WO2009122541A1 - Liquid culture media for extracorporeal fertilization and culture of human embryo and method of extracorporeal fertilization and culture of human embryo - Google Patents

Liquid culture media for extracorporeal fertilization and culture of human embryo and method of extracorporeal fertilization and culture of human embryo 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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French (fr)
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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Application filed by Inui Hiroaki, Mizuno Jinji, Nakamura Hiroko, Akaishi Kazuyuki filed Critical Inui Hiroaki
Priority to PCT/JP2008/056424 priority Critical patent/WO2009122541A1/en
Priority to JP2008534212A priority patent/JP4739420B2/en
Publication of WO2009122541A1 publication Critical patent/WO2009122541A1/en

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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

Liquid culture media for the extracorporeal fertilization and culture of a human embryo whereby a fertilized egg having a high safety and good qualities can be obtained and the pregnancy rate can be elevated. The liquid culture media are composed of a first liquid culture medium to be used in fertilizing a human ovum with human sperm, a second liquid culture medium to be used in culturing the human embryo after the fertilization to the 16-cell stage, and a third liquid culture medium to be used in culturing the human embryo from the morula stage to the blastocyst stage. The first to third liquid culture media each contains glucose, a lactic acid salt and a pyruvic acid salt and is serum-free. The first liquid culture medium has the highest glucose concentration while the second liquid culture medium has the lowest one. The third liquid culture medium has the lowest lactic acid salt and pyruvic acid salt concentrations.

Description

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In vitro fertilization and culture medium for human embryo and in vitro fertilization and culture method for human embryo
　本発明は、生殖補助医療技術（ＡＲＴ）においてヒトの不妊治療、特に体外受精治療の際にヒト胚を体外で受精及び培養するのに用いられるヒト胚の体外受精及び培養用培養液及びこれを用いてヒト胚を体外で受精及び培養するヒト胚の体外受精及び培養方法に関するものである。 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.
　不妊は、子を持つことを望む夫婦にとって深刻な問題であると共に、少子高齢化の問題にも関わるものであるため、早急に解決することが望まれている。従来、不妊治療としては、薬物療法や外科的手段による生殖細胞の通過性の回復、人工授精や体外受精（ＩＶＦ）などが行われている。このうち体外受精は、採卵から１～３時間後にシャーレの中で調整済みの精子を振りかけて受精を行い、この受精した卵を培養した後、分裂した卵（胚）を子宮内に移植することによって行われている。ここで、受精卵の培養には様々な培養液が開発されて用いられている（例えば、特許文献１参照。）。
特開２００３－２４０５５号公報 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. Conventionally, as fertility treatment, recovery of germ cell passage by drug therapy or surgical means, artificial insemination, in vitro fertilization (IVF), or the like has been performed. 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. Has been done by. Here, 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
　しかし、従来の培養液は、ヒト胚の代謝（metabolism）に適したものではないので、質の良い受精卵を得るのが難しく、妊娠率を高めることができないという問題がある。 However, since conventional culture solutions are not suitable for metabolism of human embryos, it is difficult to obtain high-quality fertilized eggs, and there is a problem that the pregnancy rate cannot be increased.
　また、近年においては安心・安全な医療が求められているが、従来の培養液には例えばヒト血清アルブミン（ＨＳＡ）等のようなヒト由来の血清成分が含まれているため、感染症の危険性が否定できないという問題もある。そして現在のところ血清成分を含まない完全無血清培養液は存在しない。 In recent years, there has been a demand for safe and secure medical care. However, since conventional culture fluid contains human-derived serum components such as human serum albumin (HSA), there is a risk of infectious diseases. There is also a problem that sex cannot be denied. And at present, there is no complete serum-free medium containing no serum component.
　本発明は上記の点に鑑みてなされたものであり、安全性が高く、質の良い受精卵を得ることができ、妊娠率を高めることができる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を提供することを目的とするものである。 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.
　本発明の請求項１に係るヒト胚の体外受精及び培養用培養液は、ヒトの精子と卵子の受精を行うのに用いられる第１培養液と、受精後から１６細胞期までのヒト胚を培養するのに用いられる第２培養液と、桑実期から胚盤胞期までのヒト胚を培養するのに用いられる第３培養液とを備え、第１～第３培養液は、グルコース、乳酸塩、ピルビン酸塩を含有し、かつ無血清であると共に、グルコース濃度は第１培養液が最も高く、第２培養液が最も低く、乳酸塩濃度及びピルビン酸塩濃度は第３培養液が最も低いことを特徴とするものである。 A culture solution for in vitro fertilization and culture of human embryo according to claim 1 of the present invention comprises a first culture solution used for fertilization of human sperm and eggs, and a human embryo from fertilization to the 16-cell stage. A second culture solution used for culturing, and a third culture solution used for culturing human embryos from the mulberry stage to the blastocyst stage, wherein the first to third culture solutions comprise glucose, It contains lactate and pyruvate and is serum-free, and the glucose concentration is the highest in the first culture solution, the second culture solution is the lowest, and the lactate and pyruvate concentrations are in the third culture solution. It is characterized by the lowest.
　請求項２に係る発明は、請求項１において、第１培養液は、グルコース濃度が２．２±１．１ｍＭ、乳酸塩濃度が２０±１０ｍＭ、ピルビン酸塩濃度が０．３±０．１５ｍＭ、かつ無血清であり、第２培養液は、グルコース濃度が０．２±０．１ｍＭ、乳酸塩濃度が２０±１０ｍＭ、ピルビン酸塩濃度が０．３±０．１５ｍＭ、かつ無血清であり、第３培養液は、グルコース濃度が２．１±１．０５ｍＭ、乳酸塩濃度が１０±５ｍＭ、ピルビン酸塩濃度が０．１５±０．０７５ｍＭ、かつ無血清であることを特徴とするものである。 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.
　請求項３に係る発明は、請求項１又は２において、第１～第３培養液の少なくともいずれかに上皮細胞成長因子及びインスリンが添加されていることを特徴とするものである。 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.
　請求項４に係る発明は、請求項１乃至３のいずれか１項において、第１～第３培養液の少なくともいずれかに完全無血清化のために血清及びアルブミン代替物質としてヒアルロナンが添加されていることを特徴とするものである。 According to a fourth aspect of the present invention, in any one of the first to third aspects, 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.
　請求項５に係る発明は、請求項１乃至４のいずれか１項において、第１～第３培養液の少なくともいずれかにはリコンビナントヒトアルブミンが添加されていることを特徴とするものである。 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.
　本発明の請求項６に係るヒト胚の体外受精及び培養方法は、グルコース、乳酸塩、ピルビン酸塩を含有し、かつ無血清である第１～第３培養液を用いてヒト胚を体外で培養する方法であって、グルコース濃度が最も高い第１培養液でヒトの精子と卵子の受精を行い、グルコース濃度が最も低い第２培養液で受精後から１６細胞期までのヒト胚を培養し、乳酸塩濃度及びピルビン酸塩濃度が最も低い第３培養液で桑実期から胚盤胞期までのヒト胚を培養することを特徴とするものである。 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. In this method, 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.
　請求項７に係る発明は、請求項６において、第１培養液として、グルコース濃度が２．２±１．１ｍＭ、乳酸塩濃度が２０±１０ｍＭ、ピルビン酸塩濃度が０．３±０．１５ｍＭ、かつ無血清であるものを用い、第２培養液として、グルコース濃度が０．２±０．１ｍＭ、乳酸塩濃度が２０±１０ｍＭ、ピルビン酸塩濃度が０．３±０．１５ｍＭ、かつ無血清であるものを用い、第３培養液として、グルコース濃度が２．１±１．０５ｍＭ、乳酸塩濃度が１０±５ｍＭ、ピルビン酸塩濃度が０．１５±０．０７５ｍＭ、かつ無血清であるものを用いることを特徴とするものである。 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. And 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.
　請求項８に係る発明は、請求項６又は７において、第１～第３培養液の少なくともいずれかに上皮細胞成長因子及びインスリンが添加されていることを特徴とするものである。 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.
　請求項９に係る発明は、請求項６乃至８のいずれか１項において、第１～第３培養液の少なくともいずれかに完全無血清化のために血清及びアルブミン代替物質としてヒアルロナンが添加されていることを特徴とするものである。 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.
　請求項１０に係る発明は、請求項６乃至９のいずれか１項において、第１～第３培養液の少なくともいずれかにはリコンビナントヒトアルブミンが添加されていることを特徴とするものである。 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.
　本発明の請求項１に係るヒト胚の体外受精及び培養用培養液によれば、安全性が高く、質の良い受精卵を得ることができ、妊娠率を高めることができるものである。 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.
　請求項２に係る発明によれば、安全性が高く、質の良い受精卵を得ることができ、妊娠率を高めることができるものである。 According to the invention of claim 2, it is possible to obtain a fertilized egg with high safety and high quality, and to increase the pregnancy rate.
　請求項３に係る発明によれば、生産効率と生産胚の品質を高めることができるものである。 According to the invention of claim 3, the production efficiency and the quality of the produced embryo can be improved.
　請求項４に係る発明によれば、卵子・精子細胞を保護したり、受精・発育を促進したりすることができるものである。 According to the invention of claim 4, it is possible to protect ovum and sperm cells and to promote fertilization and development.
　請求項５に係る発明によれば、感染の心配がなく安全であるので、第１～第３培養液を受精・培養に適したものにすることができるものである。 According to the invention of claim 5, since there is no concern about infection and it is safe, the first to third culture solutions can be made suitable for fertilization and culture.
　本発明の請求項６に係るヒト胚の体外受精及び培養方法によれば、安全性が高く、質の良い受精卵を得ることができ、妊娠率を高めることができるものである。 According to the in vitro fertilization and culture method of a human embryo according to claim 6 of the present invention, fertilized eggs with high safety and quality can be obtained, and the pregnancy rate can be increased.
　請求項７に係る発明によれば、安全性が高く、質の良い受精卵を得ることができ、妊娠率を高めることができるものである。 According to the invention of claim 7, it is possible to obtain a fertilized egg with high safety and quality, and to increase the pregnancy rate.
　請求項８に係る発明によれば、生産効率と生産胚の品質を高めることができるものである。 According to the invention of claim 8, the production efficiency and the quality of the produced embryo can be improved.
　請求項９に係る発明によれば、卵子・精子細胞を保護したり、受精・発育を促進したりすることができるものである。 According to the invention of claim 9, it is possible to protect ovum and sperm cells and to promote fertilization and development.
　請求項１０に係る発明によれば、感染の心配がなく安全であるので、第１～第３培養液を受精・培養に適したものにすることができるものである。 According to the invention of claim 10, since there is no concern about infection and it is safe, the first to third culture solutions can be made suitable for fertilization and culture.
　以下、本発明の実施の形態を説明する。 Hereinafter, embodiments of the present invention will be described.
　本発明においてヒト胚の体外受精及び培養用培養液は、第１培養液と、第２培養液と、第３培養液とを備えている。 In the present invention, 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.
　第１培養液は、ヒトの精子と卵子の受精を行うのに用いられるものであり、また第２倍溶液は、受精後（前核期）から１６細胞期までのヒト胚を培養するのに用いられるものであり、また第３培養液は、桑実期から胚盤胞期までのヒト胚を培養するのに用いられるものである。いずれの培養液も、無機塩、エネルギー源（糖、アミノ酸など）、細胞保護物質（ポリビニールアルコール、ヒアルロナンを代表とするグリコスアミノグリカンなど）、抗生物質、生理活性物質（成長因子、サイトカインなど）を超純粋又は再蒸留水に溶解させることによって調製することができる。ただし、いずれの培養液も無血清である。また第１～第３培養液においてエネルギー源の濃度は異ならせ、エネルギー源以外の成分の濃度は同じにしてある。 The first culture is used to fertilize human sperm and eggs, and 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.
　ここで、エネルギー源以外の成分としては、次のようなものを用いることができる。すなわち、塩化ナトリウム（ＮａＣｌ）を９５±４７．５ｍＭ、リン酸二水素カリウム（ＫＨ２ＰＯ４）を０．７±０．３５ｍＭ、塩化カリウム（ＫＣｌ）を２．５０±１．２５ｍＭ、塩化カルシウム（ＣａＣｌ２）を１．７０±０．８５ｍＭ、硫酸マグネシウム七水和物（ＭｇＳＯ４・７Ｈ２Ｏ）を０．２０±０．１０ｍＭ、Ｌ－アラニン（L-Alanine）を０．０５±０．０２５ｍＭ、Ｌ－アルギニン一塩酸塩（L-Arginine HCl）を０．３０±０．１５ｍＭ、Ｌ－アスパラギン一水和物（L-Asparagine H2O）を０．０５±０．０２５ｍＭ、Ｌ－アスパラギン酸（L-Aspartic acid）を０．０５±０．０２５ｍＭ、Ｌ－シスチン二塩酸塩（L-Cystine 2HCl）を０．０５±０．０２５ｍＭ、Ｌ－グルタミン酸（L-Glutamic acid）を０．０５±０．０２５ｍＭ、グリシン（Glycine）を０．０５±０．０２５ｍＭ、Ｌ－ヒスチジン塩酸塩一水和物（L-Histidine HClH2O）を０．１０±０．０５ｍＭ、Ｌ－イソロイシン（L-Isoleucine）を０．２０±０．１０ｍＭ、Ｌ－ロイシン（L-Leucine）を０．２０±０．１０ｍＭ、Ｌ－リシン一塩酸塩（L-LysineHCl）を０．２０±０．１０ｍＭ、Ｌ－メチオニン（L-Methionine）を０．０５±０．０２５ｍＭ、Ｌ－フェニルアラニン（L-Phenylalanine）を０．１０±０．０５ｍＭ、Ｌ－プロリン（L-Proline）を０．０５±０．０２５ｍＭ、Ｌ－セリン（L-Serine）を０．０５±０．０２５ｍＭ、Ｌ－トレオニン（L-Threonine）を０．２０±０．１０ｍＭ、Ｌ－トリプトファン（L-Tryptophan）を０．０２５±０．０１２５ｍＭ、Ｌ－チロシン（L-Tyrosine）を０．１０±０．０５ｍＭ、Ｌ－バリン（L-Valine）を０．２０±０．１０ｍＭ、炭酸水素ナトリウム（ＮａＨＣＯ３）を２１．４３±１０．７ｍＭ、エチレンジアミン四酢酸二ナトリウム二水和物（EDTA 2Na 2H2O）を０．０１±０．００５ｍＭ、ゲンタマイシン硫酸塩（Gentamicin Sulfate）を５．０±２．５ｍｇ／Ｌ用いることができ、また培養時の操作性を向上させるために血清の代替成分としてポリビニルアルコール（Polyvinylalcohol）を１００．０±５０．０ｍｇ／Ｌ用いることができる。またＬ－グルタミン（L-Glutamine）を１．００±０．５０ｍＭ用いることができるが、アンモニア／アンモニウムが発生するおそれがあるので、Ｌ－グルタミン（L-Glutamine）の代わりにアラニル－Ｌ－グルタミン（Alanyl-L-Glutamine）を１．００±０．５０ｍＭ用いるのが好ましい。またｐＨ調整用として塩酸（ＨＣｌ）を０．００３±０．００１５ｍＭ用いることができる。 Here, as components other than the energy source, the following can be used. That is, sodium chloride (NaCl) 95 ± 47.5 mM, potassium dihydrogen phosphate (KH 2 PO 4 ) 0.7 ± 0.35 mM, potassium chloride (KCl) 2.50 ± 1.25 mM, calcium chloride (CaCl 2 ) 1.70 ± 0.85 mM, magnesium sulfate heptahydrate (MgSO 4 .7H 2 O) 0.20 ± 0.10 mM, L-alanine (L-Alanine) 0.05 ± 0 0.025 mM, L-Arginine monohydrochloride (L-Arginine HCl) 0.30 ± 0.15 mM, L-Asparagine monohydrate (L-Asparagine H 2 O) 0.05 ± 0.025 mM, L- Aspartic acid (L-Aspartic acid) 0.05 ± 0.025 mM, L-cystine dihydrochloride (L-Cystine 2HCl) 0.05 ± 0.025 mM, L-Glutamic acid (L-Glutamic acid) 05 ± 0.025 mM, glycine (Glycine The 0.05 ± 0.025 mM, L-histidine hydrochloride monohydrate (L-Histidine HClH 2 O) to 0.10 ± 0.05 mM, L-isoleucine (L-Isoleucine) to 0.20 ± 0. 10 mM, L-leucine (L-Leucine) 0.20 ± 0.10 mM, L-lysine monohydrochloride (L-LysineHCl) 0.20 ± 0.10 mM, L-methionine (L-Methionine) 0. 05 ± 0.025 mM, L-Phenylalanine (L-Phenylalanine) is 0.10 ± 0.05 mM, L-Proline (L-Proline) is 0.05 ± 0.025 mM, and 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, L-Valine 0.20 ± 0.10 mM Sodium bicarbonate (NaHCO 3) and 21.43 ± 10.7 mM, disodium ethylenediaminetetraacetate dihydrate (EDTA 2Na 2H 2 O) to 0.01 ± 0.005 mM, gentamycin sulfate salt (Gentamicin Sulfate) 5 0.0 ± 2.5 mg / L can be used, and 100.0 ± 50.0 mg / L of polyvinyl alcohol can be used as an alternative component of serum in order to improve operability during culture. 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 preferably used at 1.00 ± 0.50 mM. Further, 0.003 ± 0.0015 mM hydrochloric acid (HCl) can be used for pH adjustment.
　また、エネルギー源としては、Ｄ－グルコース（D-Glucose）等のグルコース、ＤＬ－乳酸ナトリウム（DL-Lactate Na）等の乳酸塩（Lactate）、ピルビン酸ナトリウム（Na-pyruvate）等のピルビン酸塩（Pyruvate）を用いることができるものであるが、これらの濃度は第１～第３培養液において異ならせている。すなわち、グルコース濃度は第１培養液が最も高く、第２培養液が最も低い。また、乳酸塩濃度及びピルビン酸塩濃度は第３培養液が最も低い。 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). (Pyruvate) can be used, but these 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.
　具体的には、第１培養液は、グルコース濃度が２．２±１．１ｍＭ、乳酸塩濃度が２０±１０ｍＭ、ピルビン酸塩濃度が０．３±０．１５ｍＭである。このように第１培養液は精子のエネルギー代謝に必要なグルコースを豊富に含んでいるものである。なお、第１培養液の浸透圧は２６０～２７５ｍＯｓｍであることが好ましく、ｐＨは７．２～７．４であることが好ましい。 Specifically, 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. Thus, 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.
　また第２培養液は、グルコース濃度が０．２±０．１ｍＭ、乳酸塩濃度が２０±１０ｍＭ、ピルビン酸塩濃度が０．３±０．１５ｍＭである。第２培養液では初期胚の発育にグルコースを余り必要としないので、上記のようにグルコース濃度が低く設定されている。なお、第２培養液の浸透圧は２６０～２７５ｍＯｓｍであることが好ましく、ｐＨは７．２～７．４であることが好ましい。 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.
　また第３培養液は、グルコース濃度が２．１±１．０５ｍＭ、乳酸塩濃度が１０±５ｍＭ、ピルビン酸塩濃度が０．１５±０．０７５ｍＭである。第３培養液では後期胚の卵割に多くのエネルギーを必要とするので、上記のようにグルコース濃度が高く設定されている。また第３培養液では卵子の栄養要求性に合わせて乳酸塩濃度及びピルビン酸塩濃度が低く設定されている。なお、第３培養液の浸透圧は２６０～２７５ｍＯｓｍであることが好ましく、ｐＨは７．２～７．４であることが好ましい。 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.
　ここで、生産効率と生産胚の品質の観点から、特に第１～第３培養液の少なくともいずれかには上皮細胞成長因子（ＥＧＦ）を２０±１０ｎｇ／ｍｌ及びインスリン（Ｉｎｓｕｌｉｎ）を１０±５μｇ／ｍｌ添加しておくのが好ましい。上皮細胞成長因子（ＥＧＦ）は細胞の成長と発育を促進するものであり、またインスリン（Ｉｎｓｕｌｉｎ）は糖代謝を円滑に進めるものであり、これらのものは細胞の分割促進と受精卵の子宮内への着床を行わせるのに重要な役割を演ずる。なお、同様に第１培養液にも上皮細胞成長因子（ＥＧＦ）及びインスリン（Ｉｎｓｕｌｉｎ）を添加しておいてもよい。 Here, from the viewpoint of production efficiency and quality of the produced embryo, in particular, at least one of the first to third culture solutions contains 20 ± 10 ng / ml of epidermal growth factor (EGF) and 10 ± 5 μg of insulin (Insulin). / Ml is preferably added. Epidermal growth factor (EGF) promotes cell growth and development, and insulin (Insulin) facilitates glucose metabolism, which promotes cell division and in the uterus of fertilized eggs. Plays an important role in getting to land. Similarly, epidermal growth factor (EGF) and insulin (Insulin) may be added to the first culture medium.
　また第１～第３培養液の少なくともいずれかには完全無血清化のために血清及びアルブミン代替物質としてヒアルロナンを０．０５～７．０ｍｇ／ｍｌ添加しておくのが好ましい。これにより、卵子・精子細胞を保護したり、受精・発育を促進したりすることができるものである。なお、ヒアルロナンとはヒアルロン酸やヒアルロン酸ナトリウム等のヒアルロン酸塩のことをいう。 Moreover, it is preferable to add 0.05 to 7.0 mg / ml of hyaluronan as a serum and albumin substitute for at least one of the first to third culture solutions for complete serum-free. Thereby, an egg and a sperm cell can be protected or fertilization and development can be promoted. Hyaluronan refers to hyaluronic acid salts such as hyaluronic acid and sodium hyaluronate.
　また第１～第３培養液の少なくともいずれかにはリコンビナントヒトアルブミン（ｒ－ＨＡ）を０．１～２質量％添加しておくのが好ましい。リコンビナントヒトアルブミンは、感染の心配がなく安全であるので、第１～第３培養液を受精・培養に適したものにすることができるものである。 Further, it is preferable to add 0.1 to 2% by mass of recombinant human albumin (r-HA) to at least one of the first to third culture solutions. Recombinant human albumin is safe without worrying about infection, so that the first to third culture solutions can be made suitable for fertilization and culture.
　また各培養液の浸透圧及びｐＨは、例えば、塩化ナトリウム（ＮａＣｌ）、リン酸二水素カリウム（ＫＨ２ＰＯ４）、炭酸水素ナトリウム（ＮａＨＣＯ３）等を適宜に増量又は減量することによって調整することができる。 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.
　そしてヒトの卵子の採取は、ヒト卵子採取用培養液を用いて行うことができる。このヒト卵子採取用培養液は、第１培養液に１１±５．５ｍＭのＨｅｐｅｓ（4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid；Ｃ６Ｈ１８Ｎ２Ｏ４Ｓ）及び９±４．５ｍＭのＮａ－Ｈｅｐｅｓ（Sodium 4-(2-hydroxyethyl)piperazin-1-ylethanesulphonate；Ｃ８Ｈ１７Ｎ２ＮａＯ４Ｓ）を添加して調製することができる。このようにＨｅｐｅｓ及びＮａ－Ｈｅｐｅｓが添加されていることによって、インキュベータ外の空気中でヒトの卵子を採取する場合に、ヒト卵子採取用培養液のｐＨを安定させることができるものである。なお、ヒトの卵子の採取は、卵子採取用ツールの注射筒にあらかじめヒト卵子採取用培養液を吸引した後、超音波ガイド下で卵子採取用ツールの注射針を卵胞に穿刺し、卵胞内の卵胞液と共に卵子を注射筒内に吸引することによって行うことができる。 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). 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.
　また、不妊患者から採取した卵子は、ガラス化保存法によって、すなわち卵子凍結保存液に入れて液体窒素で凍結することによって半永久的に保存することができる。ここで、卵子凍結保存液は、ヒト卵子採取用培養液にメチルセルロースを０．１～１．０質量％添加して調製することができる。必要に応じてさらに、糖類（ラフィノース、シュークロース、トレハロース及びラクトースなど）、高分子化合物（Dextrin, PVA；Poly Vinyl Alcohol，PVP；Poly Vinyl Pyrrolidoneなど）等の毒性の低いあるいは毒性の無い凍結保護剤（エチレングリコール、DMSO:Dimethyl Sulphoxide）を１０～４０質量％添加してもよい。この凍結保護剤は、卵子の凍結保存前に卵子細胞内外の自由水及び結合水と置き換わって、卵子に耐凍能を付与することができる。 In addition, 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. Here, 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.
　また、上記のヒト卵子採取用培養液は、ヒト卵子マイクロタクタイルセンサ（ＭＴＳ）測定用培養液として用いることができる（例えば、文献日本受精着床学会雑誌 25,116-119,2008及び文献Human Cell2006,19,119-125）。このヒト卵子ＭＴＳ測定用培養液は、ヒト卵子の品質を評価するためにＭＴＳを用いて卵子透明帯の弾性率を測定するために用いられるものである。そして弾性率の測定は、インキュベータ外の空気中で行われるが、ヒト卵子ＭＴＳ測定用培養液にはＨｅｐｅｓ及びＮａ－Ｈｅｐｅｓが添加されているので、ｐＨが安定しているものである。 Further, 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). 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. However, since Hepes and Na-Hepes are added to the culture medium for measuring human egg MTS, the pH is stable.
　他方、ヒトの精子の採取は、ヒト精子採取用培養液を用いて、ヒトから採取した精液を洗浄すると共に遠心分離することによって行うことができる。ここで、ヒト精子採取用培養液は、ヒト卵子採取用培養液と同様に、第１培養液に１１±５．５ｍＭのＨｅｐｅｓ（4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid；Ｃ６Ｈ１８Ｎ２Ｏ４Ｓ）及び９±４．５ｍＭのＮａ－Ｈｅｐｅｓ（Sodium 4-(2-hydroxyethyl)piperazin-1-ylethanesulphonate；Ｃ８Ｈ１７Ｎ２ＮａＯ４Ｓ）を添加して調製することができる。このようにＨｅｐｅｓ及びＮａ－Ｈｅｐｅｓが添加されていることによって、インキュベータ外の空気中でヒトの精液を洗浄したり遠心分離したりする場合に、ヒト精子採取用培養液のｐＨを安定させることができるものである。なお、精液の洗浄・遠心分離によって、細菌や白血球などの不純物を除去し、運動性の高い精子のみを得ることができるものである。 On the other hand, human sperm can be collected by washing and centrifuging semen collected from a human using a culture solution for collecting human sperm. Here, 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. By adding Hepes and Na-Hepes in this way, 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. In addition, impurities such as bacteria and leukocytes can be removed by washing and centrifuging semen, and only sperm with high mobility can be obtained.
　また、一旦採取した精子は、精子凍結保存液に入れて液体窒素で凍結することによって半永久的に保存することができる。ここで、精子凍結保存液は、ヒト精子採取用培養液にメチルセルロースを０．１～１．０質量％、ヒアルロナンを０．１～５．０ｍｇ／ｍｌ添加して調製することができる。必要に応じてさらに、糖類（ラフィノース、シュークロース、トレハロース及びラクトースなど）、高分子化合物（Dextrin, PVA；Poly Vinyl Alcohol，PVP；Poly Vinyl Pyrrolidoneなど）等の毒性の低いあるいは毒性の無い凍結保護剤を１０～３０質量％添加してもよい。この凍結保護剤は、精子の凍結保存前に精子細胞内外の自由水及び結合水と置き換わって、精子に耐凍能を付与することができる。 In addition, once collected sperm can be stored semipermanently by placing it in a sperm cryopreservation solution and freezing with liquid nitrogen. Here, 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.
　次に、上記のように採卵・採精した後、第１～第３培養液を備える体外受精及び培養用培養液を用いてヒト胚を体外で培養するにあたっては、インキュベータ内において、まず第１培養液でヒトの精子と卵子の受精を行う。このとき特に精子の活力が芳しくなく自力受精が不可能な場合には、顕微授精法（卵細胞質内精子注入法；ＩＣＳＩ）によって受精を行うこともできる。 Next, after the eggs are collected and collected as described above, 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).
　受精確認後、第１培養液を第２培養液に交換し、この第２培養液で受精確認後から１６細胞期までのヒト胚（初期胚）を培養する。このヒト胚は、胚細胞緊密化（compaction）を起こす前のものである。 After fertilization confirmation, 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.
　なお、２～８細胞期に至ったヒト胚は、医師の判断により、第２培養液と共に移植用カテーテルによって、非外科的（経膣的）に子宮内に移植することができる。ここで、着床率をより向上させるためには、第２培養液をヒト胚移植用培養液に交換した後に移植するのが好ましい。このヒト胚移植用培養液は、第２培養液にヒアルロナンを０．１～５．０ｍｇ／ｍｌ、上皮細胞成長因子（ＥＧＦ）を１０～５０ｎｇ／ｍｌ添加して調製することができる。そしてこのヒト胚移植用培養液中のヒアルロナンが、上皮細胞成長因子（ＥＧＦ）との相乗効果により血管新生を促進させ、着床率をより向上させるものである。 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. Here, in order to further improve the implantation rate, it is preferable to transplant the second culture solution after exchanging it with a culture solution for human embryo transfer. This culture medium 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. And 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.
　ここで、受精後から１６細胞期に至るまでの間、ヒト胚の品質を評価するためにマイクロタクタイルセンサ（ＭＴＳ）を用いて透明帯の弾性率を測定することができる。この測定には初期胚ＭＴＳ測定用培養液を用いることができ、この初期胚ＭＴＳ測定用培養液は、第２培養液に１１±５．５ｍＭのＨｅｐｅｓ（4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid；Ｃ６Ｈ１８Ｎ２Ｏ４Ｓ）及び９±４．５ｍＭのＮａ－Ｈｅｐｅｓ（Sodium 4-(2-hydroxyethyl)piperazin-1-ylethanesulphonate；Ｃ８Ｈ１７Ｎ２ＮａＯ４Ｓ）を添加して調製することができる。このようにＨｅｐｅｓ及びＮａ－Ｈｅｐｅｓが添加されていることによって、インキュベータ外の空気中でヒト胚の透明帯の弾性率を測定する場合に、初期胚ＭＴＳ測定用培養液のｐＨを安定させることができるものである。 Here, during the period from fertilization to the 16-cell stage, 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. For this measurement, 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. 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.
　また、初期胚は、ガラス化保存法によって、すなわち初期胚凍結用培養液に入れて液体窒素で凍結することによって半永久的に保存することができる。ここで、初期胚凍結用培養液は、初期胚ＭＴＳ測定用培養液にメチルセルロースを０．１～１．０質量％、ヒアルロナンを０．１～５．０ｍｇ／ｍｌ添加して調製することができる。必要に応じてさらに、糖類（ラフィノース、シュークロース、トレハロース及びラクトースなど）、高分子化合物（Dextrin, PVA；Poly Vinyl Alcohol，PVP；Poly Vinyl Pyrrolidoneなど）等の毒性の低いあるいは毒性の無い凍結保護剤（エチレングリコール、DMSO:Dimethyl Sulphoxide）を１０～４０質量％添加してもよい。この凍結保護剤は、卵子の凍結保存前に精子細胞内外の自由水及び結合水と置き換わって、初期胚に耐凍能を付与することができる。 Also, 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. Here, 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. . 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 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.
　そして１６細胞期又は桑実期に至ると、第２培養液を第３培養液に交換し、この第３培養液で桑実期から胚盤胞期までのヒト胚（後期胚）を培養する。このように発育ステージに応じて第１培養液を第２培養液に交換し、さらに第２培養液を第３培養液に交換するものである。なお、受精から胚盤胞期までの培養は、インキュベータを用い、５～６％ＣＯ２を含む空気の湿度飽和気相の環境下において温度を３７．０℃に設定して行うのが好ましい。 When the 16-cell stage or the mulberry stage is reached, 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. . Thus, according to the growth stage, 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 .
　ここで、１６細胞期又は桑実期に至るまでの間、ヒト胚の品質を評価するためにマイクロタクタイルセンサ（ＭＴＳ）を用いて透明帯の弾性率を測定することができる（例えば、文献日本受精着床学会雑誌 25,116-119,2008及び文献Human Cell2006,19,119-125）。この測定には後期胚ＭＴＳ測定用培養液を用いることができ、この後期胚ＭＴＳ測定用培養液は、第３培養液に１１±５．５ｍＭのＨｅｐｅｓ（4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid；Ｃ６Ｈ１８Ｎ２Ｏ４Ｓ）及び９±４．５ｍＭのＮａ－Ｈｅｐｅｓ（Sodium 4-(2-hydroxyethyl)piperazin-1-ylethanesulphonate；Ｃ８Ｈ１７Ｎ２ＮａＯ４Ｓ）を添加して調製することができる。このようにＨｅｐｅｓ及びＮａ－Ｈｅｐｅｓが添加されていることによって、インキュベータ外の空気中でヒト胚の透明帯の弾性率を測定する場合に、後期胚ＭＴＳ測定用培養液のｐＨを安定させることができるものである。 Here, 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). For this measurement, 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. 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.
　また、後期胚は、ガラス化保存法によって、すなわち後期胚凍結用培養液に入れて液体窒素で凍結することによって半永久的に保存することができる。ここで、後期胚凍結用培養液は、後期胚ＭＴＳ測定用培養液にメチルセルロースを０．１～１．０質量％、ヒアルロナンを０．１～５．０ｍｇ／ｍｌ添加して調製することができる。必要に応じてさらに、糖類（ラフィノース、シュークロース、トレハロース及びラクトースなど）、高分子化合物（Dextrin, PVA；Poly Vinyl Alcohol，PVP；Poly Vinyl Pyrrolidoneなど）等の毒性の低いあるいは毒性の無い凍結保護剤を１０～３０質量％添加してもよい。この凍結保護剤は、卵子の凍結保存前に精子細胞内外の自由水及び結合水と置き換わって、後期胚に耐凍能を付与することができる。 Also, 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. Here, 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.
　そして、胚盤胞期に至ったヒト胚は、医師の判断により、第３培養液と共に移植用カテーテルによって、非外科的（経膣的）に子宮内に移植することができる。ここで、着床率をより向上させるためには、第３培養液をヒト胚移植用培養液に交換した後に移植するのが好ましい。このヒト胚移植用培養液は、第３培養液にヒアルロナンを０．１～５．０ｍｇ／ｍｌ、上皮細胞成長因子（ＥＧＦ）を１０～５０ｎｇ／ｍｌ添加して調製することができる。そしてこのヒト胚移植用培養液中のヒアルロナンが、上皮細胞成長因子（ＥＧＦ）との相乗効果により血管新生を促進させ、着床率をより向上させるものである。 Then, 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. Here, in order to further improve the implantation rate, 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. And 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.
　従来は受精から胚盤胞期までのヒト胚を単一の培養液で培養するのが一般的であったが、このような培養方法では質の良い受精卵を得ることが難しい。しかし、本発明では第１～第３培養液において、ヒト胚の発育ステージの栄養要求性に合わせて、グルコース濃度、乳酸塩濃度、ピルビン酸塩濃度をそれぞれ増減させているので、質の良い受精卵を得ることができるものであり、その結果、着床率が向上し、妊娠率を高めることができるものである。そして第１～第３培養液においてグルコース、乳酸塩、ピルビン酸塩以外の成分と濃度はほぼ同一であるので、患者の卵巣から卵子を採取し、精巣から精子を採取してから、受精卵の培養を経て子宮内へ受精卵を移植するまでの間、卵子、精子、受精卵に対するストレスを最小限に抑えることができるものである。従って、第１～第３培養液は、看者子宮への受精卵移植液あるいはガラス化保存液としての使用も可能である。本発明ではヒト由来の血清成分を除外しているので、病原体の侵入・感染を防止することができ、安全性を高めることができるものである。 Conventionally, 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. However, in the present invention, in the first to third culture solutions, 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. 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.
　以下、本発明を実施例によって具体的に説明する。 Hereinafter, the present invention will be specifically described by way of examples.
　下記［表１］に示す組成で実施例１、２及び比較例１の培養液を調製した。実施例１、２はそれぞれ第１～第３培養液を備えている。比較例１はＫＳＯＭ（Potassium Simplex Optimized Medium）をベースとする従来の市販の培養液（Life Global社製「Global」）である。 The culture solutions of 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 ™ Global) based on KSOM (Potassium Simplex Optimized Medium).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　次に、実施例１及び比較例１の培養液を用いてそれぞれマウス胚を体外で培養した。 Next, mouse embryos were cultured in vitro using the culture solutions of Example 1 and Comparative Example 1, respectively.
　実施例１については、まず第１培養液でマウスの精子と卵子の受精を行い、次に第２培養液で受精後から１６細胞期までのマウス胚を培養した後、第３培養液で桑実期から胚盤胞期までのマウス胚を培養した。なお、受精・培養は、ＣＯ２濃度を５～６％に保ったインキュベータ内において行った。 For 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%.
　比較例１については、上記［表１］に示す培養液のみで受精を行うと共に、受精後から胚盤胞期までのマウス胚を培養した。 For Comparative Example 1, fertilization was performed only with the culture solution shown in [Table 1] above, and mouse embryos from fertilization to the blastocyst stage were cultured.
　また、実施例１及び比較例１の第２、第３培養液のそれぞれに上皮細胞成長因子（ＥＧＦ）を２０ｎｇ／ｍｌ添加して上記と同様にマウス胚を培養した。また、実施例１及び比較例１の第２、第３培養液のそれぞれにインスリン（Ｉｎｓ）を１０μｇ／ｍｌ添加して上記と同様にマウス胚を培養した。また、実施例１及び比較例１の第２、第３培養液のそれぞれに上皮細胞成長因子（ＥＧＦ）を２０ｎｇ／ｍｌ及びインスリン（Ｉｎｓ）を１０μｇ／ｍｌ添加して上記と同様にマウス胚を培養した。 Further, 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. In addition, 10 μg / 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. In addition, 20 ng / ml of epidermal growth factor (EGF) and 10 μg / 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.
　なお、上記の培養実験では、受精から胚盤胞期までの培養は、インキュベータを用い、５～６％ＣＯ２を含む空気の湿度飽和気相の環境下において温度を３７．０℃に設定して行った。また上記の培養実験は各２回行った。 In the above-described culture experiment, 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.
　そして、ＥＧＦもＩｎｓも添加していない場合、ＥＧＦのみ添加した場合、Ｉｎｓのみ添加した場合、ＥＧＦとＩｎｓを両方添加した場合のそれぞれの場合について、胚盤胞到達率、総細胞数、総細胞数５０未満の胚盤胞の個数と割合、総細胞数１００以上の胚盤胞の個数と割合を求めた。その結果を下記［表２］に示す。なお、下記［表２］中、「Ｎ」は総細胞数を数えた胚盤胞の個数（総細胞数５０未満の胚のデータは含まない）を示し、「ＳＴＤ」は標準偏差を示す。 And when EGF and Ins are not added, when only EGF is added, when only Ins is added, and when both EGF and Ins are added, the blastocyst arrival rate, the total number of cells, the total cells The number and ratio of blastocysts less than several 50 and the number and ratio of blastocysts having a total cell number of 100 or more were determined. The results are shown in [Table 2] below. In the following [Table 2], “N” indicates the number of blastocysts counting the total number of cells (not including data on embryos having a total number of cells less than 50), and “STD” indicates standard deviation.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　一方、実施例２及び比較例１の培養液を用いてそれぞれマウス胚を体外で培養した。 Meanwhile, mouse embryos were cultured in vitro using the culture solutions of Example 2 and Comparative Example 1, respectively.
　実施例２については、まず第１培養液でマウスの精子と卵子の受精を行い、次に第２培養液で受精後から１６細胞期までのマウス胚を培養した後、第３培養液で桑実期から胚盤胞期までのマウス胚を培養した。 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.
　比較例１については、上記［表１］に示す培養液のみで受精を行うと共に、受精後から胚盤胞期までのマウス胚を培養した。 For Comparative Example 1, fertilization was performed only with the culture solution shown in [Table 1] above, and mouse embryos from fertilization to the blastocyst stage were cultured.
　また、実施例２及び比較例１の第２、第３培養液のそれぞれに上皮細胞成長因子（ＥＧＦ）を２０ｎｇ／ｍｌ添加して上記と同様にマウス胚を培養した。また、実施例２及び比較例１の第２、第３培養液のそれぞれにインスリン（Ｉｎｓ）を１０μｇ／ｍｌ添加して上記と同様にマウス胚を培養した。また、実施例２及び比較例１の第２、第３培養液のそれぞれに上皮細胞成長因子（ＥＧＦ）を２０ｎｇ／ｍｌ及びインスリン（Ｉｎｓ）を１０μｇ／ｍｌ添加して上記と同様にマウス胚を培養した。 Further, 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. In addition, 10 μg / 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. In addition, 20 ng / ml of epidermal growth factor (EGF) and 10 μg / 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.
　なお、上記の培養実験では、受精から胚盤胞期までの培養は、インキュベータを用い、５～６％ＣＯ２を含む空気の湿度飽和気相の環境下において温度を３７．０℃に設定して行った。また上記の培養実験は各２回行った。 In the above-described culture experiment, 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.
　そして、ＥＧＦもＩｎｓも添加していない場合、ＥＧＦのみ添加した場合、Ｉｎｓのみ添加した場合、ＥＧＦとＩｎｓを両方添加した場合のそれぞれの場合について、胚盤胞到達率、総細胞数、総細胞数５０未満の胚盤胞の個数と割合、総細胞数１００以上の胚盤胞の個数と割合を求めた。その結果を下記［表３］に示す。なお、下記［表３］中、「Ｎ」は総細胞数を数えた胚盤胞の個数（総細胞数５０未満の胚のデータは含まない）を示し、「ＳＴＤ」は標準偏差を示す。 And when EGF and Ins are not added, when only EGF is added, when only Ins is added, and when both EGF and Ins are added, the blastocyst arrival rate, the total number of cells, the total cells The number and ratio of blastocysts less than several 50 and the number and ratio of blastocysts having a total cell number of 100 or more were determined. The results are shown in [Table 3] below. In the following [Table 3], “N” indicates the number of blastocysts counting the total number of cells (not including data of embryos having a total number of cells less than 50), and “STD” indicates standard deviation.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　また、実施例２の培養液を用いて次のようにマウス胚を体外で培養した。 In addition, mouse embryos were cultured in vitro using the culture solution of Example 2 as follows.
　まず第１培養液にコスモ・バイオ株式会社製のヒアルロナンを０．１２５ｍｇ／ｍｌ、０．５ｍｇ／ｍｌ、２．０ｍｇ／ｍｌ、ナカライテスク株式会社製のヒアルロナンを０．１２５ｍｇ／ｍｌ、０．５ｍｇ／ｍｌ、２．０ｍｇ／ｍｌ、ヒト血清アルブミン（ＨＳＡ）を１質量％添加し、各第１培養液でマウスの精子と卵子の受精を行った。また比較のため、ヒアルロナンもＨＳＡも添加しない無添加の第１培養液でマウスの精子と卵子の受精を行った。このときの受精数及び受精率を下記［表４］に示す。 First, 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, 0.5 mg of hyaluronan manufactured by Nacalai Tesque Co., Ltd. were added to the first culture solution. / Ml, 2.0 mg / ml, 1% by mass of human serum albumin (HSA) was added, and the sperm and egg of the mouse were fertilized with each first culture solution. For comparison, mouse sperm and ovum were fertilized in the first culture solution without addition of hyaluronan or HSA. The number of fertilization and fertilization rate at this time are shown in [Table 4] below.
　次に第２、第３培養液のそれぞれに上皮細胞成長因子（ＥＧＦ）を２０ｎｇ／ｍｌ及びインスリン（Ｉｎｓ）を１０μｇ／ｍｌ添加した。さらにこの第２、第３培養液にコスモ・バイオ株式会社製のヒアルロナンを０．１２５ｍｇ／ｍｌ、０．５ｍｇ／ｍｌ、２．０ｍｇ／ｍｌ、ナカライテスク株式会社製のヒアルロナンを０．１２５ｍｇ／ｍｌ、０．５ｍｇ／ｍｌ、２．０ｍｇ／ｍｌ添加し、各第２培養液で受精後から１６細胞期までのマウス胚を培養した後、各第３培養液で桑実期から胚盤胞期までのマウス胚を培養した。また比較のため、ヒアルロナンもＨＳＡも添加しない無添加の第２培養液で受精後から１６細胞期までのマウス胚を培養した後、同様に無添加の第３培養液で桑実期から胚盤胞期までのマウス胚を培養した。 Next, 20 ng / ml of epidermal growth factor (EGF) and 10 μg / ml of insulin (Ins) were added to each of the second and third culture solutions. Furthermore, 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. , 0.5 mg / ml and 2.0 mg / ml were added, and mouse 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. For comparison, after culturing a mouse embryo from fertilization to the 16-cell stage in a second culture solution without addition of hyaluronan or HSA, similarly, from a mulberry stage with a third culture solution without addition. Mouse embryos up to the blast stage were cultured.
　なお、上記の培養実験では、受精から胚盤胞期までの培養は、インキュベータを用い、５～６％ＣＯ２を含む空気の湿度飽和気相の環境下において温度を３７．０℃に設定して行った。 In the above-described culture experiment, 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.
　そして、上記の各場合について、２ｃｅｌｌ分割数（受精後２細胞に発育した胚数）、２ｃｅｌｌ分割率（受精後２細胞に発育した胚の占める占有率）、胚盤胞到達数、胚盤胞到達率、Ｈ．ｉｎｇ数（Hatching blastocystのことであり、胚盤胞（blastocyst）に発育したものの中で透明帯から脱出を開始した胚数）、Ｈ．ｉｎｇ率（透明帯から脱出を開始した胚の占める占有率）を求めた。その結果を下記［表４］に示す。なお、下記［表４］中、「ＳＴＤ」は標準偏差を示し、「ｎ」は総細胞数を数えた胚盤胞の個数（総細胞数５０未満の胚のデータは含まない）を示す。 And in each of the above cases, 2 cell division number (number of embryos that have developed into 2 cells after fertilization), 2 cell division rate (occupation ratio of embryos that have developed into 2 cells after fertilization), blastocyst arrival number, blastocysts Reach rate, H.R. ing number (Hatching blastocyst, the number of embryos that started to escape from the zona pellucida among the blastocysts that developed), The ing rate (occupation ratio of embryos that started to escape from the zona pellucida) was determined. The results are shown in [Table 4] below. In the following [Table 4], “STD” indicates a standard deviation, and “n” indicates the number of blastocysts obtained by counting the total number of cells (data of embryos having a total number of cells less than 50 is not included).
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　また、実施例２の培養液を用いて次のようにマウス胚を体外で培養した。 In addition, mouse embryos were cultured in vitro using the culture solution of Example 2 as follows.
　まず第１培養液にリコンビナントヒトアルブミン（ｒ－ＨＡ）を０．５質量％、ヒト血清アルブミン（ＨＳＡ）を１質量％添加し、各第１培養液でマウスの精子と卵子の受精を行った。また比較のため、ｒ－ＨＡもＨＳＡも添加しない無添加の第１培養液でマウスの精子と卵子の受精を行った。さらに比較のため、株式会社ナカメディカル製「ＱＡファティリゼイション（ＨＴＦ）メディウム」にｒ－ＨＡを０．５質量％、ＨＳＡを１質量％添加し、各ＨＴＦでマウスの精子と卵子の受精を行った。このときの受精数及び受精率を下記［表５］に示す。 First, 0.5% by mass of recombinant human albumin (r-HA) and 1% by mass of human serum albumin (HSA) were added to the first culture solution, and mouse sperm and eggs were fertilized in each first culture solution. . For comparison, mouse sperm and ovum were fertilized in the first culture solution to which neither r-HA nor HSA was added. For further comparison, 0.5% by mass of r-HA and 1% by mass of HSA were added to “QA Fattytization (HTF) Medium” manufactured by Naka Medical Co., Ltd., and fertilization of mouse sperm and ovum was performed with each HTF. went. The number of fertilization and fertilization rate at this time are shown in [Table 5] below.
　次に第２、第３培養液のそれぞれに上皮細胞成長因子（ＥＧＦ）を２０ｎｇ／ｍｌ及びインスリン（Ｉｎｓ）を１０μｇ／ｍｌ添加したが、ｒ－ＨＡもＨＳＡも添加しなかった。そして第２培養液で受精後から１６細胞期までのマウス胚を培養した後、第３培養液で桑実期から胚盤胞期までのマウス胚を培養した。 Next, 20 ng / ml epidermal growth factor (EGF) and 10 μg / ml insulin (Ins) were added to each of the second and third cultures, but neither r-HA nor HSA was added. Then, mouse 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.
　なお、上記の培養実験では、受精から胚盤胞期までの培養は、インキュベータを用い、５～６％ＣＯ２を含む空気の湿度飽和気相の環境下において温度を３７．０℃に設定して行った。 In the above-described culture experiment, 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.
　そして、上記の各場合について、２ｃｅｌｌ分割数（受精後２細胞に発育した胚数）、２ｃｅｌｌ分割率（受精後２細胞に発育した胚の占める占有率）、胚盤胞到達数、胚盤胞到達率、Ｈ．ｉｎｇ数（Hatching blastocystのことであり、胚盤胞（blastocyst）に発育したものの中で透明帯から脱出を開始した胚数）、Ｈ．ｉｎｇ率（透明帯から脱出を開始した胚の占める占有率）を求めた。その結果を下記［表５］に示す。なお、下記［表５］中、「ＳＴＤ」は標準偏差を示し、「ｎ」は総細胞数を数えた胚盤胞の個数（総細胞数５０未満の胚のデータは含まない）を示す。 And in each of the above cases, 2 cell division number (number of embryos that have developed into 2 cells after fertilization), 2 cell division rate (occupation ratio of embryos that have developed into 2 cells after fertilization), blastocyst arrival number, blastocysts Reach rate, H.R. ing number (Hatching blastocyst, the number of embryos that started to escape from the zona pellucida among the blastocysts that developed), The ing rate (occupation ratio of embryos that started to escape from the zona pellucida) was determined. The results are shown in [Table 5] below. In the following [Table 5], “STD” indicates a standard deviation, and “n” indicates the number of blastocysts obtained by counting the total number of cells (data of embryos having a total number of cells less than 50 is not included).
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　次に、実施例２の第１培養液でマウスの精子と卵子の受精を行い、受精後から第２培養液で培養を行い、受精確認後２４時間目に２細胞期の胚のみを選定し、４８時間目まで第２培養液、４８時間目以降は第３培養液で継続して培養を行い、受精確認後９６時間目に２細胞期胚のうちの胚盤胞数から総胚盤胞率を求めた。 Next, fertilization of mouse sperm and ovum is performed with the first culture solution of Example 2, and after the fertilization, culture is performed with the second culture solution, and only embryos at the 2-cell stage are selected 24 hours after fertilization confirmation. Culturing continuously in the second culture solution until 48 hours and in the third culture solution after 48 hours, and from the number of blastocysts in the 2-cell stage embryos at 96 hours after fertilization confirmation, the total blastocyst The rate was determined.
　一方、比較例１の培養液でマウスの精子と卵子の受精を行い、受精後も同様の培養液で培養を行い、受精確認後２４時間目に２細胞期の胚のみを選定し、さらに同様の培養液で継続して培養を行い、受精確認後９６時間目に２細胞期胚のうちの胚盤胞数から総胚盤胞率を求めた。 On the other hand, fertilization of mouse sperm and ovum was performed with the culture solution of Comparative Example 1, and after the fertilization, culture was performed with the same culture solution, and only the 2-cell stage embryo was selected 24 hours after fertilization was confirmed. The culture was continued in the above culture solution, and the total blastocyst rate was determined from the number of blastocysts in the 2-cell embryo at 96 hours after fertilization was confirmed.
　なお、上記のいずれの場合においても、受精から胚盤胞期までの培養は、インキュベータを用い、５～６％Ｏ２を含む空気の湿度飽和気相の環境下において温度を３７．０℃に設定して行った。 In any of the above cases, 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.
　上記のようにして求めた実施例２及び比較例１の総胚盤胞率を棒グラフにして図１に示す。 The total blastocyst rate of Example 2 and Comparative Example 1 obtained as described above is shown in FIG. 1 as a bar graph.
　また、胚盤胞まで到達した胚に対して低張液にて低張処理を施し、これを固定液でスライドガラス上に固定した。引き続きＤＡＰＩ染色を施した後、蛍光顕微鏡にて観察及び撮影を行った。そして撮影した画像から全ての胚の総細胞数を数えて平均総細胞数を求めた。また、２種の蛍光色素で染色し、平均総細胞数と同様に細胞数を数えて平均ＩＣＭ（内部細胞塊）数を求めた。 Further, 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 | stained with 2 types of fluorescent dyes, the number of cells was counted like the average total cell number, and average ICM (inner cell mass) number was calculated | required.
　上記のようにして求めた実施例２及び比較例１の平均総細胞数を棒グラフにして図２に示す。 The average total cell numbers of Example 2 and Comparative Example 1 obtained as described above are shown as a bar graph in FIG.
　また実施例２及び比較例１について胚盤胞の総細胞数を数えて総細胞数が１００個以上の優良胚盤胞率を求めた。この優良胚盤胞率を棒グラフにして図３に示す。 Further, regarding Example 2 and Comparative Example 1, 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.
　また、実施例２及び比較例１について受胚雌妊娠率及び産仔生産率を求めた。 In addition, for Example 2 and Comparative Example 1, the fertilized female pregnancy rate and offspring production rate were determined.
　具体的には実施例２については、まず第１培養液でマウスの精子と卵子の受精を行い、次に第２培養液で受精後から１６細胞期までのマウス胚を培養した後、第３培養液で桑実期から胚盤胞期までのマウス胚を培養した。なお、受精から胚盤胞期までの培養は、インキュベータを用い、５～６％Ｏ２を含む空気の湿度飽和気相の環境下において温度を３７．０℃に設定して行った。次にこの胚盤胞期のマウス胚をあらかじめ精管結刹を施したＩＣＲオスマウスと自然交尾させ偽妊娠の状態にしたＩＣＲメスマウスの卵管あるいは子宮に移植した。 Specifically, for 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.
　また比較例１については、上記［表１］に示す培養液のみで受精を行うと共に、受精後から胚盤胞期までのマウス胚を培養した。なお、受精から胚盤胞期までの培養は、インキュベータを用い、５～６％Ｏ２を含む空気の湿度飽和気相の環境下において温度を３７．０℃に設定して行った。次にこの胚盤胞期のマウス胚をあらかじめ精管結刹を施したＩＣＲオスマウスと自然交尾させ偽妊娠の状態にしたＩＣＲメスマウスの卵管あるいは子宮に移植した。 In Comparative Example 1, fertilization was performed only with the culture solution shown in [Table 1] above, and mouse embryos from fertilization to the blastocyst stage were cultured. 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.
　そして、胚盤胞期のマウス胚を移植した雌のうち、妊娠成立に至った雌の頭数の割合として受胚雌妊娠率（レシピエントの妊娠率）を求めた。また、移植した胚盤胞期のマウス胚の総数のうち、産まれた匹数の割合として産仔生産率を求めた。 And, among the females transplanted with mouse embryos at the blastocyst stage, the embryo-fertilized female pregnancy rate (recipient pregnancy rate) was determined as the ratio of the number of females that became pregnant. Moreover, the offspring production rate was calculated | required as a ratio of the number of the born babies out of the total number of transplanted blastocyst stage mouse embryos.
　上記のようにして求めた実施例２及び比較例１の受胚雌妊娠率及び産仔生産率を棒グラフにして図４に示す。 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.
　このように、図１～図４にみられるように比較例１に比べて実施例２の培養液の方が質の良い受精卵を得ることができることが確認される。その結果、着床率が向上し、妊娠率を高めることができるものである。また本発明ではヒト由来の血清成分を除外しているので、病原体の侵入・感染を防止することができ、安全性を高めることができるものである。 Thus, as shown in FIGS. 1 to 4, it is confirmed that the culture solution of Example 2 can obtain a fertilized egg with better quality than Comparative Example 1. As a result, the implantation rate is improved and the pregnancy rate can be increased. Further, in the present invention, human-derived serum components are excluded, so that invasion / infection of pathogens can be prevented and safety can be improved.
実施例２及び比較例１について総胚盤胞率を示す棒グラフである。3 is a bar graph showing the total blastocyst rate for Example 2 and Comparative Example 1. 実施例２及び比較例１について平均総細胞数を示す棒グラフである。It is a bar graph which shows the average total cell number about Example 2 and Comparative Example 1. FIG. 実施例２及び比較例１について優良胚盤胞率を示す棒グラフである。It is a bar graph which shows the outstanding blastocyst rate about Example 2 and Comparative Example 1. FIG. 実施例２及び比較例１について受胚雌妊娠率（レシピエントの妊娠率）及び産仔生産率を示す棒グラフである。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. FIG.

Claims (10)

  1. 　ヒトの精子と卵子の受精を行うのに用いられる第１培養液と、受精後から１６細胞期までのヒト胚を培養するのに用いられる第２培養液と、桑実期から胚盤胞期までのヒト胚を培養するのに用いられる第３培養液とを備え、第１～第３培養液は、グルコース、乳酸塩、ピルビン酸塩を含有し、かつ無血清であると共に、グルコース濃度は第１培養液が最も高く、第２培養液が最も低く、乳酸塩濃度及びピルビン酸塩濃度は第３培養液が最も低いことを特徴とするヒト胚の体外受精及び培養用培養液。 A first culture used to fertilize human sperm and eggs, a second culture used to culture human embryos from fertilization to the 16-cell stage, and morula to blastocyst stage A third culture solution used for culturing human embryos up to and including the first to third culture solutions containing glucose, lactate, pyruvate and being serum-free, and having a glucose concentration of A culture medium for in vitro fertilization and culture of human embryo, characterized in that the first culture medium is the highest, the second culture medium is the lowest, and the lactate concentration and pyruvate concentration are the lowest in the third culture solution.
  2. 　第１培養液は、グルコース濃度が２．２±１．１ｍＭ、乳酸塩濃度が２０±１０ｍＭ、ピルビン酸塩濃度が０．３±０．１５ｍＭ、かつ無血清であり、第２培養液は、グルコース濃度が０．２±０．１ｍＭ、乳酸塩濃度が２０±１０ｍＭ、ピルビン酸塩濃度が０．３±０．１５ｍＭ、かつ無血清であり、第３培養液は、グルコース濃度が２．１±１．０５ｍＭ、乳酸塩濃度が１０±５ｍＭ、ピルビン酸塩濃度が０．１５±０．０７５ｍＭ、かつ無血清であることを特徴とする請求項１に記載のヒト胚の体外受精及び培養用培養液。 The first culture solution has a glucose concentration of 2.2 ± 1.1 mM, a lactate concentration of 20 ± 10 mM, a pyruvate concentration of 0.3 ± 0.15 mM, and is serum-free. The glucose concentration is 0.2 ± 0.1 mM, the lactate concentration is 20 ± 10 mM, the pyruvate concentration is 0.3 ± 0.15 mM, and it is serum-free. The in vitro fertilization and culture of a human embryo according to claim 1, characterized in that it is ± 1.05 mM, lactate concentration is 10 ± 5 mM, pyruvate concentration is 0.15 ± 0.075 mM, and it is serum-free. Culture fluid.
  3. 　第１～第３培養液の少なくともいずれかに上皮細胞成長因子及びインスリンが添加されていることを特徴とする請求項１又は２に記載のヒト胚の体外受精及び培養用培養液。 The culture medium for in vitro fertilization and culture of human embryo according to claim 1 or 2, wherein epidermal growth factor and insulin are added to at least one of the first to third culture solutions.
  4. 　第１～第３培養液の少なくともいずれかに完全無血清化のために血清及びアルブミン代替物質としてヒアルロナンが添加されていることを特徴とする請求項１乃至３のいずれか１項に記載のヒト胚の体外受精及び培養用培養液。 The human according to any one of claims 1 to 3, wherein hyaluronan is added as a serum and albumin substitute for complete serum-free in at least one of the first to third culture solutions. Culture fluid for in vitro fertilization and culture of embryos.
  5. 　第１～第３培養液の少なくともいずれかにはリコンビナントヒトアルブミンが添加されていることを特徴とする請求項１乃至４のいずれか１項に記載のヒト胚の体外受精及び培養用培養液。 The in vitro fertilization and culture medium for human embryo according to any one of claims 1 to 4, wherein recombinant human albumin is added to at least one of the first to third culture liquids.
  6. 　グルコース、乳酸塩、ピルビン酸塩を含有し、かつ無血清である第１～第３培養液を用いてヒト胚を体外で培養する方法であって、グルコース濃度が最も高い第１培養液でヒトの精子と卵子の受精を行い、グルコース濃度が最も低い第２培養液で受精後から１６細胞期までのヒト胚を培養し、乳酸塩濃度及びピルビン酸塩濃度が最も低い第３培養液で桑実期から胚盤胞期までのヒト胚を培養することを特徴とするヒト胚の体外受精及び培養方法。 A method for culturing human embryos in vitro using first to third culture solutions containing glucose, lactate, pyruvate and serum-free, wherein the first culture solution having the highest glucose concentration Sperm and eggs are fertilized, human embryos from fertilization to the 16-cell stage are cultured in the second culture solution with the lowest glucose concentration, and mulberry in the third culture solution with the lowest lactate concentration and pyruvate concentration. A method for in vitro fertilization and culturing of human embryos, comprising culturing human embryos from the real stage to the blastocyst stage.
  7. 　第１培養液として、グルコース濃度が２．２±１．１ｍＭ、乳酸塩濃度が２０±１０ｍＭ、ピルビン酸塩濃度が０．３±０．１５ｍＭ、かつ無血清であるものを用い、第２培養液として、グルコース濃度が０．２±０．１ｍＭ、乳酸塩濃度が２０±１０ｍＭ、ピルビン酸塩濃度が０．３±０．１５ｍＭ、かつ無血清であるものを用い、第３培養液として、グルコース濃度が２．１±１．０５ｍＭ、乳酸塩濃度が１０±５ｍＭ、ピルビン酸塩濃度が０．１５±０．０７５ｍＭ、かつ無血清であるものを用いることを特徴とする請求項６に記載のヒト胚の体外受精及び培養方法。 As the first culture solution, a glucose culture with a concentration of 2.2 ± 1.1 mM, a lactate concentration of 20 ± 10 mM, a pyruvate concentration of 0.3 ± 0.15 mM, and a serum-free one is used. As a solution, 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 a serum-free solution are used as a third culture solution. The glucose concentration is 2.1 ± 1.05 mM, the lactate concentration is 10 ± 5 mM, the pyruvate concentration is 0.15 ± 0.075 mM, and serum-free is used. In vitro fertilization and culture method of human embryo.
  8. 　第１～第３培養液の少なくともいずれかに上皮細胞成長因子及びインスリンが添加されていることを特徴とする請求項６又は７に記載のヒト胚の体外受精及び培養方法。 The in vitro fertilization and culture method for human embryos according to claim 6 or 7, wherein epidermal growth factor and insulin are added to at least one of the first to third culture solutions.
  9. 　第１～第３培養液の少なくともいずれかに完全無血清化のために血清及びアルブミン代替物質としてヒアルロナンが添加されていることを特徴とする請求項６乃至８のいずれか１項に記載のヒト胚の体外受精及び培養方法。 The human according to any one of claims 6 to 8, wherein hyaluronan is added as a serum and albumin substitute for complete serum-free in at least one of the first to third culture solutions. Embryo in vitro fertilization and culture method.
  10. 　第１～第３培養液の少なくともいずれかにはリコンビナントヒトアルブミンが添加されていることを特徴とする請求項６乃至９のいずれか１項に記載のヒト胚の体外受精及び培養方法。 10. The in vitro fertilization and culture method for human embryos according to any one of claims 6 to 9, wherein recombinant human albumin is added to at least one of the first to third culture solutions.
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