CN108949670A - A kind of preparation method of oocyte in vitro maturation culture solution - Google Patents
A kind of preparation method of oocyte in vitro maturation culture solution Download PDFInfo
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- CN108949670A CN108949670A CN201810934929.XA CN201810934929A CN108949670A CN 108949670 A CN108949670 A CN 108949670A CN 201810934929 A CN201810934929 A CN 201810934929A CN 108949670 A CN108949670 A CN 108949670A
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- 238000000338 in vitro Methods 0.000 title claims abstract description 33
- 230000035800 maturation Effects 0.000 title claims abstract description 30
- 210000000287 oocyte Anatomy 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 19
- 239000001963 growth medium Substances 0.000 claims abstract description 23
- 102000004895 Lipoproteins Human genes 0.000 claims abstract description 15
- 108090001030 Lipoproteins Proteins 0.000 claims abstract description 15
- 239000003104 tissue culture media Substances 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 6
- 210000000130 stem cell Anatomy 0.000 claims description 6
- 239000012930 cell culture fluid Substances 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 238000012549 training Methods 0.000 claims description 3
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 claims 2
- 229960005309 estradiol Drugs 0.000 claims 1
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 11
- 230000004913 activation Effects 0.000 abstract description 6
- 238000011161 development Methods 0.000 abstract description 5
- 230000018109 developmental process Effects 0.000 abstract description 5
- 230000013020 embryo development Effects 0.000 abstract description 4
- 230000008182 oocyte development Effects 0.000 abstract description 4
- 230000005058 diapause Effects 0.000 abstract description 3
- 230000035899 viability Effects 0.000 abstract description 2
- 102000002322 Egg Proteins Human genes 0.000 description 5
- 108010000912 Egg Proteins Proteins 0.000 description 5
- 210000004681 ovum Anatomy 0.000 description 5
- 238000000034 method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 3
- 230000001850 reproductive effect Effects 0.000 description 3
- 150000000307 17β-estradiols Chemical class 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000002459 blastocyst Anatomy 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010033266 Ovarian Hyperstimulation Syndrome Diseases 0.000 description 1
- GBOGMAARMMDZGR-UHFFFAOYSA-N UNPD149280 Natural products N1C(=O)C23OC(=O)C=CC(O)CCCC(C)CC=CC3C(O)C(=C)C(C)C2C1CC1=CC=CC=C1 GBOGMAARMMDZGR-UHFFFAOYSA-N 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 229960004407 chorionic gonadotrophin Drugs 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- GBOGMAARMMDZGR-JREHFAHYSA-N cytochalasin B Natural products C[C@H]1CCC[C@@H](O)C=CC(=O)O[C@@]23[C@H](C=CC1)[C@H](O)C(=C)[C@@H](C)[C@@H]2[C@H](Cc4ccccc4)NC3=O GBOGMAARMMDZGR-JREHFAHYSA-N 0.000 description 1
- GBOGMAARMMDZGR-TYHYBEHESA-N cytochalasin B Chemical compound C([C@H]1[C@@H]2[C@@H](C([C@@H](O)[C@@H]3/C=C/C[C@H](C)CCC[C@@H](O)/C=C/C(=O)O[C@@]23C(=O)N1)=C)C)C1=CC=CC=C1 GBOGMAARMMDZGR-TYHYBEHESA-N 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 210000002394 ovarian follicle Anatomy 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
Classifications
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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
- C12N5/0608—Germ cells
- C12N5/0609—Oocytes, oogonia
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/35—Polyols, e.g. glycerin, inositol
-
- 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
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- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Developmental Biology & Embryology (AREA)
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- General Health & Medical Sciences (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a kind of preparation methods of oocyte in vitro maturation culture solution, it is characterized in that: steps are as follows for the preparation method: step 1) prepares basis IVM culture medium；Step 2) adds in basic IVM culture medium lipoprotein to get oocyte in vitro maturation culture solution.Present invention improves Oocyte Development potentiality after IVM are poor, the problem of Embryo viability is relatively low after lonely female activation, embryo can be made to continue development to blastaea after addition lipoprotein, the IVM culture solution for adding lipoprotein improves embryonic development potential, effectively improves embryo diapause problem.
Description
Technical field
The present invention relates to a kind of preparation method of culture solution, especially a kind of preparation of oocyte in vitro maturation culture solution
Method.Belong to medical domain.
Background technique
The maturation in vitro (in vitro maturation, IVM) of egg mother cell refers to the ovum of GV phase or M I phase is female thin
Born of the same parents cultivate the process that the second meiotic division (M II phase) is arrived in development in vitro.IVM can be used as a technology, in conjunction with
(IVF) technology in vitro fertilization can be applied effectively in reproductive medicine field, and infertility is treated.
Oocyte in vitro maturation technology can not only improve egg mother cell benefit as assisted reproductive technology important component
With efficiency, the generation of ovarian hyperstimulation syndrome is reduced, also provides more more options to give ovum and female fertility preservation.Closely
Nian Lai, with the fast development of reproductive medicine, oocyte in vitro maturation technical research and clinical application are developed rapidly,
But still face the problems such as maturing rate is low, rate of fertilization is low, Embryo quality is low and pregnancy rate is low.
Summary of the invention
The purpose of the present invention is to provide a kind of preparation method of oocyte in vitro maturation culture solution, present invention improves
The problem of Oocyte Development potentiality are poor after IVM, and Embryo viability is relatively low after lonely female activation.
Realizing the technical solution of above-mentioned purpose is: a kind of preparation method of oocyte in vitro maturation culture solution, feature
Be: steps are as follows for the preparation method:
Step 1) prepares basis IVM culture medium；
Step 2) adds in basic IVM culture medium lipoprotein to get oocyte in vitro maturation culture solution.
The step 1) prepares basis IVM culture medium:
Step a) uses the tissue culture medium TCM199 of commercialization for basic cell culture fluid；
0.075-0.10IU/ml FSH and 0.5-0.8IU/ml HCG, 0.1-0.2 the μ g/ that step b) will be commercialized
The tissue cultures that alternative serum is added to commercialization are commercialized in 17 beta estradiols of ml and the people of 10%-20% percent by volume
In liquid TCM199；
Step c) is filtered the tissue culture medium TCM199 of the commercialization after the addition of step b)；
It saves after step d) filtering to get basic IVM culture medium.
Filtering in the step c), is filtered by filter, the filter that the filter is 0.22 μm.
It is to save in 4 DEG C of environment that preservation in the step d), which is in temperature,.
It is used in the basis step d) IVM culture medium 2 weeks.
The step 2) adds 20ug/m lipoprotein in basic IVM culture medium.
Step 2) the oocyte in vitro maturation culture solution is before cultivating egg mother cell, In vitro maturation
Liquid is placed in 37 DEG C of incubators, is balanced 12 hours or more under the conditions of 5% gas concentration lwevel.
The invention has the advantages that embryo sends out after lonely female activation present invention improves Oocyte Development potentiality after IVM are poor
The problem of ability of educating is relatively low can make embryo continue development to blastaea, add the IVM training of lipoprotein after adding lipoprotein
Nutrient solution improves embryonic development potential, effectively improves embryo diapause problem.This method can make Oocyte in Vitro at
Ripe rate has reached 68.78%, and Blastocyst formation rate has reached 16.33%.
The present invention is described in further details below by specific embodiment, but not as a limitation of the invention.
Specific embodiment
Embodiment 1
A kind of preparation method of oocyte in vitro maturation culture solution, the specific steps are as follows:
Step 1) prepares basis IVM culture medium；
Step 2) adds in basic IVM culture medium lipoprotein to get oocyte in vitro maturation culture solution.
Embodiment 2
A kind of preparation method of oocyte in vitro maturation culture solution, the specific steps are as follows:
Step 1) prepares basis IVM culture medium；
Step a) uses the tissue culture medium TCM199 of commercialization for basic cell culture fluid；
0.075-0.10IU/ml FSH and 0.5-0.8IU/ml HCG, 0.1-0.2 the μ g/ that step b) will be commercialized
The tissue cultures that alternative serum is added to commercialization are commercialized in 17 beta estradiols of ml and the people of 10%-20% percent by volume
In liquid TCM199；
Step c) carries out the tissue culture medium TCM199 of the commercialization after the addition of step b) with 0.22 μm of filter
Filtering；
It is that cryo-conservation is carried out in 4 DEG C of environment to get basic IVM culture medium in temperature after step d) filtering.Described
It is used in basic IVM culture medium 2 weeks.
Step 2) adds in basic IVM culture medium 20ug/ml lipoprotein to get oocyte in vitro maturation culture solution.
The oocyte in vitro maturation culture solution before cultivating egg mother cell, put by oocyte in vitro maturation culture solution
It is placed in 37 DEG C of incubators, is balanced 12 hours or more under the conditions of 5% gas concentration lwevel.
Wherein FSH is follicular stimulating hormone, is a kind of hormone of anterior pituitary basophil cell secretion, ingredient is glycoprotein；
HCG refers to human chorionic gonadotrophin, is made of the glycoprotein of α and β dimer.
Embodiment 3
The embodiment is the experimental example done for embodiment 2, and experimentation is as follows:
6 week old~8 week old female mice is chosen, the PMSG that 10IU is injected intraperitoneally carries out superfecundation processing, after 46-48h
Disconnected neck puts to death mouse, takes out ovary and is put into M2 culture solution, crushes ovarian follicle with needle under stereomicroscope, and it is female thin to select GV phase ovum
Born of the same parents are cultivated, control group by following grouping: basic IVM culture medium；Experimental group: 20ug/ml is added in basic IVM culture medium
Lipoprotein in each culture drop plus 10-15 egg mother cell, is cultivated in above-mentioned condition, culture statistics II incidence of M afterwards for 24 hours,
Select II ovum of M be put into balanced in advance containing 20mMSrCl2Ksom in 1h carry out lonely female activation, with 37 DEG C, 5%CO2Condition
The lower KSOM culture solution for pre-equilibrating 4h or more is washed 5 times, then ovum is moved into the pre-equilibration for containing 5 μ g/ml Cytochalasin B
KSOM culture solution, 37 DEG C, 5%CO2Under the conditions of cultivate 4h, with 37 DEG C, 5%CO2Under the conditions of pre-equilibrate 4h or more KSOM training
Nutrient solution is washed 5 times, is put into the ksom culture for pre-equilibrating 4h or more later, cleavage rates is observed after 18h, activation the same day note D0 days, D5 worked as
Its observation Blastocyst formation rate.It is shown in Table 1: table 1
As shown in table 1, present invention improves Oocyte Development potentiality after IVM are poor, embryonic development energy after lonely female activation
The problem of power is relatively low can make embryo continue base of the development to blastaea, without adding lipoprotein after adding lipoprotein
Basal culture medium cannot allow embryo to continue to develop, and show that the IVM culture solution for adding lipoprotein improves embryonic development potential, effectively
Improve embryo diapause problem.
The common knowledge of part and the english abbreviation category industry that the present embodiment does not describe in detail, is not chatted one by one here
It states.
Claims (7)
1. a kind of preparation method of oocyte in vitro maturation culture solution, it is characterized in that: steps are as follows for the preparation method:
Step 1) prepares basis IVM culture medium；
Step 2) adds in basic IVM culture medium lipoprotein to get oocyte in vitro maturation culture solution.
2. the preparation method of a kind of oocyte in vitro maturation culture solution according to claim 1, it is characterized in that: the step
It is rapid 1) to prepare basis IVM culture medium:
Step a) uses the tissue culture medium TCM199 of commercialization for basic cell culture fluid；
The 0.075-0.10IU/ml FSH and 0.5-0.8IU/mlHCG that step b) will be commercialized, the 17 of 0.1-0.2 μ g/ml
The tissue culture medium that alternative serum is added to commercialization is commercialized in the people of beta estradiol and 10%-20% percent by volume
In TCM199；
Step c) is filtered the tissue culture medium TCM199 of the commercialization after the addition of step b)；
It saves after step d) filtering to get basic IVM culture medium.
3. the preparation method of a kind of oocyte in vitro maturation culture solution according to claim 2, it is characterized in that: the step
It is rapid c) in filtering, be filtered by filter, the filter is 0.22 μm of filter.
4. the preparation method of a kind of oocyte in vitro maturation culture solution according to claim 2, it is characterized in that: the step
It is rapid d) in preservation be temperature be 4 DEG C of environment in save.
5. the preparation method of a kind of oocyte in vitro maturation culture solution according to claim 2, it is characterized in that: the step
It is used in rapid d) basis IVM culture medium 2 weeks.
6. the preparation method of a kind of oocyte in vitro maturation culture solution according to claim 1 or 2, it is characterized in that: institute
It states step 2) and adds 20ug/ml lipoprotein in basic IVM culture medium.
7. the preparation method of a kind of oocyte in vitro maturation culture solution according to claim 1, it is characterized in that: the step
For rapid 2) oocyte in vitro maturation culture solution before cultivating egg mother cell, oocyte in vitro maturation culture solution is placed in 37 DEG C of trainings
It supports in case, is balanced 12 hours or more under the conditions of 5% gas concentration lwevel.
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| CN201810934929.XA CN108949670A (en) | 2018-08-16 | 2018-08-16 | A kind of preparation method of oocyte in vitro maturation culture solution |
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| CN201810934929.XA CN108949670A (en) | 2018-08-16 | 2018-08-16 | A kind of preparation method of oocyte in vitro maturation culture solution |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628385A (en) * | 2019-01-18 | 2019-04-16 | 周桦 | A kind of human oocyte In-vitro maturation liquid and preparation method thereof and cultural method |
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|---|---|---|---|---|
| CN1620499A (en) * | 2001-12-21 | 2005-05-25 | 哥本哈根大学医院 | Igamete recruitment and developmental competence in mammals by inhibiting the de novo sterol biosynthesis and/or promoting sterol efflux |
| CN103275930A (en) * | 2013-06-20 | 2013-09-04 | 江苏皓康生物医药科技有限公司 | Oocyte in-vitro maturity nutrient solution and preparation method thereof |
| CN104312971A (en) * | 2014-11-04 | 2015-01-28 | 广西大学 | Method for promoting in-vitro buffalo oocyte maturation |
| CN105624100A (en) * | 2016-04-06 | 2016-06-01 | 云南中科胚胎工程生物技术有限公司 | Buffalo oocyte in-vitro maturation (IVM) culture method |
-
2018
- 2018-08-16 CN CN201810934929.XA patent/CN108949670A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1620499A (en) * | 2001-12-21 | 2005-05-25 | 哥本哈根大学医院 | Igamete recruitment and developmental competence in mammals by inhibiting the de novo sterol biosynthesis and/or promoting sterol efflux |
| CN103275930A (en) * | 2013-06-20 | 2013-09-04 | 江苏皓康生物医药科技有限公司 | Oocyte in-vitro maturity nutrient solution and preparation method thereof |
| CN104312971A (en) * | 2014-11-04 | 2015-01-28 | 广西大学 | Method for promoting in-vitro buffalo oocyte maturation |
| CN105624100A (en) * | 2016-04-06 | 2016-06-01 | 云南中科胚胎工程生物技术有限公司 | Buffalo oocyte in-vitro maturation (IVM) culture method |
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| Title |
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| KYLIE R DUNNING ET AL.: ""Lipids and oocyte developmental competence: the role of fatty acids and β-oxidation"", 《REPRODUCTION》 * |
| TIFFANY VON WALD, M.D. ET AL.: ""Age-related variations in follicular apolipoproteins may influence human oocyte maturation and fertility potential"", 《FERTILITY AND STERILITY》 * |
| 张晓鹰 等: ""辽宁绒山羊羔羊卵母细胞体外培养技术的研究"", 《现代畜牧兽医》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109628385A (en) * | 2019-01-18 | 2019-04-16 | 周桦 | A kind of human oocyte In-vitro maturation liquid and preparation method thereof and cultural method |
| CN109628385B (en) * | 2019-01-18 | 2022-12-23 | 周桦 | Human oocyte in-vitro maturation culture solution and preparation method and culture method thereof |
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