CN110709505A - Cytokine-free adjuvants, in particular for in vitro fertilization or for cell culture media for follicles, male germ cells or embryos - Google Patents
Cytokine-free adjuvants, in particular for in vitro fertilization or for cell culture media for follicles, male germ cells or embryos Download PDFInfo
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Abstract
Subject of the present invention are compositions that can be used as adjuvants for in vitro cell culture, characterized in that they comprise at least two compounds selected from the group consisting of zinc salts, inositol in any of its stereoisomeric forms, carnitine or carnitine derivatives, melatonin or its analogs, cystine in any of its stereoisomeric forms or its derivatives, cysteamine or cysteamine salts, and corticosteroids, preferably hydrocortisone P, said compositions being free of cytokines. The subject of the invention is also the in vitro fertilization method and the maturation method of follicles, male germ cells or embryos using this medium adjuvant, as well as the kits for carrying out these methods, comprising the above lyophilisate and an aqueous solution comprising the other components of the medium.
Description
Technical Field
The subject of the present invention is a composition for use as an adjuvant or "booster" of a cell culture medium, optionally after thawing of follicles or sperm, in particular for the maturation of developing follicles or for cells of a male germ line, and for In Vitro Fertilization (IVF) and embryo development, and in general for mammals including humans.
Background
In the prior art, in vitro culture of cells for ovarian follicles or male germ lines is described, as well as compositions for oocyte fertilization and embryo development. In some of these examples, the composition comprises one or more growth factors.
Thus, in European patent EP 1088057, the use of GM-CSF for the preparation of a medium from the early stage to the blastocyst stage of human embryonic development during IVF is described.
Compositions for in vitro culture of follicles or embryos are described in european patent EP 1176190, characterized in that they comprise a combination of at least two growth factors and at least one compound of the corticosteriod sterol family involved in energy production of mammals, and, where appropriate (i.e. optionally) at least one energy metabolism critical coenzyme, such as NAD/NADH and NADP/NADPH.
Disclosure of Invention
The present invention results from the inventors' demonstration of compositions that can be used as adjuvants, for the culture of follicles or male germ lines, for the fertilization of oocytes and the development of embryos up to the blastocyst stage, and that, when mixed with aqueous solutions comprising the ingredients commonly used in vitro fertilization or the culture of follicles, spermatozoa or embryos, are capable of increasing the yield compared to compositions currently used on the market, these new compositions being characterized in particular in that they do not comprise cytokines.
The subject of the present invention is therefore compositions in all possible forms, in particular in liquid, powder or lyophilisate form, for in vitro culture of developing follicles to mature oocytes contained in said follicles, or for maturation of cells of a male germ line, or for in vitro fertilization of oocytes by spermatozoa, or for embryonic development, optionally after thawing of the follicles or spermatozoa, said compositions being characterized in that they comprise:
at least two compounds selected from the group consisting of:
a zinc salt, preferably a water-soluble salt, such as zinc chloride or zinc sulphate, in particular in a concentration of 0.1 to 3. mu.g/mL, preferably in a concentration of 0.5 to 1.5. mu.g/mL, expressed as zinc element,
inositol, preferably cis-1, 2,3, 5-trans-4, 6-inositol (myo-inositol), in any stereoisomeric form, or its phosphate form, preferably inositol triphosphate, especially in salt form, especially at a concentration of 1.8 to 18mg/mL, preferably 5 to 10mg/mL, expressed as inositol,
carnitine, preferably L-carnitine, in the form of one of the stereoisomers, preferably the hydrochloride salt of carnitine, a carnitine derivative, preferably acetyl-carnitine, or a salt of a carnitine derivative, preferably in the form of the hydrochloride salt, in particular at a concentration of 0.1 to 2mg/mL, preferably 0.5 to 1mg/mL, expressed as carnitine base,
melatonin or an analogue, a melatonin-acceptable salt or solvate, in particular at a concentration of 10-11To 10-5M, preferably 10-10To 10-6M, especially 10-9To 10-7M, expressed as the melatonin basic structure,
cystine in the form of one of the stereoisomers, preferably levocystine, a salt of cystine, preferably the hydrochloride salt, a cystine derivative, preferably acetylcystine (acetylcystine), or a salt of a cystine derivative, preferably the hydrochloride salt, in particular at a concentration of cystine of 20 to 100. mu.g/mL, preferably 35 to 60. mu.g/mL, expressed as cystine basic structure,
cysteamine, a cysteamine salt, preferably a hydrochloride salt, a cysteamine derivative, preferably acetylcysteamine, or a salt of a cysteamine derivative, preferably in the form of a hydrochloride salt, in particular cysteamine, having a concentration of 1.5 to 15 μ g/mL, preferably 4 to 12 μ g/mL,
expressed in terms of the basic structure of cysteamine,
and, optionally,
one or more compounds from the corticosteroid family
And/or
One or more energy metabolism critical coenzymes, such as NAD/NADH and NADP/NADPH,
and the composition is free of cytokines.
Another subject of the invention are compositions in all possible forms, in particular in liquid, powder or lyophilisate form, for in vitro culture of developing follicles to mature oocytes contained in said follicles, or for maturation of cells of a male germ line, or for in vitro fertilization of oocytes by spermatozoa, or for embryonic development, optionally after thawing of the follicles or spermatozoa, said compositions being characterized in that they comprise:
at least two compounds or at least three compounds selected from the group consisting of:
a zinc salt, preferably a water-soluble salt, such as zinc chloride or zinc sulphate,
inositol in any stereoisomeric form, preferably cis-1, 2,3, 5-trans-4, 6-inositol, or its phosphate form, preferably inositol triphosphate, especially in salt form,
carnitine, preferably L-carnitine, in the form of one of the stereoisomers, a salt of carnitine, preferably the hydrochloride, a carnitine derivative, preferably acetyl-carnitine, or a salt of a carnitine derivative, preferably in the form of the hydrochloride,
melatonin or an analogue, a melatonin acceptable salt or solvate,
cystine, preferably l-cystine, in the form of one of the stereoisomers, a salt of cystine, preferably hydrochloride, a cystine derivative, preferably acetylcysteine, or a salt of a cystine derivative, preferably in the form of hydrochloride,
cysteamine, a salt of cysteamine, preferably hydrochloride, a cysteamine derivative, preferably acetylcysteamine, or a salt of a cysteamine derivative, preferably in the form of hydrochloride, one or more compounds selected from the group consisting of corticosteroids
And, optionally,
one or more energy metabolism critical coenzymes, such as NAD/NADH and NADP/NADPH,
and wherein the composition is free of cytokines.
The compositions of the present invention may be viewed as being similar to adjuvants, supplements or "enhancers". These are compositions that when combined with other ingredients enable a culture medium to be obtained.
In the composition of the invention, the concentration of zinc salt corresponds to the amount of zinc element per volume.
Preferably, the concentration of the zinc salt in the above-described composition of the invention is from 0.1. mu.g/mL to 3. mu.g/mL, preferably from 0.5. mu.g/mL to 1.5. mu.g/mL, expressed as zinc element.
The concentration of elemental zinc in the aforementioned composition of the invention is 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. mu.g/mL.
In the compositions of the invention, the inositol may especially be present in the form of a phosphate derivative of inositol, such as inositol mono-, di-or triphosphate, or in the form of a salt. The indicated inositol concentration corresponds to the amount of inositol per volume.
Preferably, the inositol concentration in the aforementioned compositions of the invention is from 1.8mg/mL to 18mg/mL, preferably from 5 to 10 mg/mL.
The concentration of inositol in the aforementioned composition of the invention is 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 composition of the present invention, carnitine is in the form of the levorotatory enantiomer, and the concentration of carnitine corresponds to the amount of basic structure (base) per volume.
Preferably, the concentration of carnitine in the aforementioned composition of the invention is from 0.1mg/mL to 2mg/mL, preferably from 0.5mg/mL to 1 mg/mL.
The concentration of carnitine in the aforesaid compositions of the invention is 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 present invention, the melatonin is in the form of a salt or solvate of melatonin.
Melatonin salts are intended to mean any salt with inorganic or organic acids, for example with hydrochloric acid, sulfuric acid, acetic acid, citric acid, fumaric acid, hemisuccinic acid or maleic acid.
Examples of solvates include hydrates and alcoholates.
Melatonin analogs include biologically active products having a chemical formula derived from melatonin.
Melatonin analogues are also described in e.g. WO 1989/001472, WO 2002/024181, EP 0820768, EP 1476152.
Preferably, the melatonin concentration in the compositions of the present invention is at 10-11To 10-5M, more preferably 10-10To 10-6M, and in particular between 10-9To 10-7Between M, the concentrations are expressed in terms of the basic structure of melatonin.
The concentration expressed as melatonin in the above-mentioned compositions of the invention is in particular about 10-11，5x10-11，10-10，5x10-10，10-9，5x10-9，10-8，5x10-8，10-7，5x10-7，10-6，5x10-6Or 10-5M。
Each of the two cysteine molecules linked by disulfide bonds to form a cystine molecule may be in either dextrorotatory or levorotatory form.
Possible stereoisomers may be used in the practice of the present invention. The L-cystine form is the preferred form.
Capable of forming cystine salts with mineral acids such as hydrochloric acid or sulfuric acid.
Among cystine derivatives, mention may be made of acetylcysteine.
Preferably, in the composition of the invention, the concentration of cystine is between 20 and 100. mu.g/mL, preferably 35 to 60. mu.g/mL, expressed as cystine basic structure.
The concentration of cystine in the aforementioned composition of the invention is in particular 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μ g/mL.
Capable of forming a cysteine salt with a mineral acid such as hydrochloric acid or sulfuric acid.
Among cysteamine derivatives, mention may be made of acetylcysteamine.
Preferably, in the composition of the invention, the concentration of cysteamine is between 1.5 and 15 μ g/m1, preferably between 4 and 12 μ g/mL, expressed as cysteamine groups.
The concentration of cysteamine in the aforementioned composition of the invention is 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. mu.g/mL.
The invention more particularly relates to a composition as described above, optionally after thawing of the follicle or sperm, for in vitro culturing of a developing follicle for maturation of an oocyte contained in said follicle, or for maturation of a cell of a male germ line, or for in vitro fertilization of an oocyte by sperm, or for embryo culture.
The compositions of the invention provide regulatory molecules for growing and differentiating cells.
Thus, these compositions are capable of maturing mammalian oocytes, particularly human immature oocytes, as well as maturing cells of male germ lines. They also ensure the development of mammalian embryos, particularly human embryos, up to the blastocyst stage, which is a higher implantation rate in utero than at lower developmental stages.
Advantageously, the compositions of the invention comprise the following concentrations of active ingredients:
the concentration of zinc, in particular in the form of zinc chloride or zinc sulphate, is approximately 1. mu.g/mL, expressed as zinc element,
the concentration of inositol, in particular in the form of cis-1, 2,3, 5-trans-4, 6-inositol, expressed as inositol, is approximately 5mg/mL,
the concentration of L-carnitine, in particular in the form of a base (base) or a hydrochloride, is about 0.6mg/mL, expressed as carnitine base,
the concentration of the melatonin basic structure (melatonin base, or melatonin base) is approximately 10-7M,
The concentration of cystine, in particular in the form of L-cystine and more particularly L-cystine hydrochloride, is approximately 50. mu.g/mL, expressed as cystine base (or cystine acid group),
the concentration of cysteamine, in particular in the form of hydrochloride, is approximately 8 μ g/mL, expressed as cysteamine base (or cysteamine group),
advantageously, the compositions according to the invention are characterized in that they comprise at least three compounds selected from the group consisting of:
a zinc salt, preferably a water-soluble salt, such as zinc chloride or zinc sulphate, in particular in a concentration of 0.1 to 3. mu.g/mL, preferably in a concentration of 0.5 to 1.5. mu.g/mL, expressed as zinc element,
inositol in any stereoisomeric form, preferably cis-1, 2,3, 5-trans-4, 6-Inositol (myo-Inositol), or its phosphate form, preferably Inositol triphosphate, especially in salt form, especially at a concentration of 1.8 to 18mg/mL, preferably 5 to 10mg/mL, expressed as Inositol,
carnitine, preferably L-carnitine, in the form of one of the stereoisomers, preferably the hydrochloride salt of carnitine, a carnitine derivative, preferably acetyl-carnitine, or a salt of a carnitine derivative, preferably in the form of the hydrochloride salt, in particular at a concentration of 0.1 to 2mg/mL, preferably 0.5 to 1mg/mL, expressed as carnitine base,
melatonin or the like, melatoninAcceptable salts or solvates of the black hormone, especially at a concentration of 10-11To 10-5M, preferably 10-10To 10-6M, especially 10-9To 10-7M, expressed as the melatonin basic structure,
cystine in the form of one of the stereoisomers, preferably levocystine, a salt of cystine, preferably a hydrochloride salt, a cystine derivative, acetylcysteine, or a salt of a cystine derivative, preferably in the form of a hydrochloride salt, in particular a concentration of cystine of 20 to 100. mu.g/mL, preferably 35 to 60. mu.g/mL, expressed as cystine basic structure,
cysteamine, a cysteamine salt, preferably a hydrochloride salt, a cysteamine derivative, preferably acetylcysteamine or a salt of a cysteamine derivative, preferably in the form of a hydrochloride salt, in particular cysteamine having a concentration of 1.5 to 15 μ g/mL, preferably 4 to 12 μ g/mL, expressed as cysteamine groups.
Advantageously, the compositions according to the invention are characterized in that they comprise the amounts preferably indicated in the present application for each of the compounds mentioned in the above group, namely zinc salts, cis-1, 2,3, 5-trans-4, 6-cyclohexanehexol optionally in the form of a phosphate salt, L-carnitine or acetyl-carnitine optionally in the form of a hydrochloride salt, melatonin, L-cystine or acetyl-cystine optionally in the form of a hydrochloride salt, and cysteamine or acetyl-cysteamine optionally in the form of a hydrochloride salt.
A preferred form of the composition according to the invention comprises about 1. mu.g/mL zinc, 5mg/mL inositol, 0.6mg/mL L-carnitine base, 10-7M melatonin basic structure, 50. mu.g/mL L-cystine basic structure and possibly 8. mu.g/mL cysteamine basic structure.
A subject of the invention is also the above-mentioned composition for in vitro culture, comprising at least one compound of the corticosteriod family, hydrocortisone, preferably in the form of a water-soluble salt, such as hydrocortisone hemisuccinate or hydrocortisone sodium succinate and hydrocortisone sodium phosphate. Preferably, in the above composition, the concentration of the cortisol salt is 5x10-7M to 10-6M, and in particular 7x10-7The amount of M, hydrocortisone hemisuccinate, was about 350ng/mL expressed as the basic structure of hydrocortisone.
A subject of the present invention is also the above-mentioned composition for in vitro culture comprising at least one zinc salt selected from the hydrated or anhydrous forms of 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.
Advantageously, the above composition comprises the above key coenzymes of energy metabolism, in particular at the following concentrations:
the concentration of NAD/NADH is approximately 1. mu.g/mL,
the concentration of NADP/NADPH was approximately 1. mu.g/mL.
A subject of the present invention is also the above composition for in vitro culture comprising at least two compounds selected from the group consisting of:
-a water-soluble salt of zinc selected from zinc sulphate or zinc chloride and/or cis-1, 2,3, 5-trans-4, 6-cyclohexanehexol and/or melatonin and/or L-carnitine hydrochloride and/or L-cystine hydrochloride and/or L-cysteamine hydrochloride and
hydrocortisone hemisuccinate, and optionally
NAD/NADH and/or NADP/NADPH.
The invention relates in particular to the composition defined above in the form of a lyophilisate, i.e. a composition whose different constituents are in the dry state and are capable of changing back to the solution state while retaining its physicochemical and biological properties.
Thus, the invention relates more particularly to a lyophilisate of the above kind, comprising:
at least two compounds selected from the group consisting of:
in particular at a concentration of 0.1 to 3. mu.g/mL, preferably at a concentration of 0.5 to 1.5. mu.g/mL, expressed as elemental zinc,
inositol in any stereoisomeric form, preferably cis-1, 2,3, 5-trans-4, 6-cyclohexanehexol, in particular in a concentration of from 1.8 to 18mg/mL, preferably in a concentration of from 5 to 10mg/mL, expressed as inositol,
carnitine, preferably L-carnitine, in the form of one of the stereoisomers, preferably the salt of carnitine, preferably the hydrochloride, a carnitine derivative, preferably acetyl-carnitine, or a salt of a carnitine derivative, in particular in a concentration of from 0.1 to 2mg/mL, preferably from 0.5 to 1mg/mL, expressed as carnitine base,
melatonin or an analogue, a melatonin-acceptable salt or solvate, in particular at a concentration of 10-11To 10-5M, preferably 10-10To 10-6M, especially 10-9To 10-7M, expressed as the melatonin basic structure,
cystine in the form of one of the stereoisomers, preferably levocystine, a salt of cystine, preferably the hydrochloride salt, a cystine derivative, preferably the acetylcysteine, or a salt of a cystine derivative, preferably the hydrochloride salt, in particular a concentration of cystine of 20 to 100. mu.g/mL, preferably 35 to 60. mu.g/mL, expressed as cystine basic structure,
and/or
Cysteamine, a cysteamine salt, preferably a hydrochloride salt, preferably a cysteamine derivative of acetylcysteamine, or a salt of a cysteamine derivative, preferably in the form of a hydrochloride salt, in particular cysteamine, having a concentration of 1.5 to 15 μ g/mL, preferably 4 to 12 μ g/mL, expressed as cysteamine basic structure,
and
at least one compound of the corticosteroid family defined above, such as hydrocortisone hemisuccinate,
-and optionally, where appropriate, or at least one of the above-defined energy metabolism-critical coenzymes, such as NAD/NADH and NADP/NADPH,
and the composition is in lyophilized form, characterized in that it is free of cytokines.
The subject of the invention is also the use of the above-mentioned composition or lyophilisate as an effector adjuvant, optionally after thawing the follicles or male gametes, for preparing a culture medium for in vitro cultureA developing follicle for maturing an oocyte contained in said follicle or for maturation of a cell of a male germ line, or for in vitro fertilization of an oocyte by spermatozoa, or for embryo culture, said in vitro culture medium comprising a composition as defined above, in admixture or in combination with an aqueous solution comprising ingredients commonly used in vitro fertilization or culture of follicles, spermatozoa or embryos. Such ingredients are known to those skilled in the art. They may in particular be selected from human or bovine serum albumin, optionally recombinant human or bovine serum albumin, and/or proteins such as KCl, NaCl, MgSO4、NaHCO3、Na2HPO4、KH2PO4And/or energetic molecules such as glucose, pyruvate, citrate and lactate, and/or amino acids for protein biosynthesis, and/or purine and pyrimidine bases for nucleic acid biosynthesis, and/or phospholipids or cholesterol for forming cell membranes, and/or vitamins, such as the B vitamins including 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 which aids in the preparation process of the culture medium, in particular one or more growth factors, in particular selected from GM-CSF, IGF-1, IGF-2, EGF, TGF or LIF, preferably GM-CSF, and/or at least one antibiotic.
In the following, the expression "mixing" means that only one solution is formed as the final culture medium in pouring the composition of the invention into an aqueous solution comprising the ingredients commonly used in vitro fertilization or in the culture of follicles, sperm or embryos; the expression "in combination" means that the two formulations, i.e. the composition of the invention and the aqueous solution, are present together, e.g. in a kit, but are not mixed prior to use.
The invention also relates to a method for preparing the above-mentioned culture medium for in vitro culture, characterized in that it comprises the step of mixing a composition according to the invention as adjuvant, supplement or "enhancer" (if present in powder or lyophilized powder form, after dissolving said composition of the invention in a suitable liquid, if appropriate) with a solution comprising the ingredients commonly used in vitro fertilization or in the culture of follicles, spermatozoa or embryos, as defined above.
Suitable liquids may be, for example, distilled water, physiological saline, recombinant or non-recombinant bovine or human serum proteins, mixtures of these different components or customary media.
The invention also relates to a culture medium, optionally after thawing of the follicles or spermatozoa, for in vitro culturing of developing follicles to mature oocytes contained in said follicles, or for maturation of cells of a male germ line, or for in vitro fertilization of oocytes by spermatozoa, or for embryo development, obtained by combining a composition of the invention in liquid, powder or lyophilisate form with an aqueous solution comprising the ingredients commonly used in vitro fertilization or culture of follicles, spermatozoa or embryos, a non-limiting list of which is indicated above.
As shown in the above-mentioned modifications, the composition according to the invention is mainly an adjuvant or "supplement" or "enhancer", which in principle is not intended to be used as a culture medium alone. They are primarily intended to be mixed with aqueous solutions comprising the components commonly used in vitro fertilization. A non-exhaustive list of these ingredients has been indicated previously.
These aqueous solutions may consist of a medium for in vitro culture which may be used as such, but the addition of adjuvants, supplements or "enhancers" according to the invention enables improved performance. Thus, these aqueous solutions may be commercial media, such as those sold under the names such as G1, G2, EmbryoGen, Blastogen, Ferticult, and HTF.
Aqueous solutions comprising ingredients commonly used in vitro fertilization or culture of follicles, sperm or embryos may also be potential media prepared by biologists in the art, and the addition of adjuvants, supplements or "enhancers" according to the present invention may improve performance and efficiency as described above and as described in the detailed description section below.
The invention more particularly relates to a culture medium, optionally after thawing of follicles or spermatozoa, for in vitro culture of developing follicles, or for maturation of oocytes contained in said follicles, or for maturation of cells of a male germ line, for in vitro fertilization of oocytes by spermatozoa, or for embryo culture, said medium being characterized in that it comprises the constituents of the composition of the invention as defined above, in combination or mixed with the aforementioned constituents as defined above, which are commonly used in vitro fertilization or in culture of follicles, spermatozoa or embryos.
The subject of the invention is also a method for maturing a mammalian developing ovarian follicle for maturing an oocyte in said follicle (more particularly a human follicle), where appropriate after thawing of a previously frozen follicle, characterized in that it comprises the step of culturing in vitro a follicle taken from a female, more particularly from a female, advantageously for about 24 to 48 hours, in a medium according to the invention as defined above.
The invention also relates to a method for maturation of cells of a mammalian, more particularly human, male germ line, where appropriate after thawing of previously frozen cells, characterized in that it comprises a step of culturing said cells in vitro in a medium according to the invention as defined above, in particular for several days, advantageously for 1 to 5 days.
The subject of the invention is also a method for in vitro fertilization of oocytes, in particular of spermatozoa of human origin and oocytes by spermatozoa, where appropriate after thawing of previously frozen spermatozoa, characterized in that it comprises a step of in vitro culturing of the above mentioned oocytes and spermatozoa in a medium according to the invention as defined above, advantageously for about 1 to about 2 days.
The invention also relates to a method for developing mammalian embryos, in particular human embryos, characterized in that it comprises a step of culturing said embryos in vitro in a medium according to the invention as defined above, preferably within 5 to 6 days after in vitro fertilization, advantageously obtaining embryos at the blastocyst stage.
In a non-limiting manner, the invention relates in particular to a method as defined above, wherein the mammal may be selected from the group consisting of primates, including human, ovine, bovine, porcine and dying animal species.
Advantageously, the composition of the invention can be used simultaneously, after optionally thawing of the follicles or spermatozoa, for maturation of cells of the follicular or male germ line, for in vitro fertilization of oocytes and for maturation of embryos resulting from said fertilization, in particular up to the blastocyst stage.
The subject of the present invention is also a kit (or kit) for the extemporaneous preparation of a culture medium as defined above, according to the invention, in particular in the case of implementation of the above-mentioned method, comprising:
-a composition according to the invention, optionally in the form of a lyophilisate, and
an aqueous solution as defined above comprising the components commonly used in vitro fertilization or in the culture of follicles, spermatozoa or embryos, in particular a commercially available medium.
The invention will be further illustrated by the following detailed description of examples of media prepared according to the invention.
As with the aqueous solution defined above, the culture medium prepared from the composition according to the invention may essentially comprise a compound selected from: such as KCl, NaCl, MgSO4、NaHCO3、Na2HPO4、KH2PO4The inorganic salt of (1); essential amino acids and other amino acids such as glutamic acid, glycine, taurine, cysteine, and glutamine; sugar and metabolic derivatives such as glucose, pyruvate, lactate, acetate; vitamins, particularly the vitamin B group and vitamin C; purine and pyrimidine bases; albumin; a phospholipid; cholesterol; insulin and/or at least one substance contributing to the process, in particular a growth factor; antibiotics such as penicillin G, streptomycin, gentamicin, or other aminoglycosides.
The invention, which can be present in various forms (liquid, powder or lyophilisate), can be mixed, before conditioning, with a liquid solution comprising the ingredients commonly used in vitro fertilization or in the culture of follicles, spermatozoa or embryos, which solution can therefore itself be a ready-to-use culture medium. The improved medium resulting from the mixture will be intended for use in culturing developing follicles to mature oocytes, optionally after thawing of follicles or sperm, or for cell maturation of male germ lines, or for in vitro fertilization of oocytes by sperm, or for embryo culture. The composition according to the invention may also be temporarily mixed with a liquid solution comprising ingredients commonly used in vitro fertilization or culture of follicles, sperm or embryos. The resulting solution was the culture medium.
Detailed Description
The following describes the formulation of compositions of the effector adjuvants of the invention that can be used to prepare culture media for in vitro culture of developing follicles to mature oocytes in follicles, or for maturation of cells of the male germ line, or for in vitro fertilization of oocytes by sperm, or for culture of embryos, optionally after thawing of follicles or sperm.
In all the following experiments, the active ingredients were finally used at the following concentrations:
-zinc expressed as element 1 μ g/mL
-inositol expressed as a molecule 5mg/mL
-l-carnitine expressed as basic structure 0.6mg/mL
-melatonin ═ 10 expressed in the basic structure-7M
Cystine expressed as basic structure 50 μ g/mL
-cysteamine expressed as basic structure 8 μ g/mL
Hydrocortisone expressed as basic structure 7x10-7M
We chose to pre-prepare the composition at a concentration 10 times higher than the above. Therefore, in carrying out the method for its application, these solutions should be temporarily diluted in a normal medium in a proportion of one tenth.
All manipulations were performed in sterile raw materials and sterile media.
Composition 1
The solution prepared extemporaneously by mixing a culture medium with the composition comprising zinc salt and melatonin according to the invention is distributed in 1mL flasks, so that each flask comprises 10 μ g of elemental zinc and 10-6M melatonin.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 2
The solution prepared extemporaneously by mixing a culture medium with a composition comprising zinc salt, inositol and melatonin according to the invention is dispensed in 1mL flasks, so that each flask comprises 10 μ g of zinc element, 50mg of inositol and 10mg of melatonin-6M melatonin.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 3
The solution prepared extemporaneously by mixing one medium with the composition comprising zinc salt, inositol, melatonin and cystine according to the invention was dispensed in 1mL flasks, so that each flask contained 10 μ g of zinc element, 50mg of inositol, 10mg of cystine-6M melatonin and 0.5mg cystine basic structure.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 4
The solution prepared extemporaneously by mixing one medium with the composition comprising zinc salt, inositol, melatonin, cystine and carnitine according to the invention was dispensed in 1mL flasks, such that each flask contained 10 μ g of zinc element, 50mg of inositol, 10mg of carnitine-6M melatonin, 0.5mg cystine basic structure and 6mg L-carnitine basic structure.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 5
By mixing a culture medium with a medium according to the invention containing zinc salt, inositol, melatonin, cystineThe solution prepared extemporaneously by mixing the composition of acid, cysteamine and carnitine was dispensed into 1mL flasks such that each flask contained 10 μ g of elemental zinc, 50mg of inositol, 10mg of carnitine-6Melatonin of M, a cystine basic structure of 0.5mg, a cysteamine basic structure of 80. mu.g, and a carnitine basic structure of 6 mg.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 6
The solution prepared extemporaneously by mixing one medium with the composition comprising zinc salt, inositol and carnitine according to the invention is distributed in 1mL flasks, so that each flask contains 10 μ g of zinc element, 50mg of inositol and 6mg of l-carnitine basic structure.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 7
The solution prepared extemporaneously by mixing a culture medium with a composition comprising inositol, melatonin and cystine according to the invention was dispensed in 1mL flasks, so that each flask contained 50mg inositol, 10mg cystine-6M melatonin and 0.5mg cystine basic structure.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 8
The solution prepared extemporaneously by mixing one culture medium with the composition comprising zinc salt, inositol, melatonin and carnitine according to the invention was dispensed in 1mL flasks, such that each flask contained 10 μ g of zinc element, 50mg of inositol, 10mg of carnitine-6Melatonin at M and the basic structure of 6mg l-carnitine.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 9
The solution prepared extemporaneously by mixing one culture medium with the composition comprising inositol, melatonin, cystine and carnitine according to the invention is distributed in 1mL flasks, so that each flask comprises50mg inositol, 10-6M melatonin, 0.5mg cystine basic structure and 6mg L-carnitine basic structure.
In practice, the solution should be diluted in the culture medium in a tenth ratio.
Composition 10
A solution prepared extemporaneously by mixing a medium with a composition comprising hydrocortisone salts, zinc salts and melatonin according to the invention was dispensed in 1mL flasks such that each flask contained 7x10-6M hydrocortisone, 10. mu.g zinc and 10-6M melatonin.
In carrying out the process, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 11
The solution prepared extemporaneously by mixing one medium with a composition comprising hydrocortisone salt, zinc salt, inositol and carnitine according to the invention was distributed in 1mL flasks, such that each flask contained 7x10-6M hydrocortisone, 10. mu.g of zinc, 50mg of inositol and 6mg of L-carnitine.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 12
The solution prepared extemporaneously by mixing one medium with a composition comprising hydrocortisone salt, zinc salt and inositol according to the invention was dispensed in 1mL flasks, such that each flask contained 7x10-6M hydrocortisone, 10. mu.g of Zinc and 50mg of inositol.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 13
A solution prepared extemporaneously by mixing a medium with a composition comprising a zinc salt and inositol according to the present invention was dispensed into 1mL flasks such that each flask contained 10 μ g of zinc element and 50mg of inositol.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 14
The solution prepared extemporaneously by mixing one medium with a composition comprising hydrocortisone salt, zinc salt and carnitine according to the invention was distributed in 1mL flasks, such that each flask contained 7 × 10-6M hydrocortisone, 10. mu.g zinc element and 6mg L-carnitine.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 15
The solution prepared extemporaneously by mixing one medium with the composition comprising zinc salt and carnitine according to the invention is distributed in 1mL flasks, so that each flask contains 10 μ g of zinc element and 6mg of l-carnitine basic structure.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 16
The solution prepared extemporaneously by mixing one medium with a composition comprising hydrocortisone salt, inositol and cystine according to the invention was dispensed in 1mL flasks, such that each flask contained 7 × 10-6M hydrocortisone, 50mg inositol and 0.5mg cystine basic structure.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Composition 17
The solution prepared extemporaneously by mixing one medium with a composition comprising inositol and cystine according to the invention was dispensed in 1mL flasks, so that each flask contained 50mg inositol and 0.5mg cystine basic structure.
In practicing the method, the solution should be diluted in the culture medium in a proportion of one tenth.
Each flask of each of the compositions from 1 to 17 was stored at +4 ℃ or below-20 ℃ when the contents of these compositions were lyophilized.
Testing 1) bodies of mammalian oocytesExo-maturation
Depending on the stage of oocyte maturation, the "blastocyst stage oocytes" or "metaphase I stage oocytes" are cultured in conventional in vitro maturation medium supplemented with two or more substances.
Test 1a) oocytes at the foaming (VG) stage
The number of VG oocytes evolved to the Metaphase I (MI) or metaphase II (MII) stage was determined.
In this experiment, 10 groups were used.
Group 1 (control), VG oocytes were placed in wells containing medium only.
Group 2, VG oocytes were placed in wells containing culture medium and a composition according to the invention comprising melatonin and zinc.
Group 3, VG oocytes are placed in wells containing culture medium and a composition according to the invention comprising melatonin, zinc and inositol.
Group 4, VG oocytes are placed in wells containing culture medium and a composition according to the invention comprising melatonin, zinc, inositol and cystine.
Group 5, VG oocytes were placed in wells containing medium and a composition according to the invention comprising melatonin, zinc, inositol, cystine, cysteamine and carnitine.
Group 6, VG oocytes were placed in wells containing medium and a composition according to the invention comprising melatonin, zinc and hydrocortisone.
Group 7 VG oocytes are placed in wells containing culture medium and a composition according to the invention comprising zinc and inositol.
Group 8, VG oocytes were placed in wells containing medium and a composition according to the invention comprising zinc, inositol and hydrocortisone.
Group 9, VG oocytes were placed in wells containing culture medium and a composition according to the invention comprising zinc and carnitine.
Group 10 VG oocytes were placed in wells containing medium and a composition according to the invention comprising zinc, carnitine and hydrocortisone.
Different groups of oocytes were placed in the same oven at 37 ℃ in a medium containing 5% CO2Is cultured in the atmosphere of (2).
Results
It was demonstrated that the addition of a composition comprising substances such as hydrocortisone, zinc, inositol, carnitine, melatonin, cystine and/or cysteamine according to the present invention resulted in an improved maturation of VG oocytes in the MI and mil phases at the end of 24 and 48 hours of culture relative to VG oocytes cultured in medium only.
Test 1b) metaphase I stage oocytes
In this experiment, the number of oocytes that evolved to MII was determined. By selecting oocytes in the MI stage, the same protocol as described above was used.
Results
It was demonstrated that the addition of the composition according to the invention leads to an improved maturation of MI oocytes into the MII stage in the presence of the composition according to the invention comprising substances such as hydrocortisone, zinc, inositol, carnitine, melatonin, cystine and/or cysteamine.
In both mentioned experiments, it was also demonstrated that the addition of hydrocortisone leads to an improved maturation of the oocytes compared to the absence of hydrocortisone.
Test 2) in vitro fertilization
In order to evaluate the effect of the various substances (hydrocortisone, zinc, inositol, l-carnitine, melatonin, cystine and cysteamine) comprised in the composition according to the invention on in vitro fertilization, each experiment was carried out in a mammal.
To this end, the most mature eggs and best sperm were selected to promote fertilization and the number of embryos formed after 48 hours of culture was evaluated.
As with the assessment of in vitro maturation, 10 groups were used in this assay.
In each group, mature oocytes were placed in the presence of about 106Viable sperm alone, according to the same as beforeIs added to a culture medium according to the present invention comprising a composition of substances such as hydrocortisone, zinc, inositol, carnitine, melatonin, cystine and/or cysteamine.
Results
It was demonstrated that the addition of a composition comprising substances such as hydrocortisone, zinc, inositol, carnitine, melatonin, cystine and/or cysteamine according to the invention to the culture medium increased the percentage of fertilized eggs relative to the group of eggs present in the culture medium alone.
It was also demonstrated that the addition of hydrocortisone leads to improved fertilization results compared to the absence of hydrocortisone.
Test 3) early embryo development
To assess the effect of compositions according to the invention comprising substances such as hydrocortisone, zinc, inositol, carnitine, melatonin, cystine and/or cysteamine on early embryo development, mouse CF-1 strain may be used, since the number of embryos that evolve to the blastocyst stage in normal medium is small. The model allows assessment of the beneficial effects of these substances on early embryo development.
In this experiment, 10 groups of embryos were cultured under different conditions using the same protocol.
Results
It was demonstrated that the addition of a composition comprising substances such as hydrocortisone, zinc, inositol, carnitine, melatonin, cystine and/or cysteamine according to the present invention leads to an improved development of the embryos at different stages with respect to the group comprising only the culture medium.
It was also demonstrated that the addition of hydrocortisone results in improved early embryo development compared to the absence of hydrocortisone.
Claims (10)
1. A composition in the form of a liquid, powder or lyophilisate, optionally after thawing of follicles or sperm, for in vitro culture of developing follicles to mature oocytes contained in said follicles, or for maturation of cells of a male germ line, or for in vitro fertilization of oocytes by sperm, or for embryonic development, characterised in that said composition comprises:
at least two or at least three compounds selected from the group consisting of:
a zinc salt, preferably a water-soluble salt, such as zinc chloride or zinc sulphate,
inositol, preferably cis-1, 2,3, 5-trans-4, 6-inositol in any stereoisomeric form, or in the form of its phosphate, preferably inositol triphosphate, in particular in the form of a salt,
carnitine, preferably l-carnitine, in the form of one of the stereoisomers, preferably the salt of carnitine, preferably the hydrochloride, a carnitine derivative, preferably acetyl-carnitine, or
The salts of carnitine derivatives are preferably in the form of the hydrochloride,
melatonin or an analogue, an acceptable salt or solvate of melatonin,
cystine, preferably l-cystine, in the form of one of the stereoisomers, a salt of cystine, preferably hydrochloride, a cystine derivative, preferably acetylcysteine, or a salt of a cystine derivative, preferably in the form of hydrochloride,
cysteamine, a cysteamine salt, preferably a hydrochloride salt, a cysteamine derivative, preferably acetylcysteamine, or a salt of a cysteamine derivative, preferably in the form of a hydrochloride salt,
one or more compounds from the corticosteroid family
And, optionally,
one or more of the energy metabolism-critical coenzymes, such as NAD/NADH and NADP/NADPH,
and wherein the composition is free of cytokines.
2. A composition in liquid form, optionally after thawing of follicles or spermatozoa, for in vitro culture of developing follicles to mature oocytes contained in said follicles, or for maturation of cells of a male germ line, or for in vitro fertilization of oocytes by spermatozoa, or for embryonic development, characterized in that the composition comprises:
at least two or at least three compounds selected from the group consisting of:
a zinc salt, preferably a water-soluble salt, such as zinc chloride or zinc sulphate, in particular in a concentration of 0.1 to 3. mu.g/mL, preferably in a concentration of 0.5 to 1.5. mu.g/mL, expressed as zinc element,
inositol in any stereoisomeric form, preferably cis-1, 2,3, 5-trans-4, 6-Inositol (myo-Inositol), or its phosphate form, preferably Inositol triphosphate, especially in salt form, especially at a concentration of 1.8 to 18mg/mL, preferably 5 to 10mg/mL, expressed as Inositol,
carnitine, preferably L-carnitine, in the form of one of the stereoisomers, preferably the hydrochloride salt of carnitine, a carnitine derivative, preferably acetyl-carnitine, or a salt of a carnitine derivative, preferably in the form of the hydrochloride salt, in particular at a concentration of 0.1 to 2mg/mL, preferably 0.5 to 1mg/mL, expressed as carnitine base,
melatonin or an analogue, an acceptable salt or a melatonin solvate, in particular at a concentration of 10-11To 10-5M, preferably 10-10To 10-6M, especially 10-9To 10-7M, expressed as the melatonin basic structure,
cystine in the form of one of the stereoisomers, preferably levocystine, a salt of cystine, preferably a hydrochloride salt, a cystine derivative, preferably acetylcysteine, or a salt of a cystine derivative, preferably in the form of a hydrochloride salt, in particular cystine at a concentration of 20 to 100. mu.g/mL, preferably 35 to 60. mu.g/mL, expressed as cystine basic structure,
cysteamine, a cysteamine salt, preferably a hydrochloride salt, a cysteamine derivative, preferably acetylcysteamine, or a salt of a cysteamine derivative, preferably in the form of a hydrochloride salt, in particular cysteamine, having a concentration of 1.5 to 15 μ g/mL, preferably 4 to 12 μ g/mL, expressed as cysteamine basic structure,
one or more compounds from the corticosteroid family
And, optionally,
one or more of the energy metabolism-critical coenzymes, such as NAD/NADH and NADP/NADPH,
and the composition is free of cytokines.
3. Composition according to claim 1 or 2, characterized in that said at least one compound of the corticosteriodal steroid family is preferably chosen from: hydrocortisone in the form of a water soluble salt, such as hydrocortisone hemisuccinate, hydrocortisone sodium succinate and hydrocortisone sodium phosphate, wherein the concentration of the salt of hydrocortisone in the aforementioned composition is 5x10-7M to 10- 6M, especially 7x10-7M, 350ng/mL for hydrocortisone hemisuccinate, expressed as hydrocortisone basic structure.
4. A composition according to claim 1,2 or 3, characterized in that it comprises at least one zinc salt selected from the hydrated or anhydrous forms of 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. The composition according to claim 1, characterized in that it is in the form of a lyophilisate.
6. Use of a composition according to any one of claims 1-5 as an effector adjuvant for the preparation of a culture medium for in vitro culturing of a developing follicle to mature an oocyte contained in the follicle, or for maturation of cells of a male germ line, or for in vitro fertilization of an oocyte by spermatozoa, or for embryo development, optionally after thawing of the follicle or spermatozoa, the in vitro cultured medium comprising a composition according to any one of claims 1-5, mixed with an aqueous solution comprising ingredients commonly used in vitro fertilization or culture of follicles, spermatozoa or embryos, in particular selected from human or bovine serum albumin, optionally recombinant human or bovine serum albumin, and/or inorganic salts, and/or high energy molecules such as glucose, pyruvate, citrate and lactate, and/or amino acids for the biosynthesis of proteins, and/or purine and pyrimidine bases for the biosynthesis of nucleic acids, and/or phospholipids or cholesterol for the formation of cell membranes, and/or vitamins, such as the B vitamins including 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 one or more growth factors, in particular selected from GM-CSF, IGF-1, IGF-2, EGF, TGF or LIF, preferably GM-CSF, and/or at least one antibiotic.
7. A culture medium, optionally after thawing of follicles or spermatozoa, for in vitro culture of developing follicles to mature oocytes contained in said follicles, or for maturation of cells of a male germ line, or for in vitro fertilization of oocytes by spermatozoa, or for embryo development, obtained by a process of combining a composition according to any one of claims 1 to 5 in liquid, powder or lyophilisate form with an aqueous solution comprising ingredients commonly used in vitro fertilization or culture of follicles, spermatozoa or embryos, in particular selected from the ingredients of claim 6.
8. A culture medium for in vitro culture of a developing follicle for maturation of an oocyte contained in the follicle, or for maturation of a cell of a male germ line, or for in vitro fertilization of an oocyte by spermatozoa, or for embryo culture, optionally after thawing of the follicle or of the spermatozoa, characterized in that it comprises the constituents of a composition according to any one of claims 1 to 5 in combination with the constituents as defined in claim 6 as being commonly used in vitro fertilization or in culture of follicles, spermatozoa or embryos.
9. An in vitro method for maturing a developing ovarian follicle of a mammal in order to mature an oocyte in said follicle, more particularly a human ovarian follicle, optionally after thawing of a previously frozen follicle, characterized in that it comprises the step of culturing an ovarian follicle taken from a female, more particularly from a female, in vitro in a medium according to claim 8, advantageously for about 24 to 48 hours.
10. Kit for the temporary preparation of a culture medium according to claim 8, in particular in the case of carrying out a method according to claim 9, characterized in that it comprises:
-a composition according to any one of claims 1-5 in the form of a liquid, powder or lyophilizate;
-an aqueous solution as defined in claim 6 comprising ingredients commonly used in the culture of follicles, spermatozoa or embryos.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR17/53233 | 2017-04-12 | ||
| FR1753233A FR3065225B1 (en) | 2017-04-12 | 2017-04-12 | ADJUVANTS FREE OF CYTOKINES FOR CELL CULTURE MEDIA, PARTICULARLY FOR IN VITRO FERTILIZATION, OR FOR THE CULTURE OF FOLLICLES, MALE GERM CELLS OR EMBRYOS |
| PCT/FR2018/000089 WO2018189436A1 (en) | 2017-04-12 | 2018-04-12 | Cytokine-free adjuvants for cell culture media, in particular for in vitro fertilisation, or for the culture of follicles, male germ cells or embryos |
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| CN110709505A true CN110709505A (en) | 2020-01-17 |
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| CN111280163A (en) * | 2020-02-12 | 2020-06-16 | 天津博裕力牧科技有限公司 | Egg extracting liquid for egg taking of live cattle and preparation method and application thereof |
| CN111280163B (en) * | 2020-02-12 | 2021-11-09 | 天津博裕力牧科技有限公司 | Egg extracting liquid for egg taking of live cattle and preparation method and application thereof |
| CN111235092A (en) * | 2020-03-06 | 2020-06-05 | 广州裕康生物科技有限公司 | Blastocyst culture solution and preparation method thereof |
| CN111235092B (en) * | 2020-03-06 | 2022-03-25 | 佛山辅康生物科技有限公司 | Blastocyst culture solution and preparation method thereof |
| CN111548987A (en) * | 2020-05-14 | 2020-08-18 | 温氏食品集团股份有限公司 | Pig oocyte in-vitro maturation culture solution and application thereof |
| CN111548987B (en) * | 2020-05-14 | 2021-07-09 | 温氏食品集团股份有限公司 | Pig oocyte in-vitro maturation culture solution and application thereof |
| CN114836373A (en) * | 2022-05-30 | 2022-08-02 | 上海洲康医疗器械有限公司 | Oocyte activation culture medium and culture method and application thereof |
| CN114836373B (en) * | 2022-05-30 | 2023-11-10 | 上海洲康医疗器械有限公司 | Egg cell activation culture medium and cultivation method and application thereof |
| CN115029303A (en) * | 2022-06-22 | 2022-09-09 | 安徽第肆纪医学科技有限公司 | IVM energy culture solution for human immature oocyte in COH cycle |
| CN115029303B (en) * | 2022-06-22 | 2023-08-15 | 安徽第肆纪医学科技有限公司 | Human immature oocyte IVM energy culture solution for COH period |
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| Publication number | Publication date |
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| WO2018189436A1 (en) | 2018-10-18 |
| FR3065225A1 (en) | 2018-10-19 |
| FR3065225B1 (en) | 2024-04-05 |
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