JP7240659B2 - Spermatogenic induction medium - Google Patents

Description

The present application relates to a spermatogenesis-inducing medium, a spermatogenesis-inducing method using the same, and the like.

Sato et al. (Non-Patent Document 1) used KnockOut ^(trademark) Serum Replacement (KSR) (Gibco ^(trademark) ) or bovine serum as a serum substitute in a testicular organ culture method for culturing neonatal mouse testes on an agarose gel. They reported successful differentiation from spermatogonial stem cells to complete spermatozoa using AlbuMAX ^™ (Gibco ^™ ), an albumin-derived preparation. However, the production methods and components of these products have not been completely elucidated, and research continues on chemically-defined media (CDM) with a clear chemical composition. Sanjo et al. (Non-Patent Document 2) succeeded in in vitro spermatogenesis using CDM, using mouse testis tissue after 4.5 days of age to differentiate into round spermatids. In the process, we discovered that albumin is one of the important ingredients for the testicular organ culture method in CDM. However, differentiation to complete spermatozoa has not been obtained with CDM, and further research is ongoing.
Albumin can be obtained by purifying plasma or the like, but the albumin thus obtained tends to form polymers and is unstable. 3, etc.). Among fatty acids, caprylic acid is commonly used as an albumin stabilizer (Non-Patent Document 4, etc.).

In vitro production of functional sperm in cultured neonatal mouse testes, T. Sato, Nature 471, 504-507, 2011 In vitro mouse spermatogenesis with an organ culture method in chemically defined medium, H. Sanjo, PLoS One. 2018 Feb 12;13(2):e0192884. doi: 10.1371/journal.pone.0192884. Biochim. Biophys. Acta (2004) 1702(1): 9-17. U.S. Pharmacopoeia Albumin Human

The object of the present application is to provide methods and means for improving spermatogenesis induction in vitro.

The present inventors conducted intensive studies to solve the above problems, and found that since caprylic acid inhibits the induction of spermatogenesis, the induction of spermatogenesis was improved by using albumin with a low caprylic acid content in testis organ culture. The inventors have found that and have completed the present invention.
The present application provides:

[1] An agent containing caprylic acid-low content albumin for use in a medium used for in vitro spermatogenesis induction.
[2] The agent of [1], wherein the in vitro spermatogenesis induction comprises testicular organ culture.
[3] The agent according to [1] or [2], wherein the low caprylic acid content albumin contains 70 μmol or less of caprylic acid in free form per 1 g of albumin.

[4] an agent containing albumin with low caprylic acid content for use in a medium used for in vitro spermatogenesis induction; and a description of the use of the agent in a medium used for in vitro spermatogenesis induction;
A kit for media in in vitro spermatogenesis induction, comprising:
[5] A medium containing caprylic acid-low albumin for use in in vitro spermatogenesis induction.
[6] The medium of [5], wherein the in vitro spermatogenesis induction comprises testicular organ culture.
[7] The agent according to [5] or [6], wherein the albumin with low caprylic acid content contains 70 μmol or less of free form caprylic acid per 1 g of albumin.
[8] A medium containing low caprylic acid albumin for use in in vitro spermatogenesis induction; and a description of the use of said medium for in vitro spermatogenesis induction;
A kit for media in in vitro spermatogenesis induction, comprising:
[9] A method for inducing spermatogenesis in vitro, characterized by using a medium containing albumin with a low caprylic acid content.

According to the present invention, in vitro induction of spermatogenesis is improved by using a medium containing albumin with low caprylate content.

Morphology (top) and fluorescence image (bottom) of cultured testicular tissue. "Before treatment" means CDM containing albumin before purification treatment of Test Example 2. "Post-treatment" means CDM containing albumin after purification treatment of Test Example 2. “Post-treatment +1000” means CDM containing albumin and 1000 μmol/L caprylic acid after purification treatment of Test Example 2. "Post-treatment +3000" means CDM containing albumin and 3000 µmol/L caprylic acid after purification treatment in Test Example 2. “Post-treatment +5000” means CDM containing albumin and 5000 μmol/L caprylic acid after purification treatment in Test Example 2. Spermatogenesis-inducing ability of testis tissue after culture. Average + S.E. Relationship between caprylic acid concentration and ability to induce spermatogenesis. Average + S.E.

In one aspect, the present application provides an agent containing caprylate-low albumin for use in media used for in vitro spermatogenesis induction. The agent of the present application can be used, for example, as a raw material for medium production, or as a supplement that is appropriately added to a basal medium for inducing spermatogenesis in vitro.

In another aspect, the present application provides a medium containing low caprylate albumin for use in in vitro spermatogenesis induction.

In still another aspect, the present application provides a method for inducing spermatogenesis in vitro, characterized by using a medium containing albumin with a low caprylic acid content.

In the present application, the term "medium" is not particularly limited as long as it can be applied to cells or the like in spermatogenesis induction. For example, the medium in the present application can be used for culturing cells, washing cells, suspending cells, and preserving cells.

In the present application, spermatogenic induction means complete or partial induction of a series of steps seen in the process of sperm formation from stem cells (eg, spermatogonial stem cells, ES cells, iPS cells).

In the present application, in vitro spermatogenesis induction means spermatogenesis induction performed in vitro. In vitro spermatogenesis induction may be performed in combination with in vivo spermatogenesis induction. Examples of in vitro spermatogenesis induction include testicular organ culture.

Testis tissue usually contains spermatogonial progenitor cells such as spermatogonial stem cells, spermatogonia, spermatocytes and spermatids. Testicular organ culture is a method of culturing testicular tissue pieces in vitro to induce spermatogenesis. The testis organ culture method is not particularly limited, and can be carried out by a conventional method in the technical field. For example, testicular tissue pieces can be cultured by placing them on a gel (eg, agarose gel) half-immersed in a culture medium (eg, αMEM medium, RPMI medium supplemented with albumin (preferably bovine serum albumin)).

In the present application, whether or not spermatogenesis is induced is determined by, for example, histological observation in light of the standard in the technical field. When in vitro spermatogenesis induction is performed in experimental animals, it may be observed using transgenic animals designed to express GFP during spermatogenesis.

In the present application, the target for in vitro spermatogenesis induction is not particularly limited, but preferably mammals (humans and mammals other than humans (e.g., cows, horses, pigs, dogs, cats, mice, rats, rabbits, monkeys)), Humans are more preferred.

In the present application, albumin with low caprylic acid content means albumin that does not contain caprylic acid to such an extent that spermatogenesis induction is adversely affected.

The amount of caprylic acid that can be contained in albumin with low caprylic acid content is not particularly limited as long as it does not adversely affect the induction of spermatogenesis. Below, more preferably 40 μmol or less, still more preferably 36 μmol or less, even more preferably 30 μmol or less, even more preferably 28 μmol or less, still more preferably 26 μmol or less. Furthermore, as an example of the amount of caprylic acid-low content albumin, caprylic acid (as a free form) is 0.01 to 70 μmol, preferably 0.1 to 50 μmol, more preferably 0.5 to 40 μmol, and more preferably 0.5 to 40 μmol per 1 g of albumin. Albumin containing more preferably 1 to 36 μmol, still more preferably 2 to 305 μmol, still more preferably 3 to 28 μmol, more preferably 5 to 26 μmol. Alternatively, a preferred range of caprylic acid (as free form) contained in 1 g of albumin is a lower limit selected from 0.01 μmol, 0.1 μmol, 0.5 μmol, 1 μmol, 2 μmol, 3 μmol, 4 μmol, and 5 μmol; an upper limit selected from 60 μmol, 50 μmol, 40 μmol, 36 μmol, 30 μmol, 28 μmol, 26 μmol, 24 μmol, 22 μmol, and 20 μmol;

The method for measuring the amount of albumin is not particularly limited, and it can be measured by a method commonly used in the art. For example, the amount of albumin can be measured as the amount of protein, eg, by the Bradford method.

The method for measuring caprylic acid (as free form) is not particularly limited, and it can be measured by a method commonly used in the art. For example, the albumin-containing fluid can be extracted (eg, by Bligh-Dyer extraction) and the resulting lipid fraction (preferably after trimethylsilylation) subjected to gas chromatography-mass spectrometry for measurement.

A preferred method for measuring caprylic acid (as free form) in albumin is illustrated below.
(1) A solvent (eg, water) is added to albumin (eg, so that the protein concentration (eg, according to the Bradford method) is 4 w/v%).
(2) The resulting albumin-containing liquid is extracted (for example, Bligh-Dyer extraction), and the resulting lipid fraction (for example, after trimethylsilylation) is subjected to a gas chromatography-mass spectrometer to measure the amount of free caprylic acid.

The albumin with low caprylic acid content of the present application may be a naturally-derived albumin (e.g., ovalbumin, porcine-derived albumin, bovine-derived albumin, or human-derived albumin), or a genetic recombinant such as bovine, porcine, or human. albumin, preferably bovine-derived albumin (eg, bovine serum albumin).

In the present application, the method for producing albumin is not particularly limited, and albumin can be produced by a conventional method. For example, albumin is purified from plasma, or produced in yeast or the like using genetic recombination techniques and purified. Such purification methods include, for example, a low-temperature ethanol fractionation method, a heat treatment method, and a chromatographic purification method.

Caprylic acid (free form or salt form) may be added since albumin after purification (particularly after purification by cold ethanol fractionation and heat treatment methods) is prone to polymerization and unstable. When caprylic acid is added to the extent that it adversely affects the induction of spermatogenesis, the low caprylic acid content albumin of the present application can be obtained by removing the caprylic acid.

In one embodiment, the caprylic acid-low content albumin used in the present application is purified by a low-temperature ethanol fractionation method (eg Cohn's Fraction V) or a heat treatment method, preferably a low-temperature ethanol fractionation method, and caprylic acid Albumin to which caprylic acid has been removed, as appropriate, can be used from albumin to which (free form or salt form) has been added.

Examples of commercially available albumins include the following, which can be used as low caprylic acid content albumin of the present application by appropriately removing caprylic acid.
Bovine serum albumin: Bovine Serum Albumin, lyophilized powder, BioReagent, suitable for cell culture, 96% (SIGMA-ALDRICH), albumin, derived from bovine serum (Wako Pure Chemical Industries), Bovine Albumin Fraction V (ThermoFisher Scientific)
Recombinant human albumin: CellPrime rAlbumin AF-s (Merck)
Human plasma albumin: Human Serum Albumin Solution (Irvine Scientific), HUMAN SERUM ALBUMIN (InVitroCare)

A method for removing caprylic acid is not particularly limited, and a method commonly used for removing fatty acids (for example, a method using activated charcoal) can be used. A method (Iscove treatment) in which lipids and metal ions are removed by deionization after treatment with activated carbon in an acidic environment may also be used.

Alternatively, the caprylic acid-low content albumin of the present application can be obtained by removing artificially added caprylic acid using a method for removing caprylic acid using ion exchange treatment. The method of ion exchange treatment is not particularly limited, but both cation exchange treatment and anion exchange treatment are preferably performed.

The cation exchange treatment may be weakly acidic cation exchange treatment (for example, using carboxylic acid groups as exchange groups) or strongly acidic cation exchange treatment (for example, using sulfonic acid groups as exchange groups). However, it is preferably a strongly acidic cation exchange treatment (for example, using a sulfonic acid group as an exchange group). The ionic form of the exchange group is not particularly limited, and examples thereof include hydrogen, sodium salts, potassium salts, etc. Hydrogen is preferably used. The cation exchange treatment can be carried out according to conventional methods using, for example, cation exchange resins (eg, weakly acidic cation exchange resins, strongly acidic cation exchange resins).

The anion exchange treatment is a weakly basic anion exchange treatment (for example, using a primary to tertiary amino group as an exchange group) or a strongly basic anion exchange treatment (for example, using a quaternary ammonium group as an exchange group). However, it is preferably a strongly basic anion exchange treatment. Examples of quaternary ammonium groups include trimethylammonium groups and dimethylethanolammonium groups, preferably trimethylammonium groups are used. The ionic form of the exchange group is not particularly limited, and examples thereof include chloride, hydroxide, acetate and formate, preferably hydroxide. The anion exchange treatment can be carried out according to a conventional method using, for example, an anion exchange resin (eg, weakly basic anion exchange resin, strongly basic anion exchange resin).

The base of the ion exchange resin is not particularly limited, and those commonly used as bases of ion exchange resins (eg, styrenedivinylbenzene) can be used.

From the viewpoint of workability, preferred ion exchange resins include mixed bed resins containing both a cation exchange resin and an anion exchange resin (for example, AG 501-X8 (D) (BIO-RAD) and AG 501-X8 ( BIO-RAD) is preferably used.

The caprylic acid removal method using the ion exchange treatment is, for example, mixing an ion exchange resin and albumin that may contain caprylic acid in a suitable solvent (eg water) (preferably at a low temperature (eg 1 to 10° C., preferably 2 to 6° C.) under light shielding, for example, 1 to 48 hours, preferably 3 to 36 hours, more preferably 5 to 30 hours).

The resulting albumin-containing solution is left as it is, or is appropriately subjected to albumin stabilization techniques known in the art, such as adjustment of pH and addition of a stabilizer (for example, a fatty acid other than caprylic acid), to obtain an albumin with a low caprylic acid content. It can be used as an inclusion agent.

Alternatively, the resulting albumin-containing liquid may be concentrated by freeze-drying, ultrafiltration, or the like. Viruses, bacteria, and the like may also be removed by filtration. In this case also, other stabilization techniques for albumin known in the art (for example, addition of fatty acids other than caprylic acid) may be applied as appropriate.

The low caprylic acid content albumin of the present application may be albumin to which caprylic acid is not artificially added. For example, albumin with low caprylic acid content is albumin purified by a low-temperature ethanol fractionation method (eg Cohn's Fraction V) or a heat treatment method, preferably a low-temperature ethanol fractionation method, to which caprylic acid is artificially added. albumin that is not Since albumin purified by these purification methods may be unstable, it is preferable to apply stabilization techniques (for example, addition of fatty acids other than caprylic acid) before use.

Albumin is a protein that has a high ability to bind to various substances. For example, serum-derived albumin binds to various substances contained in serum. Therefore, even if caprylic acid is not artificially added, albumin may carry bio-derived caprylic acid, but albumin may contain caprylic acid in an amount that does not adversely affect the induction of spermatogenesis. If it contains an amount that can adversely affect spermatogenesis induction, caprylic acid is removed by a conventional method such as a method of removing caprylic acid using ion exchange treatment or a method using activated carbon, and appropriate stabilization techniques (e.g., caprylic acid addition of fatty acid other than acid) can be used.

In the present application, the amount of caprylic acid-low albumin contained in the medium is not particularly limited, and can be appropriately selected within a range normally used according to the purpose of use of the medium in in vitro spermatogenesis induction. For example, the concentration of caprylic acid-low content albumin contained in the medium is 0.1 to 10 w/v%, preferably 1 to 8 w/v%, more preferably 2 to 6 w/v%, still more preferably 3 to It may be 5 w/v %, or even more preferably 4 w/v %. Alternatively, the preferred range of the amount of albumin with low caprylic acid content contained in the medium is 0.1 w/v%, 1 w/v%, 2 w/v%, 3 w/v%, and 4 w/v%. and an upper limit selected from 10 w/v%, 8 w/v%, 6 w/v%, 5 w/v%, and 4 w/v%.

In the present application, the fatty acid that can be used for stabilizing the albumin with a low caprylic acid content (for example, preventing the formation of a polymer) is preferably a fatty acid other than caprylic acid, such as a saturated or unsaturated fatty acid having 10 to 20 carbon atoms, or a fatty acid thereof. salts (e.g. lauric acid, myristic acid, pentadecanoic acid, palmitic acid, palmitoleic acid, margaric acid, stearic acid, oleic acid, linoleic acid, salts thereof (e.g. alkali metal salts, alkaline earth metal salts, and mixtures thereof); and mixtures thereof). Although the amount thereof is not particularly limited, for example, 1 g of albumin contains 10 to 100 μmol, preferably 20 to 80 μmol, more preferably 30 to 50 μmol of the fatty acid or salt thereof in free form.

The form of the agent containing albumin with low caprylic acid content for use in the medium used for the in vitro induction of spermatogenesis of the present application is not particularly limited. It can be a liquid (adjusted to a preferred pH (for example, pH 7-8)) or a freeze-dried solid. The agent of the present application may contain known additives as medium constituents as long as they do not adversely affect the induction of spermatogenesis. For example, but not limited to, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, cholesterol, sphingomyelin, phosphatidylcholine, retinoic acid, retinol, testosterone, triiodothyronine, luteinizing hormone, follicle-stimulating hormone, Examples include NaHCO ₃ , antibacterial agents, antifungal agents, and the like. In addition, other additives that have been conventionally used for culture and the like in in vitro spermatogenesis induction can also be included as appropriate. Additives are preferably contained within known concentration ranges, as long as they do not adversely affect in vitro spermatogenesis induction or product stability.

The form of the medium containing albumin with low caprylic acid content for use in the in vitro spermatogenesis induction of the present application is not particularly limited. ), etc., can be appropriately selected according to the purpose of use of the medium in in vitro spermatogenesis induction, and can be produced using a conventional method. The medium of the present application may contain additives known as medium constituents within a known concentration range, as well as the agent of the present application described above, as long as they do not adversely affect the induction of spermatogenesis. For example, the medium containing albumin with low caprylic acid content for use in in vitro spermatogenesis induction of the present application is a known medium known for in vitro spermatogenesis induction (e.g., αMEM medium, RPMI medium (optionally, essential fatty acids (e.g., linoleic acid, linolenic acid), retinol, retinoic acid, follicle-stimulating hormone, luteinizing hormone, testosterone, triiodothyronine, etc., and palmitic acid, stearic acid, and oleic acid may be added as appropriate)) to caprylyl It can be prepared by adding acid-low content albumin.

Examples of components that may be added to media containing caprylic acid-low albumin for use in in vitro spermatogenic induction herein include, but are not limited to, fatty acids other than caprylic acid (e.g. /L, preferably 1000-4000 μmol/L, more preferably 1500-2500 μmol/L: For example, palmitic acid (for example, the concentration in the medium is 200-1800 μmol/L, preferably 300-1000 μmol/L, more preferably 500-700 μmol /L), stearic acid (e.g. medium concentration 200-1800 μmol/L, preferably 300-1000 μmol/L, more preferably 500-700 μmol/L), oleic acid (e.g. medium concentration 200-1800 μmol/L, preferably 300 to 1000 μmol/L, more preferably 500 to 700 μmol/L), linoleic acid (for example, the concentration in the medium is 30 to 300 μmol/L, preferably 50 to 150 μmol/L, more preferably 80 to 120 μmol/L); and linolenic acid (for example, any combination of fatty acids selected from the group consisting of medium concentrations of 10 to 150 μmol/L, preferably 20 to 80 μmol/L, more preferably 30 to 70 μmol/L), cholesterol (for example, medium medium concentration of 1 to 10 μg/mL, preferably 2 to 5 μg/mL, more preferably 2.5 to 4 μg/mL), sphingomyelin (for example, medium concentration of 1 to 10 μg/mL, preferably 2 to 5 μg/mL) , more preferably 2.5 to 4.5 μg/mL), phosphatidylcholine (for example, the concentration in the medium is 5 to 60 μg/mL, preferably 10 to 40 μg/mL, more preferably 15 to 25 μg/mL), retinoic acid (for example, medium concentration of 0.3 to 3 μmol/L, preferably 0.5 to 2 μmol/L, more preferably 0.7 to 1.5 μmol/L), retinol (for example, medium concentration of 0.3 to 3 μmol/L , preferably 0.5 to 2 μmol/L, more preferably 0.7 to 1.5 μmol/L), testosterone (for example, the concentration in the medium is 0.3 to 3 μmol/L, preferably 0.5 to 2 μmol/L, more preferably 0.7 to 1.5 μmol / L), triiodothyronine (for example, the concentration in the medium is 0.5 to 6 ng / mL, preferably 1 to 4 ng / mL, more preferably 1.5 to 2.5 ng / mL), luteinizing hormone (e.g. medium concentration is 0.3 to 3 μg/mL, preferably 0.5 to 2 μg/mL, more preferably 0.7 to 1.5 μg/mL), follicle-stimulating hormone (for example, the concentration in the medium is 0.3 to 3 μg/mL, preferably 0.5 to 2 μg/mL, more preferably 0.7 to 1.5 μg/mL), NaHCO ₃ (for example, the concentration in the medium is 0.5 to 5 g/L, preferably 1 to 4 g/L, more preferably is 1.2 to 2.5 g/L), antibacterial agents, antifungal agents, and the like. All or part of the fatty acid other than caprylic acid may be added to the medium in a state in which it is previously mixed with albumin as an albumin stabilizer.

The medium containing low caprylic acid albumin for use in in vitro spermatogenesis in the present application preferably does not contain components capable of supplying caprylic acid other than low caprylic acid 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 the induction of spermatogenesis. For example, the amount of caprylic acid contained per 1000 g of the medium is 2800 μmol or less, preferably 2500 μmol or less, in free form. , more preferably 2000 μmol or less, still more preferably 1800 μmol or less, still more preferably 1500 μmol or less, even more preferably 1400 μmol or less, still more preferably 1300 μmol or less. Furthermore, examples of the amount of caprylic acid (as free form) contained in 1000 g of medium are 0.01 to 2800 μmol, preferably 0.1 to 2500 μmol, more preferably 0.5 to 2000 μmol, still more preferably 1 to 1800 μmol, Still more preferably 2 to 1500 μmol, still more preferably 3 to 1400 μmol, more preferably 5 to 1300 μmol. Alternatively, a preferred range for the amount of caprylic acid (as free form) contained in 1000 g of medium is a lower limit selected from 0.01 μmol, 0.1 μmol, 0.5 μmol, 1 μmol, 2 μmol, 3 μmol, 4 μmol, and 5 μmol; and an upper limit selected from 2800 μmol, 2500 μmol, 2000 μmol, 1800 μmol, 1500 μmol, 1400 μmol, 1300 μmol, 1200 μmol, 1100 μmol, and 1000 μmol;

In another aspect of the present application, kits are provided comprising agents or media of the present application and instructions for their use in in vitro spermatogenesis induction. The form of the explanation is not particularly limited, and it may be an instruction manual or may include access information (URL, QR code, etc.) for viewing the contents of the explanation on the Internet.

EXAMPLES The present invention will be described below with reference to examples and test examples, but the present invention is not limited to these.

Test Example 1 Preparation of Experimental Animals
Acr-Gfp transgenic mice (C57BL/6 strain) obtained from RIKEN BRC were mated, and Arc-Gfp (+/+) or (+/-) mice aged 4.5 to 5.5 days after birth were used for the culture test. Sperm cells of Acr-Gfp mice express GFP from the mid-pachytene stage and accumulate in the acrosome of sperm cells. All animal experiments were carried out in accordance with the animal experiment guidelines under the approval of the Yokohama City University Animal Care and Use Committee.

Test Example 2 Purification of albumin
2.1 Activated Carbon and Ion Exchange Treatment As bovine serum albumin (BSA), Lot. Dextran (D299100, TRC Canada) was dissolved in 200 mL of distilled water, 2 g of activated charcoal (Wako) was added and incubated with gentle agitation for 30 minutes at room temperature. 10 g of BSA was added thereto and adjusted to pH 3.0 with HCl. The resulting solution was incubated at 60° C. for 60 minutes with stirring, centrifuged at 2700 g and the supernatant collected. The resulting solution was adjusted to pH 5.5 by adding 1M NaOH and passed through a 0.22 μm filter (Millipore Express ^™ PLUS). An ion-exchange resin (Bio-Rex ^(trademark) MSZ 501(D) Resin) was added to the filtrate and incubated overnight at 4°C. was measured. The resulting solution was sterile filtered and stored at 4°C until use.
2.2 Measurement of free fatty acids Bovine serum albumin before and after treatment was prepared with ultrapure water to a protein concentration of 4 w/v%, subjected to Bligh-Dyer extraction, and the obtained lipid fraction was subjected to trimethylsilylation, followed by gas chromatography mass spectrometry (7890A GC & 5975C GC/MSD, Agilent) to measure the free fatty acid concentration. Table 1 shows the results obtained.
Table 1. Fatty acid concentrations in BSA before and after activated carbon and ion exchange treatment (μmol/L per 4w/v% BSA)

Even in the same product, the amount of fatty acid contained in each lot varied. Most of the fatty acids could be removed by activated carbon and ion exchange treatment.

Test Example 3 Cultivation method of testicular tissue Free fatty acids (palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid), cholesterol, sphingomyelin, and phosphatidylcholine were dissolved in ethanol, and the final concentrations were as shown in Table 2. After adding to a glass beaker, the ethanol was evaporated. When adding caprylic acid (159-02382, Wako), after preparing a 6.31 mmol/L stock solution, it was added to a glass beaker so that the final concentrations were 1000 μmol/L, 3000 μmol/L and 5000 μmol/L, respectively. BSA and AlbuMAX ^(trademark) (Gibco ^(trademark) ) before and after treatment in Test Example 2, which had been adjusted to 8 w/v% with distilled water, were added thereto, and stirred at room temperature for 4 hours. An equal volume of 2-fold concentrated αMEM was added to this solution, and retinoic acid, retinol, testosterone, triiodothyronine, LH, FSH, NaHCO ₃ , and antibacterial-antimycotic agents were added to the final concentrations shown in Table 2. It was added to prepare CDM and used for culture of testis tissue.

Table 2. Composition of CDM (containing 4w/v% BSA)

Agarose gel for tissue culture was prepared by adding agarose to distilled water to 1.5 w/v %, autoclaving, and pouring 33 mL into a 10 cm dish before hardening to form a 5 mm thick gel. The resulting gel was cut to a size of approximately 10 mm ² . At least 6 hours before the start of culture, the gel was placed in a 12-well microplate, 0.5 mL of medium was added, and the bottom half of the gel was submerged, and equilibrated at 34°C and 5% CO ₂ . Two to four testis tissues were placed on each gel, and the culture was continued while changing the medium once a week.

Test Example 4 Toxicity Evaluation of Caprylic Acid On day 10 from the start of culture, testis tissue was observed with a fluorescence stereoscopic microscope (Leica M205 FA, Leica). GFP expression along the seminiferous tubules was used as a sign of spermatogenesis induction, and the GFP expression rate was scored. However, the central part of the tissue was excluded from the evaluation because it was not well supplied with nutrients and often did not express GFP. The results obtained are shown in FIGS. 1 and 2. FIG.

As shown in Figures 1 and 2, spermatogenesis induction was inhibited in the presence of high concentrations of caprylic acid.

Test example 5:
Using the bovine serum albumin (Lot. SLBJ3866V) before treatment used in Test Example 2, the caprylic acid-free group (0 μmol/L), 1000 μmol/L, 2000 μmol/L, and 4000 μmol/L. The testicular tissues of the added groups were cultured in the same manner as in Test Example 3, respectively. At the same time, testis tissue was cultured in the same manner as in Test Example 3 using AlbuMAX ^(trademark) (Gibco ^(trademark) ). GFP-expressing regions were measured on the 8th, 16th, 22nd and 25th days from the start of the culture.

As shown in FIG. 3, the ability to induce spermatogenesis decreased in a caprylic acid concentration-dependent manner.

Test example 6. Removal of caprylic acid by ion exchange treatment

6.1 Caprylic acid removal treatment Ion-exchange resin (AG 501-X8 (D), BIO-RAD) washed with ultrapure water in advance and 10% recombinant human albumin (rHA, Merck) or human serum albumin (HSA, Irvine Scientific ), and then gently stirred for 24 hours at 4°C in the dark using a rotator (RT50, TAITEC). The albumin solution after the treatment was recovered, and the protein concentration was quantified by the Bradford method, and prepared with ultrapure water and 2 mol/L NaOH so as to be 5 w/v% (pH 7.4).

6.2 Free fatty acid measurement Lipid content obtained by Bligh-Dyer extraction of rHA (Comparative Example 1) and HSA (Comparative Example 2) before treatment, and rHA (Example 1) and HSA (Example 2) after treatment, respectively. After trimethylsilylation, the fraction was subjected to gas chromatography-mass spectrometry (7890A GC & 5975C GC/MSD, Agilent) to measure free fatty acid concentration. Table 3 shows the results obtained.

Table 3. Free fatty acid concentration (μmol/L) in 5w/v% albumin solution before and after ion exchange treatment

As shown in Table 3, the ion exchange treatment was able to specifically remove caprylic acid among the free fatty acids contained in the albumin solution.

Spermatogenesis induction can be improved using the agent or medium of the present application.

Claims (11)

An agent containing low caprylic acid albumin for use in a medium used for in vitro spermatogenesis induction, comprising:
The amount of caprylic acid contained in 1000 g of the medium is 2000 μmol or less as a free form,
The low caprylic acid content albumin contains 50 μmol or less of caprylic acid in free form per 1 g of albumin. 2. The agent of claim 1, wherein said in vitro spermatogenesis induction comprises testicular organ culture. an agent containing caprylate-depleted albumin for use in a medium used for in vitro spermatogenesis induction; and a description of the use of the agent in a medium used for in vitro spermatogenesis induction;
A kit for media in in vitro spermatogenesis induction, comprising
The amount of caprylic acid contained in 1000 g of the medium is 2000 μmol or less as a free form,
The kit, wherein the low caprylic acid content albumin contains 50 μmol or less of caprylic acid in free form per 1 g of albumin. 1. A medium containing low caprylate albumin for use in in vitro spermatogenesis induction, comprising:
The amount of caprylic acid contained in 1000 g of the medium is 2000 μmol or less as a free form,
A medium in which the low caprylic acid content albumin contains 50 μmol or less caprylic acid in free form per 1 g of albumin. 5. The medium of claim 4, wherein said in vitro induction of spermatogenesis comprises testicular organ culture. A medium containing low caprylic acid albumin for use in in vitro spermatogenesis induction; and a description of the use of said medium for in vitro spermatogenesis induction;
A kit for media in in vitro spermatogenesis induction, comprising
The amount of caprylic acid contained in 1000 g of the medium is 2000 μmol or less as a free form,
The kit, wherein the low caprylic acid content albumin contains 50 μmol or less of caprylic acid in free form per 1 g of albumin. A method for inducing spermatogenesis in vitro, characterized by using a medium containing albumin with a low caprylic acid content,
The amount of caprylic acid contained in 1000 g of the medium is 2000 μmol or less as a free form,
The method, wherein the low caprylic acid content albumin contains 50 μmol or less caprylic acid in free form per gram of albumin. The agent according to claim 1 or 2, wherein the medium is a chemically-defined medium with a clear chemical composition. The kit according to claim 3 or 6, wherein the medium is a synthetic medium with a defined chemical composition. The medium according to claim 4 or 5, wherein the medium is a synthetic medium with a clear chemical composition. 8. The method according to claim 7, wherein the medium is a synthetic medium with a defined chemical composition.

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