CN114934011A - In-vitro culture method for high-quality culture of oocytes - Google Patents
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
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Abstract
The invention relates to the technical field of technical embryo culture, in particular to an in-vitro culture method for culturing oocytes with high quality, which comprises the steps of preparing a basic culture solution and an additive in proportion to obtain an oocyte culture solution; transplanting oocytes into the oocyte culture solution and cleaning the oocytes once to obtain sterile oocytes; transplanting the sterile oocyte to a new oocyte culture solution to obtain a primary culture dish; and placing the preliminary culture dish into an incubator for culturing to obtain mature oocytes. The in vitro culture method for culturing the oocyte with high quality adopts a serum-free in vitro culture method, uses the serum-free basic culture solution and the additive to form the oocyte culture solution, cultures the oocyte under the condition of not using serum, improves the maturation rate of the oocyte, avoids giant embryo disease, and effectively improves the early embryo development rate of in vitro fertilization embryos and somatic cell nucleus transfer embryos.
Description
Technical Field
The invention relates to the technical field of embryo culture, in particular to an in-vitro culture method for culturing oocytes with high quality.
Background
In vitro fertilization (InVitroFertilization) or (externalization) refers to a technique in which mammalian sperm and eggs complete the fertilization process in an in vitro manually controlled environment, abbreviated IVF. Because it is inseparable from the embryo transfer technique (ET), also referred to as IVF-ET for short. In biology, an animal obtained after an in vitro fertilized embryo is transferred to a mother is called a test-tube animal (test-tube). The technology is successful in the 50 s of the 20 th century, develops rapidly in the last 20 years, and is mature day by day to become an important and conventional animal reproduction biotechnology
The in vitro maturation culture of oocytes is the basis and key link of embryo engineering technologies such as in vitro fertilization, somatic cell nuclear transfer, in vitro production of animal embryos, transgenic cloning and the like. The in vitro maturation of oocytes is not only influenced by many factors such as ovarian cycle, animal species, follicle size, hormones, serum, etc., but also closely related to the procedure and method of in vitro maturation of oocytes and the composition of the culture medium, especially serum, which can lead to the syndrome of a large fetus with embryos produced in vitro. Most of the existing oocyte in vitro culture methods adopt artificial serum to carry out oocyte culture, which is easy to cause giant embryo disease and is very unfavorable for subsequent culture.
Disclosure of Invention
The invention aims to provide an in vitro culture method for culturing oocytes with high quality, and aims to solve the problem that giant embryo disease is easy to occur in the existing in vitro culture method for oocytes.
To achieve the above objects, the present invention provides an in vitro culture method for high-quality culturing of oocytes, comprising:
preparing a basic culture solution and an additive according to a ratio to obtain an oocyte culture solution;
transplanting the oocyte into the oocyte culture solution and cleaning once to obtain a sterile oocyte;
transplanting the sterile oocyte to a new oocyte culture solution to obtain a primary culture dish;
and putting the preliminary culture dish into an incubator for culturing to obtain mature oocytes.
Wherein the basic culture solution is mature follicular fluid, and is extracted from animal reproductive cavity.
Wherein, the components of the additive comprise fatty acid-free BSA-6mg/ml, HMG-0.075IU/ml, 17 beta-estradiol-1 mug/ml, EGF-60ng/ml, L-cysteine-0.57mM, bFGF-40ng/ml, Glutamax (100x) -1.6 mug/ml, folic acid 50 mug M, cholic acid-2 mug/ml and CXCL12-50 ng/ml.
Wherein the specific steps of oocyte cleaning comprise:
placing the oocyte into the oocyte culture solution, and standing for 1-2 hours;
adding potassium chloride and sodium chloride sterilization liquid into the oocyte culture solution for sterilization;
and taking out the oocyte to obtain a sterile oocyte.
Wherein, the concrete step of putting the preliminary culture dish into the incubator to cultivate includes:
placing the preliminary culture dish into the incubator and standing for 1-3 hours at normal temperature;
adjusting the incubator to a proper temperature and humidity and starting culture;
maintaining the temperature of the incubator and the carbon dioxide saturation humidity for continuous culture for 44 hours to obtain mature oocytes.
Wherein the suitable temperature and humidity is 38.5 ℃ and 5% carbon dioxide saturation humidity.
The invention relates to an in vitro culture method for high-quality culture of oocytes, which adopts a serum-free in vitro culture method, uses a serum-free basic culture solution and additives comprising fatty acid-free BSA, HMG, 17 beta-estradiol, EGF, L-cysteine, bFGF, Glutamax (100x), folic acid, cholic acid and CXCL to form an oocyte culture solution, and cultures the oocytes under the condition of not using serum, thereby improving the maturation rate of the oocytes, avoiding giant embryo diseases and effectively improving the early embryo development rate of in vitro fertilized embryos and somatic cell nucleus transferred embryos.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of an in vitro culture method for high-quality culturing of oocytes according to the present invention.
FIG. 2 is a flow chart showing the specific steps for washing oocytes.
FIG. 3 is a flow chart showing the specific steps of placing the preliminary culture dish in an incubator for incubation.
FIG. 4 is a flow chart showing the detailed steps of preparing oocyte culture solution.
FIG. 5 is a flow chart of the specific steps for obtaining oocytes.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Please refer to, the present invention provides an in vitro culture method for high quality culture of oocytes, comprising:
s101, preparing a basic culture solution and an additive in proportion to obtain a first oocyte culture solution and a second oocyte culture solution;
the first oocyte culture solution is used for cleaning oocytes, the second oocyte culture solution is used for culturing the oocytes, the two components are the same, the processing operation is different, the serum-free basic culture solution is adopted for configuration, the maturation rate of the oocytes can be improved, and the development problem of subsequent embryo culture is avoided.
S102, transferring the oocyte to the oocyte culture solution I, and cleaning once to obtain a sterile oocyte;
the cleaning is carried out once for the purpose of disinfection and sterilization, and meanwhile, the oocyte is enabled to generate affinity to the oocyte culture solution, so that rejection reaction is avoided.
S103, transplanting the sterile oocyte to the second oocyte culture solution to obtain a primary culture dish;
the preliminary culture dish is the first step of oocyte in vitro development, and the preliminary culture is carried out on the oocytes.
S104, placing the preliminary culture dish into an incubator for cultivation to obtain mature oocytes.
Wherein the basic culture solution is mature follicular fluid, and is extracted from animal reproductive cavity.
Wherein, the components of the additive comprise fatty acid-free BSA-6mg/ml, HMG-0.075IU/ml, 17 beta-estradiol-1 mug/ml, EGF-60ng/ml, L-cysteine-0.57mM, bFGF-40ng/ml, Glutamax (100x) -1.6 mug/ml, folic acid 50 mug M, cholic acid-2 mug/ml and CXCL12-50 ng/ml.
Wherein the specific steps of oocyte washing comprise:
s201, placing the oocyte into the first oocyte culture solution, and standing for 1-2 hours;
s202, adding a potassium chloride and sodium chloride sterilizing solution into the oocyte culture solution for sterilization;
s203, taking out the oocyte, and obtaining a sterile oocyte.
Wherein, the concrete step of putting the preliminary culture dish into the incubator to cultivate includes:
s401, placing the preliminary culture dish into the incubator and standing for 1-3 hours at normal temperature;
s402, adjusting the incubator to a preset temperature and humidity and starting culture;
s403, maintaining the temperature of the incubator and the carbon dioxide saturation humidity for continuous culture for 44 hours to obtain mature oocytes.
Wherein the preset temperature and humidity are 38.5 ℃ and 5% of carbon dioxide saturation humidity.
Wherein, the specific steps of preparing the oocyte culture solution comprise:
s111, extracting mature follicular fluid to obtain a basic culture fluid;
s112, sequentially adding additives into the basic culture solution in an aseptic environment to obtain a normal-temperature culture solution;
s113, the normal-temperature culture solution is placed in a constant temperature box and heated to the animal body temperature, and the constant-temperature culture solution is stored for later use to obtain an oocyte culture solution.
Wherein the specific steps of obtaining the oocyte comprise:
s121, taking the ovary out of the animal and placing the ovary in physiological saline containing antibiotics;
s122 removing the follicle with a needle syringe;
s123, picking up the oocyte by using an egg picking needle under the assistance of a microscope.
The invention relates to an in vitro culture method for high-quality culture of oocytes, which adopts a serum-free in vitro culture method, uses a serum-free basic culture solution and additives comprising fatty acid-free BSA, HMG, 17 beta-estradiol, EGF, L-cysteine, bFGF, Glutamax (100x), folic acid, cholic acid and CXCL to form an oocyte culture solution, and cultures bovine oocytes under the condition of not using serum, thereby improving the maturation rate of the bovine oocytes, avoiding giant embryo disease and effectively improving the early embryo development rate of in vitro fertilized embryos and somatic cell nucleus transferred embryos.
The following are examples of the present invention:
treatment group: selected COCs were washed twice in serum-free bovine oocyte in vitro maturation medium (ex vivo maturation medium from example 1, as treatment group) and transferred to 3cm dishes containing 3mL of ex vivo maturation medium from example 1 (equilibrated one hour in the incubator in advance), at 38.5 deg.C, 5% CO 2 And culturing for 18-22 h under the saturated humidity condition. Will be cultured into
The cooked COCs were washed 3 times with PBS and Ca-free containing 0.1% hyaluronidase 2+ 、Mg 2+ And digesting in PBS for 1-2 min, and repeatedly blowing and beating by using a 1000mL pipette gun to remove cumulus cells diffused outside the oocytes. And (3) after blowing and beating, washing in PBS for 3 times, poking the oocyte by a foreign body needle under an entity microscope, and selecting the oocyte with a polar body for standby.
Control group 1: selected COCs were washed twice in bovine oocyte serum in vitro maturation medium (ex vivo maturation medium of comparative example 1, as control 1), transferred to 3cm dishes containing 3mL of in vitro maturation medium of comparative example 1 (equilibrated in incubator for one hour in advance), incubated at 38.5 ℃ with 5% CO 2 And culturing for 18-22 h under the saturated humidity condition. Will cultivate
For mature COCsWashing with PBS solution for 3 times, adding Ca-free solution containing 0.1% hyaluronidase 2+ 、Mg 2+ And (3) digesting in PBS for 1-2 min, and repeatedly blowing and beating by using a 1000mL pipette gun to remove cumulus cells diffused outside the oocytes. After the oocytes are blown and beaten cleanly, the oocytes are washed in PBS for 3 times, then the oocytes are stirred by a foreign body needle under a solid sight microscope, and the oocytes with polar bodies are selected for standby.
Control group 2: selected COCs were washed twice in bovine oocyte serum-free in vitro maturation medium (ex vivo maturation medium of comparative example 2, as control 2), transferred to 3cm dishes containing 3mL of in vitro maturation medium of comparative example 2 (equilibrated in the incubator for one hour in advance), incubated at 38.5 ℃ with 5% CO 2 And culturing for 18-22 h under the saturated humidity condition. Washing mature COCs with PBS solution for 3 times, adding Ca-free solution containing 0.1% hyaluronidase 2+ 、Mg 2+ And (4) digesting in PBS for 1-2 min, and repeatedly blowing and beating by using a 1000mL pipette gun to remove cumulus cells diffused outside the oocytes. And (3) after blowing and beating, washing in PBS for 3 times, poking the oocyte by a foreign body needle under an entity microscope, and selecting the oocyte with a polar body for standby.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (6)
1. An in vitro culture method for high-quality culture of oocytes,
the method comprises the following steps: preparing a basic culture solution and an additive in proportion to obtain a first oocyte culture solution and a second oocyte culture solution;
transplanting the oocyte into the oocyte I culture solution and cleaning once to obtain a sterile oocyte;
transplanting the sterile oocyte to the second oocyte culture solution to obtain a primary culture dish;
and putting the preliminary culture dish into an incubator for culturing to obtain mature oocytes.
2. The in vitro culture method of oocytes in high quality culture according to claim 1,
the basic culture solution is mature follicular fluid, and is extracted from an animal reproductive cavity.
3. The in vitro culture method of high quality cultured oocytes according to claim 1,
the components of the additive comprise fatty acid-free BSA-6mg/ml, HMG-0.075IU/ml, 17 beta-estradiol-1 mu g/ml, EGF-60ng/ml, L-cysteine-0.57mM, bFGF-40ng/ml, Glutamax (100x) -1.6 mu L/ml, folic acid 50 mu M, cholic acid-2 mu g/ml and CXCL12-50 ng/ml.
4. The in vitro culture method of high quality cultured oocytes according to claim 1,
the specific steps of oocyte washing include:
placing the oocyte into the oocyte I culture solution, and standing for 1-2 hours;
adding a potassium chloride and sodium chloride sterilizing solution into the oocyte culture solution for sterilization;
and taking out the oocyte to obtain a sterile oocyte.
5. The in vitro culture method of high quality cultured oocytes according to claim 1,
the specific steps of putting the primary culture dish into an incubator for cultivation comprise:
placing the preliminary culture dish into the incubator and standing for 1-3 hours at normal temperature;
adjusting the incubator to a preset temperature and humidity and starting to cultivate;
maintaining the temperature of the incubator and the carbon dioxide saturation humidity for continuous culture for 44 hours to obtain mature oocytes.
6. The in vitro culture method of high quality cultured oocytes according to claim 5,
the preset temperature and humidity are 38.5 ℃ and 5% of carbon dioxide saturation humidity.
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