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Method for in vitro culture of ovarian follicles

A technology of in vitro culture and follicles, applied in the culture process, tissue culture, cell culture active agent, etc., can solve the problem of impossible evaluation and so on

Inactive Publication Date: 2004-03-10
艾格森特瑞斯有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing techniques cannot detect any one of the discrete stages of folliculogenesis, and it is generally not possible to assess the effect of external influences on follicle cells at a particular stage of development, as follicle cultures usually try to mimic the natural conditions of different stages of follicular development

Method used

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  • Method for in vitro culture of ovarian follicles
  • Method for in vitro culture of ovarian follicles
  • Method for in vitro culture of ovarian follicles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Isolation and preculture of secondary follicles ( figure 1 Step 1) in:

[0041] Ovarian tissue was isolated from 12- to 14-day-old F1 generation C57B1 / j6XCBAca hybrid mice and placed in GlutaMAX I TM Petri dishes of Leibovitz's L15 medium (Gibco BRL 31415-029) containing 10% HIA FBS (heat-inactivated fetal bovine serum) and 0.1% penicillin-streptomycin mixture (Gibco BRL 3032908). The ovaries remove the fallopian tubes and fat.

[0042] The surface of the ovary is scratched with a needle and the follicles are released in suspension in L15 medium. Under normal conditions, 30 to 40 suitable follicles can be obtained from each ovary by this isolation method.

[0043] To isolate primary follicles, use 7- to 8-day-old mice. In this case 10 to 15 suitable follicles can be obtained from each ovary.

Embodiment 2

[0045] The method of the present invention selects follicles ( figure 1 Step 2) in:

Embodiment 3

[0049] The establishment and application of follicular storage ( figure 1 Step 3) in:

[0050] Follicles can be stored using cryopreservation techniques known to those skilled in the art. The next paragraph describes an embodiment of this technique.

[0051] The follicles obtained in Example 2 were collected into plastic cryovials (Simport, Quebec, Canada). Twenty-five follicles per vial were suspended in 150 microliters of L15 medium supplemented with 10% heat-inactivated FCS and 1.5M DMSO. Slow freezing was performed using a programmable freezer (Cell Freezer R204, Planer, Sunbury-on-Thames, UK) that could be controlled. Follicles were equilibrated in the 4°C freezing mixture for 15 min and then cooled to -7°C at a rate of 2°C / min. After manual inoculation, the temperature was lowered to -40°C at a rate of -0.3°C / min. Follicles were rapidly cooled to -110°C at a rate of -50°C / min before storage in liquid nitrogen.

[0052] Follicles were ultra-rapidly thawed by heating...

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Abstract

The present invention is related to a method for in vitro culture of mammalian ovarian follicles for bioassay purposes, comprising the following subsequent steps: - providing a suitable container for the in vitro culture, - selecting a follicle from an ovary of a mammal, said follicle comprising at least a theca or pre-theca cell, a granulosa cell and an oocyte, - an optional first cultur e step using an attachment prohibiting first medium free from oil, - a second culture step using an attachment promoting second medium free from oil arranged to, and - the retrieval of the matured follicle.

Description

field of invention [0001] The invention relates to a method for culturing follicles in vitro. More specifically, the present invention relates to methods for producing large numbers of mature or semi-mature follicles and / or oocytes from mammalian ovarian tissue. Background technique [0002] Several problems have been identified in the field of studying folliculogenesis and oocyte maturation in humans and non-human mammals. [0003] With the widespread acceptance and use of oocyte maturation for animal reproduction and for in vitro fertilization in humans, large quantities of spermatozoa are usually collected and banked for future use, allowing an essentially unlimited supply of spermatozoa. However, until now there has been no practical way to collect and store large quantities of fertilizable eggs from females. The reason for this involves the reproductive biology of female animals. In female mammals, only certain cells in the ovary mature into eggs. These germ cells, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02A61D19/04C12N5/07C12N5/075G01N33/50
CPCC12N2500/25C12N2502/243C12N5/0609C12N2517/10C12N2501/11C12N2501/31
Inventor R·库特夫林特J·史密兹
Owner 艾格森特瑞斯有限公司
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