte maturation solution in vitro. Background technique [0002] Since the 1930s, many scientific researchers have done a lot of work in the field of bovine oocyte growth and development in vitro. Mother cell culture conditions have been studied extensively and deeply, and great progress has been made. At present, the bovine oocyte maturation in vitro (invitro matrue, IVM) culture technology system has been established, but due to the oocyte maturation mechanism, maturation regulation There are still many problems that have not been elucidated and some problems need to be solved urgently, so that the current method has problems such as high requirements for oocyte quality, low in vitro fertilization rate and blastocyst formation rate. [0003] Amniotic membrane is formed on the 8th day of fertilization before gastrulation, and the amniotic membrane tissue cells maintai...

Claims

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Application Information

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IPC IPC(8): C12N5/06
Inventor 李荣旗
Owner 北京科润维德生物技术有限责任公司
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