FR3065225A1 - ADJUVANTS FREE OF CYTOKINES FOR CELL CULTURE MEDIA, IN PARTICULAR FOR IN VITRO FERTILIZATION, OR FOR THE CULTIVATION OF FOLLICLES, MALE GERMINAL CELLS OR EMBRYOS - Google Patents

Abstract

The subject of the present invention is compositions which can be used as adjuvants for the in vitro culture of cells, characterized in that they comprise at least two compounds chosen from the group consisting of a zinc salt, inositol under any one of its stereoisomeric forms, carnitine or a carnitine derivative, melatonin or an analogue, cystine in one of its stereoisomeric forms or as a derivative, cysteamine or a cysteamine salt, said compositions being free of cytokines. The invention also relates to in vitro fertilization processes and processes for the maturation of follicles, male germ cells, or embryos using such culture medium adjuvants, as well as kits for the implementation of these methods. methods and comprising a aforementioned lyophilizate and an aqueous solution, containing the other constituents of said culture media.

Description

Holder (s): PATRICK CHOAY SAS Simplified joint-stock company.

Extension request (s)

Agent (s): GROSSET FOURNIER AND DEMACHY Limited liability company.

FR 3 065 225 - A1

104 / ADJUVANTS WITHOUT CYTOKINES FOR CELL CULTURE MEDIA, ESPECIALLY FOR IN VITRO FERTILIZATION, OR FOR CULTURE OF FOLLICLES, MALE GERMIN CELLS OR EMBRYOS.

The present invention relates to compositions which can be used as adjuvants for the in vitro culture of cells, characterized in that they comprise at least two compounds chosen from the group consisting of a zinc salt, inositol in one any of its stereoisomeric forms, carnitine or a carnitine derivative, melatonin or the like, cystine in one of its stereoisomeric forms or in the form of a derivative, cysteamine or a cysteamine salt, said compositions being devoid of cytokines .

The subject of the invention is also methods of in vitro fertilization and methods of maturing follicles, male germ cells, or embryos using such adjuvants from culture media, as well as kits for the implementation of these methods. methods and comprising a lyophilisate mentioned above and an aqueous solution, containing the other constituents of said culture media.

ADDITIVES WITHOUT CYTOKINES FOR CELL CULTURE MEDIA, PARTICULARLY FOR IN VITRO FERTILIZATION, OR FOR CULTIVATION OF FOLLICLES, MALE GERM CELLS OR EMBRYOS

The present invention relates to compositions which can be used as adjuvants or “boosters” for cell culture media, in particular for follicles under development or for the maturation of cells of the male germ line, as well as for fertilization in in vitro (IVF) and the development of embryos, and more generally in mammals, in particular humans, possibly after thawing of follicles or male cells.

In the prior art are described examples of compositions for the in vitro culture of follicles or cells of the male germ line, as well as for the fertilization of the oocyte and the development of the embryo. In some of these examples, the compositions contain one or more growth factors.

Thus, in European patent EP 1.088.057, the use of GM-CSF is described for the preparation of a medium intended for the development of human embryos at an early stage up to the blastocyst stage in an IVF program. .

In European patent EP 1,176,190 are described compositions for the in vitro culture of follicles or embryos, characterized in that they comprise at least two growth factors in association with at least one compound from the family of corticosteroids involved in energy production in mammals, and, where appropriate, with at least one key coenzyme of energy metabolism, such as NAD / NADH and NADP / NADPH.

The present invention follows from the discovery by the inventors of compositions which can be used as adjuvants both for the cultivation of follicles or cells of the male germ line, as well as for the fertilization of the oocyte and the development of the embryo up to at the blastocyst stage and allowing when mixed with an aqueous solution containing the elements conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos to improve the yield compared to that obtained with the compositions currently on the market, these new compositions being in particular characterized in that they do not comprise cytokines.

The present invention therefore relates to compositions in all the possible forms and in particular in the form of liquid, powder or lyophilisate for the in vitro culture of follicles under development with a view to the maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germ line, or for the in vitro fertilization of oocytes by spermatozoa, or for the development of embryos, possibly after thawing of follicles or spermatozoa characterized in that they comprise:

at least two compounds chosen from the group consisting of:

a zinc salt, preferably a water-soluble salt such as zinc chloride or zinc sulphate, in particular at a concentration of 0.1 to 3 μg / ml, preferably at a concentration of 0.5 to 1.5 pg / ml, expressed as elemental zinc,

- Inositol in any of its stereoisomeric forms, preferably myo-inositol, or in the form of phosphate, preferably inositol triphosphate, in particular in the form of salt, in particular at a concentration of 1.8 to 18 mg / ml, preferably at a concentration of 5 to 10 mg / ml, expressed in inositol,

- camitine in one of its stereoisomeric forms, preferably Lcamitine, a camitine salt preferably the hydrochloride, a carnitine derivative preferably acetylcarnitine or a salt of a camitine derivative, preferably in the form hydrochloride in particular at a concentration of 0.1 to 2 mg / ml, preferably 0.5 to 1 mg / ml, expressed as carnitine base,

- melatonin or an analog, a salt or a melatonin solvated in particular at a concentration of 10 ^'11 10' ⁵ M, preferably of 10 ^'1θ 10 ^-6 M and in particular of 10' ⁹ to 10 ^-7 M expressed in melatonin base,

cystine in one of its stereoisomeric forms, preferably L-cystine, a cystine salt preferably the hydrochloride, a cystine derivative preferably acetylcystine or a salt of a cystine derivative, preferably in the hydrochloride form, in particular at a concentration of 20 to 100 pg / ml of cystine and preferably of 35 to 60 pg / ml expressed as cystine base,

- cysteamine, a salt of cysteamine preferably the hydrochloride, a derivative of cysteamine preferably acetyl cysteamine or a salt of a derivative of cysteamine, preferably in the form of hydrochloride, in particular at a concentration of

1.5 to 15 pg / ml of cysteamine and preferably 4 to 12 pg / ml expressed as cysteamine base, and, if appropriate, one or more compounds chosen from the compounds of the corticosteroid family and / or one or more key coenzymes of energy metabolism, such as NAD / NADH and N ADP / N ADPH and compositions characterized in that they lack cytokines.

The compositions of the invention can be assimilated to adjuvants, supplements or boosters. These are compositions which, when combined with other elements, make it possible to obtain culture media.

In the compositions of the invention, the zinc salt concentrations correspond to the quantity of element per volume.

Preferably, the zinc salt concentrations in the above-mentioned compositions of the invention are from 0.1 pg / ml to 3 pg / ml, preferably from 0.5 pg / ml to 1.5 pg / ml expressed as zinc element.

The concentrations of elemental zinc in the above-mentioned compositions of the invention are in particular 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1.0; 1.1;

1.2; 1.3; 1.4; 1.5; 2; 2.5 or 3 pg / ml.

In the compositions of the invention, inositol can in particular be present in the form of phosphate derivatives of inositol, such as inositol mono-, di- or triphosphate, or in the form of salts. The indicated concentrations of inositol correspond to the quantity of inositol by volume.

Preferably, the concentrations of inositol in the above-mentioned compositions of the invention are from 1.8 mg / ml to 18 mg / ml, preferably from 5 to 10 mg / ml.

The inositol concentrations in the above-mentioned compositions of the invention are in particular 1.8; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11; 12; 13; 14; 15; 16; 17 or 18 mg / ml.

In the compositions of the invention, carnitine is in the form of an L enantiomer and the carnitine concentrations correspond to the amount of base per volume.

Preferably, the carnitine concentrations in the above-mentioned compositions of the invention are from 0.1 mg / ml to 2 mg / ml, preferably from 0.5 mg / ml to 1 mg / ml.

The carnitine concentrations in the above-mentioned compositions of the invention are in particular 0.1; 0.2; 0.3; 0.4; 0.5; 0.6; 0.7; 0.8; 0.9; 1.0; 1.1;

1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9 or 2.0 mg / ml.

In the compositions of the invention, melatonin is in the form of a melatonin salt or solvate.

By melatonin salt is meant any salt with a mineral or organic acid such as the salts with hydrochloric, sulfuric, acetic, citric, fumaric, hemisuccinic or maleic acid.

Examples of solvates include hydrates and alcoholates.

Melatonin analogs include biologically active products whose chemical formula derives from that of melatonin.

Melatonin analogs are also, for example, described in patents WO 1989/001472, WO 2002/024181, EP 820768, EP 1476152.

Preferably, in the compositions of the invention, the concentrations of melatonin are between 10 'and ¹¹ 10' ⁵ M more preferably between 10 ^'1θ and 10 ^-6 M and in particular of 10' ⁹ and 10 ^-7 M concentrations expressed in melatonin base.

The concentrations expressed in melatonin in the aforementioned compositions of the invention are in particular approximately 10 ^'11 5.10' ^11, 10 ^'1θ, 5.10' ^10, 10 ^'9 5.10' ^9, 10 ^'8 5.10' ^8, 10 ' ⁷ , 5.10' ⁷ , 10 ' ⁶ , 5.10' ⁶ or 10 ' ⁵ M.

Each of the two cysteine molecules which are linked by a disulfide bridge to form the cystine molecule can be in the D or L form.

The possible stereoisomers can be used for the implementation of the present invention. The L-cystine form is the preferred form.

Cystine salts can be formed with mineral acids such as hydrochloric acid or sulfuric acid.

Among the cystine derivatives that may be mentioned are acetylcystine.

Preferably, in the compositions of the invention, the cystine concentrations are between 20 and 100 pg / ml of cystine and preferably from 35 to 60 pg / ml expressed as cystine base.

The cystine concentrations in the above-mentioned compositions of the invention are in particular 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 pg / ml.

Cysteamine salts can be formed with mineral acids such as hydrochloric acid or sulfuric acid.

Among the cysteamine derivatives include acetylcysteamine.

Preferably, in the compositions of the invention, the cysteamine concentrations are between 1.5 and 15 pg / ml and preferably between 4 and 12 pg / ml expressed as cysteamine base.

The cysteamine concentrations in the above-mentioned compositions of the invention are in particular 1.5; 2; 2.5; 3; 3.5; 4; 4.5; 5; 5.5; 6; 6.5; 7; 7.5; 8; 8.5; 9; 9.5; 10; 10.5; 11; 11.5; 12; 12.5; 13; 13.5; 14; 14.5 or 15 pg / ml.

A more particular subject of the invention is the above-mentioned compositions for the in vitro culture of follicles under development with a view to the maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germ line, or for fertilization in vitro oocytes by spermatozoa, or for the culture of embryos, possibly after thawing of follicles or male cells.

The compositions of the invention provide regulatory cells with growing and differentiating cells.

Thus, these compositions are capable of allowing the maturation of mammalian oocytes and in particular human immature oocytes as well as the maturation of cells of the male germ line. They can also ensure the development of the mammalian embryo and in particular human embryos up to the blastocyst stage, this stage having a higher percentage of implantation in the uterus than when the embryo is at a lesser stage. advanced.

Advantageously, the compositions of the invention contain the following concentrations of active principles:

the zinc concentration, particularly in the form of zinc chloride or sulphate is approximately 1 pg / ml expressed as an element,

the concentration of inositol, particularly in the form of myo-inositol, is approximately 5 mg / ml expressed as inositol,

- the concentration of L-carnitine, particularly in the form of the base or the hydrochloride, is approximately 0.6 mg / ml, expressed as camitine base.

- the concentration of melatonin base is around 10 ' ⁷ M.

the concentration of cystine, particularly in the form of L-cystine and more particularly in the form of L-cystine hydrochloride is approximately 50 μg / ml expressed as cystine base,

- The concentration of cysteamine, particularly in the form of hydrochloride is about 8 pg / ml, expressed as cysteamine base.

Advantageously, the compositions according to the invention are characterized in that they comprise at least three compounds chosen from the group consisting of:

a zinc salt, preferably a water-soluble salt such as zinc chloride or zinc sulphate, in particular at a concentration of 0.1 to 3 μg / ml, preferably at a concentration of 0.5 to 1.5 pg / ml, expressed as elemental zinc,

- Inositol in any of its stereoisomeric forms, preferably myo-inositol, or in the form of phosphate, preferably inositol triphosphate, in particular in the form of salt, in particular at a concentration of 1.8 to 18 mg / ml, preferably at a concentration of 5 to 10 mg / ml, expressed in inositol,

- Camitine in one of its stereoisomeric forms, preferably Lcamitine, a salt of camitine preferably the hydrochloride, a derivative of camitine preferably Tacetylcamitine or a salt of a derivative of camitine, preferably in the form of hydrochloride in particular to a concentration of 0.1 to 2 mg / ml, preferably 0.5 to 1 mg / ml, expressed as base camitine,

- melatonin or an analog, a salt or a melatonin solvated in particular at a concentration of 10 ^'11 10' ⁵ M, preferably of 10 ^'1θ 10 ^-6 M and in particular of 10' ⁹ to 10 ^-7 M expressed in melatonin base.

- Cystine in one of its stereoisomeric forms, preferably L-cystine, a cystine salt preferably the hydrochloride, a cystine derivative preferably T acetyl cystine or a salt of a cystine derivative, preferably in the form hydrochloride in particular at a concentration of 20 to 100 pg / ml of cystine and preferably of 35 to 60 pg / ml expressed as cystine base,

- Cysteamine, a salt of cysteamine preferably the hydrochloride, a derivative of cysteamine preferably acetylcysteamine or a salt of a derivative of cysteamine, preferably in the form of hydrochloride in particular at a concentration of 1.5 to 15 μg / ml of cysteamine and preferably from 4 to 12 pg / ml expressed as cysteamine base.

Advantageously, the compositions according to the invention are characterized in that they comprise an amount preferentially indicated in the present application of each of the compounds mentioned in the group indicated above, namely a zinc salt, myo-ionositol optionally in the form phosphate, L-camitine or acetylcarnitine optionally in the form of hydrochloride, melatonin, L-cystine or acetylcystine optionally in the form of hydrochloride and cysteamine or acetyl cysteamine optionally form of hydrochloride.

A preferred form of composition according to the invention comprises approximately 1 pg / ml of zinc, 5 mg / ml of inositol, 0.6 mg / ml of L-carnitine base, 10 ' ⁷ M of melatonin base, 50 pg / ml of L-cystine base and possibly 8 pg / ml of cysteamine base.

A subject of the invention is also the above-mentioned compositions for in vitro culture, comprising at least one compound of the corticosteroid family, preferably hydrocortisone in the form of water-soluble salt such as hydrocortisone hemisuccinate, sodium succinate hydrocortisone and hydrocortisone sodium phosphate. Preferably, the concentration of the hydrocortisone salt in the abovementioned compositions is between 5 × 10 ′ ⁷ M and 10 ′ ⁶ M, and is in particular from 7 × 10 ′ ⁷ M, or approximately 350 ng / ml for hemisuccinate hydrocortisone expressed as hydrocortisone base.

A subject of the invention is also the above-mentioned compositions for in vitro culture, comprising at least one zinc salt in hydrated or anhydrous form chosen from the following salts: zinc chloride, zinc sulfate, zinc gluconate, zinc picolinate, citrate zinc, zinc acetate, zinc lactate, zinc stearate, preferably a water-soluble salt such as zinc sulfate or zinc chloride.

Advantageously, the compositions defined above contain key coenzymes of the energy metabolism mentioned above, in particular at concentrations such as:

- the concentration of NAD / NADH is approximately 1 pg / ml,

- the concentration of NADP / NADPH is approximately 1 pg / ml.

The subject of the invention is also the above-mentioned compositions for in vitro culture, comprising at least two compounds chosen from the group consisting of:

a water-soluble zinc salt chosen from zinc sulphate or zinc chloride and / or myo-inositol and / or melatonin and / or L-carnitine or L-carnitine hydrochloride and / or L-cystine or L-cystine hydrochloride and / or Lcysteamine or L-cysteamine hydrochloride and optionally

- hydrocortisone hemisuccinate, and

- NAD / NADH and / or NADP / NADPH.

The invention relates in particular to the compositions defined above which are in the form of lyophilisate, namely in the form of compositions in which the various constituents are brought to the dry state, and are capable of being put back into solution while retaining their properties. physico-chemical and biological.

As such, the subject of the invention is more particularly the lyophilizates mentioned above, containing:

at least two compounds chosen from the group consisting of:

a zinc salt, in particular at a concentration of 0.1 to 3 pg / ml, preferably at a concentration of 0.5 to 1.5 pg / ml, expressed in element zinc,

- Inositol in any of its stereoisomeric forms, preferably myo-inositol, in particular at a concentration of 1.8 to 18 mg / ml, preferably at a concentration of 5 to 10 mg / ml, expressed in inositol,

carnitine in one of its stereoisomeric forms, preferably Lcamitine, a carnitine salt preferably the hydrochloride, a carnitine extract preferably acetylcarnitine or a salt of a carnitine extract, in particular at a concentration of 0.1 to 2 mg / ml, preferably 0.5 to 1 mg / ml, expressed in carnitine base,

- melatonin or an analog, an acceptable salt or a melatonin solvated in particular at a concentration of 10 ^'11 10' ⁵ M, preferably of 10 ^'1θ 10 ^-6 M and in particular of 10' ⁹ to 10 ^-7 M expressed as melatonin base,

cystine in one of its stereoisomeric forms, preferably L-cystine, a cystine salt preferably the hydrochloride, a cystine derivative preferably acetylcystine or a salt of a cystine derivative, preferably in the hydrochloride form, in particular at a concentration of 20 to 100 pg / ml of cystine and preferably of 35 to 60 pg / ml expressed as cystine base and / or

- Cysteamine, a salt of cysteamine preferably the hydrochloride, a derivative of cysteamine preferably acetylcysteamine or a salt of a derivative of cysteamine, preferably in the form of hydrochloride in particular at a concentration of 1.5 to 15 μg / ml of cysteamine and preferably at 4 to 12 pg / ml expressed as cysteamine base and optionally, if necessary,

- at least one compound from the corticosteroid family defined above, such as hydrocortisone hemisuccinate,

- And / or at least one key coenzyme of the energy metabolism defined above, such as NAD / NADH and NADP / NADPH and compositions in the form of lyophilisate characterized in that they are devoid of cytokines.

A subject of the invention is also the application of the above-mentioned compositions or lyophilisates, as effector adjuvants for the preparation of media for the in vitro culture of follicles under development with a view to the maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germ line, or for the in vitro fertilization of oocytes by spermatozoa, or for the culture of embryos, optionally after thawing of the male follicles or gametes, said culture media in vitro comprising a composition as defined above, in mixture or in association with an aqueous solution containing the elements conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos. Said elements are known to those skilled in the art. They can in particular be chosen from human or bovine serum albumin, where appropriate recombinant, and / or mineral salts such as KC1, NaCl, MgSO4, NaHCO3, Na2HPO4, KH2PO4. and / or energy molecules such as glucose, pyruvate, citrate and lactate, and / or amino acids for protein biosynthesis, and / or purine and pyrimidine bases for biosynthesis of nucleic acids, and / or phospholipids or cholesterol for the formation of cell membranes, and / or vitamins, such as group B vitamins, in particular vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9, vitamin B12 and / or vitamin C and / or vitamin E, and optionally insulin and / or optionally at least one other substance contributing to the process for preparing the culture medium, in particular one or more factors of growth chosen in particular from GM-CSF, IGF-1, IGF-2, EGF, TGF or FIF and preferably GM-CSF and / or at least one antibiotic.

In what follows, the expression “in mixture” means that the compositions of the invention and the aqueous solution containing the elements conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos form only one solution which is the final culture medium after one has been poured into the other whereas the expression “in combination” means that the two formulations, namely the invention and the aqueous solution , are present together, for example in a kit, but will not be mixed until they are used.

The invention also relates to a process for the preparation of media for in vitro culture defined above, characterized in that it comprises a step of mixing a composition according to the invention acting as an adjuvant, supplement or “booster” ( where appropriate after dissolving in a suitable liquid said composition according to the invention if it is in the form of powder or lyophilisate) with a solution containing the elements conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos, said elements being as defined above.

The appropriate liquid Fe can be, for example, distilled water, physiological saline, a solution of bovine or human serum albumin, recombinant or not, a mixture of these various ingredients or a culture medium conventionally used.

The invention also relates to media for the in vitro culture of follicles under development for the maturation of oocytes contained in said follicles, or for the maturation of cells of the male germ line, or for the in vitro fertilization of oocytes by spermatozoa, or for the cultivation of embryos, optionally after thawing of follicles or male cells, said media for in vitro culture being as obtained by the process consisting in combining a composition of the invention in the form of a liquid , powder or lyophilisate with an aqueous solution containing the elements conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos, a non-limiting list of said elements being indicated above.

As it appears in the developments indicated above, the compositions according to the invention mainly constitute adjuvants or supplements or "booster" which are not, in principle, intended to be used alone as culture media. They are mainly intended to be mixed with aqueous solutions containing the elements conventionally used in the context of in vitro fertilization. A non-exhaustive list of these elements has been indicated previously.

These aqueous solutions can be constituted by media for in vitro culture which could be used as such but for which the addition of adjuvants, supplements or "booster" according to the invention makes it possible to improve performance. These aqueous solutions can therefore be commercial culture media such as those sold under the names such as Gl, G2, EmbryoGen, BlastoGen, Ferticult and HTF.

These aqueous solutions containing the elements conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos can also be potential culture media prepared by biologists of the trade and for which the adjuvants, supplements or "booster" according to the invention allow, as indicated above and shown below in the experimental part, to improve performance and efficiency.

A more particular subject of the invention is the media for the in vitro culture of follicles under development with a view to the maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germ line, or for the fertilization in in vitro oocytes by spermatozoa, or for the culture of embryos, optionally after thawing of follicles or male cells, said media being characterized in that they comprise the constituent elements of a composition according to the invention as defined herein above, in combination or in mixture with the abovementioned elements conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos as defined above.

The subject of the invention is also a method of maturing mammalian follicles under development with a view to the maturation of oocytes contained in said follicles, and more particularly of human follicles, where appropriate after thawing of the previously frozen follicles, characterized in what it includes a step of in vitro culture of follicles taken from the female, and more particularly from the woman, in a culture medium as defined above according to the invention, advantageously for approximately 24 to 48 hours .

The invention also relates to a method for maturing cells of the male germ line of mammals, and more particularly human, if appropriate after thawing of previously frozen cells, characterized in that it comprises a step of culturing said cells in vitro. cells, in a culture medium as defined above according to the invention, in particular for several days, advantageously from 1 to 5 days.

The subject of the invention is also a method of in vitro fertilization of oocytes by spermatozoa, and more particularly of oocytes and spermatozoa of human origin, where appropriate after thawing of the previously frozen sperm, characterized in that it comprises a step of in vitro culture of the abovementioned oocytes and spermatozoa, in a culture medium as defined above according to the invention, advantageously for approximately 1 day to approximately 2 days.

The invention also relates to a process for developing mammalian embryos, and more particularly human embryos, characterized in that it comprises a step of culturing said embryos in vitro, in a culture medium as defined above. above according to the invention, preferably during the 5 to 6 days which follow in vitro fertilization, advantageously so as to obtain embryos at the blastocyst stage.

Without limitation, the invention relates in particular to the methods as defined above in which the mammals can be chosen from primates, including human beings, sheep, cattle, pigs and in the animal species in process of disappearance ...

Advantageously, the compositions of the invention can be used both for the maturation of follicles or that of the cells of the male germ line, the in vitro fertilization of oocytes, and the maturation of embryos resulting from said fertilization, in particular up to blastocyst stage, possibly after thawing of follicles or male cells.

The invention also relates to a kit (or kit) for the extemporaneous preparation of a culture medium as defined above according to the invention, in particular in the context of the implementation of a process mentioned above, kit including:

- a composition according to the invention optionally in the form of lyophilisate and

- an aqueous solution containing the elements conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos, such as the elements defined above, in particular a marketed medium.

The present invention will be further illustrated with the aid of the following detailed description of examples of culture media prepared according to the invention.

As an aqueous solution as defined above, a culture medium prepared from the compositions according to the invention can mainly comprise the compounds chosen from mineral salts such as KC1, NaCl, MgSO ₄ , NaHCCh, Na2HPO ₄ , KH ₂ PO ₄ ; essential amino acids as well as other amino acids such as glutamic acid, glycine, taurine, cysteine and glutamine; oses and metabolic derivatives, such as glucose, pyruvate, lactate, acetate; vitamins, including group B vitamins and vitamin C; purine and pyrimidine bases; albumin; phospholipids; cholesterol; insulin and / or at least one other process-contributing substance, in particular a growth factor; antibiotics, such as penicillin G, streptomycin, gentamycin or another aminoglycoside.

This invention, which can be in different forms (liquid, powder, or lyophilisate), can be mixed before packaging with a liquid solution comprising the elements conventionally used in the context of in vitro fertilization, or the cultivation of follicles, of male cells, or of embryos, this solution can thus be itself a culture medium ready for use. The improved medium resulting from the mixture will be intended for the cultivation of follicles under development for the maturation of oocytes, or for the maturation of cells of the male germ line, or for the in vitro fertilization of oocytes by sperm or else for the cultivation of embryos, possibly after thawing of the follicles, or of the male cells. The compositions according to the invention can also be mixed extemporaneously with a liquid solution comprising the elements conventionally used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos. The solution thus obtained is a culture medium.

EXPERIMENTAL PART :

The following are compositions of formulations of the effector adjuvant which are the subject of the present invention which can be used for the preparation of media for the in vitro culture of follicles under development with a view to the maturation of the oocytes contained in said follicles , or for the maturation of cells of the male germ line, or for the in vitro fertilization of oocytes by spermatozoa, or for the culture of embryos, possibly after thawing of the follicles or of the male cells.

In all of the following experiments, the active ingredients are finally used at the following concentrations:

Zinc expressed in element = 1 pg / ml Inositol expressed in molecule = 5 mg / ml L-camitine expressed in base = 0.6 mg / ml Melatonin expressed in base = 10 ' ⁷ M Cystine expressed in base = 50 pg / ml Cysteamine expressed in base = 8 pg / ml

We have chosen to prepare compositions 100 times more concentrated than the concentrations mentioned above beforehand. As a result, these solutions are to be diluted extemporaneously to ^1/100 in a usual culture medium when carrying out the methods for their application.

All operations are carried out with sterile raw materials and in a sterile environment.

Composition 1

A solution prepared extemporaneously by mixing a culture medium with a composition according to the invention containing a zinc salt and melatonin is distributed in 1 ml flasks, so that each flask contains 100 μg of elemental zinc and 10 ′ ⁵ M of melatonin.

This solution is to be diluted at ^1/100 in the culture medium during the production processes.

Composition 2

A solution prepared extemporaneously by mixing a culture medium with a composition according to the invention containing a zinc salt, inositol and melatonin is distributed in 1 ml flasks, so that each flask contains 100 μg of elemental zinc, 500 mg of inositol and 10 ^-5 M melatonin.

This solution is to be diluted at ^1/100 in the culture medium during the production processes.

Composition 3

A solution prepared extemporaneously by mixing a culture medium with a composition according to the invention containing a zinc salt, inositol, melatonin and cystine is distributed in 1 ml flasks, so that each flask contains 100 µg of elemental zinc, 500 mg of inositol, 10 ' ⁵ M of melatonin and 5 mg of cystine base.

This solution is to be diluted at ^1/100 in the culture medium during the production processes.

Composition 4

A solution prepared extemporaneously by mixing a culture medium with a composition according to the invention containing a zinc salt, inositol, melatonin, cystine and carnitine is distributed in 1 ml flasks, such so that each vial contains 100 µg of elemental zinc, 500 mg of inositol, 10 ' ⁵ M of melatonin, 5 mg of cystine base and 60 mg of L-carnitine base.

This solution is to be diluted at ^1/100 in the culture medium during the production processes.

Composition 5

A solution prepared extemporaneously by mixing a culture medium with a composition according to the invention containing a zinc salt, inositol, melatonin, cystine, cysteamine and carnitine is distributed in vials of 1 ml, so that each vial contains 100 pg of elemental zinc, 500 mg of inositol, 10 ' ⁵ M of melatonin, 5 mg of cystine base, 800 pg of cysteamine base and 60 mg of Lcamitine base.

This solution is to be diluted at ^1/100 in the culture medium during the production processes.

Composition 6

A solution prepared extemporaneously by mixing a culture medium with a composition according to the invention containing a zinc salt, inositol and carnitine is distributed in 1 ml flasks, so that each flask contains 100 μg of elemental zinc, 500 mg of inositol and 60 mg of L-camitine base.

This solution is to be diluted at ^1/100 in the culture medium during the production processes.

Composition 7

A solution prepared extemporaneously by mixing a culture medium with a composition according to the invention containing inositol, melatonin and cystine is distributed in 1 ml flasks, so that each flask contains 500 mg of inositol, 10 ' ⁵ M melatonin and 5 mg cystine base.

This solution is to be diluted at ^1/100 in the culture medium during the production processes.

Composition 8

A solution prepared extemporaneously by mixing a culture medium with a composition according to the invention containing a zinc salt, inositol, melatonin and carnitine is distributed in 1 ml flasks, so that each flask contains 100 µg of elemental zinc, 500 mg of inositol, 10 ' ⁵ M of melatonin and 60 mg of L-camitine base.

This solution is to be diluted at ^1/100 in the culture medium during the production processes.

Composition 9

A solution prepared extemporaneously by mixing a culture medium with a composition according to the invention containing inositol, melatonin, cystine and carnitine is distributed in 1 ml flasks, so that each flask contains 500 mg of inositol, 10 ' ⁵ M of melatonin, 5 mg of cystine base and 60 mg of Lcamitine base.

This solution is to be diluted at ^1/100 in the culture medium during the production processes.

The bottles of the different compositions from 1 to 9 are stored at + 4 ° C or when their content is lyophilized it is stored at a temperature below - 20 ° C.

Test 1) In vitro maturation of mammalian oocytes

Depending on the stage of oocyte maturation, "oocytes at the germinal vesicle stage" or "oocytes at metaphase I stage" are cultured in a conventional in vitro maturing medium supplemented with two or more substances.

Test la) Oocytes at the stage of the germinal vesicle (LV)

The number of LV oocytes that evolve in metaphase I (MI) or metaphase II (Mil) stages is determined.

In this experiment, 5 groups are used

Group No. 1 (control), the VG oocytes are placed in wells containing only the culture medium.

Group No. 2, the VG oocytes are placed in wells containing the culture medium and a composition according to the invention containing melatonin and zinc.

Group No. 3, the LV oocytes are placed in wells containing the culture medium and a composition according to the invention containing melatonin, zinc and inositol. Group No. 4, the VG oocytes are placed in wells containing the culture medium and a composition according to the invention containing melatonin, zinc, inositol and cystine.

Group No. 5, the LV oocytes are placed in wells containing the culture medium and a composition according to the invention containing melatonin, zinc, inositol, cystine, cysteamine and carnitine.

The different groups of oocytes are cultured in the same oven at 37 ° C in an atmosphere containing 5% CO2.

Results

It is checked whether the addition of the compositions according to the invention which contain substances such as zinc, inositol, carnitine, melatonin, cystine and / or cysteamine leads to an improvement in the maturation of LV oocytes at the MI and Mil stage after 24 hours and 48 hours of culture compared to VG oocytes cultured in the culture medium alone.

Test lb) Metaphase I stage oocytes

In this experiment, we determine the number of oocytes that evolve in Μ II. The same protocol as above is used when selecting oocytes at the MI stage.

Results

It is checked whether the addition of the compositions according to the invention brings improvements in the maturation of MI oocytes evolving at the Mil stage in the presence of the compositions according to the invention which contain substances such as zinc, inositol, carnitine, melatonin, cystine and / or cysteamine

Test 2) In vitro fertilization

To evaluate the influence of the various substances (zinc, inositol, L-camitine, melatonin, cystine and cysteamine) contained in the compositions according to the invention, on in vitro fertilization, the experiments are carried out in a mammal.

To do this, we select the most mature eggs and the best sperm to facilitate fertilization and we assess the number of embryos formed after 48 hours of culture.

As for the evaluation of maturation in vitro, in this experiment 5 groups are used.

In each group, the mature oocytes are placed in the presence of approximately 10 ⁶ competent spermatozoa in a culture medium to which are added compositions according to the invention which comprise substances such as zinc, inositol, camitine, melatonin, cystine and / or cysteamine according to the same protocol as that used previously.

Results

It is checked whether the addition of the compositions according to the invention which contain substances such as zinc, inositol, carnitine, melatonin, cystine and / or cysteamine to the culture medium improves the percentage of fertilized ova compared to the group of ova present in the culture medium alone.

Trial 3) Early embryonic development

To evaluate the effects of the compositions according to the invention which contain substances such as zinc, inositol, carnitine, melatonin, cystine and / or cysteamine on early embryonic development, the CF-1 strain of mice is optionally used, because the number of embryos evolving to the blastocyst stage in a usual culture medium is weak. This model allows us to appreciate the beneficial effects of these substances on early embryonic development.

In this experiment, the same protocol is used: 5 groups of embryos are cultured under different conditions.

Results

It is checked whether the addition of the compositions according to the invention which contain substances such as zinc, inositol, camitine, melatonin, cystine and / or cysteamine leads to an improvement in the development of embryos at different stages compared to the group containing only the medium of culture.

Claims (10)

1. Composition in the form of a liquid, powder or lyophilisate for the in vitro culture of follicles under development with a view to the maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germ line, or for in vitro fertilization of oocytes by spermatozoa, or for the development of embryos, possibly after thawing of follicles or male cells characterized in that it comprises: at least two compounds or at least three compounds chosen from the group consisting of: a zinc salt, preferably a water-soluble salt such as zinc chloride or zinc sulphate, inositol in any of its stereoisomeric forms, preferably myo-inositol, or in the form of phosphate, preferably inositol triphosphate, in particular in the form of a salt, camitine in one of its stereoisomeric forms, preferably Lcamitine, a camitine salt preferably the hydrochloride, a camitine derivative preferably acetylcamitine or a salt thereof a camitine derivative, preferably in the form of the hydrochloride, melatonin or the like, an acceptable salt or a solvate of melatonin, cystine in one of its stereoisomeric forms, preferably L-cystine, a salt of preferably cystine hydrochloride, a cystine derivative preferably acetylcystine or a salt of a cystine derivative, preferably in the form of hydrochloride, cysteamine, a cysteamine salt e preferably the hydrochloride, a cysteamine derivative preferably acetylcysteamine or a salt of a cysteamine derivative, preferably in the form of the hydrochloride, and, where appropriate, one or more compounds chosen from the compounds of the family corticosteroids and / or one or more key coenzymes of energy metabolism, such as NAD / NADH and NADP / NADPH and in that it is devoid of cytokine. 2. Composition in the form of a liquid for the in vitro culture of follicles under development with a view to the maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germ line, or for the in vitro fertilization of oocytes by spermatozoa, or for the development of embryos, possibly after thawing of the follicles or male cells, characterized in that it comprises: at least two compounds or at least three compounds chosen from the group consisting of: a zinc salt, preferably a water-soluble salt such as zinc chloride or zinc sulfate, in particular at a concentration of 0.1 to 3 pg / ml, preferably at a concentration of 0.5 to 1.5 pg / ml, expressed as elemental zinc, inositol in any of its stereoisomeric forms, preferably myo-inositol, or in the form of phosphate, preferably inositol triphosphate, in particular in the form of a salt, in particular a concentration of 1.8 to 18 mg / ml, preferably at a concentration of 5 to 10 mg / ml, expressed in inositol, carnitine in one of its stereoisomeric forms, preferably Lcamitine, a salt of camitine preferably the hydrochloride, a camitine derivative preferably acetylcarnitine or a salt of a camitine derivative, preferably in the form of hydrochloride, in particular at a concentration of 0.1 to 2 mg / ml, preferably of 0.5 at 1 mg / ml, expressed as camitine base, melatonin or an analog, a acceptable salt or melatonin solvate, in particular at a concentration of 10 ' ^1! at 10 ' ^{4 5} M preferably from 10 ¹⁰ to 10' ^{6 * *} M and in particular from 10 'to 10' M expressed as melatonin base, cystine in one of its stereoisomeric forms, preferably L-cystine, a cystine salt preferably the hydrochloride, a cystine derivative preferably acetylcystine or a salt of a cystine derivative, preferably in the form of hydrochloride, in particular at a concentration of 20 to 100 μg / ml of cystine and preferably 35 to 60 pg / ml expressed as cystine base, cysteamine, a cysteamine salt preferably the hydrochloride, a cysteamine derivative preferably acetyl cysteamine or a salt of a cysteamine derivative, preferably under the form of hydrochloride, especially at a concentration of 1.5 to 15 pg / ml of cysteamine and preferably 4 to 12 pg / ml expressed as cysteamine base, and, if appropriate, one or more compounds chosen from the compounds of the corticosteroid family and / or one or more key coenzymes of energy metabolism, such as NAD / NADH and NADP / NADPH and in that it is devoid of cytokine. 3. Composition according to claim 1 or 2 characterized in that it comprises at least one compound of the family of corticoids, preferably hydrocortisone in the form of water-soluble salt such as hydrocortisone hemisuccinate, sodium succinate hydrocortisone and hydrocortisone sodium phosphate. 4. Composition according to claim 1, 2 or 3 characterized in that it comprises at least one zinc salt in hydrated or anhydrous form chosen from the following salts: zinc chloride, zinc sulfate, zinc gluconate, zinc picolinate , zinc citrate, zinc acetate, zinc lactate, zinc stearate, preferably a water-soluble salt such as zinc sulfate or zinc chloride. 5. Composition according to claim 1 characterized in that it is in the form of lyophilisate. 6. Application of the compositions according to one of claims 1 to 5, as effector adjuvants for the preparation of media for the in vitro culture of follicles under development with a view to the maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germ line, or for the in vitro fertilization of oocytes by sperm, or for the culture of embryos, optionally after thawing of follicles or male cells, said culture media in vitro comprising a composition according to one of claims 1 to 5 mixed with an aqueous solution containing the elements used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos, said elements being chosen in particular from albumin serums human or bovine, optionally recombinant, and / or mineral salts, and / or energy molecules such as gluc ose, pyruvate, citrate and lactate, and / or amino acids for protein biosynthesis, and / or purine and pyrimidine bases for biosynthesis of nucleic acids, and / or phospholipids or cholesterol for the formation of membranes cells, and / or vitamins, such as group B vitamins, especially vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6, vitamin B9, vitamin B12, and / or vitamin C and / or vitamin E, and optionally insulin and / or optionally one or more growth factors chosen in particular from GM-CSF, IGF-1, IGF-2, EGF, TGF or LIF and preferably GM-CSF and / or at least one antibiotic. 7. Media for the in vitro culture of follicles under development with a view to the maturation of oocytes contained in said follicles, or for the maturation of cells of the male germ line, or for the in vitro fertilization of oocytes by spermatozoa, or for the culture of embryos, optionally after thawing of the follicles or of the male cells, said culture media in vitro being as obtained by the process consisting in associating a composition in the form of liquid, powder or lyophilisate according to the one of claims 1 to 5 with an aqueous solution containing the elements used in the context of in vitro fertilization, or the culture of follicles, male cells, or embryos, said elements being chosen in particular from the elements described in claim 6. 8. Media for the in vitro culture of follicles under development with a view to the maturation of the oocytes contained in said follicles, or for the maturation of cells of the male germ line, or for the in vitro fertilization of the oocytes by sperm, or for the culture of embryos, optionally after thawing of the follicles or of the male cells, the said media being characterized in that they comprise the elements of a composition according to one of claims 1 to 5 in admixture with the elements used in within the framework of in vitro fertilization, or the culture of follicles, male cells, or embryos, said elements being as defined in claim 6. 9. In vitro method for maturing mammalian follicles under development with a view to maturing the oocytes contained in said follicles and more particularly human follicles, where appropriate after thawing of the previously frozen follicles, characterized in that it comprises a step of in vitro culture of follicles taken from the female and more particularly from the woman, in a culture medium according to claim 8, advantageously for approximately 24 to 48 hours. 10. Kit for the extemporaneous preparation of a medium according to claim 8, in particular in the context of the implementation of a method according to claim 9, characterized in that it comprises: - a composition in the form of liquid, powder or lyophilisate according to any one of claims 1 to 5, - And an aqueous solution containing the elements used in the context of the cultivation of follicles, male cells or embryos, as defined in claim 6.