CN104531612A - Oocyte culture fluid and culture method thereof - Google Patents
Oocyte culture fluid and culture method thereof Download PDFInfo
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- CN104531612A CN104531612A CN201510020208.4A CN201510020208A CN104531612A CN 104531612 A CN104531612 A CN 104531612A CN 201510020208 A CN201510020208 A CN 201510020208A CN 104531612 A CN104531612 A CN 104531612A
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- ovocyte
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Abstract
The invention provides oocyte culture fluid and a culture method of the oocyte culture fluid. The oocyte culture fluid is easy to prepare and low in cost and comprises G-IVF culture fluid, follicular fluid and granular cells. According to the oocyte culture fluid and the culture method of the oocyte culture fluid, the follicular fluid extracted from the follicle of a patient and granular cells around the oocyte are treated specially and added into the culture fluid, so that in-vitro maturity culturing is carried out on the immature egg of the remaining ICSI period, then fertilization, embryonic development and pregnancy results are analyzed, a simple, convenient and effective in-vitro maturity culture scheme is set up, the effective utilization rate of the immature egg is increased, and more pregnancy opportunities can be provided for the infertility patient.
Description
Technical field
The present invention relates to a kind of cell culture fluid and cultural method thereof, especially a kind of ovocyte nutrient solution and cultural method thereof.
Background technology
All the time, " Infertility " is that part is ashamed to mention, compares the thing of privacy in the subconsciousness of people.Along with the development of auxiliary procreation technology, people progressively understand IVF-ET Technique, and increasing patient Mr. and Mrs start pay close attention to and implement this technology.IVF-ET Technique refers to and take out ovum in the ovary on the wife's side, allows them be combined with the sperm on the bridegroom's or husband's side in the incubator in laboratory, forms body early embryo, then it is implanted into the intrauterine a kind of auxiliary procreation technology in the wife's side.In recent years, in vitro maturation cultivates the focus that (in vitro maturation, IVM) becomes field of reproduction research.IVM is the one of auxiliary procreation technology, it is the ripe environment by ovocyte in analogue body, in vitro immature oocyte is cultivated, make it grow the ovocyte of the MII (M II) for maturation, there is fertilization, growth is the ability of fetal tissues.In the ICSI cycle of the conventional ovulation treatment of application, first the ovocyte taken out will remove the granulosa cell around ovocyte, M II ovocyte is selected to carry out ICSI, remaining immature oocyte is dropped because using usually, if this part immature egg is carried out IVM, the quantity of portable embryo will be increased, improve accumulation pregnancy rate, especially the less patient of ovum is obtained to some, advantageously in helping pregnant final result.
The composition of IVM nutrient solution is most important, and early stage IVM nutrient solution generally comprises TC M199, ox chorionic-gonadotropin hormone (PMSG), foetal calf serum, pregnant mare serum gonadotrop(h)in (PMSG).Existing company have developed the nutrient solution system of people's in vitro maturation in the world at present, and in commercialization in 2005, but still do not break away from costly, the defects such as low, unstable, the difficult popularization of success ratio.
Summary of the invention
The invention provides and a kind ofly prepare easy, that cost is low ovocyte nutrient solution and cultural method thereof.
Realize the ovocyte nutrient solution of one of the object of the invention, comprise G-IVF nutrient solution, liquor folliculi and granulosa cell.
The volume ratio of described G-IVF nutrient solution and liquor folliculi is 1:1.
Containing the HAS (human serum albumin) of 10% volume ratio in described G-IVF nutrient solution.
Realize the ovocyte cultural method of the object of the invention two, comprise the steps:
(1) preparation of human mature follicle fluid: the chief liquor folliculi having picked up corolla mound mixture is collected in conical centrifuge tube, red corpuscle in centrifugal segregation liquor folliculi and impurity, get supernatant liquor metre filter, be put in incubator inner equilibrium in small test tube and preserve;
(2) preparation of granulosa cell: before use Unidasa removes ovocyte neighboring particles cell, first cuts the most of granulosa cell around ovocyte under the microscope with two 1ml syringes and to be put in the 3037 culture dish middle cover oil that G-IVF nutrient solution is housed for subsequent use;
(3) remainder particulate cell is divested: the ovocyte containing a small amount of granulosa cell is moved to pressure-vaccum several in Unidasa, then use the special pipe of the Bath of different diameter clean for Denudation of oocytes remaining around ovocyte, take out M II phase ovocyte, remaining M I phase and GV phase ovocyte are put in the culture dish containing granulosa cell;
(4) liquor folliculi is added: the liquor folliculi that absorption is handled well adds in the culture dish containing immature oocyte to be cultivated.
The volume ratio of described G-IVF nutrient solution and liquor folliculi is 1:1.
Containing the HAS (human serum albumin) of 10% volume ratio in described G-IVF nutrient solution.
In described step (1), remove red corpuscle in liquor folliculi and impurity by having picked up corolla mound mixture at the centrifugal 10min of 3000r/min.
For several times, the time is 30S in described step (3), the ovocyte containing a small amount of granulosa cell to be moved to pressure-vaccum in Unidasa.
In described step (4), the culture dish containing immature oocyte is put in 6.0% CO2gas incubator, 37 DEG C of cultivations.
The beneficial effect of ovocyte nutrient solution of the present invention and cultural method thereof is as follows:
1, ovocyte nutrient solution of the present invention and cultural method thereof, adds mature follicle fluid in nutrient solution and granulosa cell is simple and convenient, and general reproductive center laboratory all possesses correlated condition.
2, the nutrient solution adding mature follicle fluid and granulosa cell use in nutrient solution is that common G-IVF is subject to seminal fluid, does not need to buy commercial immature egg nutrient solution separately, avoids the waste of reagent, for patient reduces cost.
3, add mature follicle fluid and granulosa cell in nutrient solution to re-use the liquor folliculi of patient and granulosa cell, the two is containing multiple somatomedin (GF), gonad-stimulating hormone and the steroid hormone etc. needed for Growth of Oocytes growth, be conducive to the further maturation of immature oocyte, impel the synchronization of kytoplasm and Nuclear maturity.
4, add mature follicle fluid in nutrient solution and granulosa cell can re-use the immature egg collected in conventional ICSI or the IVF cycle, avoid the waste of immature oocyte, also provide chance for the freezing of patient's ovum simultaneously.
5, add mature follicle fluid and granulosa cell in nutrient solution and can significantly improve the maturing rate and stability that immature egg cultivates, further increase the spilting of an egg of embryo and the chance of Blastocyst formation, more pregnant chance is provided to infertile patient.
6, add the quantity that mature follicle fluid and granulosa cell can increase patient's portable embryo in nutrient solution, especially ovum is obtained to some less and have the patient of immature oocyte, its psychological burden can be alleviated, be conducive to pregnancy outcome.
Embodiment
Ovocyte cultural method of the present invention, comprises the steps:
Patient selection: accept in Shijiazhuang City the 4th Reproductive Medicine Center of hospital 138 patients that ICSI helps pregnant treatment during selecting in November ,-2013 in October, 2012, the mean age (31.56 ± 3.29) year, obtain ovum sum <15 piece.
1, the preparation of human mature follicle fluid: the chief liquor folliculi having picked up corolla mound mixture is collected in conical centrifuge tube, red corpuscle in 3000r/min centrifugal 10min removal liquor folliculi and impurity, get supernatant liquor 2ml metre filter, be put in incubator inner equilibrium in 5ml small test tube and preserve.
2, the preparation of granulosa cell: cut the most of granulosa cell around ovocyte under the microscope with two 1ml syringes and to be put in 3037 ware middle cover oil of the G-IVF nutrient solution of the HSA be equipped with containing 10% volume ratio for subsequent use;
3, remainder particulate cell is divested: the ovocyte containing a small amount of granulosa cell is moved to pressure-vaccum several in Unidasa, time is that about 30S is advisable, then use the special pipe of the Bath of different diameter clean for Denudation of oocytes remaining around ovocyte, take out M II phase ovocyte and carry out ICSI, leave and take remaining M I phase and GV phase ovocyte for subsequent use.
4, liquor folliculi is added: the liquor folliculi that absorption 0.6ml handles well adds in 3037 culture dish containing immature oocyte, is put in by the culture dish containing immature oocyte in 6.0% CO2gas incubator, 37 DEG C of cultivations.
Experimental data: get following nutrient solution and test
A group: containing the G-IVF nutrient solution of 10%HSA,
B group: containing G-IVF nutrient solution and the liquor folliculi of 10%HSA, volume ratio is 1:1,
C group: containing the G-IVF nutrient solution+granulosa cell of 10%HSA,
D group: B group+granulosa cell.
Immature ovocyte is put into respectively 3037 culture dish containing above-mentioned nutrient solution, and culture dish is put in 6.0% CO2gas incubator, 37 DEG C of cultivations.After 24 hours, observe the developmental state of immature oocyte, to the capable ICSI of ovocyte of growing for ripe M II phase, then be put in the droplet that G-1 cleavage stage nutrient solution makes and cultivate in incubator, 22 hours observe fertilization situation and record, the spilting of an egg situation of 48 hours observed and recorded embryos, draws following data:
The 24h maturing rate of D group, Embryo quality, available embryo lead all higher than other three groups, all have statistical significance.
Explanation of nouns:
Granulosa cell: be divided into two portions, a part is parietal layer granulosa cell, is connected with basement membrane; Another part is the cumulus granulosa cells surrounding ovocyte.Granulosa cell can carry out intracellular signaling by gap connection, for ovocyte provides nutritive substance, regulates growth and the maturation of ovocyte.(in this patent, indication is cumulus granulosa cells)
G-IVF nutrient solution: a kind of nutrient solution that Vitrolife company (Yu Bo bio tech ltd, Shanghai) produces, is mainly used in the fertilization of sperm and ovum.
M II phase ovocyte: ovocyte is in initial meiosis mid-term, hypochromatosis, first polar body discharges.
M I phase ovocyte: ovocyte is in initial meiosis mid-term, and hypochromatosis, first polar body is not yet discharged.
GV phase ovocyte: ovocyte is in the germinal vesicle phase, Visible Core structure in endochylema.
Embodiment recited above is only be described the preferred embodiment of the present invention; not scope of the present invention is limited; do not departing under the present invention designs spiritual prerequisite; the various distortion that the common engineering technical personnel in this area make technical solution of the present invention and improvement, all should fall in protection domain that claims of the present invention determine.
Claims (9)
1. an ovocyte nutrient solution, comprises G-IVF nutrient solution, liquor folliculi and granulosa cell.
2. ovocyte nutrient solution according to claim 1, is characterized in that: the volume ratio of described G-IVF nutrient solution and liquor folliculi is 1:1.
3. ovocyte nutrient solution according to claim 1 and 2, is characterized in that: containing the human serum albumin of 10% volume ratio in described G-IVF nutrient solution.
4. an ovocyte cultural method, comprises the steps:
(1) preparation of human mature follicle fluid: the chief liquor folliculi having picked up corolla mound mixture is collected in conical centrifuge tube, red corpuscle in centrifugal segregation liquor folliculi and impurity, get supernatant liquor and with metre filter, be put in incubator inner equilibrium in small test tube and preserve;
(2) preparation of granulosa cell: cut the most of granulosa cell around ovocyte under the microscope with two syringes, and the culture dish middle cover oil being put in the G-IVF nutrient solution be equipped with is for subsequent use;
(3) remainder particulate cell is divested: the ovocyte containing a small amount of granulosa cell is moved to pressure-vaccum several in Unidasa, then use the special pipe of the Bath of different diameter clean for Denudation of oocytes remaining around ovocyte, take out M II phase ovocyte, remaining M I phase and GV phase ovocyte are put in the culture dish containing granulosa cell;
(4) liquor folliculi is added: draw the liquor folliculi handled well and add cultivating containing in the culture dish of immature oocyte of preparing.
5. ovocyte nutrient solution according to claim 4, is characterized in that: the volume ratio of described G-IVF nutrient solution and liquor folliculi is 1:1.
6. ovocyte nutrient solution according to claim 4, is characterized in that: containing the human serum albumin of 10% volume ratio in described G-IVF nutrient solution.
7. according to the arbitrary described ovocyte cultural method of claim 4 ~ 6, it is characterized in that: in described step (1), removing red corpuscle in liquor folliculi and impurity by having picked up corolla mound mixture at the centrifugal 10min of 3000r/min.
8. according to the arbitrary described ovocyte cultural method of claim 4 ~ 6, it is characterized in that: in described step (3), for several times, the time is 30S the ovocyte containing a small amount of granulosa cell to be moved to pressure-vaccum in Unidasa.
9., according to the arbitrary described ovocyte cultural method of claim 4 ~ 6, it is characterized in that: in described step (4), the culture dish containing immature oocyte is put in 6.0% CO2gas incubator, 37 DEG C of cultivations.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105018418A (en) * | 2015-07-17 | 2015-11-04 | 浙江大学 | Human oocyte in vitro maturation culture solution containing Endothelin-1 and application of human oocyte in vitro maturation culture solution |
| CN106701659A (en) * | 2016-10-31 | 2017-05-24 | 四川农业大学 | Porcine ovarian granulosa cell primary culture method |
| CN109628386A (en) * | 2019-01-18 | 2019-04-16 | 周桦 | A kind of the In-vitro maturation liquid and preparation method thereof and cultural method of human oocyte |
| CN112680406A (en) * | 2021-01-12 | 2021-04-20 | 艾尔斯（浙江）医学科技有限公司 | Fertility preservation method for egg production through caesarean section |
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| WO2005019440A1 (en) * | 2003-08-20 | 2005-03-03 | Northern Sydney And Central Coast Area Health Service | Methods for enhancing embryo viability |
| CN1800371A (en) * | 2005-12-19 | 2006-07-12 | 浙江大学 | In vitro maturing culture method for human immature ovum |
| CN101851606A (en) * | 2010-04-07 | 2010-10-06 | 魏红江 | Cumulus oophorus-oocyte compound cutting filter method |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105018418A (en) * | 2015-07-17 | 2015-11-04 | 浙江大学 | Human oocyte in vitro maturation culture solution containing Endothelin-1 and application of human oocyte in vitro maturation culture solution |
| CN106701659A (en) * | 2016-10-31 | 2017-05-24 | 四川农业大学 | Porcine ovarian granulosa cell primary culture method |
| CN109628386A (en) * | 2019-01-18 | 2019-04-16 | 周桦 | A kind of the In-vitro maturation liquid and preparation method thereof and cultural method of human oocyte |
| CN112680406A (en) * | 2021-01-12 | 2021-04-20 | 艾尔斯（浙江）医学科技有限公司 | Fertility preservation method for egg production through caesarean section |
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Application publication date: 20150422 |