WO2019240255A1 - 生殖補助医療用培地 - Google Patents
生殖補助医療用培地 Download PDFInfo
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- WO2019240255A1 WO2019240255A1 PCT/JP2019/023628 JP2019023628W WO2019240255A1 WO 2019240255 A1 WO2019240255 A1 WO 2019240255A1 JP 2019023628 W JP2019023628 W JP 2019023628W WO 2019240255 A1 WO2019240255 A1 WO 2019240255A1
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- caprylic acid
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- 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
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0608—Germ cells
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- 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
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- 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
- C12N2500/36—Lipids
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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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
Definitions
- This application relates to a assisted reproduction medical medium and a assisted reproduction medical method using the same.
- the treatment method is stepped up as follows: timing method ⁇ ovulation induction method ⁇ artificial insemination ⁇ in vitro fertilization / microinsemination.
- In vitro fertilization is a method of fertilizing eggs and sperm outside the body.
- Medium used for egg and sperm preculture, sperm, and embryo culture usually contains inorganic salts, energy sources, proteins / polymers, antibiotics, and albumin is widely used as proteins / polymers Has been.
- Albumin can be obtained by purifying plasma or the like, but the obtained albumin is unstable and easily forms a polymer, and it is known that addition of a fatty acid is effective for its stabilization (non-patent document). 1).
- caprylic acid is usually used as a stabilizer for albumin (Non-Patent Document 2, etc.).
- Microinsemination is a method of injecting sperm into an egg under a microscope to achieve fertilization.
- the fertilization rate is improved by microinsemination compared to in vitro fertilization, the lack of the normal fertilization process in which fertilization is performed only with the ability of each sperm and egg, physical damage, decreased embryo development rate, and increased epigenetic disease Problems such as suspicion are pointed out.
- improvement of fertilization rate in other assisted reproduction is required.
- improvement of the fertilization rate is desired in any of the fertilization methods described above.
- the problem of the present application is to provide a method and means for improving the fertilization rate in assisted reproduction.
- the present inventors have used a culture solution prepared using albumin obtained by removing caprylic acid from caprylic acid-added albumin. As a result, the inventors have found that the present invention has been improved. In addition, the present inventors added various fatty acids (not caprylic acid) as stabilizers to albumin from which caprylic acid had been removed, so that in vitro fertilization can be achieved compared to the case of using caprylic acid-added albumin. It has been found that the fertilization rate is improved and has reached the present invention. Furthermore, the present inventors have found that caprylic acid is specifically removed by purifying caprylic acid-added albumin with an ion exchange resin, and have reached the present invention. The present application provides the following.
- the reproductive assistance medicine includes artificial insemination, in vitro fertilization, or microinsemination.
- the reproductive support medicine includes in vitro fertilization.
- the saturated or unsaturated fatty acid having 10 to 20 carbon atoms or a salt thereof is lauric acid, myristic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid, stearic acid, oleic acid, linoleic acid, or a salt thereof
- a medium containing albumin containing low caprylic acid for use in assisted reproduction [9] The medium according to [8], wherein the assisted reproduction includes artificial insemination, in vitro fertilization, or microinsemination. [10] The medium according to [9], wherein the reproductive support medicine includes in vitro fertilization. [11] The medium according to any one of [8] to [10], further containing a saturated or unsaturated fatty acid having 10 to 20 carbon atoms or a salt thereof.
- the saturated or unsaturated fatty acid having 10 to 20 carbon atoms or a salt thereof is lauric acid, myristic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid, stearic acid, oleic acid, linoleic acid, or a salt thereof
- the medium according to any one of [8] to [11] which is selected from the group consisting of a mixture thereof.
- the agent according to any one of [8] to [12] wherein the low-caprylic acid-containing albumin contains 50 ⁇ mol or less of caprylic acid as free form per gram of albumin.
- a method for assisting reproduction which uses a medium containing albumin containing a low amount of caprylic acid.
- An in vitro fertilization method comprising using a medium containing albumin containing a low amount of caprylic acid.
- a method for stabilizing albumin containing low caprylic acid for use in assisted reproduction comprising adding a saturated or unsaturated fatty acid having 10 to 20 carbon atoms or a salt thereof.
- [20] A method for removing caprylic acid from a protein, characterized by using an ion exchange treatment. [21] The method according to [20], wherein the protein is albumin.
- the fertilization rate in assisted reproduction medicine is improved by using a medium containing albumin containing low caprylic acid.
- the present application provides an agent containing low-caprylic acid-containing albumin for use in a medium used for assisted reproduction.
- the agent of the present application can be used, for example, as a raw material for medium production or as a supplement used by appropriately adding to a basal medium for assisted reproduction or the like.
- the present application provides a medium containing albumin containing a low amount of caprylic acid for use in assisted reproduction.
- the present application provides a method for assisting reproduction, which uses a medium containing albumin containing low caprylic acid.
- the “medium” is not particularly limited as long as it can be applied to gametes / embryos and the like.
- the medium in the present application preserves gametes / embryos, etc., for culturing gametes / embryos, etc., for washing gametes / embryos, etc., for suspending gametes / embryos, etc. Can be used for
- assisted reproduction means a treatment method including an operation for artificially manipulating a gamete to fertilize (artificial fertilization) to reach pregnancy.
- artificial fertilization is not necessarily performed, and operations performed for preparation of artificial fertilization (for example, egg collection and ovum cryopreservation) are also included in the assisted reproduction.
- artificial insemination include artificial insemination, in vitro fertilization, and microinsemination.
- the target of assisted reproduction is not particularly limited, but preferably mammals (human and non-human mammals (eg, cows, horses, pigs, dogs, cats, mice, rats, rabbits, monkeys)), and Preferably it is a human.
- assisted reproduction medicine may include treatments performed for industrial purposes such as improvement of production or breed improvement of the mammals.
- fertility-related research using human and non-human mammal gametes can also be included in assisted reproduction.
- artificial insemination means an operation of artificially injecting collected sperm into a female genital organ.
- the collected sperm is usually washed and suspended before injection, but the medium of the present application can also be used as a solution for such washing and suspension.
- in vitro fertilization means an operation in which an egg is fertilized and fertilized outside the body.
- the medium of the present application can be used as a medium for the sperm.
- the medium of the present application can be used as a retentate for embryos / fertilized eggs or the like during transplantation into the uterus, or as a cryopreservation solution for embryos / fertilized eggs.
- sperm with high motility is selected and used from the collected sperm.
- the medium of the present application can be used for washing, sorting, etc. of collected sperm.
- the collected egg and sperm are each pre-cultured before the nymph.
- the medium of the present application can be used for such preculture.
- microinsemination is a method of injecting sperm into an egg under a microscope to achieve fertilization. After fertilization, the cells are cultured for a certain period of time in the same manner as in vitro fertilization, and embryos / fertilized eggs are transplanted into the uterus or cryopreserved.
- the medium of the present application can also be used in such operations. Usually, in microinsemination, sperm selection and preculture, egg preculture, and the like are performed in the same manner as in vitro fertilization. The medium of the present application can also be used in operations accompanying before and after such microinsemination.
- cryopreservation of eggs / sperm in addition to artificial fertilization (artificial fertilization, in vitro fertilization, micro insemination, etc.) or in preparation for artificial fertilization, cryopreservation of eggs / sperm, in vitro embryo culture, embryo / blastocyst Operations such as transplantation, hatching support method, cryopreservation of embryos, in vitro culture of eggs, and the like are performed in an appropriate combination according to the patient's condition and symptoms.
- the medium of the present application can also be used in operations associated with such artificial fertilization.
- whether an egg and a sperm have been fertilized is determined in accordance with a normal standard in the technical field. For example, fertilization can be determined when the second polar body and the male and female pronuclei can be observed.
- the medium containing albumin containing low caprylic acid of the present application is used before fertilization and / or during fertilization in assisted reproduction. Since the medium suitable for fertilization and the medium suitable for embryo development are generally different, the medium is usually exchanged after fertilization.
- the medium containing albumin containing a low amount of caprylic acid of the present application can be used for preculture before fertilization and the like during fertilization and embryo development after fertilization, but is preferably used before fertilization and during fertilization.
- the present application provides an in vitro fertilization method characterized by using a medium containing albumin containing low caprylic acid.
- the in vitro fertilization of the present application can be performed, for example, in infertility treatment or in an experiment related to fertilization.
- low-caprylic acid-containing albumin means albumin that does not contain caprylic acid to the extent that it adversely affects assisted reproduction (eg, fertilization).
- the amount of caprylic acid that can be contained in low-caprylic acid-containing albumin is preferably small in order to improve the fertilization rate.
- the amount of caprylic acid contained in the low-caprylic acid-containing albumin is not particularly limited as long as it does not adversely affect assisted reproduction (for example, fertilization).
- the amount of caprylic acid contained in 1 g of albumin is 50 ⁇ mol or less as a free form, It is preferably 45 ⁇ mol or less, more preferably 40 ⁇ mol or less, still more preferably 30 ⁇ mol or less, still more preferably 20 ⁇ mol or less, even more preferably 15 ⁇ mol or less, and even more preferably 10 ⁇ mol or less.
- caprylic acid (as a free form) per 1 g of albumin is 0.01 to 50 ⁇ mol, more preferably 0.01 to 45 ⁇ mol, still more preferably 0.01 to 40 ⁇ mol, even more Preferred is albumin containing 0.01 to 30 ⁇ mol, still more preferably 0.1 to 20 ⁇ mol, still more preferably 1 to 15 ⁇ mol, and more preferably 2 to 10 ⁇ mol.
- a preferable range of caprylic acid (as a free form) contained in 1 g of albumin is a lower limit selected from 0.01 ⁇ mol, 0.1 ⁇ mol, 1 ⁇ mol, 2 ⁇ mol, and 5 ⁇ mol; and 50 ⁇ mol, 45 ⁇ mol, 40 ⁇ mol, 30 ⁇ mol, 20 ⁇ mol,
- the upper limit value selected from 15 ⁇ mol and 10 ⁇ mol can be used.
- the method for measuring the amount of albumin is not particularly limited, and can be measured by a method commonly used in the technical field.
- the amount of albumin can be measured as protein mass by, for example, the Bradford method.
- the method for measuring caprylic acid is not particularly limited, and can be measured by a method usually used in the technical field.
- the albumin-containing liquid can be extracted (for example, by Bligh-Dyer extraction), and the obtained lipid fraction can be measured by using a gas chromatography mass spectrometer (preferably after trimethylsilylation).
- a preferred method for measuring caprylic acid (as a free form) contained in albumin is exemplified below.
- a solvent for example, water
- albumin for example, so that the protein concentration (for example, by Bradford method) is 5 w / v%), and adjusted to, for example, pH 7 to 8 (preferably pH 7.4).
- the obtained albumin-containing liquid is extracted (for example, Bligh-Dyer extraction), and the obtained lipid fraction (for example, after trimethylsilylation) is subjected to a gas chromatography mass spectrometer to measure the amount of free caprylic acid.
- the low-caprylic acid-containing albumin of the present application may be a naturally occurring (eg, ovalbumin, porcine-derived albumin, bovine-derived albumin, human-derived albumin) albumin, a bovine, porcine, or human-type recombinant.
- the albumin may also be used.
- HSA human serum albumin
- rHSA recombinant human albumin
- the method for producing albumin is not particularly limited and can be produced by a conventional method.
- albumin is produced by purification from plasma or by producing and purifying in yeast or the like using a gene recombination technique.
- the purification method include a low temperature ethanol fractionation method, a heat treatment method, and a chromatographic purification method.
- caprylic acid free form or salt form
- the caprylic acid-containing albumin of the present application can be obtained by removing caprylic acid.
- the low-caprylic acid-containing albumin used in the present application is purified by a low-temperature ethanol fractionation method (for example, Fraction IV of corn) or a heat treatment method, preferably a low-temperature ethanol fractionation method, and caprylic acid Albumin from which caprylic acid has been removed from albumin to which (free form or salt form) has been added can be used.
- a low-temperature ethanol fractionation method for example, Fraction IV of corn
- a heat treatment method preferably a low-temperature ethanol fractionation method
- caprylic acid Albumin from which caprylic acid has been removed from albumin to which (free form or salt form) has been added can be used.
- albumin examples include the following. Caprylic acid can be appropriately removed and used as the low-caprylic acid-containing albumin of the present application.
- Recombinant human albumin Cell Prime rAlbumin AF-s (Merck)
- Human plasma-derived albumin Human Serum Albumin Solution (Irvine Scientific), HUMAN SERUM ALBUMIN (InVitroCare)
- the method for removing caprylic acid is not particularly limited, and a method usually used for removing fatty acid (for example, a method using activated carbon) can be used. Or the caprylic acid low content albumin of this application can be obtained by removing the caprylic acid added artificially using the caprylic acid removal method using the ion exchange process described below.
- the present application provides a method for specifically removing caprylic acid from a protein (eg, albumin) using ion exchange treatment.
- a protein eg, albumin
- fatty acids may remain after purification, and the method of the present application can be used to remove caprylic acid from a biological protein.
- the methods of the present application can also be used to remove caprylic acid from proteins that have been artificially supplemented with caprylic acid (free or salt form).
- the method of ion exchange treatment used in the caprylic acid removal method of the present application is not particularly limited, but it is preferable that both cation exchange treatment and anion exchange treatment are performed.
- the cation exchange treatment may be a weak acid cation exchange treatment (for example, using a carboxylic acid group as an exchange group) or a strong acid cation exchange treatment (for example, using a sulfonic acid group as an exchange group). Is preferably a strongly acidic cation exchange treatment (for example, using a sulfonic acid group as an exchange group).
- the ion form of the exchange group is not particularly limited, and examples thereof include hydrogen, sodium salt, potassium salt and the like, and preferably hydrogen is used.
- the cation exchange treatment can be performed according to a usual method using, for example, a cation exchange resin (for example, a weak acid cation exchange resin or a strong acid cation exchange resin).
- the anion exchange treatment is a weak basic anion exchange treatment (for example, a primary to tertiary amino group is used as an exchange group), a strong basic anion exchange treatment (for example, quaternary ammonium is used as an exchange group). However, it is preferably a strong basic anion exchange treatment. Examples of the quaternary ammonium include a trimethylammonium group and a dimethylethanolammonium group, and a trimethylammonium group is preferably used.
- the ion form of the exchange group is not particularly limited, and examples thereof include chloride, hydroxide, acetate, and formate, and hydroxide is preferably used.
- the anion exchange treatment can be performed according to a normal method using, for example, an anion exchange resin (for example, a weakly basic anion exchange resin or a strong basic anion exchange resin).
- the base of the ion exchange resin is not particularly limited, and those usually used as the base of the ion exchange resin (for example, styrenedivinylbenzene) can be used.
- mixed bed resins containing both cation exchange resins and anion exchange resins for example, AG 501-X8 (D) (BIO-RAD), and AG 501-X8 ( BIO-RAD) is preferably used.
- an ion exchange resin and a protein (albumin) that may contain caprylic acid are mixed in an appropriate solvent (eg, water) (preferably, at a low temperature (eg, 1 to 10 ° C., preferably 2 To 6 degrees) under light shielding, for example, for 1 to 48 hours, preferably 3 to 36 hours, more preferably 5 to 30 hours.
- an appropriate solvent eg, water
- a low temperature eg, 1 to 10 ° C., preferably 2 To 6 degrees
- light shielding for example, for 1 to 48 hours, preferably 3 to 36 hours, more preferably 5 to 30 hours.
- the obtained protein-containing solution can be used as it is, or by appropriately performing protein stabilization techniques known in the art, such as pH adjustment and addition of stabilizers, and can be used as a protein containing low caprylic acid. .
- the obtained protein-containing solution may be concentrated by lyophilization or ultrafiltration. Moreover, you may remove a virus, bacteria, etc. by filtration. In this case as well, other stabilization techniques for proteins known in the art may be applied as appropriate.
- the low-caprylic acid-containing albumin of the present application may be albumin to which caprylic acid is not artificially added.
- low-caprylic acid-containing albumin is albumin purified by a low-temperature ethanol fractionation method (for example, Fraction V of Corn) or a heat treatment method, preferably a low-temperature ethanol fractionation method, and caprylic acid is artificially added.
- Non-albumin Since albumin purified by these purification methods may be unstable, it is preferable to use it with appropriate stabilization techniques.
- Albumin is a protein having a high ability to bind to various substances.
- serum-derived albumin is bound to various substances contained in serum. Therefore, even if no caprylic acid is artificially added, albumin may carry caprylic acid derived from a living body, but may be contained in an amount that does not adversely affect assisted reproduction (eg fertilization). .
- caprylic acid is removed by a conventional method such as a caprylic acid removal method using the ion exchange treatment or a method using activated carbon of the present application. Can be used with stabilization technology.
- the amount of low-caprylic acid-containing albumin contained in the medium is not particularly limited, and can be appropriately selected within a range that is usually used according to the purpose of use of the medium in assisted reproduction medicine.
- the concentration of low-caprylic acid-containing albumin contained in the medium is 0.001 to 5 w / v%, preferably 0.01 to 3 w / v%, more preferably 0.05 to 2 w / v%, even more Preferably, it may be 0.1-1 w / v%.
- a preferable range of the amount of low-caprylic acid-containing albumin contained in the medium is selected from 0.001 w / v%, 0.01 w / v%, 0.05 w / v%, and 0.1 w / v% A lower limit; and an upper limit selected from 5 w / v%, 3 w / v%, 2 w / v%, and 1 w / v%.
- an agent containing low-caprylic acid-containing albumin for use in a medium used for assisted reproduction of the present application and low-caprylic acid-containing albumin for use in assisted reproduction of the present application
- Each of the contained media may further contain a saturated or unsaturated fatty acid having 10 to 20 carbon atoms or a salt thereof.
- assisted reproduction for example, fertilization rate
- albumin is stabilized compared with the case of using caprylic acid-added albumin. (For example, prevention of polymer formation).
- the low-caprylic acid-containing albumin and the fatty acid or salt thereof are intimately mixed and mixed with other optional components so as to provide efficient albumin stabilization. Before the preparation, it is preferable that the low-caprylic acid-containing albumin and the fatty acid or salt thereof are mixed.
- the present application provides a method for stabilizing albumin containing low caprylic acid for use in assisted reproduction, which comprises adding a saturated or unsaturated fatty acid having 10 to 20 carbon atoms or a salt thereof. To do.
- albumin can be stabilized (for example, prevention of polymer formation) while improving the effect (for example, fertilization rate) of assisted reproduction medicine compared with the case of using caprylic acid-added albumin.
- a mixture containing the fatty acid or a salt thereof and low-caprylic acid-containing albumin may be formed during assisted reproduction.
- the fatty acid or salt thereof and low caprylic acid-containing albumin may be mixed prior to assisted reproduction, or the fatty acid and low caprylic acid-containing albumin may be mixed during assisted reproduction. It is preferable that albumin is stabilized early, and it is preferable that the fatty acid or a salt thereof is added promptly after production of low-caprylic acid-containing albumin.
- the saturated or unsaturated fatty acid having 10 to 20 carbon atoms or a salt thereof improves the effect of assisted reproduction (for example, fertilization rate) and stabilizes albumin (for example, heavy weight) compared with the case of using caprylic acid-added albumin. There is no particular limitation as long as it can prevent formation of coalescence.
- the saturated or unsaturated fatty acid having 10 to 20 carbon atoms or a salt thereof may be a single fatty acid and / or a salt thereof, or a mixture of a plurality of fatty acids and / or a salt thereof.
- Preferred examples of the fatty acid or salt thereof include lauric acid, myristic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid, stearic acid, oleic acid, linoleic acid, their salts, and mixtures thereof. More preferably, the mixture contains at least palmitic acid or a salt thereof. Further preferred examples include lauric acid, myristic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid, stearic acid, their salts, and mixtures thereof. Particularly preferred examples include palmitic acid and its salts.
- the salt of the saturated or unsaturated fatty acid having 10 to 20 carbon atoms is a physiologically acceptable salt, for example, an alkali metal salt such as sodium salt or potassium salt; an alkaline earth such as calcium salt, magnesium salt or barium salt Metal salts; basic amino acid salts such as arginine and lysine; ammonium salts such as ammonium salt and tricyclohexylammonium salt; monoethanolamine salt, diethanolamine salt, triethanolamine salt, monoisopropanolamine salt, diisopropanolamine salt and triisopropanol Various alkanolamine salts such as amines are preferable. More preferred examples of the fatty acid salts of the present invention include alkali metal salts, alkaline earth metal salts, and mixtures thereof.
- the amount of the saturated or unsaturated fatty acid having 10 to 20 carbon atoms or a salt thereof used in the present application improves the effect of assisted reproduction (for example, fertilization rate) compared to the case of using caprylic acid-added albumin,
- the amount is not particularly limited as long as albumin can be stabilized (for example, prevention of polymer formation).
- the fatty acid or salt thereof is contained in a free form of 10 to 100 ⁇ mol, preferably 20 to 80 ⁇ mol, more preferably 30 to 50 ⁇ mol per 1 g of albumin.
- the preferred range of the amount of the fatty acid or salt thereof per gram of albumin is a lower limit selected from the range of 5 ⁇ mol to 100 ⁇ mol, for example, 5 ⁇ mol, 10 ⁇ mol, 20 ⁇ mol, and 30 ⁇ mol as free form; and 100 ⁇ mol, 90 ⁇ mol, 80 ⁇ mol , 70 [mu] mol, 60 [mu] mol, and 50 [mu] mol.
- the form of the agent containing albumin containing low caprylic acid albumin for use in the medium used for assisted reproduction of the present application is not particularly limited.
- a liquid mixed with an appropriate solvent for example, water, buffer
- It can be a solid (appropriately prepared to a preferred pH (for example, pH 7 to 8)), freeze-dried or the like.
- the agent of the present application may contain a known additive as a medium component as long as it does not adversely affect assisted reproduction (for example, fertilization).
- inorganic salts NaCl, KCl, MgSO 4 , KH 2 PO 4 , CaCl 2 , NaHCO 3 etc.
- saccharides eg glucose etc.
- organic acids eg pyruvic acid, lactic acid etc.
- amino acids for example, L-glutamine and the like
- vitamins for example, ascorbic acid and the like
- antibiotics for example, gentamicin and the like
- cultivation etc. in reproduction assistance medical treatment can also be included suitably.
- Additives are preferably included in known concentration ranges as long as they do not adversely affect fertilization, product stability, and the like.
- the medium containing the low-caprylic acid-containing albumin for use in assisted reproduction of the present application does not contain any component capable of supplying caprylic acid other than the low-caprylic acid-containing albumin.
- the amount of caprylic acid that can be contained in the medium is not particularly limited as long as it does not adversely affect assisted reproduction (for example, fertilization).
- the amount of caprylic acid contained per 1000 g of the medium is 2500 ⁇ mol or less as a free form, It is preferably 2200 ⁇ mol or less, more preferably 2000 ⁇ mol or less, further preferably 1800 ⁇ mol or less, still more preferably 1500 ⁇ mol or less, even more preferably 1400 ⁇ mol or less, and even more preferably 1300 ⁇ mol or less.
- caprylic acid (as a free form) contained in 1000 g of the medium, 0.01 to 2500 ⁇ mol, preferably 0.1 to 2200 ⁇ mol, more preferably 0.5 to 2000 ⁇ mol, even more preferably 1 to 1800 ⁇ mol, Still more preferably, it is 2 to 1500 ⁇ mol, more preferably 3 to 1400 ⁇ mol, and still more preferably 5 to 1300 ⁇ mol.
- the preferred range of the amount of caprylic acid (as free form) contained in 1000 g of medium is a lower limit selected from 0.01 ⁇ mol, 0.1 ⁇ mol, 0.5 ⁇ mol, 1 ⁇ mol, 2 ⁇ mol, 3 ⁇ mol, 4 ⁇ mol, and 5 ⁇ mol; and
- the upper limit value selected from 2500 ⁇ mol, 2000 ⁇ mol, 1800 ⁇ mol, 1500 ⁇ mol, 1400 ⁇ mol, 1300 ⁇ mol, 1200 ⁇ mol, 1100 ⁇ mol, and 1000 ⁇ mol;
- the shape of the medium containing albumin containing low caprylic acid-containing albumin for use in the assisted reproduction of the present application is not particularly limited, and a solid medium (such as an agar medium), a liquid medium, or a powder medium (for example, dissolved in water as a liquid medium) Etc.) and the like, and can be produced using a conventional method.
- the medium of the present application may contain a known additive in a known concentration range as a medium component, as long as it does not adversely affect fertilization, as with the agent of the present application.
- the medium containing albumin containing low caprylic acid for use in the assisted reproduction of the present application is a known medium known for use in assisted reproduction (eg, HTF medium, KSOM AA medium, HFF99 medium, HiGROW IVF medium). It can be produced by adding albumin containing low caprylic acid.
- kits in another aspect of the present application, includes an agent or medium of the present application and instructions for their use in assisted reproduction.
- the form of the explanation is not particularly limited, and may be an instruction manual or access information (URL, QR code, etc.) for browsing the contents of the explanation on the Internet.
- Test example 1 Caprylic acid removal by ion exchange treatment
- caprylic acid could be specifically removed from the free fatty acid contained in the albumin solution by performing the ion exchange treatment.
- Test Example 2 Fertilization inhibition ability of caprylic acid 2.1 Preparation of Medium
- an albumin solution (Example 2 + 2000 caprylic acid, Example 2 + 4000 caprylic acid) supplemented with 4,000 ⁇ mol / L sodium caprylate was prepared and added to the HTF medium at 0.5 w / v%. 200 ⁇ L of these media were overlaid with mineral oil and equilibrated overnight under conditions of 37 ° C. and 6 v / v% CO 2 and used for sperm preculture and in vitro fertilization.
- sperm collected in 2.2 was introduced into the medium prepared in 2.1 so that the concentration was 2 ⁇ 10 6 mice / mL, and pre-cultured at 37 ° C. and 6v / v% CO 2 for 1 hour until in vitro fertilization. .
- Example 3 Stabilization of albumin from which caprylic acid was removed Albumin from which caprylic acid was removed was effective in improving the in vitro fertilization rate, but on the other hand, it was unstable and easily polymerized by heating. Therefore, fatty acids other than caprylic acid were added to Example 2 to examine the concentration at which polymerization can be suppressed under heating conditions.
- Palmitic acid, stearic acid, oleic acid, and linoleic acid are adjusted to 1 mg / mL with ethanol, and 19.2 ⁇ L of palmitic acid, 21.3 ⁇ L of stearic acid, 21.2 ⁇ L of oleic acid, and 21.0 ⁇ L of linoleic acid are contained in a container. Then, nitrogen gas was blown to dryness. 100 ⁇ L of the albumin of Example 2 was added thereto and stirred at 37 ° C. for 2 hours to prepare albumin to which four kinds of mixed free fatty acids (FFA MIX) having a total concentration of 3000 ⁇ mol / L were added.
- FFA MIX mixed free fatty acids
- Test Example 4 Fertilization rate of albumin added with various free fatty acids 2000 ⁇ mol / L of various free fatty acids were added to 5 w / v% albumin of Example 2 in the same manner as in Test Example 3 (Table 3) and added to the HTF medium. After addition to 0.5 w / v% albumin, examination was performed under the same conditions as the in vitro fertilization experiment described in Test Example 2. The obtained results are shown in FIG.
- Test Example 5 Concentration of palmitate-added albumin in culture medium
- Example 5 shows the result of comparison with the case where albumin of Comparative Example 2 was added to the HTF medium at 0.5 w / v%.
- the results for 0.5 w / v% (10-fold dilution, Example 6) are as shown in FIG.
- Test Example 6 Addition concentration of palmitic acid to albumin Double amount (4000 ⁇ mol / L) of palmitic acid as in Example 6 was added to Example 2 and 0.5 w / v% (10-fold dilution, Example 17), 0.25 After adding w / v% (20-fold dilution, Example 18) and 0.125 w / v% (40-fold dilution, Example 19) to the HTF medium, study under the same conditions as in vitro fertilization experiments described in Test Example 2 Went.
- FIG. 6 shows the result of comparison with the case where the albumin of Comparative Example 2 was added to the HTF medium at 0.5 w / v%.
- the albumin of Example 17 to which 4000 ⁇ mol / L of palmitic acid was added after the ion exchange treatment had all medium concentrations (0.125 w / v%-0.5 w / v% (10-fold-40-fold dilution)) ) Produced an effect of improving the fertilization rate as compared with the albumin of Comparative Example 2.
- Fertilization rate can be improved by using the agent or medium of the present application.
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Abstract
Description
加えて、良好な不妊治療結果を得るために、上記のいずれの受精方法においても、受精率の向上が望まれている。
本願は下記を提供する。
[2] 該生殖補助医療が人工授精、体外受精、または顕微授精を含む、[1]に記載の剤。
[3] 該生殖補助医療が体外受精を含む、[2]に記載の剤。
[4] 炭素数10~20の飽和または不飽和脂肪酸またはその塩をさらに含有する、[1]~[3]のいずれか1項に記載の剤。
[5] 該炭素数10~20の飽和または不飽和脂肪酸またはその塩が、ラウリン酸、ミリスチン酸、ペンタデカン酸、パルミチン酸、パルミトレイン酸、マルガリン酸、ステアリン酸、オレイン酸、リノール酸、それらの塩、およびそれらの混合からなる群から選択される、[1]~[4]のいずれか1項に記載の剤。
[6] 該カプリル酸低含有アルブミンが、アルブミン1gあたりカプリル酸を遊離形態として50μmol以下含む、[1]~[5]のいずれか1項に記載の剤。
[7] 生殖補助医療に使用される培地に使用するための、カプリル酸低含有アルブミンを含有する剤;および
該剤を生殖補助医療に使用される培地に使用することの説明;
を含む、生殖補助医療における培地用キット。
[9] 該生殖補助医療が人工授精、体外受精、または顕微授精を含む、[8]に記載の培地。
[10] 該生殖補助医療が体外受精を含む、[9]に記載の培地。
[11] 炭素数10~20の飽和または不飽和脂肪酸またはその塩をさらに含有する、[8]~[10]のいずれか1項に記載の培地。
[12] 該炭素数10~20の飽和または不飽和脂肪酸またはその塩が、ラウリン酸、ミリスチン酸、ペンタデカン酸、パルミチン酸、パルミトレイン酸、マルガリン酸、ステアリン酸、オレイン酸、リノール酸、それらの塩、およびそれらの混合からなる群から選択される、[8]~[11]のいずれか1項に記載の培地。
[13] 該カプリル酸低含有アルブミンが、アルブミン1gあたりカプリル酸を遊離形態として50μmol以下含む、[8]~[12]のいずれか1項に記載の剤。
[14] 生殖補助医療に使用するための、カプリル酸低含有アルブミンを含有する培地;および
該培地を生殖補助医療に使用することの説明;
を含む、生殖補助医療における培地用キット。
[16] カプリル酸低含有アルブミンを含有する培地を使用することを特徴とする、インビトロ受精方法。
[18] カプリル酸低含有アルブミンおよび炭素数10~20の飽和または不飽和脂肪酸またはその塩の存在下に、生殖補助医療に使用される培地を製造する、該培地の製造方法。
[21] 該タンパク質がアルブミンである、[20]に記載の方法。
受精に適する培地と胚発生に適する培地は一般に異なるため、通常受精後には培地は交換される。本願のカプリル酸低含有アルブミンを含有する培地は、受精前の前培養等に、受精時、および受精後の胚発生時に使用されうるが、好ましくは受精前および受精時に使用される。
(1)アルブミンに、溶媒(例えば水)を加え(例えば、蛋白質濃度(例えばブラッドフォード法による)として5w/v%となるように)、適宜例えばpH7~8(好ましくはpH7.4)に調整する。
(2)得られたアルブミン含有液を抽出(例えばBligh-Dyer抽出)し、得られた脂質分画を(例えばトリメチルシリル化後に)ガスクロマトグラフィー質量分析計に供して遊離カプリル酸量を測定する。
組換えヒトアルブミン:CellPrime rAlbumin AF-s(Merck)
ヒト血漿由来アルブミン:Human Serum Albumin Solution(Irvine Scientific)、HUMAN SERUM ALBUMIN(InVitroCare)
本発明のより好ましい当該脂肪酸の塩の例としては、アルカリ金属塩、アルカリ土類金属塩、およびその混合が挙げられる。
あらかじめ超純水で洗浄したイオン交換樹脂(AG 501-X8 (D), BIO-RAD)とヒト血清アルブミン(HSA, Irvine Scientific)もしくは10%遺伝子組換えヒトアルブミン(rHA, Merck)を混合後、ローテーター(RT50, TAITEC)を用いて4℃遮光下で24時間穏やかに撹拌した。処理後のアルブミン溶液を回収し、ブラッドフォード法により蛋白質濃度を定量し、5w/v%(pH7.4)となるよう超純水と2mol/L NaOHで調製した。
処理前のHSA(比較例1)とrHA(比較例2)、処理後のHSA(実施例1)とrHA(実施例2)をそれぞれBligh-Dyer抽出し、得られた脂質分画をトリメチルシリル化後ガスクロマトグラフィー質量分析計(7890A GC & 5975C GC/MSD, Agilent)に供し遊離脂肪酸濃度を測定した。得られた結果を表1に示す。
2.1 培地の調製
前記表1で示した4種のアルブミン溶液、および実施例1に4,000μmol/Lのカプリル酸ナトリウムを添加したアルブミン溶液(実施例1+4000カプリル酸)、もしくは実施例2に2,000μmol/L、4,000μmol/Lのカプリル酸ナトリウムを添加したアルブミン溶液(実施例2+2000カプリル酸、実施例2+4000カプリル酸)を作製し、HTF培地に0.5w/v%となるよう添加した。これらの培地200μLにミネラルオイルを重層し、37℃、6v/v%CO2条件下で一晩平衡化したものを精子の前培養と体外受精に使用した。
C57BL/6N系雄性マウス(日本SLC)を屠殺後、精巣上体尾部を切り出した。その精巣上体尾部中央の精巣上体管を切開して得られた精子塊を、ミネラルオイル下に作製した0.1w/v%ポリビニルアルコール(PVA)含有HTF培地200μL中に採取した。採取した精子塊を37℃、6v/v%CO2条件下で10分間培養し精子を均一に分散させた。
2.1で調製した培地に2.2で回収した精子を2×106匹/mLとなるよう導入し、体外受精まで37℃、6v/v%CO2条件下で1時間前培養した。
C57BL/6N系雌性マウス(日本SLC)に、PMSG(妊馬血清性性腺刺激ホルモン、あすか製薬、セロトロピン(商標))を7.5国際単位腹腔内に投与し、48時間後にhCG(ヒト絨毛性性腺刺激ホルモン、あすか製薬、ゴナトロピン(商標))を7.5国際単位腹腔内に投与した。hCG投与15時間後に当該マウスを屠殺し、すぐに卵管を切り出した。卵管膨大部から卵子-卵丘細胞集合体を、2.1で調製した培地に回収した。回収した卵子-卵丘細胞集合体は、体外受精まで37℃、6v/v%CO2条件下で培養した。
2.4で準備した卵子-卵丘細胞集合体の入っている培地に2.3で準備した精子を100匹/μLとなるように加えた。37℃、6v/v%CO2条件下で6時間媒精した後、第2極体および雌雄前核が観察できた卵を受精卵と判定し、以下の式に当てはめ受精率を算出した。
受精率(%) = 受精卵/正常な形態の卵子数×100
得られた結果を図1、図2に示す。
カプリル酸を除去したアルブミンは体外受精率の向上に有効であったが、その半面、不安定で加温によって容易に重合化する。そこで、カプリル酸以外の脂肪酸を実施例2に加え、加温条件下で重合体化を抑制できる濃度を検討した。
パルミチン酸、ステアリン酸、オレイン酸、リノール酸をエタノールで1mg/mLに調製し、パルミチン酸 19.2μL、ステアリン酸 21.3μL、オレイン酸 21.2μL、リノール酸 21.0μLを容器にとり、窒素ガスを吹き付け乾固させた。そこに実施例2のアルブミンを100μL加え37℃で2時間撹拌することにより総濃度3000μmol/Lの4種混合遊離脂肪酸(FFA MIX)を付加したアルブミンを作製した。0.2μmのシリンジフィルターでろ過滅菌し、使用時まで4℃遮光下で保存した。同様の方法で、表2に示す濃度のFFA MIXを実施例2のアルブミンに付加し試験に供した。
表2の(1) - (8)を37℃で7日間保管し、0.5w/v%に希釈後、9μLを非変性ポリアクリルアミドゲル電気泳動(Native PAGE)用 sample buffer 3μLと混合し、7.5w/v%ゲルに10μLずつアプライし、Native PAGEを行った。クマシーブリリアントブルーで蛋白質を染色し、アルブミンの重合の有無を評価した。結果を図3に示す。
2000μmol/Lの各種遊離脂肪酸を実施例2の5w/v%アルブミンに試験例3と同様の方法で付加し(表3)、HTF培地に0.5w/v%アルブミンとなるよう添加後、試験例 2に記載の体外受精実験と同様の条件で検討を行った。得られた結果を図4に示す。
試験例4において最も受精率の高かった実施例6のパルミチン酸付加アルブミンを、0.5w/v%(10倍希釈、実施例6)、0.25w/v%(20倍希釈、実施例13)、0.125w/v%(40倍希釈、実施例14)、0.0625w/v%(80倍希釈、実施例15)、0.03125w/v%(160倍希釈、実施例16)でHTF培地に添加し、試験例2に記載の体外受精実験と同様の条件で検討を行った。比較例2のアルブミンを0.5w/v%でHTF培地に添加した場合と比較した結果を図5に示す。なお、0.5w/v%(10倍希釈、実施例6)の結果は図4に示すとおり。
実施例6の倍量(4000μmol/L)のパルミチン酸を実施例2に付加し、0.5w/v%(10倍希釈、実施例17)、0.25w/v%(20倍希釈、実施例18)、0.125w/v%(40倍希釈、実施例19)でHTF培地に添加後、試験例 2に記載の体外受精実験と同様の条件で検討を行った。比較例2のアルブミンを0.5w/v%でHTF培地に添加した場合と比較した結果を図6に示す。
Claims (19)
- 生殖補助医療に使用される培地に使用するための、カプリル酸低含有アルブミンを含有する剤。
- 該生殖補助医療が人工授精、体外受精、または顕微授精を含む、請求項1に記載の剤。
- 該生殖補助医療が体外受精を含む、請求項2に記載の剤。
- 炭素数10~20の飽和または不飽和脂肪酸またはその塩をさらに含有する、請求項1~3のいずれか1項に記載の剤。
- 該炭素数10~20の飽和または不飽和脂肪酸またはその塩が、ラウリン酸、ミリスチン酸、ペンタデカン酸、パルミチン酸、パルミトレイン酸、マルガリン酸、ステアリン酸、オレイン酸、リノール酸、それらの塩、およびそれらの混合からなる群から選択される、請求項1~4のいずれか1項に記載の剤。
- 生殖補助医療に使用される培地に使用するための、カプリル酸低含有アルブミンを含有する剤;および
該剤を生殖補助医療に使用される培地に使用することの説明;
を含む、生殖補助医療における培地用キット。 - 生殖補助医療に使用するための、カプリル酸低含有アルブミンを含有する培地。
- 該生殖補助医療が人工授精、体外受精、または顕微授精を含む、請求項7に記載の培地。
- 該生殖補助医療が体外受精を含む、請求項8に記載の培地。
- 炭素数10~20の飽和または不飽和脂肪酸またはその塩をさらに含有する、請求項7~9のいずれか1項に記載の培地。
- 該炭素数10~20の飽和または不飽和脂肪酸またはその塩が、ラウリン酸、ミリスチン酸、ペンタデカン酸、パルミチン酸、パルミトレイン酸、マルガリン酸、ステアリン酸、オレイン酸、リノール酸、それらの塩、およびそれらの混合からなる群から選択される、請求項7~10のいずれか1項に記載の培地。
- 生殖補助医療に使用するための、カプリル酸低含有アルブミンを含有する培地;および
該培地を生殖補助医療に使用することの説明;
を含む、生殖補助医療における培地用キット。 - カプリル酸低含有アルブミンを含有する培地を使用することを特徴とする、生殖補助医療方法。
- カプリル酸低含有アルブミンを含有する培地を使用することを特徴とする、インビトロ受精方法。
- カプリル酸低含有アルブミンおよび炭素数10~20の飽和または不飽和脂肪酸またはその塩の存在下に、生殖補助医療に使用される培地に使用するための剤を製造する、該剤の製造方法。
- カプリル酸低含有アルブミンおよび炭素数10~20の飽和または不飽和脂肪酸またはその塩の存在下に、生殖補助医療に使用される培地を製造する、該培地の製造方法。
- 炭素数10~20の飽和または不飽和脂肪酸またはその塩を添加することを特徴とする、生殖補助医療に使用されるためのカプリル酸低含有アルブミンの安定化方法。
- イオン交換処理を用いることを特徴とする、タンパク質からカプリル酸を除去する方法。
- 該タンパク質がアルブミンである、請求項18に記載の方法。
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JP7364560B2 (ja) | 2023-10-18 |
EP3808837A1 (en) | 2021-04-21 |
CN112368368A (zh) | 2021-02-12 |
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