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In-vitro culture method and culture medium for embryonic cells containing quercetin

A technology of embryonic cells and in vitro culture, applied to embryonic cells, culture process, animal cells, etc., can solve problems such as slow development, insufficient developmental ability of oocytes and embryos, and pregnancy failure

Active Publication Date: 2021-11-26
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing technology has insufficient development ability of oocytes and embryos, which causes slow development and even pregnancy failure after early embryo transfer

Method used

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  • In-vitro culture method and culture medium for embryonic cells containing quercetin
  • In-vitro culture method and culture medium for embryonic cells containing quercetin
  • In-vitro culture method and culture medium for embryonic cells containing quercetin

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Patient recruitment and ethics review

[0047] The research for this application was approved by the Review Board of the Institute of Reproductive Medicine, Shandong University ([2018] Ethics Approval #41). All methods described in this application were performed in accordance with the approved guidelines and regulations of the Institute of Reproductive Medicine, Shandong University. A formal informed consent was obtained from each patient before the experiments related to human beings.

Embodiment 2

[0048] Embodiment 2 experimental method and reagent

[0049] Oocyte and Embryo Collection

[0050]9-10-month-old female Kunming mice (the fertility declines rapidly at this stage) were used as a natural aging mouse model; 6-8-week-old female mice were also used in the experiment. They were housed in a room with temperature control and a setting of 12D:12L (dark and light), with free access to water and food. To obtain intact GV oocytes, mice were injected intraperitoneally with 10 IU pregnant horse serum gonadotropin (PMSG) (purchased from Ningbo Sansheng Biotechnology Co., Ltd., China) to stimulate follicles, and ruptured antral follicles were collected after 46-48 hours Oocytes with closed cumulus. Then place the oocytes in 6% CO 2 , 5% O 2 , 90% N 2 , cultured in M16 medium (Sigma-Aldrich) at 37°C, covered with mineral oil, and cultured for 4h or 16h to determine the ratio of GVBD and the rate of first polar body (PB1) excretion. To induce superovulation, 10 IU of hum...

Embodiment 3

[0079] Example 3 In vitro experiments show that treating oocytes from aged mice with quercetin improves the maturation and early embryonic development of oocytes

[0080] figure 1 A is a schematic diagram of the experimental procedure and method.

[0081] 1066 GV stage oocytes from 9–10 month old female mice were collected and cultured for 16 hours in M16 medium supplemented with different concentrations (0 μM, 5 μM, 10 μM or 20 μM) of quercetin. The results showed that compared with the control, the extrusion rate of GVBD and Pb1 increased with different concentrations of quercetin. Among them, compared with the control, the GVBD rate (86.6%, n=284vs.79.7%, n=286, pfigure 1 B), and it is the highest in each concentration treatment group. 10 μM quercetin treatment was selected for further study.

[0082] To further determine the role of quercetin, the effect of quercetin treatment on early embryonic development in aged mice after IVF was examined. MII stage oocytes were cr...

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Abstract

The invention provides an in-vitro cultivation method of embryonic cells of mammals, especially humans, wherein quercetin is added into a culture medium. The invention also provides culture medium for embryonic cells of mammals, especially humans, containing quercetin. The in-vitro culture method and culture medium provided by the invention are particularly suitable for culturing embryo cells of mammals of middle or high childbearing age, including female humans.

Description

technical field [0001] The present invention relates to the field of assisted reproduction of mammals, especially humans. Specifically, the present invention relates to a method and culture medium for embryonic cells of mammals including humans in vitro. Background technique [0002] The phenomenon of decreased fertility due to increasing age is called reproductive senescence. Reproductive aging leads to a gradual decline in the number and quality of oocytes within ovarian cortical follicles. Age-related fertility decline is not only due to a reduced pool of oocytes but also to decreased oocyte quality. Oocytes originate from primordial germ cells, stagnate in the germinal vesicle stage (GV stage), and need to undergo chromatin condensation and nuclear membrane separation to enter the germinal vesicle rupture stage to obtain the ability of fertilization and embryogenesis. After chromosome segregation, the oocyte extrudes the first polar body (PB1), then stops at the secon...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
CPCC12N5/0603C12N2500/30
Inventor 赵跃然陈子江曹永芝赵海滨王钊张倡铭边月红
Owner SHANDONG UNIV
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