WO2009122541A1 - Milieux de culture liquides pour la fertilisation et la culture extracorporelle d’embryon humain et procédé de fertilisation et de culture extracorporelle d’embryon humain - Google Patents

Milieux de culture liquides pour la fertilisation et la culture extracorporelle d’embryon humain et procédé de fertilisation et de culture extracorporelle d’embryon humain Download PDF

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WO2009122541A1
WO2009122541A1 PCT/JP2008/056424 JP2008056424W WO2009122541A1 WO 2009122541 A1 WO2009122541 A1 WO 2009122541A1 JP 2008056424 W JP2008056424 W JP 2008056424W WO 2009122541 A1 WO2009122541 A1 WO 2009122541A1
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culture
human
concentration
fertilization
serum
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PCT/JP2008/056424
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English (en)
Japanese (ja)
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裕昭 乾
仁ニ 水野
寛子 中村
一幸 赤石
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Inui Hiroaki
Mizuno Jinji
Nakamura Hiroko
Akaishi Kazuyuki
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Priority to JP2008534212A priority Critical patent/JP4739420B2/ja
Priority to PCT/JP2008/056424 priority patent/WO2009122541A1/fr
Publication of WO2009122541A1 publication Critical patent/WO2009122541A1/fr

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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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • 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
    • C12N2500/34—Sugars
    • 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
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components

Definitions

  • the present invention relates to an in vitro fertilization and culture medium for human embryos used for fertilization and culture of human embryos in vitro in the treatment of human infertility, particularly in vitro fertilization treatment, in assisted reproduction technology (ART),
  • the present invention relates to a method for in vitro fertilization and culture of a human embryo that is used to fertilize and culture a human embryo in vitro.
  • Infertility is a serious problem for couples who want to have children, and it is also related to the problem of declining birthrate and aging.
  • In vitro fertilization involves fertilizing sprinkled sperm in a petri dish 1 to 3 hours after egg collection, culturing the fertilized egg, and then transferring the divided egg (embryo) into the uterus.
  • various culture solutions have been developed and used for culturing fertilized eggs (see, for example, Patent Document 1).
  • Japanese Patent Laid-Open No. 2003-24055 Japanese Patent Laid-Open No. 2003-24055
  • the present invention has been made in view of the above points, and is capable of obtaining fertilized eggs with high safety and quality and capable of increasing the pregnancy rate. It is an object of the present invention to provide an in vitro fertilization and culture method for embryos.
  • a culture solution for in vitro fertilization and culture of human embryo comprises a first culture solution used for fertilization of human sperm and eggs, and a human embryo from fertilization to the 16-cell stage.
  • the invention according to claim 2 is the invention according to claim 1, wherein the first culture solution has a glucose concentration of 2.2 ⁇ 1.1 mM, a lactate concentration of 20 ⁇ 10 mM, and a pyruvate concentration of 0.3 ⁇ 0.15 mM. And the second culture solution has a glucose concentration of 0.2 ⁇ 0.1 mM, a lactate concentration of 20 ⁇ 10 mM, a pyruvate concentration of 0.3 ⁇ 0.15 mM, and is serum-free.
  • the third culture solution has a glucose concentration of 2.1 ⁇ 1.05 mM, a lactate concentration of 10 ⁇ 5 mM, a pyruvate concentration of 0.15 ⁇ 0.075 mM, and is serum-free. It is.
  • the invention according to claim 3 is characterized in that, in claim 1 or 2, an epidermal growth factor and insulin are added to at least one of the first to third culture solutions.
  • hyaluronan is added as a serum and albumin substitute for at least one of the first to third culture solutions for complete serum-free. It is characterized by being.
  • the invention according to claim 5 is characterized in that, in any one of claims 1 to 4, recombinant human albumin is added to at least one of the first to third culture solutions.
  • a method for in vitro fertilization and culture of a human embryo according to claim 6 of the present invention comprises using a first to a third culture solution containing glucose, lactate, and pyruvate and being serum-free.
  • human sperm and eggs are fertilized in a first culture solution having the highest glucose concentration, and human embryos from fertilization to the 16-cell stage are cultured in a second culture solution having the lowest glucose concentration.
  • the human embryo from the mulberry stage to the blastocyst stage is cultured in the third culture solution having the lowest lactate concentration and pyruvate concentration.
  • the invention according to claim 7 is the invention according to claim 6, wherein, as the first culture solution, the glucose concentration is 2.2 ⁇ 1.1 mM, the lactate concentration is 20 ⁇ 10 mM, and the pyruvate concentration is 0.3 ⁇ 0.15 mM.
  • the serum-free solution is used as the second culture solution, with a glucose concentration of 0.2 ⁇ 0.1 mM, a lactate concentration of 20 ⁇ 10 mM, a pyruvate concentration of 0.3 ⁇ 0.15 mM, and no Using a serum, the third culture solution has a glucose concentration of 2.1 ⁇ 1.05 mM, a lactate concentration of 10 ⁇ 5 mM, a pyruvate concentration of 0.15 ⁇ 0.075 mM, and serum-free. It is characterized by using a thing.
  • the invention according to claim 8 is characterized in that, in claim 6 or 7, an epidermal growth factor and insulin are added to at least one of the first to third culture solutions.
  • the invention according to claim 9 is the method according to any one of claims 6 to 8, wherein hyaluronan is added as a serum and albumin substitute substance to at least one of the first to third culture solutions for complete serum-free. It is characterized by being.
  • the invention according to claim 10 is characterized in that, in any one of claims 6 to 9, recombinant human albumin is added to at least one of the first to third culture solutions.
  • in vitro fertilization and culture medium for human embryo According to the in vitro fertilization and culture medium for human embryo according to claim 1 of the present invention, fertilized eggs with high safety and quality can be obtained, and the pregnancy rate can be increased.
  • the production efficiency and the quality of the produced embryo can be improved.
  • the first to third culture solutions can be made suitable for fertilization and culture.
  • fertilized eggs with high safety and quality can be obtained, and the pregnancy rate can be increased.
  • the production efficiency and the quality of the produced embryo can be improved.
  • the first to third culture solutions can be made suitable for fertilization and culture.
  • the culture medium for in vitro fertilization and culture of a human embryo includes a first culture liquid, a second culture liquid, and a third culture liquid.
  • the first culture is used to fertilize human sperm and eggs
  • the second solution is used to culture human embryos from post-fertilization (pronuclear stage) to the 16-cell stage.
  • the third culture solution is used for culturing human embryos from the mulberry stage to the blastocyst stage. All cultures include inorganic salts, energy sources (sugars, amino acids, etc.), cytoprotective substances (polyvinyl alcohol, glycosaminoglycans such as hyaluronan), antibiotics, bioactive substances (growth factors, cytokines, etc.) ) Can be prepared by dissolving in ultrapure or double distilled water. However, any culture solution is serum-free. In the first to third culture solutions, the concentration of the energy source is different, and the concentrations of the components other than the energy source are the same.
  • L-leucine L-Leucine 0.20 ⁇ 0.10 mM
  • L-lysine monohydrochloride L-LysineHCl
  • L-methionine L-Methionine
  • L-Phenylalanine is 0.10 ⁇ 0.05 mM
  • L-Proline is 0.05 ⁇ 0.025 mM
  • L-Serine is 0 0.05 ⁇ 0.025 mM
  • L-Threonine 0.20 ⁇ 0.10 mM L-Tryptophan 0.025 ⁇ 0.0125 mM
  • L-Tyrosine 0.10 ⁇ 0.05 mM
  • Sodium bicarbonate NaHCO 3
  • disodium ethylenediaminetetraacetate dihydrate EDTA 2Na 2H 2 O
  • gentamycin sulfate salt Gentamicin Sulfate 5 0.0 ⁇ 2.5 mg / L can be used, and 100.0 ⁇ 50.0 mg / L of
  • L-glutamine (L-Glutamine) can be used at 1.00 ⁇ 0.50 mM, but ammonia / ammonium may be generated, so alanyl-L-glutamine is used instead of L-Glutamine.
  • Alanyl-L-Glutamine is used instead of L-Glutamine.
  • Alanyl-L-Glutamine is preferably used at 1.00 ⁇ 0.50 mM.
  • 0.003 ⁇ 0.0015 mM hydrochloric acid (HCl) can be used for pH adjustment.
  • Energy sources include glucose such as D-glucose, lactate such as DL-sodium lactate (DL-Lactate Na), and pyruvate such as sodium pyruvate (Na-pyruvate).
  • glucose such as D-glucose
  • lactate such as DL-sodium lactate (DL-Lactate Na)
  • pyruvate such as sodium pyruvate (Na-pyruvate).
  • concentrations are different in the first to third culture solutions. That is, the glucose concentration is highest in the first culture solution and lowest in the second culture solution. The lactate concentration and pyruvate concentration are the lowest in the third culture solution.
  • the first culture solution has a glucose concentration of 2.2 ⁇ 1.1 mM, a lactate concentration of 20 ⁇ 10 mM, and a pyruvate concentration of 0.3 ⁇ 0.15 mM.
  • the first culture solution contains abundant glucose necessary for sperm energy metabolism.
  • the osmotic pressure of the first culture solution is preferably 260 to 275 mOsm, and the pH is preferably 7.2 to 7.4.
  • the second culture solution has a glucose concentration of 0.2 ⁇ 0.1 mM, a lactate concentration of 20 ⁇ 10 mM, and a pyruvate concentration of 0.3 ⁇ 0.15 mM. Since the second culture solution does not require much glucose for the development of the early embryo, the glucose concentration is set low as described above.
  • the osmotic pressure of the second culture solution is preferably 260 to 275 mOsm, and the pH is preferably 7.2 to 7.4.
  • the third culture solution has a glucose concentration of 2.1 ⁇ 1.05 mM, a lactate concentration of 10 ⁇ 5 mM, and a pyruvate concentration of 0.15 ⁇ 0.075 mM. Since the third culture solution requires a lot of energy for cleavage of the late embryo, the glucose concentration is set high as described above. In the third culture solution, the lactate concentration and the pyruvate concentration are set low in accordance with the auxotrophy of the egg.
  • the osmotic pressure of the third culture solution is preferably 260 to 275 mOsm, and the pH is preferably 7.2 to 7.4.
  • At least one of the first to third culture solutions contains 20 ⁇ 10 ng / ml of epidermal growth factor (EGF) and 10 ⁇ 5 ⁇ g of insulin (Insulin). / Ml is preferably added.
  • Epidermal growth factor (EGF) promotes cell growth and development
  • insulin (Insulin) facilitates glucose metabolism, which promotes cell division and in the uterus of fertilized eggs. Plays an important role in getting to land.
  • epidermal growth factor (EGF) and insulin (Insulin) may be added to the first culture medium.
  • Hyaluronan refers to hyaluronic acid salts such as hyaluronic acid and sodium hyaluronate.
  • r-HA recombinant human albumin
  • the osmotic pressure and pH of each culture solution are adjusted by appropriately increasing or decreasing sodium chloride (NaCl), potassium dihydrogen phosphate (KH 2 PO 4 ), sodium hydrogen carbonate (NaHCO 3 ), etc., for example. be able to.
  • NaCl sodium chloride
  • KH 2 PO 4 potassium dihydrogen phosphate
  • NaHCO 3 sodium hydrogen carbonate
  • the collection of human eggs can be performed using a culture solution for collecting human eggs.
  • This human ovum collection culture solution was prepared by adding 11 ⁇ 5.5 mM Hepes (4- (2-Hydroxyethyl) -1-piperazineethanesulfonic acid; C 6 H 18 N 2 O 4 S) and 9 ⁇ 4. It can be prepared by adding 5 mM Na-Hepes (Sodium 4- (2-hydroxyethyl) piperazin-1-ylethanesulphonate; C 8 H 17 N 2 NaO 4 S).
  • Hepes and Na-Hepes By adding Hepes and Na-Hepes in this way, the pH of the culture fluid for collecting human eggs can be stabilized when collecting human eggs in the air outside the incubator.
  • Human eggs are collected in advance by sucking the human egg collection medium into the syringe of the egg collection tool, and then puncturing the follicle with the needle of the egg collection tool under an ultrasonic guide. This can be done by sucking the ovum with the follicular fluid into the syringe.
  • an egg collected from an infertile patient can be stored semipermanently by vitrification, that is, by freezing it in liquid nitrogen in an egg cryopreservation solution.
  • the ovum cryopreservation solution can be prepared by adding 0.1 to 1.0% by mass of methylcellulose to a culture solution for collecting human eggs.
  • Low-toxic or non-toxic cryoprotectants such as sugars (raffinose, sucrose, trehalose, lactose, etc.) and polymer compounds (Dextrin, PVA; Poly Vinyl Alcohol, PVP; Poly Vinyl Pyrrolidone, etc.) (Ethylene glycol, DMSO: Dimethyl Sulphoxide) may be added in an amount of 10 to 40% by mass.
  • This cryoprotectant can provide freezing resistance to the ovum by replacing it with free water and bound water inside and outside the ovum cells before cryopreservation of the ovum.
  • the above-described culture fluid for collecting human ovum can be used as a culture fluid for measuring a human ovum microtactile sensor (MTS) (for example, Japanese Journal of Fertilization and Implantation, Journal 25,116-119,2008 and literature Human Cell2006,19,119). -125).
  • MTS human ovum microtactile sensor
  • This culture medium for human egg MTS measurement is used to measure the elastic modulus of the egg zona pellucida using MTS in order to evaluate the quality of the human egg.
  • the elastic modulus is measured in the air outside the incubator.
  • Hepes and Na-Hepes are added to the culture medium for measuring human egg MTS, the pH is stable.
  • human sperm can be collected by washing and centrifuging semen collected from a human using a culture solution for collecting human sperm.
  • the culture solution for collecting human sperm is 11 ⁇ 5.5 mM Hepes (4- (2-Hydroxyethyl) -1-piperazineethanesulfonic acid; C 6 H) in the first culture solution, similar to the culture solution for collecting human ovum. 18 N 2 O 4 S) and 9 ⁇ 4.5 mM Na-Hepes (Sodium 4- (2-hydroxyethyl) piperazin-1-ylethanesulphonate; C 8 H 17 N 2 NaO 4 S) it can.
  • the pH of the culture solution for collecting human sperm can be stabilized when washing or centrifuging human semen in the air outside the incubator. It can be done.
  • impurities such as bacteria and leukocytes can be removed by washing and centrifuging semen, and only sperm with high mobility can be obtained.
  • the sperm cryopreservation solution can be prepared by adding 0.1 to 1.0% by mass of methylcellulose and 0.1 to 5.0 mg / ml of hyaluronan to a culture solution for collecting human sperm.
  • Low-toxic or non-toxic cryoprotectants such as sugars (raffinose, sucrose, trehalose, lactose, etc.) and polymer compounds (Dextrin, PVA; Poly Vinyl Alcohol, PVP; Poly Vinyl Pyrrolidone, etc.) May be added in an amount of 10 to 30% by mass.
  • This cryoprotectant can replace free water and bound water inside and outside sperm cells before cryopreservation of sperm, and can impart frost resistance to sperm.
  • the human embryo is cultured in vitro using the in vitro fertilization and culture medium including the first to third culture solutions. Fertilize human sperm and eggs in the culture solution. At this time, particularly when sperm vitality is not good and self-fertilization is impossible, fertilization can also be performed by microinsemination (intracytoplasmic sperm injection method; ICSI).
  • ICSI intracytoplasmic sperm injection method
  • the first culture solution is replaced with the second culture solution, and human embryos (early embryos) from the confirmation of fertilization to the 16-cell stage are cultured with this second culture solution.
  • This human embryo is before germ cell compaction.
  • the human embryo that has reached the 2-8 cell stage can be transplanted non-surgically (transvaginally) into the uterus by a transplantation catheter together with the second culture medium at the discretion of the doctor.
  • a culture solution for human embryo transfer can be prepared by adding hyaluronan 0.1-5.0 mg / ml and epidermal growth factor (EGF) 10-50 ng / ml to the second culture.
  • EGF epidermal growth factor
  • the hyaluronan in this culture medium for human embryo transfer promotes angiogenesis by a synergistic effect with epidermal growth factor (EGF), and further improves the implantation rate.
  • the elastic modulus of the zona pellucida can be measured using a micro tactile sensor (MTS) in order to evaluate the quality of the human embryo.
  • MTS micro tactile sensor
  • a culture medium for measuring early embryo MTS can be used, and this culture medium for measuring early embryo MTS is added to the second culture medium with 11 ⁇ 5.5 mM Hepes (4- (2-Hydroxyethyl) -1- Piperazineethanesulfonic acid (C 6 H 18 N 2 O 4 S) and 9 ⁇ 4.5 mM Na-Hepes (Sodium 4- (2-hydroxyethyl) piperazin-1-ylethanesulphonate; C 8 H 17 N 2 NaO 4 S) Can be prepared.
  • Hepes and Na-Hepes By adding Hepes and Na-Hepes in this way, when measuring the elastic modulus of the zona pellucida of the human embryo in the air outside the incubator, the pH of the culture medium for measuring the initial embryo MTS can be stabilized. It can be done
  • the early embryo can be stored semipermanently by vitrification preservation method, that is, by placing it in a culture solution for freezing early embryo and freezing with liquid nitrogen.
  • the culture medium for freezing early embryos can be prepared by adding 0.1 to 1.0% by mass of methylcellulose and 0.1 to 5.0 mg / ml of hyaluronan to the culture medium for measuring early embryo MTS. .
  • cryoprotectants such as sugars (raffinose, sucrose, trehalose, lactose, etc.) and polymer compounds (Dextrin, PVA; Poly Vinyl Alcohol, PVP; Poly Vinyl Pyrrolidone, etc.) (Ethylene glycol, DMSO: Dimethyl Sulphoxide) may be added in an amount of 10 to 40% by mass.
  • This cryoprotectant can replace the free water and bound water inside and outside the sperm cells prior to cryopreservation of the ovum, thereby imparting freezing tolerance to the early embryo.
  • the second culture solution is replaced with the third culture solution, and human embryos (late embryos) from the mulberry stage to the blastocyst stage are cultured in this third culture solution.
  • the first culture solution is exchanged with the second culture solution, and the second culture solution is exchanged with the third culture solution.
  • the culture from fertilization to the blastocyst stage is preferably performed by using an incubator and setting the temperature to 37.0 ° C. in a humidity saturated gas phase atmosphere of air containing 5 to 6% CO 2 .
  • the elastic modulus of the zona pellucida can be measured using a micro tactile sensor (MTS) to evaluate the quality of the human embryo during the 16-cell stage or the mulberry stage (for example, Japanese literature) Fertilization and Implantation Society Journal 25,116-119,2008 and literature Human Cell 2006,19,119-125).
  • MTS micro tactile sensor
  • a culture medium for measuring late embryo MTS can be used, and this culture medium for measuring late embryo MTS is added to 11 ⁇ 5.5 mM Hepes (4- (2-Hydroxyethyl) -1- Piperazineethanesulfonic acid (C 6 H 18 N 2 O 4 S) and 9 ⁇ 4.5 mM Na-Hepes (Sodium 4- (2-hydroxyethyl) piperazin-1-ylethanesulphonate; C 8 H 17 N 2 NaO 4 S) Can be prepared.
  • Hepes and Na-Hepes By adding Hepes and Na-Hepes in this way, when measuring the elastic modulus of the zona pellucida of human embryos in the air outside the incubator, the pH of the culture medium for late embryo MTS measurement can be stabilized. It can be done.
  • late embryos can be stored semipermanently by vitrification preservation method, that is, by putting them in a culture solution for freezing late embryos and freezing them with liquid nitrogen.
  • the culture medium for freezing late embryos can be prepared by adding 0.1 to 1.0% by mass of methylcellulose and 0.1 to 5.0 mg / ml of hyaluronan to the culture medium for measuring late embryo MTS.
  • Low-toxic or non-toxic cryoprotectants such as sugars (raffinose, sucrose, trehalose, lactose, etc.) and polymer compounds (Dextrin, PVA; Poly Vinyl Alcohol, PVP; Poly Vinyl Pyrrolidone, etc.) May be added in an amount of 10 to 30% by mass. This cryoprotectant can replace the free water and bound water inside and outside the sperm cells before cryopreserving the ovum, and can impart freezing tolerance to the late stage embryo.
  • human embryos that have reached the blastocyst stage can be transplanted into the uterus non-surgically (transvaginally) by a transplantation catheter together with the third culture solution at the discretion of the doctor.
  • the third culture solution is preferably replaced with a culture solution for human embryo transfer and then transplanted.
  • This culture medium for human embryo transfer can be prepared by adding hyaluronan 0.1 to 5.0 mg / ml and epidermal growth factor (EGF) 10 to 50 ng / ml to the third culture.
  • EGF epidermal growth factor
  • the hyaluronan in this culture medium for human embryo transfer promotes angiogenesis by a synergistic effect with epidermal growth factor (EGF), and further improves the implantation rate.
  • human embryos from fertilization to the blastocyst stage are generally cultured in a single culture solution, but it is difficult to obtain a fertilized egg of good quality by such a culture method.
  • the glucose concentration, lactate concentration, and pyruvate concentration are increased / decreased according to the nutritional requirements of the developmental stage of the human embryo, so that fertilization with good quality is performed. Eggs can be obtained. As a result, the implantation rate can be improved and the pregnancy rate can be increased.
  • the egg Since the concentrations of the first to third culture solutions are almost the same as those of components other than glucose, lactate, and pyruvate, the egg is collected from the ovary of the patient, the sperm is collected from the testis, and then the fertilized egg is collected. The stress on the ovum, sperm, and fertilized egg can be minimized until the fertilized egg is transplanted into the uterus after culturing. Therefore, the first to third culture solutions can also be used as a fertilized egg transplantation solution or vitrification preservation solution for the uterus of the observer. In the present invention, since serum components derived from humans are excluded, invasion / infection of pathogens can be prevented and safety can be improved.
  • Examples 1 and 2 and Comparative Example 1 were prepared with the compositions shown in [Table 1] below. Examples 1 and 2 have first to third culture solutions, respectively. Comparative Example 1 is a conventional commercially available culture solution (“Global” manufactured by Life TM Global) based on KSOM (Potassium Simplex Optimized Medium).
  • mice embryos were cultured in vitro using the culture solutions of Example 1 and Comparative Example 1, respectively.
  • Example 1 fertilization of mouse sperm and ovum was first performed in the first culture solution, then mouse embryos from fertilization to the 16-cell stage were cultured in the second culture solution, and then mulberry in the third culture solution. Mouse embryos from the real stage to the blastocyst stage were cultured. Fertilization and culture were performed in an incubator where the CO 2 concentration was maintained at 5 to 6%.
  • Example 1 20 ng / ml of epidermal growth factor (EGF) was added to each of the second and third culture solutions of Example 1 and Comparative Example 1, and mouse embryos were cultured in the same manner as described above.
  • 10 ⁇ g / ml of insulin (Ins) was added to each of the second and third culture solutions of Example 1 and Comparative Example 1, and mouse embryos were cultured in the same manner as described above.
  • 20 ng / ml of epidermal growth factor (EGF) and 10 ⁇ g / ml of insulin (Ins) were added to each of the second and third culture solutions of Example 1 and Comparative Example 1, and mouse embryos were obtained in the same manner as described above. Cultured.
  • the culture from fertilization to the blastocyst stage is carried out by using an incubator and setting the temperature to 37.0 ° C. in a humidity saturated gas phase atmosphere of air containing 5 to 6% CO 2. I went.
  • the above culture experiment was performed twice.
  • mice embryos were cultured in vitro using the culture solutions of Example 2 and Comparative Example 1, respectively.
  • Example 2 For Example 2, first, mouse sperm and eggs were fertilized in the first culture solution, then mouse embryos from fertilization to the 16-cell stage were cultured in the second culture solution, and then mulberry in the third culture solution. Mouse embryos from the real stage to the blastocyst stage were cultured.
  • Example 2 and Comparative Example 1 20 ng / ml of epidermal growth factor (EGF) was added to each of the second and third culture solutions of Example 2 and Comparative Example 1, and mouse embryos were cultured in the same manner as described above.
  • 10 ⁇ g / ml of insulin (Ins) was added to each of the second and third culture solutions of Example 2 and Comparative Example 1, and mouse embryos were cultured in the same manner as described above.
  • 20 ng / ml of epidermal growth factor (EGF) and 10 ⁇ g / ml of insulin (Ins) were added to each of the second and third culture solutions of Example 2 and Comparative Example 1, and mouse embryos were obtained in the same manner as described above. Cultured.
  • the culture from fertilization to the blastocyst stage is carried out by using an incubator and setting the temperature to 37.0 ° C. in a humidity saturated gas phase atmosphere of air containing 5 to 6% CO 2. I went.
  • the above culture experiment was performed twice.
  • mice embryos were cultured in vitro using the culture solution of Example 2 as follows.
  • EGF epidermal growth factor
  • Ins insulin
  • 0.15 mg / ml, 0.5 mg / ml, 2.0 mg / ml of hyaluronan manufactured by Cosmo Bio Co., Ltd. and 0.125 mg / ml of hyaluronan manufactured by Nacalai Tesque Co., Ltd. were added to the second and third culture solutions.
  • mice embryos from fertilization to 16-cell stage were cultured in each second culture solution, and then from each morula stage to blastocyst stage in each third culture solution Until then, mouse embryos were cultured.
  • mouse embryos up to the blast stage were cultured.
  • the culture from fertilization to the blastocyst stage is carried out by using an incubator and setting the temperature to 37.0 ° C. in a humidity saturated gas phase atmosphere of air containing 5 to 6% CO 2. I went.
  • mice embryos were cultured in vitro using the culture solution of Example 2 as follows.
  • r-HA recombinant human albumin
  • HSA human serum albumin
  • mice embryos from fertilization to the 16-cell stage were cultured in the second culture solution, and then mouse embryos from the mulberry stage to the blastocyst stage were cultured in the third culture solution.
  • EGF epidermal growth factor
  • Ins 10 ⁇ g / ml insulin
  • the culture from fertilization to the blastocyst stage is carried out by using an incubator and setting the temperature to 37.0 ° C. in a humidity saturated gas phase atmosphere of air containing 5 to 6% CO 2. I went.
  • the culture from fertilization to the blastocyst stage is carried out using an incubator at a temperature of 37.0 ° C. in a humidity saturated gas phase of air containing 5 to 6% O 2. Set and went.
  • Example 2 The total blastocyst rate of Example 2 and Comparative Example 1 obtained as described above is shown in FIG. 1 as a bar graph.
  • hypotonic treatment was performed on the embryo reaching the blastocyst with a hypotonic solution, and this was fixed on a slide glass with a fixing solution. Subsequently, after DAPI staining, observation and photographing were performed with a fluorescence microscope. Then, the total number of cells of all embryos was counted from the photographed images to obtain the average total number of cells. Moreover, it dye
  • Example 2 The average total cell numbers of Example 2 and Comparative Example 1 obtained as described above are shown as a bar graph in FIG.
  • Example 2 the total number of blastocysts was counted to determine the ratio of excellent blastocysts having a total cell number of 100 or more. This excellent blastocyst rate is shown as a bar graph in FIG.
  • Example 2 In addition, for Example 2 and Comparative Example 1, the fertilized female pregnancy rate and offspring production rate were determined.
  • Example 2 First, fertilization of mouse sperm and ovum was performed in the first culture solution, and then mouse embryos from fertilization to the 16-cell stage were cultured in the second culture solution. Mouse embryos from the mulberry stage to the blastocyst stage were cultured in the culture solution. Cultivation from fertilization to the blastocyst stage was performed using an incubator at a temperature of 37.0 ° C. in a humidity saturated gas phase of air containing 5 to 6% O 2 . Next, the mouse embryo at the blastocyst stage was transplanted to the oviduct or uterus of an ICR female mouse that had been spontaneously mated with an ICR male mouse that had been previously ligated to the vas deferens.
  • the embryo-fertilized female pregnancy rate was determined as the ratio of the number of females that became pregnant.
  • the offspring production rate was calculated
  • FIG. 4 shows the fertilized female pregnancy rate and offspring production rate of Example 2 and Comparative Example 1 obtained as described above in a bar graph.
  • Example 2 can obtain a fertilized egg with better quality than Comparative Example 1.
  • the implantation rate is improved and the pregnancy rate can be increased.
  • human-derived serum components are excluded, so that invasion / infection of pathogens can be prevented and safety can be improved.
  • FIG. 3 is a bar graph showing the total blastocyst rate for Example 2 and Comparative Example 1. It is a bar graph which shows the average total cell number about Example 2 and Comparative Example 1. FIG. It is a bar graph which shows the outstanding blastocyst rate about Example 2 and Comparative Example 1. FIG. It is a bar graph which shows a fertilized female pregnancy rate (recipient pregnancy rate) and a litter production rate about Example 2 and Comparative Example 1.

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Abstract

La présente invention concerne des milieux de culture liquides pour la fertilisation et la culture extracorporelle d’embryon humain selon lequel un ovule fertilisé présentant une sécurité élevée et de bonnes qualités peut être obtenu et le taux de conception peut être élevé. Les milieux de culture liquide sont constitués d’un premier milieu de culture liquide destiné à être utilisé dans la fertilisation d’un ovule humain avec du sperme humain, un second milieu de culture destiné à être utilisé dans la culture de l’embryon humain après la fertilisation jusqu’au stade 16 cellules, et un troisième milieu de culture destiné à être utilisé dans la culture de l’embryon humain depuis le stade morula jusqu’au stade blastocyte. Chacun des premier jusqu’au troisième milieux de culture liquides contient du glucose, un sel d’acide lactique et un sel d’acide pyruvique et est exempt de sérum. Le premier milieu de culture liquide contient la concentration la plus élevée de glucose tandis que le second milieu de culture liquide en contient la plus faible. Le troisième milieu de culture liquide contient les concentrations les plus faibles de sel d’acide lactique et de sel d’acide pyruvique.
PCT/JP2008/056424 2008-03-31 2008-03-31 Milieux de culture liquides pour la fertilisation et la culture extracorporelle d’embryon humain et procédé de fertilisation et de culture extracorporelle d’embryon humain WO2009122541A1 (fr)

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JP2008534212A JP4739420B2 (ja) 2008-03-31 2008-03-31 ヒト胚の体外受精及び培養用培養液及びヒト胚の体外受精及び培養方法
PCT/JP2008/056424 WO2009122541A1 (fr) 2008-03-31 2008-03-31 Milieux de culture liquides pour la fertilisation et la culture extracorporelle d’embryon humain et procédé de fertilisation et de culture extracorporelle d’embryon humain

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JP2020028248A (ja) * 2018-08-22 2020-02-27 国立大学法人京都大学 胚培養培地、及び胚培養方法
CN115161265A (zh) * 2022-08-04 2022-10-11 山东大学 一种获得相同遗传背景的哺乳动物胚胎的方法

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016526573A (ja) * 2013-07-10 2016-09-05 マトリックス バイオロジー インスティテュート 高い弾性を有するヒアルロナンの組成物およびその使用
JP2019502372A (ja) * 2015-11-24 2019-01-31 ザ ユニバーシティー オブ アデレード 胚を培養するための方法、培地および製品
JP2020028248A (ja) * 2018-08-22 2020-02-27 国立大学法人京都大学 胚培養培地、及び胚培養方法
CN115161265A (zh) * 2022-08-04 2022-10-11 山东大学 一种获得相同遗传背景的哺乳动物胚胎的方法
CN115161265B (zh) * 2022-08-04 2023-11-24 山东大学 一种获得相同遗传背景的哺乳动物胚胎的方法

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