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Feeder cells using in immatural ovocyte in-vitro cultivating matural

A feeder cell, in vitro culture technology, applied in the field of reproductive medicine and cell biology, can solve the problems that limit the exertion and application of the advantages of IVM technology, the maturation rate of 90% of oocytes, and the inconsistency of oocyte nuclear and cytoplasmic maturation. , to achieve easy-to-obtain, stable results, and minimal trauma

Inactive Publication Date: 2007-07-18
ANHUI PROVINCIAL HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But these substances are very expensive and unstable, prone to inconsistent nucleoplasmic maturation of oocytes
Cambridge, England "Reproduction" magazine (Reproduction, 2001, 121: 925) reported that monkey kidney cells, granulosa cells and oviduct epithelial cells were used as feeder cells, and the secretion of cells was used to reduce the addition of substances and improve the maturation rate of oocytes. The 90% maturation rate of oocytes during ovulation stimulation has not been reached, and there is also the risk of contamination by pathogens and foreign genes
[0004]The existence of the above reasons greatly limits the use and application of the advantages of IVM technology

Method used

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Effect test

Embodiment 1

[0027] Using MSCs as feeder cells for the culture of immature oocytes separated by stimulation

[0028] 8-10 weeks old female Kunming mice, intraperitoneal injection of 10IU PMSG, 48 hours later intraperitoneal injection of 10IU HCG, 16-20 hours later cut the oviduct, at 37 ℃, volume ratio of 5% CO 2 Equilibrate overnight in the BSS to pick eggs under a stereomicroscope. Oocytes below MII stage were incubated at 37°C in 5% CO by volume. 2 Under the conditions, a part was cultured with the second-generation MSCs, and a part was cultured with the conditioned medium for 40 minutes to obtain mature oocytes (shown in FIG. 2 ).

[0029] Experiments have shown that oocytes below the MII stage were cultured with second-generation MSCs or conditioned medium at 37°C with a volume ratio of 5% CO 2 The maturation rate reaches 95% after 40 minutes of culture under the condition. In order to avoid the mature oocytes from continuing to divide in MSC or conditioned medium, the mature oocyte...

Embodiment 2

[0032] Using MSCs as feeder cells for the culture of immature oocytes obtained by aspirating from ovarian tissue

[0033] Under aseptic conditions, remove both ovaries of 8-10 week-old female Kunming mice, and place them in 37°C, 5% CO by volume. 2 Equilibrate overnight in DMEM containing 10% fetal bovine serum by volume, and obtain free antral follicles and preantral follicles by aspirating with a 1ml 25G syringe needle under a stereomicroscope. 2 Under the conditions, a part was cultured with the second-generation MSCs, and a part was cultured with the conditioned medium for 24 hours to 7 days to obtain mature oocytes (as shown in FIG. 3 ).

[0034] Experiments have shown that, depending on the developmental stage of the follicles, in order to obtain mature oocytes, the culture time varies from 24 hours to 7 days. The maturation rate of mother cells is 93%; after being cultured with conditioned medium for 7 days, the maturation rate of oocytes is 85%. In order to prevent m...

Embodiment 3

[0038] Using MSCs as feeder cells for the cultivation of ovarian tissue blocks

[0039] Prepare agar scaffolds with a weight-to-volume ratio of 0.4%: second-generation MSCs in 1×105 Inoculate a 6-well plate at a cell density of / ml, pour off the culture medium after the cells adhere to the wall; prepare 0.4% agar with the conditioned culture medium, wait until it is cooled to 37°C-42°C, suck 3ml and spread it on the MSC, at 37°C, the volume ratio is 5 %CO 2 Equilibrated overnight for ovarian tissue block culture.

[0040] Under aseptic conditions, take out both ovaries of 8-10 weeks old female Kunming mice, place them in 4°C pre-cooled DMEM containing 10% fetal bovine serum by volume, and quickly make 1 -2mm action sheet, attached to 37°C, 5% CO by volume 2 On overnight MSC-containing agar with a weight-to-volume ratio of 0.4%, lightly press the tissue block to embed in the agar, and at the same time add HCG at 3 international units per milliliter; some ovarian tissue slices...

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Abstract

The present invention is feeder cell for extracorporeal culture of immature oocyte and features that marrow mesenchyme stem cell MSC of female mouse is used as the feeder cell. The feeder cell is easy to obtain, can secrete several kinds of cell factors and growth factors, promote over 90 % of the immature oocyte to become mature and promote the growth, ovulation and maturing of the ovarian follicles in the ovary tissue block under extracorporeal culture. The present invention is significant in surviving rear endangered species of animals, breeding fine variety of animal, cell biological research of embryo stem cell, etc.

Description

technical field [0001] The present invention relates to the fields of reproductive medicine and cell biology, more specifically relates to the feeder cells used in immature oocytes cultured and matured in vitro. Background technique [0002] Immature oocyte in vitro maturation (IVM) technology emerged with the development of artificial assisted reproductive technology (ART). IVM can shorten the reproductive cycle, maximize the use of good genetic material, and be used for artificial reproduction of rare or endangered animals. and the cultivation and commercial production of livestock breeds; it is convenient to study the development mechanism of oocytes, combined with in vitro fertilization or parthenogenetic activation technology to obtain a large number of embryonic stem cells for scientific research; IVM broadens the source of oocytes in the field of human reproduction and immature eggs Mother cells are easier to cryopreserve and help create an "egg bank" that provides re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12N5/0775
Inventor 冯定庆凌斌高婷周颖沈国栋朱园园姚凤球陈峥峥
Owner ANHUI PROVINCIAL HOSPITAL
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