CN110669719A - In-vitro follicle culture method - Google Patents
In-vitro follicle culture method Download PDFInfo
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- CN110669719A CN110669719A CN201810727872.6A CN201810727872A CN110669719A CN 110669719 A CN110669719 A CN 110669719A CN 201810727872 A CN201810727872 A CN 201810727872A CN 110669719 A CN110669719 A CN 110669719A
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- 108090001061 Insulin Proteins 0.000 claims description 3
- 229940125396 insulin Drugs 0.000 claims description 3
- 238000002955 isolation Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 2
- 241000699670 Mus sp. Species 0.000 claims description 2
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- 239000013028 medium composition Substances 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 229960005322 streptomycin Drugs 0.000 claims description 2
- 238000012258 culturing Methods 0.000 abstract description 5
- 241000124008 Mammalia Species 0.000 abstract description 4
- 210000002503 granulosa cell Anatomy 0.000 abstract 1
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- 229920006395 saturated elastomer Polymers 0.000 description 1
- 229940082569 selenite Drugs 0.000 description 1
- MCAHWIHFGHIESP-UHFFFAOYSA-L selenite(2-) Chemical compound [O-][Se]([O-])=O MCAHWIHFGHIESP-UHFFFAOYSA-L 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
Abstract
The invention relates to a method for culturing in vitro follicles of mammals, which comprises the following continuous operations: selecting a follicle in a suitable diameter range from the ovary of a mammal, said follicle comprising at least the outer layer of theca cells, the middle layer of ovarian granulosa cells, and the inner layer of oocytes; culturing the follicle meeting the requirements in a 96-well plate; the first phase of culture, i.e., the first three days, uses the first medium, and the second phase of culture, i.e., the fourth day, uses the second medium. Mature follicles can be obtained finally by culturing.
Description
Technical Field
The invention relates to a method for culturing in vitro follicles of mammals, which lasts for 4 days, and after a culture period, the follicles cultured in vitro can successfully discharge cumulus-oocyte complexes (COCs).
Background
The number of follicles gradually decreases with age. 70-200 ten thousand primordial follicles are in the bilateral ovarian cortex of the newborn, and the number of primordial follicles is reduced to about 30 ten thousand in the age of 7-9, about 4 ten thousand in the beginning of puberty, and only hundreds of primordial follicles remain in the period of menopause. During the whole growth period (30-40 years) from puberty to menopause, the ovary generally has 15-20 follicles growing and developing every 28 days or so under the influence of gonadotropins secreted by the pituitary cycle, but usually only 1 follicle is mature and ovulates. In one lifetime, ovaries can ovulate a total of 400-. To obtain more mature follicles, we established a culture model of follicles.
Disclosure of Invention
The invention overcomes the defect of longer follicle culture period in the traditional method and establishes a short-term in-vitro follicle culture model.
In order to achieve the above object, the method of the present invention mechanically isolates the mammalian ovarian follicles in accordance with the requirements, cultures the follicles in vitro for a total of 4 days, and induces ovulation by changing the culture medium on the 3 rd day of culture.
The method of the invention is operated according to the following steps (all operating in a sterile super clean bench): step one, opening the abdomen, taking out a tissue part containing the ovary and the oviduct under the kidney, and placing the tissue part in a sterilized PBS buffer solution; secondly, removing other parts except ovaries by using micro forceps under a microscope, and transferring the ovarian tissues to another culture dish filled with the alpha MEM culture medium; in the third step, the appropriate follicles in the ovaries were mechanically isolated under a microscope and transferred to a 96-well plate previously filled with medium in an amount of 200. mu.l per well. Approximately 20-30 follicles per ovary can be isolated for subsequent culture; and fourthly, replacing the culture medium on the third day of culture, inducing ovulation and observing the discharge condition of the COCs.
Drawings
FIG. 1 is a schematic representation of the procedures involved in the present invention from the isolation of follicles from a mammal to the development of maturity and ovulation.
Fig. 2 is an initial isolated follicle from a mammalian ovary.
FIG. 3 shows the luminal follicles generated during the culture. There is a single large vacuolar antral follicle and there is a prominent cumulus structure in the follicle.
FIG. 4 shows the ovarian follicles after ovulation induction, with the discharged cumulus-oocyte complexes (COCs) visible.
Detailed Description
Examples
An in vitro mammalian follicle culture method is characterized in that the culture method comprises the following specific steps:
1) isolation of ovarian follicles ovarian tissue was isolated from 35 day old ICR mice and placed in a Petri dish containing PBS (phosphate buffer saline) containing a 0.1% penicillin-streptomycin mixture. Ovarian follicles in the ovaries were isolated with an insulin needle, and generally 20 to 30 qualifying follicles were isolated per ovary.
2) Appropriate mouse follicles were isolated from the ovaries and placed in 96-well plates previously filled with medium for four days. The first three days of culture were with the first medium, the third day with the medium changed, and the second medium was used. The medium composition is shown in Table 1.
Preferably, the size of the follicles isolated in step 1) ranges from 200-.
Preferably, the specific method of step 2) is: a first medium was prepared by adding 5% fetal bovine serum FBS to a minimal medium α MEM, and adding follicle stimulating hormone FSH at a concentration of 5IU/ml and 1 XTS [1.0mg/ml recombinanthuman insulin, 0.55mg/ml human transferrin (substential iron-free), 0.5. mu.g/ml of plasmodium selenite](ii) a The obtained follicles were washed in sterilized PBS and transferred to a 96-well plate containing the first medium at 37 ℃ with 5% CO2Culturing in a carbon dioxide incubator with saturated humidity; preparing a second culture medium, and adding 1.5IU/mL HCG and 5ng/mL EGF on the basis of the first culture medium; on the third day of culture, the first medium in the 96-well plate was changed to the second medium; on the fourth day of culture, COCs excretion was observed.
Preferably, FSH is added to the first medium at a concentration of 5 IU/ml.
Preferably, EGF is added to the second medium at a concentration of 5 ng/ml.
Specific media functions and compositions are shown in the following table:
FBS ═ fetal bovine serum, ITS ═ insulin-transferrin-sodium selenite, FSH ═ follicle stimulating hormone, HCG ═ human chorionic gonadotropin, EGF ═ epidermal growth factor, α MEM may be replaced with any suitable basal cell culture medium, such as Waymouth medium.
The invention has been tested and implemented for many times, the result of the test is successful, achieve the goal of the invention.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (3)
1. An in vitro mammalian follicle culture method is characterized in that the culture method comprises the following specific steps:
1) isolation of ovarian follicles ovarian tissue was isolated from 35 day old ICR mice and placed in a Petri dish containing PBS (phosphatebuffer saline) containing a 0.1% penicillin-streptomycin mixture. Ovarian follicles in the ovaries were isolated with an insulin needle, and generally 20 to 30 qualifying follicles were isolated per ovary.
2) Appropriate mouse follicles were isolated from the ovaries and placed in 96-well plates previously filled with medium for four days. The first three days of culture were with the first medium, the third day with the medium changed, and the second medium was used. The medium composition is shown in Table 1.
2. The method according to claim 1, wherein the diameter of the isolated follicle in step 1) is in the range of 200-400 μm.
3. The culture method according to claim 1 or 2, wherein the FSH is added to the first medium in step 2) at a concentration of 5IU/ml and the EGF is added to the second medium at a concentration of 5 ng/ml.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810727872.6A CN110669719A (en) | 2018-07-02 | 2018-07-02 | In-vitro follicle culture method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810727872.6A CN110669719A (en) | 2018-07-02 | 2018-07-02 | In-vitro follicle culture method |
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| CN110669719A true CN110669719A (en) | 2020-01-10 |
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| CN201810727872.6A Pending CN110669719A (en) | 2018-07-02 | 2018-07-02 | In-vitro follicle culture method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112980773A (en) * | 2021-03-15 | 2021-06-18 | 中山大学附属第六医院 | Method for culturing mouse secondary follicle in vitro |
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|---|---|---|---|---|
| CN100999723A (en) * | 2007-01-08 | 2007-07-18 | 安徽省立医院 | In-vitro cultivating matural process of immatural ovocyte through promoting discharging separating or sucting obtained in ovarium organized |
| CN101180399A (en) * | 2005-01-26 | 2008-05-14 | 莱顿大学 | Means and methods for improving the development and maturation of eggs and/or sperm in fish using hormones produced by transplanted cells |
| WO2012115885A1 (en) * | 2011-02-22 | 2012-08-30 | Caris Life Sciences Luxembourg Holdings, S.A.R.L. | Circulating biomarkers |
| US20140079836A1 (en) * | 2008-12-01 | 2014-03-20 | LifeSpan Extension, LLC | Methods and compositions for altering health, wellbeing, and lifespan |
| CN107475178A (en) * | 2016-06-08 | 2017-12-15 | 江苏齐氏生物科技有限公司 | A kind of mouse granulosa cells separation and cultural method |
-
2018
- 2018-07-02 CN CN201810727872.6A patent/CN110669719A/en active Pending
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|---|---|---|---|---|
| CN101180399A (en) * | 2005-01-26 | 2008-05-14 | 莱顿大学 | Means and methods for improving the development and maturation of eggs and/or sperm in fish using hormones produced by transplanted cells |
| CN100999723A (en) * | 2007-01-08 | 2007-07-18 | 安徽省立医院 | In-vitro cultivating matural process of immatural ovocyte through promoting discharging separating or sucting obtained in ovarium organized |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112980773A (en) * | 2021-03-15 | 2021-06-18 | 中山大学附属第六医院 | Method for culturing mouse secondary follicle in vitro |
| CN112980773B (en) * | 2021-03-15 | 2023-11-17 | 中山大学附属第六医院 | Method for in vitro culture of mouse secondary follicles |
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Application publication date: 20200110 |