FR2910024A1 - Cell culture medium comprises a defined amino acid mixture, mineral salts, sugars, organic acids, nitrogen sources and vitamins and/or antioxidants - Google Patents

Abstract

Cell culture medium comprises an amino acid mixture, mineral salts, sugars, organic acids, nitrogen sources and vitamins and/or antioxidants. The amino acid mixture comprises (mole% +- 20%): alanine (9.93), arginine (5.54), asparagine (5.09), aspartic acid (1.7), cystine (4.13), optionally glutamine (16.35), glutamic acid (2.29), glycine (6.2), histidine (4.58), isoleucine (1.96), leucine (3.71), lysine (5.61), methionine (0.81), phenylalanine (1.59), proline (9.52), serine (9.96), threonine (5.68), tryptophan (0.52), tyrosine (1.11) and valine (3.74). Independent claims are also included for: (1) cell multiplication by culturing cells in a medium as above; (2) producing a protein of interest (I) by culturing (I)-expressing cells in a medium as above and optionally harvesting (I); (3) producing a virus (II) by culturing (II)-infected cells in a medium as above and optionally harvesting (II).

Description

The invention relates to a synthetic cell culture medium,

  chemically defined, preferably serum-free and aprotic, a cell culture method in this synthetic medium, chemically defined, and a method for producing recombinant proteins, in particular monoclonal antibodies, interferon gamma or viruses. The first cultures of vertebrate cells were made using biological fluids (chorioallantoic liquid, serum), then using such fluids diluted in a buffer solution (eg Earle salts) supplemented with hydrolyzate of lactalbumin and extract yeast.

  Cell culture, in particular mammalian cells, is now commonly done in a so-called basic or minimum medium supplemented with biological products such as serum (in particular fetal bovine serum, SFB) and / or growth factors, and / or cytokines.  This methodology has a long history of success.  However, it has disadvantages: the serum added has no chemically defined composition and can vary from one batch to another, and on the other hand the risk factor (probability of the presence of a pathogen, of a toxic derivative ,. . . ) is not defined.  With the advent of recombinant protein production technologies, the use of semi-synthetic media has developed.  There are many formulations of chemically defined semi-synthetic media.  However, for the most part, these formulations contain added recombinant proteins such as, for example, insulin, or peptide growth factors.  Only a few formulations are really protein-free.  This is the case of Darfler's ABC medium (Darfler FJ, 1990, In Vitro Cell Dev Biol. ; 26 (8): 769-78; Darfler FJ, 1991 US Patent No. 5045468), Schneider Modified Sato Medium (BDM Medium, Schneider YJ, 1989, J Immunol Methods. ; 116 (1): 65-77).  These media make it possible to cultivate hybridomas and some other cell lines.  However, the ABC medium is very complex since it consists of 83 components.  It is prepared from 3 solutions stored separately (A: acid, B: basic, C: concentrated).  It allows the same level of monoclonal antibody production by hybridoma L243 as that obtained with DMEM medium + 10% SFB (5 μg / ml).  Schneider's BDM medium is derived from Sato's medium, i.e. it is a mixture of DMEM Iscove, F12 Ham and NCTC 135 media.  The BDM medium also makes it possible to culture hybridomas.  The level of production of monoclonal antibody by hybridoma 7F11 C7 obtained in BDM medium is identical to that obtained in DMEM medium + 10% SFB (60 μg / ml).  However, the BDM medium may comprise albumin carrying linoleic acid.  European Patent Application EP 1482031 also describes a defined medium, serum-free and aprotic, particularly intended for epithelial cell cultures and fibroblasts.  However, this medium includes an iron chelator and a zinc salt which are believed to replace transferrin and heparin, respectively, thereby allowing the medium to facilitate the growth of mammalian cells.  The long-term culture of cells in a chemically defined and aprotic synthetic medium, however, remains very little developed, whereas it has many advantages: pathogens that are not detectable by conventional methods, transported by the serum, are absent (securing ).  This concerns the unconventional agents, but also the conventional agents present at too low concentration.  For example, if a serum lot contains 100 mycoplasma per liter, the contamination will be detectable only once in ten with a test sample of 1 milliliter.  On the other hand cells regularly cultured in a medium supplemented with this serum will inevitably be infected.  - The production, in cell culture, of certain viruses, including Rotavirus, is low in medium supplemented with serum.  This production is considerably increased in the absence of serum, and in the presence of trypsin.  This involves washing the cells to remove serum inhibitors of trypsin.  In this case the serum-free culture saves time and allows a lower cost (less medium, no serum).  - The stress induced in the cell by the deprivation of serum is suppressed.  The secreted protein of interest in the culture supernatant is relatively clean.  In particular, the protein of interest, which may be a monoclonal antibody secreted by a hybridoma or a recombinant protein secreted by a transfected cell line, is immediately found in an environment which is not contaminated with other undesirable proteins.  The use of an aprotic synthetic medium therefore greatly facilitates subsequent purification operations of the protein of interest over a 10% serum medium which contains about 7.5 mg / ml of protein, or a semi-synthetic medium that contains insulin, transferrin, and sometimes albumin.  In addition, the use of an aprotic medium makes it possible to analyze, without radiolabeling, the secreted proteins in the culture supernatants (for example after viral infection).  This analysis is otherwise virtually impossible even if the cells, initially cultured in serum medium, are then replaced in an aprotic medium.  Indeed, when they have been previously cultured in the presence of serum, the cells placed in serum-free medium release the serum proteins that they have adsorbed; these serum proteins mix with secreted proteins that can not be distinguished.  A defined medium has a chemically defined and constant formulation, in contrast to formulations containing serum or added proteins for which the batches of sera or proteins differ from each other, thereby achieving reproducibility of the experiments. laboratory or industrial productions.  - A lower cost: the addition of serum multiplies by 3 to 8 the costs of production.  Moreover, it is necessary to test several batches of fetal serum or added proteins before acquiring a lot whose characteristics are as close as possible to those of the previous batch.  This necessary procedure is long, tedious and expensive.  - The suppression of pharmacological effects that can be induced by serum proteins (fetal and bovine) on cells in culture.  The invention thus provides a chemically defined synthetic medium which makes it possible to culture mammalian or non-mammalian cells, in particular hybridomas, but also numerous non-lymphoid cell lines.  The establishment of the chemically defined formula of this medium is described hereinafter, as is the use of this medium for the production of viruses, recombinant proteins, and monoclonal antibodies by hybridomas.  The results obtained show that the medium according to the invention provides performance that is as good or better than that obtained with the serum-free, aprotic media of the prior art.  Synthetic and Aproteic Defined Medium The invention provides a chemically defined, synthetic cell culture medium whose base formulation is devoid of serum and protein.  The medium may however contain serum, proteins, hormones, growth factors, if the user deems it necessary for the intended use.  By aprotic medium is meant a cell culture medium that does not contain protein at the time of seeding with the cells.  However, once the culture of the primed cells, the medium may contain proteins; it will then be proteins that have been synthesized by the cells in 10 cultures.  By protein is meant indifferently peptides or polypeptides, that is to say sequences of at least two amino acids linked by a peptide bond.  The culture medium according to the invention is a chemically defined synthetic medium, i.e. the nature and concentration of the constituents of the medium are exactly known.  Thus the invention provides a chemically defined synthetic cell culture medium which comprises: - a mixture of natural amino acids; At least one mineral salt; - at least one sugar; at least one organic acid; at least one nitrogenous nutrient; and at least one vitamin and / or antioxidant.  This medium allows the culture of a very large number of cells without the need to add serum, hormone (insulin for example), or growth factors.  Advantageously, this medium is therefore serum-free and aprotic.  To determine the amino acid composition of the medium, the inventor relied on mammalian cell usage codon data (i.e., the frequency of cell use of the different codons) that have been transformed into the proportions of amino acids needed for protein synthesis.  Furthermore Franek and Sramkova (1996, Immunol.  Lett. ; 52 (2-3): 139-44) demonstrated that cells in culture require high concentrations of hydrophilic amino acids.  On these bases, an original dosage of 20 amino acids has been established to take into account the amino acid requirements of cells in culture for the synthesis of proteins but also their amino acid requirements as metabolites.  This dosage can be varied as to the overall amounts of amino acids, but it is preferable to respect the proportions between amino acids.  Thus, in the cell culture medium according to the invention, each amino acid is present in the mixture of 20 natural amino acids according to the percentage (in molarity / total molarity) indicated in Table 1.  Table 1: Percentage of each amino acid in the mixture of the 20 naturally occurring amino acids Amino acids Ala Arg, Asn Asp Leu, Lys Met Phe% (molarity / total molarity of the mixture) Preferred embodiment (molarity / total molarity of the mixture) 9.39 0.99 5.54 0.55 5.09 0.51 1.70 0.17 4.13 0.41 16.35 1.63 2.29 0.23 6.20 0.62 4, 58 0.46 1.96 0.20 3.71 0.37 5.61 0.56 0.81 0.08 1.59 0.16 9.52 0.95 9.96 1.00 5.68 0 , 57 0.52 0.05 1.11 0.11 3.74 0.37 Still preferred embodiment% (molarity / total molarity of the mixture) 9 93 0 50 5, 541 0.28 5.09 0.25 1.70 0.08 4.13 0.21 16.35 0.82 2.29 0.11 6.20 0.31 4 58 0 23 1.96 0.10 3.71 0.19 5.61 0 , 28 0.81 0.04 1.59 0.08 9.52 0.48 9.96 0.50 5.68 0.28 0.52 0.03 111 + 006 3.74 0.19 9.93 1.99 5.54 1.11 5.09 1.021 1.70 0.34 4.13 0.83 16.35 3.27 2.29 0.46 620 124 4.58 0.92 1.96 0, 39 3.71 0.74 5.61 1.12 0.81 0.16 159.032 9.52 1,90 9,96 1,99 5,68 1,14 Trp 0,52 0,10 Tyr 1,11 0,22 Val 3,74 0,75: cysteine is preferentially supplied in the form of cystine.  Cystine may, however, be replaced by cysteine, which is then given in double the amount of cystine.  #: possibly absent because unstable in solution; it can be added extemporaneously by the user to the indicated quantity.  It has been found that the overall amount of amino acids in the medium can vary up to a factor of 7.5 while maintaining satisfactory culture results.  Indeed, if the cultured cells are not very demanding (for example, if they are non-secretory cells), the overall amount of amino acids can be reduced in relation to the overall amount that would be used for more demanding cells such as secretory cells.  Thus, in the culture medium according to the invention, the overall concentration of amino acids can vary from about 8.16 x 10 -3 M to about 61.22 x 10 -3 M.  The ranges of possible concentrations, or preferred concentrations, for each of the amino acids in the medium are shown in Tables 2-4.  The overall quantity of amino acids determined for the medium designated H-MSM-1 corresponds to a quantity suitable for undemanding cells, for example cells of the BoSk, CV1 or L929 lines.  The overall amount of amino acids determined for the medium designated H-MSM-2 corresponds to an amount close to the optimum amount for most of the lines studied (Table 20).  Table 2: Concentration of Amino Acids in Culture Medium H-MSM-1 Concentration in Medium H-MSM-1 (10-3 M) 1.345 0.269 0.75 0.150 0.69 0.138 0.23 0.046 0.56 0.112 2,215 0.443 0.31 0.062 0.84 0.168 0.62 0.124 0.265 0.502 0.76 0.11 0.215 1.29 1.35 Amino acid (Cys-Cys) Gln Gly His lily Leu Lys Met Phe Pro Preferably, preferably more preferably 0.265 0.502 0.76 0.11 0.215 1.29 1.35 1.345 0.135 0.75 0.075 0.69 0.069 0.23 0.023 0.56 0.056 2.215 0.222 0.31 0.031 0.84 0.084 0.62 0.062 1.345 0.067 1.345 0.75 0.038 0.75 0 69 0.035 0.69 0.23 0.012 0.23 0 56 0.028 0.56 2215 0.11 2.215 0.31 0.016 0.31 0.84 0.042 0, 84 0.62 0.031 0.62 0.053 0.265 0.027 0.265 0'013 0.100 0.502 0.050 0.502 0.025 0.152 0.76 0.076 0.76 0.038 0.022 0.11 0.011 0.11 0.006 0.043 0.215 0.022 0.215 0.011 0.258 1.29 0.129 1 , 29 0.065 0.270 1.35 0.135 1.35 0 068 2910024 7 still Thr 0 , 77 0.154 0.77 _ + 0.077 0.77 0.039 0.77 Trp 0.07 0.014 0.07 0.007 0.07 0.004 0.07 Tyr 0.15 0.030 0.15 0.015 0.15 0.008 0.15 Val 0.507 0.101 0.507 0.051 0, 507 0.025 0.507 Total molarity 13.549 2.710! 13,549 1,355 13,549 0,677 13,549 *: the cysteine is preferentially brought in the form of cystine in the medium.  However, cystine can be replaced by cysteine, which is then added twice as much as cystine.  #: possibly absent because unstable in solution; it can be added extemporaneously by the user at the indicated amount.  It is possible to vary the concentration of all the amino acids by a factor of between about 0.75 and 3.75, relative to the defined amino acid concentrations for the H-MSM-1 medium, while retaining satisfactory culture results (Table 3).  The concentration of each of the amino acids in the culture medium is then equal to the concentrations shown in Table 2, multiplied by a factor of between 0.75 and 3.75 (inclusive).  Table 3: Recommended minimum and maximum concentrations for the 15 amino acids in the culture medium according to the invention Concentration in the medium (10 -3 M) Amino acid Concentration in the H-MSM-1 medium (10 -3 M) preferably, preferably Amino acid Ala Arg Minimum concentration Maximum concentration 0.81 6.05 0.45 3.38 Asn 0.11 3.31 Asp 0.14 1.04 (Cys-Cys) * 0.34 2 , 52 Gln # 1.33 9.97 Glu 0.19 1.40 Gly 0.50 3.78 His 0.37 2.79 Island 0.16 1.19 Leu 0.30 2.26 Lilies 0.46 3 , 42 Met 0, 07 0,50 Phe 0,13 0,97 Pro 0,77 5,81 Ser 0,81 6,08 2910024 8 Concentration in the medium (10 "3 M) Minimum concentration Maximum concentration 0, 46 3 0.04 0.32 0.09 0.68 0.30 2.28 8. 13 60.97 *: the cysteine is preferentially brought in the form of cystine in the medium.  However, cystine can be replaced by cysteine, which is then added twice as much as cystine.  #: possibly absent because unstable in solution; it can be added extemporaneously by the user at the indicated amount.  Table 4: Concentration of amino acids in the culture medium H-MSM-2 Concentration in the medium H-MSM-2 (10-3 M) Amino acid preferably, preferably, preferably. Total Molarity Ala Arg Asn Asp (Cys-Cys) * Gln #, Glu Leu Lys Met Phe Pro 2.69 0.54 1.5 0.30 1.38 0.28 0.46 0.09 1.12 0, 22 4.43 0.89 0.62 0.12 Gly 1.68 0.34 His 1.24 0.25 Ile 053 + 011 1 04 + 0 20 1.52 0.30 0.22 0.04 0, 43 0.09 2.58 0.52 Ser 2.7 0.54 Thr 1.54 0.31 Trp 0.14 0.03 Tyr 0.3 0.06 2.69 0.27 1.5 0.15 1.38 0.14 1.68 0.17 1.24 0.12 0.53 0.05 1.04 0.10 1.52 0.15 0.22 0.02 0.43 0.04 2 58 0.26 2.7 0.27 1.54 0.15 0.14 0.01 0.3 0.03 0.3 0.02 0.3 1.141 0.10 1.14 0.05 1.14 0.62 0.03 1.68 0.08 1.24 + 0.06 0.53 0.03 1.04 0.05 1.52 0.08 022 001 0.43 0.02 258 013 2.7 0.14 2.7 1.54 0.08 1.54 0.14 0.01 0.14 0.43 2.58 Val 1.14 0.20 Total molarity 27.10 5.42 27.10 2, 71 27.10 1.35 27.10 *: the cysteine is preferentially provided in the form of cystine in the medium.  However, cystine can be replaced by cysteine, which is then added twice as much as cystine.  #: possibly absent because unstable in solution; it can be added extemporaneously by the user to the indicated quantity.  Since glutamine is very unstable in solution, it may possibly be absent from the amino acid mixture present in the culture medium according to the invention.  In these cases, it can be added in the medium by the end user, at the recommended concentrations, before use of the medium.  The inorganic salts which can be used in the culture medium according to the invention include sodium, potassium, calcium, magnesium, phosphate, carbonate and iron salts, and trace elements such as zinc, cobalt, molybdenum, selenium, iodine, boron, manganese, tin and lanthanum, which can be supplied in the form of salts as well.  Preferably, the inorganic salts contained in the medium include sodium, potassium, calcium, magnesium, phosphate, carbonate, iron, zinc, cobalt, molybdenum, selenium, iodine, boron, manganese, tin and lanthanum.  More preferably, the medium is free of copper since it may be toxic to certain cells at concentrations greater than 10 -8 M.  Advantageously, the iron may be provided in the form of iron phosphate (Fe4 (P2O7) 3) which is more stable than the ferrous sulfate used in the culture media of the prior art.  This form is furthermore preferred to the nitrate form which may be at the origin of free radical formation.  In addition, the iron citrate form is very popular with some authors (Schneider YJ. , 1989, J. Immunol Methods. ; 116 (1): 65-77) can under certain conditions be hardly soluble.  Boron, in the form of borate or boric acid, is a microelement described for culture media of bacteria and yeasts but has only recently been described in culture media for vertebrate cells. European patent EP 1482031).  It is likely to slightly enhance the growth of mammalian cells, as well as lanthanum in chloride form at a concentration of 10 -9 M (Haase et al. , 2001, Biot Chem. , 382 (8): 1227-34), and cobalt at the concentration of 10 7 5M.  The nature of the inorganic salts which may be contained in the culture medium according to the invention as well as the ranges of possible or preferred concentrations for each of these salts are shown in Table 5.  Table 5: Concentrations of inorganic salts in the culture medium according to the invention Mineral salts Concentration range Embodiment NaCl 11.5-115 × 10 -3 M 115 × 10 -3 M KCl 0.537-5.37 × 10 -M 5, 37 x 100-3M CaCl2.2H2O 0.18-1.8 x10-3M 1.8x10-3M MgSO4. 7H2O 0.18-1.8 x10-3M 1.8 x10-3M NaH2PO4. H2O 0.2-2 x1 0 "3M 2 x10-3M NaHCO3 2.62-26.2 x1 0" 3M 26.2 x10-3M Fe4O21 P6 (Fe4 (P207) 3) 0.1-1 x10-5 ' 5M 10-5'5M * Zn SO4. 7H2O 0.1-1 x10-6. 5M 10-65M * CoCl2. 6H2O 0.1-1 x10-7'5M 10-7, bM * MoO4Na2. H2O 0.1-1 x10-8M 10-8M * SeO3Na2 0.1-1 x10-8M 10-8M * IK 0.1-1 x10-8M 10-8M BO3H3 0.1-1 x1 0-8M 10- 8M MnCl2. 4H2O 0.1-1 x1 0 "9M 10" 9M * SnCl2. 2H2O 0.1-1 x10-9M 10 "9M * LaCI3. The culture medium according to the invention also contains at least one sugar, in particular glucose, fructose, galactose, mannose and inositol, at least one organic acid, for example sodium. pyruvate and sodium butyrate, and at least one nitrogen compound, such as choline, ethanolamine and optionally alaninol.  The amounts of sugars, organic acids and nitrogen elements used have not been optimized for a given cell line, but correspond to conditions adapted to the largest number of cell lines.  The amount of sugar energy suppliers (glucose, fructose) in the medium can be modulated so as to provide a more or less rich medium depending on the type of cell to be cultivated and its energy requirements.  The culture medium may further contain alaninol which has been shown to have a significant trophic effect for certain cells such as CV1 cells.  Previous work had demonstrated the trophic effect of ethanolamine (another amino alcohol, corresponding to glycine) on hybridoma cells (Murakami et al. , 1982, Proc Natl Acad Sci U S A.  79 (4): 1158-1162).  Alanine has been described as having properties similar to those of glycine in cell culture (Franek and Sramkova, 1996, Immunol.  Lett. ; 52 (2-3): 139-44), the inventor sought to characterize the properties of alaninol, the alcoholamine corresponding to alanine.  The trophic properties of alaninol have thus been demonstrated by placing in low alanine culture conditions, the trophic effect of alaninol being strongly masked in an alanine enriched medium.  The nature of the sugars, organic acids and nitrogen elements which may be contained in the culture medium according to the invention as well as the ranges of possible or preferred concentrations for each of these constituents are shown in Table 6.  Table 6: Concentrations of sugars, organic acids and nitrogenous elements in the culture medium according to the invention Sugars Fashion range of other mode of concentrations embodiment embodiment Glucose 2.5-10 × 10 -3 M 5 × 10 -3 M 10 × 10 -3 M Fructose 0.79-3.16 x1 03M 1.58 x10-3M 3.16 x10-3M Galactose 10 "3'5-10" 2.5M 10 "3M 10-3M Mannose 10" 3'5-10-2 5M 10 "3M 10-3M Inositol o-4. 5-1 0 3'5M 10 "4M 10-4M Organic Acids Sodium Pyruvate 10-3'5-10" 2M 10-3M 10-3M Sodium Butyrate 10-6-10 "5M 10-5.5M 10-5 ' 5M Nitrogen compounds Choline HCl 10-5-10'4M 10-4M 10 "4M Ethanolamine 10-5'5-10 4'5M 10-4'5M 10 4.5M Possibly Alaninol 10--10-4M 10-5 ' 5M 10 "5. 5M Vitamins and / or antioxidants that may be added in the culture medium include, for example, biotin, D-Ca-pantothenate, nicotinamide, pyridoxine, thiamine, thioctic acid, folic acid and optionally its precursor P-amino benzoic acid retinylacetate, riboflavin, ethyl linoleate, vitamin B12, and optionally ascorbic acid.  Ascorbic acid which is very unstable in solution may possibly be absent from the medium, in which case it will be added by the end user just before using the medium according to the invention.  It has been found that biotin has a significant effect on the cloning efficiency of cells in culture, and that this effect is even more pronounced that biotin is present in the medium at a concentration about 100 times greater than that vitamin B12 and approximately 10-fold higher than that of retinylacetate.  Thus, according to one embodiment of the invention, the culture medium comprises biotin, vitamin B12 and retinylacetate, the ratio of biotin: vitamin D 12 concentrations being between 80: 1 and 120: 1, preferably between 90: 1 and 110: 1, and the ratio of biotin: retinyl acetate concentrations being between 8: 1 and 12: 1, preferably between 9: 1 and 11: 1.  In a preferred embodiment, the medium contains 10 -5 M biotin, 10 -7 M-vitamin B12, and 10 -6 M retinyl acetate.  Finally, linoleic acid is advantageously present in the medium as a growth factor for hybridomas.  The nature of the vitamins and antioxidants that may be contained in the culture medium according to the invention as well as the ranges of possible or preferred concentrations for each of these constituents are shown in Table 7.  Table 7: Concentrations of vitamins and antioxidants in the culture medium according to the invention Preferred range of embodiment Concentrations Biotin 10 "6-10" 4M 10 "5M D-Ca-pantothenate 10" 6-10 "4M 10- 5M Nicotinamide 10 "6-10" 4M 10-5M Pyridoxine 10-6-10-4M 10-5M Thiamine 10 "6-10" 4M 10-5M 10 "Thioctic Acid b-10-4M 10" 5M 10 "Folic Acid ~ 5-10 "5 5M 10-5. 5M Retinylacetate 10 -10 "M 10-6M Possibly Ac.  P. amino 10 "'- 10" 5M 10-6M benzoic Riboflavin 10 -'- 10-6M 10 "6 ~ 5M Ethyl linoleate 10" - 10 "6M 10-" 5M Vitamin B12 10 "6-10" 6M 10- M Possibly Acid 10-6-10 "3M 104M LAscorbic Table 8 summarizes the concentration of the various constituents that may be present in the culture medium according to the invention (H-MSM) and in two particular embodiments ( H-MSM1 and H-MSM2).  Table 8: Concentrations of natural amino acids, mineral salts, sugars, organic acids, nitrogen nutrients, vitamins and antioxidants H-MSM H-MSM-1 H-MSM-2 Mineral salts NaCl 11.5-115x10 "3M 115x10" 3M 115x1 0 "3M KCI 0.537-5.37 xl0" 3M 5.37x1 0 "3M 5.37x10-3M 2910024 13 H-MSM H-MSM-1 H-MSM-2 CaCl2.2H2O 018-1.8 x10-3M 1 , 8 x10-3M 1.8 x10-3M MgSO4. 7H20 0.18-1.8 x10-3M 1.8x10 "3M 1.8x10-3M NaH2PO4. H20 0.2-2 x10-3M 2x1 0 "3M 2x10-3M NaHCO3 2.62-26.2 x10-3M 26.2x10-3M 26.2x10" 3M Fe4O21P6 (0.1-1 x10-5'5M) -5. 5M 10-5'5M (Fe4 (P207) 3) * Zn SO4. 7H20 0.1-1 x10 "5'5M 10" ~ 5M 10-b'5M * CoCl2. 6H2O 0.1-1 x1 0 "7'5M 10" 7'5M 10 "7'5M * MoO4Na2.  H2O 0.1-1 x1 0 "8M 10-8M 10" 8M * SeO3Na2 0.1-1 x1 0 "8M 10" 8M 10 "8M * IK 0.1-1 x1 0" 8M 10-8M 10 "8M B03H3 0.1-1 x10-8M 10-8M10-8MMnCl2. 4H20 0.1-1 x10-9M 10 "9M 10-9M * SnCl2. 2H2O 0.1-1 x10-9M 10-9M 1 x10-9M * LaCI3. 7H2O 0.1-1 x10-9M 10 "9M 1 x1 0" 9M Sugars Glucose 2.5-10 x10-3M 5 x1 0 "3M 10 x10-3M Fructose 0.79-3.16 x10-3M 1.58 x10-3M 3,16 x1 0 "3M Galactose 10" 3'5-10 "2'5M 10" 3M 10 "3M Mannose 10" 3'5-10 "2'5M 10" 3M 10 "3M Inositol 10" 4 5-10 3'5M 10 "4M 10" 4M Organic Acids Sodium Pyruvate 10 "3.5-10" 2M 10 "3M 10" 3M Sodium Butyrate 10-6-10-5M 10 "5. 5M 10-5'5M Nitrogen compounds Choline HCl 10 "5-10" 4M 10-4M 10-4M Ethanolamine 10-5'5-10 "4'5M 10" 4'5M 10 "4'5M Possibly Alaninol 10" b -10-4M 10 "5. 5M 10 "5'5M Amino acids Alanine 1,01-5,04x1 0" 3M 1,345 x1 0 "3M 2,69 x10-3M Arginine 0.56-2.81 x10-3M 0.75 x10-3M 1.50 x10-3M Asparagine 0.52-2.59 x10-3M 0.69 x10-3M 1.38 x10-3M Aspartic Acid 0.17-0.86 x1 0 "3M 0.23 x10-3M 0.46 x1 0 "3M Cystine 0.42-2.10 x1 0" 3M 0.56 x10-3M 1.12 x10-3M Glutamic Acid 0.23-1.16 x10-3M 0.31 x10-3M 0.62 x10-3M Possibly Glutamine 1.66-8.31 x1 0 "3M 2,215 x1 0" 3M 4,43 x10-3M Glycine 0,63-3,15 x1 0 "3M 0,84 x10-3M 1,68 x10-3M Histidine 0 , 47-2.33 x10-3M 0.62 x10-3M 1.24 x10-3M Isoleucine 0.20-0.99 x10-3M 0.265 x10-3M 0.53 x10-3M Leucine 0.38-1.88 x1 0 "3M 0.502 x1 0" 3M 1.04 x1 0 "3M Lysine 0.57-2.85 x1 0" 3M 0.76 x10-3M 1.52 x10-3M Methionine 0.08-0.41 x10- 3M 0.11 x1 0 "3M 0.22 x10-3M Phenylalanine 0.16-0.81 x10-3M 0.215 x10-3M 0.43 x10-3M Proline 0.97-4.84 x1 0" 3M 1.29 x1 0 "3M 2.58 x1 0" 3M 2910024 14 H-MSM H-MSM-1 H-MSM-2 Serum 1.01-5.06 x10-3M 1.35 x10-3M 2.70 x1 0 "3M Threonine 0.58-2.89 x1 0 "3M 0.77 x10-3M 1.54 x10-3M Tryptophan 0.05-0.26 x10-3M 0.07 x10-3M 0.14 x1 0 "3M Tyrosine 0.11-0.56 x10-3M 0.15 x10-3M 0.30 x10-3M Valine 0.38-1.90 x10-3M 0.507 x10- 3M 1,14 x10-3M Total Biotin Vitamins 10 "6-10-4M 10-5M 10" 5M D-Ca-Pantothenate 10 "6-10-4M 10" 5M 10 "5M N icoti nam ide 10" 6-10 "4M 10" 5M 10 "5M Pyridoxine 10" 6-10 "4M 10-5M 10" 5M Thiamine 10-6-10 "4M 10-5M 10" 5Midic acid 10-6-10-4M 10-5M 10 "5M Folic acid 10-b'5-10 5'5M 10 "5'5M 10" 5'5M Retinylacetate 10 "b'5-10 5'5M 10-6M 10-6M Possibly Ac.  P. 10 "-10" 5M 10-6M 10 "6M amino benzoic Riboflavin 10" 7-10-6M 10-6'5M 10- "m Ethyl linoleate 10" 7-10 "6M 10" 6'5M 10 "6 5M Vitamin B12 10 "6-10-bM 10 -'M 10" 7M Possibly Acid 10 "-10" 3M 10 "4M 10" 4M Ascorbic Preferably, the medium is serum-free and / or aprotic-free.  The H-MSM medium consists of only 63 chemically defined molecules (mineral salts, amino acids, vitamins, sugars and other nutrients).  Of these 63 components composing EMEM, the H-MSM medium described in Table 8 can easily be derived from EMEM.  It contains vitamin C and glutamine, which are unstable in solution.  These two components are preferentially kept separately, frozen, and added to the medium at the time of use, as is the case for glutamine in all media intended for the culture of vertebrate cells.  The medium may further contain phenol red as a pH indicator.  Phenol red is typically present in the medium at a concentration of 0.026 x 10-3M.  The medium according to the invention can be formulated at the 1X concentration (i.e. all the constituents of the medium are at the working concentration) or formulated at higher concentrations, for example 5X (ie that is, the concentration of the constituents of the medium is five times higher than that in the 1X formulation or 10X (10 times higher concentration than in the 1X formulation), the medium being optionally diluted before use by the user to return the medium to a 1X formulation.  The medium allows the culture of vertebrate cells (mammalian or non-mammalian), or nonvertebrate cells, for example yeasts, bacteria or insect cells.  The cells may be cell lines, or primary cells.  They may be epithelial, endothelial, fibroblast, hybridoma cells, hematopoietic cells, in particular lymphocytes, macrophages / monocytes, dendritic cells, hepatocytes or chondrocytes.  The culture medium can be used to grow adherent cells, which form a monolayer, or cells in suspension.  In particular, it has been observed that the adherent cells do not die once they have reached confluence, which is an advantage of the medium according to the invention.  The medium according to the invention is extremely flexible in use.  Indeed, no adaptation of the cells, prior to growth in the synthetic medium, is necessary to cultivate indefinitely in the experimental conditions described.  On the other hand, cells usually cultured in the presence of serum can withstand up to 4 passages in hormone-free and protein-free media (1/4 subculture ratio, 1/44 dilution factor = 1/256), without their growth characteristics being clearly modified.  It is only beyond this dilution factor that the effects due to deficiencies caused by the absence of serum can possibly become sensitive.  For most cells, the growth in the medium according to the invention is not very different from that which can be observed in EMEM medium supplemented with 3% fetal bovine serum.  In most cases, the mean doubling time is increased by a factor of 1.1 to 1.2 compared to the doubling time observed with EMEM culture with serum.  It has been observed that cells HEK293, HT29 and CEV1 form cell mats transiently.  These cells, when they come to confluence, have adhesion properties for themselves that are stronger than for the support.  They then aggregate into clusters.  This phenomenon may be due either to the inadequacy of the support or to a lack of extracellular matrix synthesis by these cells.  This phenomenon seems to be of little importance, however, as it does not affect the production of recombinant protein by transfected CHO cells (Houdebine LM, personal communication).  Table 9 shows the overall compositions of the main commercially available media currently compared to that of the H-MSM medium.  The concentration of each of the 20 amino acids, glucose and fructose of the H-MSM-2 medium is twice that of the H-MSM-1 medium, the composition as for the other constituents being unchanged.  Table 9: Comparative Elements of the Main Media Cited Medium 199 EMEM RPMI Ham D-MEM Alpha-ABC H-MSM 1640 F12 MEM (Darfler) Number of 62 30 39 47 33 42 81 63 Components Acids 758 851 970 590 1581 1136 1427 18208 Amines 3 640b total (mg / L) Additives 10% SFB 10% SFB 10% SFB 10% SFB 10% SFB - - 7500mg / l 7500mg / l 7500mg / l 7500mg / l 7500mg / l Protects 8257.50 8351 8470 9081 8636 1427 1820a mg / L / medium 3640b complete a: H-MSM-1b: H-MSM-2 The chemically defined synthetic culture medium can also be supplied in powder form, the medium then being reconstituted in liquid form by the user by adding a suitable volume of filtered distilled water to the mixture of powdery constituents.  Table 10 illustrates the amounts of components that can be used to obtain the two embodiments H-MSM1 and H-MSM-2 in powder form, and to reconstitute 1 liter of liquid medium.  Table 10: Contents of H-MSM1 and H-MSM-2 media in powder form for reconstituting 1 liter of liquid medium H-MSM-1 H-MSM-2 M Molarity Amount Molarity Amount per liter for 1 liter (mg / I) (mg / I) Mineral Salts NaCl 58.44 115x10-3M 6720 115x10-3M 6720 2910024 17 H-MSM-1 H-MSM-2 M * Molarity Amount Molarity Amount per 1 liter per 1 liter (mg / I) (mg / I) KCl 74.5 5.37x10 -3M 400 5.37x10.0 3M 400 CaCl2.2H2O 147 1.8 x10-3M 265 1.8 x10-3M 265 MgSO4. 7H2O 246.5 1.8x10-3M 443.7 1.8x10 -3M 443.7 NaH2PO4. H2O 138 2x10 "3M 276 2x1 0" 3M 276 NaHCO3 84 26.2x10-3M 2200 26.2x10-3M 2200 Fe4O21P6 (Fe4 (P2O7) 3) 745 10-5'5M 2.36 10 "5'5M 2.36 * Zn SO4. 7H2O 287.5 10 "6'5M 0.090 10" 6'5M 0.090 * CoCl2. 6H2O 238 10 "7'5M 0.00752 10-7'5M 0.00752 * MoO4Na2. H2O 242 10 "8M 0.00242 10-8M 0.00242 * SeO3Na2 173 10-8M 0.00173 10" 8M 0.00173 * 1K 166 10 "8M 0.00166 10" 8M 0.00166 BO3H3 61.83 10 " 8M 0.00062 10-8M 0.00062 MnCl2. 4H2O 198 10-9M 0.000198 10-9M 0.000198 * SnCl2. 2H2O 225.5 10-9M 0.000225 1 x10 "9M 0.000225 * LaCI3. 7H2O 371 10 "9M 0.000371 1 x10-9M 0.000371 Glucose Sugars 180 5 x10-3M 900 10 x10-3M 1800 Fructose 180 1.58 x1 0" 3M 284 _ 3.16 x10-3M 569 Galactose 180 10 " 3M 180 _ 10-3M 180 Mannose 180 10-3M 180 10-3M 180 Inositol 180 10-4M 18 10-4M 18 Organic acids _ Sodium Pyruvate 110 10 "3M 110 10-3M 110 Sodium Butyrate 110 10" 5'5M 0.347 10 "5'5M 0.347 Nitrogen compounds Choline HCl 139 10-4M 13.9 10-4M 13.9 Ethanolamine 61 10" 4'5M 1.93 10 "4'5M 1.93 Possibly 75 10-5'5M 0, 24 10-5'5M 0.24 Alaninol Amino acids Alanine 89 1.345 x10-3M 119.7 2.69 x10-3M 239.4 Arginine 174 0.75 x10-3M 130.5 1.50 x10-3M 261 Asparagine 132 91.1 1.38x10-3M_ 182.2 0.69 x10-3M Aspartic Acid 133 0.23 x1 0 "3M 30.1 0.46 x10" 3M 60.2 Cystine 240 0.56 x1 0 "3M 147.6 1.12 x10-3M 295.2 Glutamic acid 147 _ 0.31 x10-3M 45 0.62 x10-3M 90 Possibly 146 2.215 x10-3M 323.4 4.43 x10-3M 646.8 Glutamine _ Glycine 77 0.84 x 100 0M 3M 64.7 1.68 x 10-3M 129.4 Histidine 155C), 62 x103M 96.1 1.24 x10-3M 192.2 Isoleucine 131 0.26 5 x10-3M 34.7 0.53 x10-3M 69.4 2910024 18 H-MSM-1 H-MSM-2 M * Molarity Quantity Molarity Quantity for 1 liter for 1 liter (mg / I) (mg / I) Leucine 131 0.502 x10-3M 65.8 1.04 x10- 3M 131.6 Lysine 146 0.76 x10 "3M 111 1.52 x10-3M 222 Methionine 153 0.11 x10-3M 16.8 0.22 x10- 3M 33.6 Phenylalanine 165 0.215 x 10-3M 35.5 0.43 x 10 -3 M Proline 115 1.29 x 10 -3 M 148.3 2.58 x 10 -3 M 296.6 Serine 105 1.35 x 10 -3 M 141, 7 2.70 x 10 -3 M 283.4 Threonine 119 0.77 x 1.03M 91.6 1.54 x103M 183.2 Tryptophan 204 0.07 x 1.03M 14.3 0.14 x10-3M 28.6 Tyrosine 182 0, 15 x10-3M 27.3 0.30 x10-3M 54.6 Valine 117 0.507 x10-3M 59.3 1.14 x10-3M 118.6 Vitamins Biotin 244 10 "5M 2.44 10" 5M 2.44 D-Ca-pantothenate 476 10 -5M 4.76 10-5M 4.76 Nicotinamide 122 10-5M 1.22 10 -5M 1.22 Pyridoxine 169 10 -5M 1.69 10-5M 1.69 Thiamine 337 10 "5M 3.37 10" 5M 3.37 Thioctic acid 206 10 "5M 2.06 10" 5M 2.06 Folic acid 477 10 "5.5M 1.5 10" 5.5M Retinylacetate 328 10 -6M 0.328 10- I 0.328 Possibly Ac.  159 10-6M 0.16 10- 0.16 P. amino benzoic Riboflavin 376 10 "6.5M 0.12 10" 6'5M 0.12 Ethyl linoleate 308.5 10 "6. 5M 0.975 10 "6. 5M 0.975 Vitamin B12 1355 10-7M 0.135 10-TM 0.135 Possibly Acid 176 10 "4M 17.6 10" 4M 17.6 Ascorbic M *: Molecular weight.  Applications The invention also relates to a cell multiplication method comprising the steps of: a) contacting the culture medium according to the invention with at least one cell; and b) cultivating said at least one cell for a time and under conditions allowing multiplication of said cell.  The cells thus cultured can be adherent or in suspension.  The multiplied cells may be cells expressing a protein, in particular a recombinant protein, or producing a virus.  The invention thus further provides a method of producing a protein of interest comprising the steps of: a) contacting the culture medium of the invention with at least one cell expressing a protein of interest; b) cultivating said at least one cell for a time and under conditions allowing the expression of the protein of interest; and c) optionally harvesting the expressed protein of interest.  The cell expressing a protein of interest may for example be a cell transformed with a nucleic acid comprising a sequence encoding a protein of interest which is a recombinant protein.  According to a particular embodiment, said cell is a hybridoma and the protein of interest is a monoclonal antibody.  The protein of interest can be harvested by collecting the culture supernatant if the protein is secreted, or by collecting cells in culture to lyse and harvest the protein in the cell lysate.  Any methodology known to those skilled in the art can be employed.  The medium according to the invention has the advantage of allowing the preparation of proteins of interest, such as monoclonal antibodies, free of protein contaminants, in particular serum contaminants.  The protein of interest can then be easily purified.  However, the addition of factors that may increase recombinant protein yields (eg, growth factor, hormone, such as TGF, insulin, IRTKA, IGF, etc. . . ) may be considered.  The invention further provides a method of producing a virus comprising the steps of: a) contacting the culture medium according to the invention with at least one cell infected with a virus; b) cultivating said at least one cell for a time and under conditions enabling the replicative cycle of said virus to be carried out; and c) eventually harvest the viruses produced.  The virus may be any virus such as, for example, rotavirus, Aujesky's disease virus, respiratory syncytial virus (RSV), influenza virus, herpesvirus, etc.  The medium according to the invention makes it possible to obtain production yields of 5 viruses equivalent to those obtained in medium with serum.  In addition, in the case of rotavirus produced in H-MSM medium, a better morphogenesis of the virus has been observed, in particular with regard to the spicules which appear complete under observation by electron microscopy.  Monoclonal antibody productions are equivalent in H-MSM-2 media (i.e. medium with high levels of amino acids, glucose and fructose) and in RPMI1640 + 10% SFB medium.  But the advantage of the medium according to the invention is to obtain a very clean protein without contamination by serum proteins.  Better production was observed with H-MSM-1 medium (lower levels of amino acids, glucose and fructose), probably because the growth rate of hybridomas was then slower.  The use of the culture medium according to the invention is therefore an interesting alternative for hybridoma culture: the monoclonal antibodies can thus be produced at a lower cost (no purchase of fetal serum or growth factors) and already pre-purified ( no added protein).  The other method of producing antibodies

  so-called in vivo monoclonal antibodies, that is to say in ascites liquid makes it possible to produce highly concentrated solutions of these antibodies. But this in vivo method has the disadvantage of inducing suffering in the experimental animal. As a result, the production of monoclonal antibodies is subject to very strong restrictions in France (ethical regulation in animal experimentation), and is even prohibited in many countries, including countries of northern Europe. In addition, ascites fluids contain serum proteins, including immunoglobulins. It is impossible to separate monoclonal immunoglobulins carrying the antibody activity of interest from immunoglobulins of the host animal with which they are mixed.

The use of the medium according to the invention makes it possible to circumvent these difficulties. As for the hybridomas which are not able to grow spontaneously in H-MSM medium, they can be put into production in this H-MSM medium supplemented with the growth factor (IL-6, IL-4, etc.) provided that ( s) Critical growth factor (s) known for the clone under consideration.

The level of recombinant protein produced by RK13 cells secreting porcine interferon gamma was found to be slightly better in H-MSM-2 medium than in EMEM + 10% SFB medium. Here again, the protein produced with the medium according to the invention is much cleaner.

The medium according to the invention can also be used for the storage of cells in liquid nitrogen, in particular in a mixture with DMSO, for example in medium containing 5.0% DMSO + 0.25%. % (volume / volume), ie 5.5% +/- 0.28% w / v, or DMSO at 0.704 M +/- 0.035M. The resulting mixture has an osmolarity of about 1000 mOsM (milliOsMole).

The following Figures and Examples illustrate the invention without limiting its scope. FIGURES Figure 1 shows the growth results of F17 hybridomas in RPMI1640 medium + 10% SFB or in H-MSM-2 medium alone. Figure 2 shows the growth results of G16 hybridomas in RPMI1640 medium + 10% SFB or in H-MSM-2 medium alone. Figure 3 shows a 4-20% polyacrylamide gel (SDS) analysis: A: 20 Size markers, B: 2 μ SFB, C: 1 μl ascites G16, D: 1 μl ascites F17, E: -, F: 20 μl of G16 hybridoma culture crude supernatant grown in RPMI + 10% SFB, G: -, H: 20 μl of F17 hybridoma culture crude supernatant grown in RPMI + 10% SFB, I: - 500 μg Mouse IgG, K: 20 μl of G16 hybridoma culture crude supernatant grown in H-MSM, L: 20 μl of F17 hybridoma culture crude supernatant grown in H-MSM. FIG. 4 represents the neutralizing activity of the culture supernatants of hybridomas G16 and F17 after 7 days of culture in RPMI 1640 medium + 10% SFB or in H-MSM-2 alone. Figure 5 shows the results of ELISA quantitation of porcine interferon gamma in culture supernatants of Al2RK13 cells after 4, 7, 10, and 14 days of culture in EMEM + 10% SFB, H-MSM- 2 alone, and H-MSM-2 + 10% SFB.

FIG. 6 shows the antiviral activity of culture supernatants of Al2RK13 cells after 4, 7, 10 and 14 days of culture in EMEM + 10% SFB medium, or in H-MSM-2 medium, or in H medium. Example 1: Determination of the composition of the medium H-MSM A cell line of bovine skin (BoSk cells, for Bovine Skin, L'Haridon, R unpublished) established in the years 1978 / 1979 has the original property of being able to propagate in standard aprotic media (eg RPMI 1640, or EMEM alpha) supplemented only with iron salts. This result led the inventor to investigate whether other cells commonly used in the laboratory, notably CV1 from which the COS7, PK15, MA 104, RK13 and MDBK cells are derived, had similar properties. The results were a little ambiguous. The use of a Sato formulation (DMEM + F12 mixture) or other mixtures including the 199 medium did not lead to satisfactory cell growth either. Moreover, the large distortions that exist between the media, such as EMEM, DMEM, 199, or RPMI 1640, as regards the concentration of vitamins have led to the determination of the dose / response effect of the main vitamins and antioxidants (vitamins A, H, B1, B2, B6, B7, B12, Bx, C, M, thioctic acid).

The formulation of the medium according to the invention was established by measuring the effect of the different components of the culture medium on the clonal efficiency of a number of cell lines. Clonal efficiency is measured by the ratio of number of colonies / number of cells seeded after a given time of culture. • Determination of the vitamin and antioxidant formulation The consensus concentrations of vitamins and antioxidants were determined experimentally. The concentrations used are the non-toxic trophic minimum concentrations. It was thus determined that biotin (vit.H), D-Ca-pantothenate (vit.B7), nicotinamide (vit.B3), pyridoxine (vit.B6), thiamine (vit B1), and thioctic acid have an optimal trophic effect for all cells tested at 10-5M concentration. Ascorbic acid has optimal trophic properties at the 10-4M concentration. Its effect is complementary to that of thioctic acid. In contrast, its effect is not complementary to that of alpha tocopherol (vit.E), which is why alpha tocopherol has not been retained in the formulation of the medium according to the invention. In the absence of protein, folic acid (vit.M) is toxic at a concentration of 10-5M and riboflavin (vit.B2) is toxic at the concentration of 10-6M. 10 "6.5M, respectively, were therefore chosen for these two vitamins. For many cell lines the maximum concentration of linoleic acid is 10-6.5M.

Cyanocobalamin (Vit.B12) and Retinol Acetate (Vit A) have optimal trophic properties at 10-7M and 10-6M, respectively, and the results obtained by the inventor led to the definition of optimum composition of vitamins and antioxidants defined in Table 11 below.

Table 11: Optimal Vitamin and Antioxidant Concentrations Vitamin or Antioxidant Optimal Concentration Biotin (Vitamin H) 10-5M D-Ca-Pantothenate (Vitamin B7) 10-5M Nicotinamide (Vitamin B3) 10-5M Pyridoxine (Vitamin B6) 10- 5M Thiamine (Vitamin B1) 10 "5M Thioctic Acid 10" 5M Folic Acid (Vitamin M) 10-5'5M Retinyl Acid (Vitamin A) 10-6M Possibly Ac. P-amino benzoic acid (vitamin Bx) 10-6M Riboflavin (vitamin B2) 10-5'5M Ethyl linoleate 10-6'5M cyanocobalamin (vitamin B12) 10 7M Ascorbic acid 10-4M P-amino benzoic acid, which is a precursor of folic acid, may optionally be present in the formulation at an optimum concentration of 10-6M.

The formulation thus defined contains a particularly high biotin content relative to the media of the prior art where it is generally present at a concentration of 10-7M. Indeed, considering the test used to determine the composition of the culture medium, it has been noticed that biotin has a determining effect on the cloning efficiency. The 10 -5 M concentration appeared to be close to optimum, and it was found that Eagle's EMEM medium supplemented with non-essential amino acids and these vitamins allows for satisfactory growth of 4 cell lines: BoSk, CV1, PK15 , RK13 • Determination of the formulation in mineral elements The concentrations of the various salts including macroelements (Mg ", PO4" -), and microelements Fe ", Zn ++ Co ++, SeO3" MoO4--, I-, BO--, Sn ++, Mn ++, +++ were determined experimentally and a microelement composition was deduced (Table 12) Table 12: Optimal concentrations of microelements Mineral salts Optimal concentration NaCl 115 x10-3M KCl 5.37x1 0 "3M CaCl2, 2H20 1 , 8x10 "3M MgSO4.7H2O 1.8x10" 3M NaH2PO4.H2O 2 x10-3M NaHCO3 26.2 x10-3M Fe4O21P6 (Fe4 (P207) 3) 10 5'5M * Zn SO4.7H20 10-6.5M * CoCl2.6H2O 10 "7'5M * MoO 4Na2.H2O 10-8M * SeO3Na2 10" 5M * IK 10-8M B03H3 10-3M MnCl2.4H2O 10 "9M * SnCl2.2H2O 10-9M * LaCI3.7H2O 9M In this form ulation, iron is brought in an original form: iron phosphate, which is more stable than ferrous sulfate. Cobalt is brought to a relatively high concentration while copper, toxic to many cells, is absent. The composition thus determined has been improved, in particular for the purpose of cultivating hybridomas. These improvements relate to the assay of: 5 sugars 2910024 (glucose, fructose, mannose, galactose, inositol), a short chain fatty acid (butyrate), and 2 amino alcohols (ethanolamine, alaninol). • Determination of the formulation of sugars, organic acids and nitrogen nutrients The different assays are not optimized for a given cell line, but are the conditions which allow a maximum number of cell lines to multiply suitably. The sugars involved in the supply of energy are provided in a scalable amount depending on whether the medium is more or less rich. The sugars that are components of the glycoconjugates are added in fixed amounts. Sugars added in modulable amounts: The glucose concentration was chosen by reference to the average blood concentration in humans: 0.9 g / l (H-MSM-1 medium). This amount was doubled for the medium called H-MSM-2, ie 1.8 g / I (10 -2M) .Fructose, whose metabolism is not modulated by insulin, was tested with satisfaction at the concentration of 1/3 and 1/2 of that of glucose The concentration of 1/3 of that of glucose was retained and reduced to 1 / 3,17 (= 1k10) or 0.28 g / I for the medium. H-MSM-1, and 0.57 g / I for H-MSM-2 medium Sugar added in fixed amounts The optimal concentration of inositol was determined experimentally (10-4M) .Mannose and galactose components of the glycosylated portion of the glycoproteins are added at 10 -3 M (Minamoto Y. et al., 1991, Cytotechnology Suppl 2: S35-51). Organic acids. Pyruvic acid (which is also an antioxidant) is used at the concentration of 10 -3 M, as in most standard media, butyric acid is used at the maximum non-toxic trophic concentration: 10-5.5M.

2910024 26 Nitrogenous nutrients. The consensus concentrations of choline, ethanolamine and alaninol were determined experimentally.

Table 13: Formulation of sugars, organic acids and nitrogen nutrients Medium H-MSM-1 Medium H-MSM-2 Sugars Glucose 5 x1 0 "3M 10 x10-3M Fructose 1.58 x10-3M 3.16 x1 0" 3M Galactose 10-3M 10 "3M Mannose 10" 3M 10-3M Inositol 10-4M 10 "4M Organic Acids Sodium Pyruvate 10-3M 10" 3M Sodium Butyrate 10 "5'5M 10-5'5M Nitrogen compounds Choline HCI 10-4M 10 "4M Ethanolamine 10-4'5M 10-4'5M Possibly Alaninol 10" 5.5M 10 "5'5M • Determination of the amino acid formulation The amino acid concentrations were calculated from the needs of the cell for to synthesize its proteins but also energy needs. The use codon gives a good idea of the amino acid requirements for protein synthesis. The energy requirements of the cell that are covered by amino acids have been the subject of an excellent study by Franek and Sramkova (1996, Immunol Lett, Sep, 52 (2-3): 139-44).

These amino acids are supplied to the cells in the proportions necessary for protein synthesis, but the amounts intended to cover the energy requirements of the cell in culture are provided in addition. These amino acid energy suppliers have been classified into four categories based on an experimental approach based on the analysis of clonal efficiency on the one hand, and the longevity of cell mats (delay in onset apoptosis) on the other hand: - Large feeder: glutamine, is superadded at a rate of 2x1 0 "3M for H-MSM-1 medium 2910024 27 - Medium feeder: alanine, arginine, cysteine (which is substituted cystine at a concentration less than half), proline, serine are superadded to 10 "3M for H-MSM-1 medium. - Small feeder: asparagine, glycine, histidine, lysine, threonine, are superadjusted at a rate of 0.5x1 0 "3M and valine at a rate of 0.25x1 0" 3M for the medium H -MSM-1. - Non-feeder: the other amino acids that are only supplied in the quantities required to meet the needs of the cell to synthesize proteins (see Table 14: codon usage). These are: aspartic acid, glutamic acid, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine. Table 14: Usage codon of Sus scrofa (frequency of use per thousand of different codons by pig) codon frequency _AA codon frequency AA codon frequency AA codon frequency AA UUU 16,3 F UCU 11,2 S UAU 11,0 Y UGU 9.2 C UUC 26.4 F UCC 18.4 S UAC 20.5 Y UGC 14.0 C UUA 6.3 L UCA 10.1 S UAA 0.7 Stop UGA 3.5 Stop UUG 10.7 L UCG 4.2 S UAG 0.5 Stop UGG 14.1 W CUU 10.6 L CCU 14.3 P CAU 8.1 H TU 3.7 R CUC 22.5 L CCC 20.8 P CAC 15.7 H CGC 11.1 R CUA 11.6 L CCA 15.5 P CAA 10.4 Q CGA 5.9 R CUG 43.5 L CCG 7.9 P CAG 32.5 Q CGG 10.1 R AUC 15.8 I ACU 11.0 T .MU 14.4 N AGU 8.2 S AUC 28.4 I ACC 23.2 T AAC 23.7 N AGC 18.5 S AUA 9.3 I ACA 14.4 T AAA 20.6 K AGA 9.5 R AUG 21.9 M ACG 7.4 T AAG 31.7 K AGG 10.2 R GUU 9.0 V GCU 16.6 A GAU 17.7 D GGU 9.5 _ G GUC 17, 1 V GCC 31.1 A GAC 28.4 D GGC 24.9 G GUA 7.0 V GCA 13.7 A GAA 23.3 E GGA 17.0 G GUG 31.3 V GCG 7.9 A GAC 38, 4 E GGG 16.7 G Table 15: Codon use of Gallus gallus (frequencies of use per thousand of the different codons by the hen) codon frequency AA codon frequency AA codon frequency AA codon frequency AA _ UUU 16.9 F UCU 14.1 S UAU 12.0 Y UGU 8.8 C UUC 20.1 F UCC 15.6 S UAC 17, 8 Y UGC 12.8 C UUA 7.1 L UCA 11.5 S UAA 0.7 Stop UGA 1.0 Stop UUG 12.6 L UCG 5.2 S UAG 0.5 Stop UGG 11.8 W 2910024 28 codon frequency AA codon frequency AA codon frequency M codon frequency AA CUU 12.5 L CCU 15.3 P CAU 9.6 H TOS 5.4 R CUC 16.6 L CCC 16.5 P CAC 14.2 H CGC 10.3 R CUA 6.0 L CCA 15.6 P CM 12.2 Q CGA 5.3 R CUG 38.2 L CCG 7.6 P AGC 32.6 Q CGG 9.6 R AUU 16.9 I ACU 13.3 T MU 17.0 N AGU 11.3 AUC 22.0 I ACC 16.4 T AAC 22.6 N AGC 20.0 S AUA 8, 8 I ACA 16.1 T AAA 27.7 K AGA 12.3 R AUG 23.3 M ACG 7.6 T MG 34.9 K AGG 11.7 R GUU 13.2 V GCU 20.9 A GAU 25.6 D GGU 11.5 G GUC 13.5 V GCC 22.6 A GAC 25.1 D GGC 19.7 G GUA 7.9 V GCA 19.0 A GAA 31.6 E GGA 17.7 G GUG 28.0 V GCG 9.0 A GAC 41.3 E GGG 15.6 Table 16: Codon use of Cyprinus carpio (frequency of use per thousand of different codons by the ca rpe) codon frequency AA codon frequency M codon frequency AA codon frequency AA UUU 17.4 F UCU 15.8 S UAU 12.8 Y UGU 12.5 C UUC 24.3 F UCC 14.6 S UAC 20.0 Y UGC 12.3 C UUA 5.6 L UCA 12.3 S UAA 1.2 Stop UGA 1.0 Stop UUG 11.5 L UCG 4.3 S UAG 0.5 Stop UGG 11.5 W CUU 11.8 LCCU 15 , 2 P CAU 10.0 H TU 7.0 R CUC 17.0 L CCC 12.6 P CAC 13.6 H CGC 7.4 R CUA 5.1 L CCA 14.7 P CAA 12.0 Q CGA 4 , 9 R CUG 36.7 L CCG 6.6 P AGC 32.4 Q CGG 4.8 R AUU 17.1 I ACU 15.8 T AAU 16.2 N AGU 11, 6 S AUC 26.4 I ACC 17 , 8 T AAC 25.0 N AGC 17.7 S AUA 7.3 I ACA 16.0 T AAA 28.5 K AGA 13.6 R AUG 27.1 M ACG 6, 5 T AG 34.3 K AGG 9 15.4 GUU 15.4 V GCU 23.3 A GAU 26.5 D GGU 16.3 G GUC 17.4 V GCC 20.6 A GAC 28.5 D GGC 18.0 G GUA 6.4 V GCA 15 A GAA 23.5 E GGA 21.6 G GUG 29.8 V GCG 7.6 A GAC 40.8 E GGG 9.2 G From these codon data use of mammalian cells, birds and fish, the proportions of amino acids needed for protein synthesis have been deduced. These proportions vary little from one group of vertebrates to another.

TABLE 17 Amino acid ratios required for protein synthesis in a fish, a bird and a mammal. Mammal: Bird: Fish: Sus scrofa Gallus gallus Cyprinus carpio AA% Concentrations% Concentrations% Molecular Molecular Molecular Molecules A Ale 6,93 0,693x10 3M 7,15 0,715x10-3M 6,68 0,668x10 "3M _ R Arg 5,10 0.51 x10-3M 5.46 0.546x1 0 "3-M 4.72 0.472x10-3M N Asn 3.81 0.381 x10-3M _ 3.96 0.396x10" 3-M 4.12 0.412 x1OE-3M _J D Asp 4.61 0.461 x1 0 "3M _ 0.507x10-3M 5.50 0.550 x10-3M 5.07 _ C CYs 2.32 0.23 x10-3M 2.16 0.216x1 0" 3-M 2.48 0.248 x10-3M Q Gln 4.29 0.43 xl0-3M 4.47 0.447x1 0-3-M 4.44 0.444 x10-3M E Glu 6.17 0.62x10 "3M-0.729x10" 3-M 6 , 43 0.643 x1 0 "3M 7.29 G GIY 6.81 0.68x1 0" 3M 6.45 0.645x1 0 "M 6.51 0.651x10-3M H His 2.38 0.24x10" 3M 2.38 0.238x10-3M 2.36 0.236 x 10 -3 I Ie 5.35 0.53x1 0 "3M 4.77 0.477x10" 3M 5.08 0.508 x 1.0 "3M L Leu 10.52 1.05x10 -3 M 9, 30 0.930x10 "3M 8.77 0.877 x10-3M K Lys 5.23 0.52x10-3M 6.26 0.626x1 0" 3M 6.28 0.628 x10-3M M Met 2.19 0.22x10-3M 2.33 0,233x10 "3M 2,71 0,271 x10-3M F Phe 4,27 0,43x1 0" 3M 3,70 0,370x10 "3M 4,17 0,417 x1 0 "3M P Pro 5.85 0.58x10-3M 5.50 0.550x10" 3M 4.91 0.491 x10-3M S Ser 7.06 0.70x10-3M 7.77 0.777x10-3M 7.63 0.763 x10- 3M T Thr 5.40 0.54x10-3M 5.34 0.534x10 "3M 4.61 0.461x10-3M W Trp 1.41 0.14x10" 3M 1.18 0.118x10 "3M 1.15 0.115 x1 0" 3M Y Tire 3.15 0.31x10 "3M 2, 98 0.298x10" 3M 3.28 0.328 x10-3M V val 6.44 0.64x1 0 "3M _ 0.626x1 0" 3-M 6.90 0.690 x1 0 " 3M 6.26 stop 0.47 0.27 0.22 99.76 9.93x10-3M 100 99 The amino acid composition of the medium according to the invention was established by adding the amino acid concentrations necessary to protein synthesis, and determined using the use codon, with the necessary amino acid concentrations as energetic elements, as described by Franek and Sramkova (1996, Immunol. Lett. 52 (2-3): 139-44).

More specifically, the leucine concentration required for protein synthesis was set at 0.5 x 10-3 M (commonly accepted in the state of the art). The concentration of the other amino acids was deduced as a function of the relative frequency of the other amino acids according to the use codon (Table 18).

Table 18: Amino Acid Concentrations Required for Protein Synthesis (Usage Codon), Amino Acid Concentrations Required as Energetic Elements, and Concentrations in Medium H-MSM AA% Concentrations Nutrients * Based on Medium H-MSM-1 according to codon Franek and Sramkova use of Sus scrofa A Ala 6.93 0.346x10 "3M 10" 3M (2) 1.345x10 "3M R Arg 5, 10 0.250 x1 0" 3M 0.5x10 "3M (') 0.75x1 0" 3M N Asn 3.81 0.190 x10-3M 0.5x10 "3M (3) 0.69x10" 3M D Asp 4.61 0.230 x1 0 "3M - 0.23x10-3M C Cys 2.32 0.115 x 10-3M 10-3M (3) 1.115x10 "3M (3) Or cys- / / 0.56x10-3M cys Q Gln 4, 29 0.215x10-3M 2x10" 3M (') 2.215x10 " 3M E Glu 6.17 0.310x10 "3M - 0.31x10-3M G Gly 6.81 0.340x10-3M 0.5x10_3M (2) 0.84x10" 3M H His 2.38 0.120x10 "3M 0.5x1 0" 3M (2) 0.62x10-3M 1 IIe 5.35 0.265x10 "3M - 0.265x1 0" 3M L Leu 10.52 0.502x10-3 M - 0.502 x1 0 "3M K Lys 5.23 0.260x1 0" 3M 0.5x10 "3M (2) 0.76x10-3M M Met 2.19 0.110x10" 3M - 0.11x10 "3M F Phe 4.27 0.215x1 0" 3M -0.215x1 0 "3M P Pro 5.85 0,290x1 0-3M 10_3M (2) 1.29x10 "3M S Ser _7.06 0.350x10-3M 10-3M (2) 1.35x10" 3M T Thr 5.4 0.270x10 "3M 0.5x1 0" 3M ( 2) 0.77 x 1.0 "3M W Trp 1.4 0.070x10" 3M - 0.07x10-3M Y Tyr 3.15 _ - 0.15 x10-3M 0.155x1 0 "3M V val 6.44 0.320x1 ## EQU1 ## The sugars alone are not sufficient to meet the energy requirements of the cell in culture: Glutamine and I arginine are added at high concentrations in the main traditional media (EIVIEM, DMEM, RPMI, ...). (2) The water-soluble amino acids, most often considered non-essential in the Eagle formula, are, according to the works of Franek and Sramkova, determinants for cell growth in culture. (3) The trophic properties of cysteine are known (Franek and Sramkova 1996). It is preferable to provide cysteine in the form of oxidized dimer (more stable cystine in solution). The cystine is thus brought to a concentration equal to 2 times the concentration of cysteine which it replaces.

From the above results, the formulation of H-MSM-1 medium was established. The H-MSM2 medium has unchanged concentrations of mineral salts, added sugars in fixed amounts, organic acids, nitrogen compounds, vitamins and antioxidants. In contrast, glucose, fructose and amino acid concentrations are doubled in H-MSM2 medium. Table 19: Detailed formulations of H-MSM-1 medium and H-MSM2 medium. H-MSM-1 H-MSM-2 M Molarity mg / I Molarity mg / I Mineral salts NaCl 58.44 115x10-3M 6720 KCl 74.5 5.37x10-3M 400 unchanged CaCl2 2H2O 147 1.8 x10-3M 265 unchanged MgSO4.7H20 246.5 1.8x10 -3M 443.7 unchanged NaH2PO4.H20 138 2x1 0 "3M 276 _ unchanged NaHCO3 84 26.2x1 0" 3M 2200 Unchanged Fe4O21P6 745 10-5.5M 2.36 Unchanged (Fe4 (P207) 3) * Zn SO4.7H20 287.5 10 -6.5M 0.090 unchanged * CoCl2.6H2O 238 10-7.5m 0.00752 unchanged * MoO4Na2.H2O 242 1.x10-8M 0.00242 _ Unchanged * SeO3Na2 173 1x10-8M 0, 00173 Unchanged * IK 166 1 x10-9M 0.00166 Unchanged B03H3 161.83 1 x10 8M 0, 00062 Unchanged MnCl2.4H20 198 1 x1 0 "9M 0.000198 unchanged * SnCl2.2H2O 225.5 1x10-9M 0.000225 Unchanged * LaCI3.7H2O 371 1 x1 0" 9M 0, 000371 unchanged Glucose Sugars 180 5x10-3M 900 10x10 "3M 1800 _ Fructose 180 51 8x10-3M 284 3.16x10-3M 569 Galactose _ 180 10-3M 180 Unchanged Mannose 180 10-3M 180 Unchanged Inositol _ 180 10-4M 18 Unchanged Acids organic Sodium Pyruvate 110 10-3M 110 _ unchanged Sodium Butyrate 110 10-5.5M 0.347 unchanged Nitrogen compounds CholineHCI 139 10 "4M 13.9 Unchanged Ethanolamine 61 10-4.5M 1.93 Unchanged Alaninol 75 10-5.5M 0.24 unchanged Amino acids Alanine 89 1.345x10-3M 119.7 2.69 x10-3M 239.4 Arginine 174 5x1 0 "3M 130, 5 1.50 x10-3M 261 Asparagine 132 0.69x10-3M 91.1 1.38 x10M 182.2 Aspartic Acid 133 0.23x10-3M 30.1 _ 0.46 x10-3M 60 , 2 Cystine 240 0.56x10-3M 147.6 1.12 x10-3M 295.2 Glutamic acid 147 0.31 x1 0 "3M 45 0.62 x10-3M 90 _ Glutamine 146 2.215x10-3M 323.4 4.43 x10" 3M 646 , 8 Glycine 77 0.84 x10-3M 64.7 1.68 x10-3M 129.4 2910024 32 H-MSM-1 H-MSM-2 M Molarity mg / I Molarity mg / I Histidine 155 0.62x10-3M 96.1 1.24 x10-3M 192.2 Isoleucine 131 0.265x10-3M 34.7 0.53 x10-3M 69.4 Leucine 131 0.502x10 "3M 65.8 1.04 x10-3M 131.6 Lysine 146 0.76x10-3M 111 '1.52 x10-3M 222 Methionine 153 0.11 x10-3M 16.8 0.22 x10-3M 33.6 Phenylalanine 165 0.215x1 0 "3M 35.5 0. 43 x10-3M 71 Proline 115 1.29 x10-3M 148.3 2.58 x10-3M 296.6 Serine 105 1.35 x10-3M 141.7 2.70 x10-3M 283.4 Threonine 119 0.77x10-3M 91.6 1.54 x10-3M 183.2 Tryptophan 204 0.07x10-3M 14.3 0.14 x10-3M 28.6 Tyrosine 182 0.15x10-3M 27.3 0.30 x10- 3M 54.6 Valine 117 0.507x1 0 "3M 59.3 1.14 x10-3M 118.6 total 1819.3 Vitamins Biotin 244 10-5M 2.44 Unchanged D-Ca-pantothenate 476 10-5M 4.76 Unchanged Nicotinamide 122 "'10-5M 1.22 Unchanged Pyridoxine 169! 10-5M 1.69 Unchanged Thiamine 337 10 "5M 3.37 Unchanged Thioctic acid 206 10-5M 2.06 Unchanged Folic acid 477 10-5.5M 1.5 Unchanged Retinylacetate 328 10-6M 0.328 Unchanged Ac. P.-amino 159 10-6M 0.16 Unchanged benzoic Riboflavin 376 10 "6.5M 0.12 Unchanged Ethyl linoleate 308.5 10" 6.5M 0.975 Unchanged Vitamin B12 1355 10-7M 0.135, Unchanged Ascorbic acid 176 10-4M 17.6 Unchanged Phenol Red 376 0.026 x10-3M 10.00 optional Example 2: Ability of different cell lines to multiply indefinitely in a synthetic medium. A total of 25 cell lines were tested for their ability to propagate in H-MSM-2 medium (Table 20). The traditional technique of subculturing the cell mats with a trypsin / EDTA mixture was dispersed. However it is quite possible to propagate the cells by other methods. The collection of mitotic cells which are suspended in the supernatant gives good results but few cells. The advantage in this case is to keep the cells continuously in an environment free of proteins. The dispersion of the cells with a more versene papain mixture is under study, but the first results are encouraging.

Table 20: Culture characteristics of different cell lines grown in H-MSM-2 medium Cells Species Tissues Subcultures TMD Carpet (c) cells MDBK (a) Bovine Kidney 80 36.5 Yes (g) BoSk (a, f) Cattle Skin 80 ND yes CHO s Hamster ovary 22 ND suspension BHK21 Hamster Kidney <2 Not applicable / HEK-293 (a) Human Kidney 35 ND Yes (g) HT29 (a) Human Intestine 22 N / A Yes CaCo2 (a) Human Colon 6 ND yes RK13 (a) Rabbit Kidney 50 ND yes PmMou (a, d, Sheep Lung 7 ND yes f) RTG-2 Fish / tru Gonads <2 Not applicable / ite EPC (a) Fish / ca 12 ND yes rpe PK15 ( a) Porcine Kidney 60 29 yes TrphPo (a, t) Porcine Trophoblast 25 ND yes CEV1 (a) Chicken Viscera 25 22 non-LSCC-RP9 Chicken Lymphoma <2 Not applicable suspension B MDCK (a) (e) Dog Kid In progress ND yes CV1 (a) Monkey Kidney 50 40 yes MA 104 (a) Monkey Kidney 50 ND yes L929 (a) Mouse Areola 35 31 yes MOV (a, f) Mouse Cel. Schann 22 33 yes SP20 (a) Mouse Myeloma 6 months 21 suspension Hyb.G16 (b, f) Mouse Hybridoma 2 years ND suspension Hyb. K9 (b, 0 Mouse Hybridoma 2 years ND suspension Hyb F17 (b, t) Mouse Hybridoma 6 months ND suspension Hyb R29 (b, f) Mouse Hybridoma <3 Not applicable suspension 5 a: Transplanting was done in ratios ranging from 1/4 to 1/10 for cells that are not of lymphoid origin b: For non-adherent lymphoid cells (SP2O, hybridomas), subculture ratios are high 1/1000 to 1 This is the time during which the cells were cultured is indicated (600 days equals about 50 passages every 12 days with a subculturing ratio of 1/1000), c: Mean Time to Doubling (TMD) was measured as follows: at time TO, 104 cells per cm 2 were seeded (NO) After 8 days (192h) of culture, cells were harvested (trypsin for adherent cells) and counted: NX: TMD = 192 / log2 NX / NO; N / A: indicates cells do not grow d: These cells have not been stored e: Serial pass tests are in progress. 2910024 34 f: BoSk, PmMou: The Haridon R., unpublished. ûTrphPo: Bonnardiere C, et al., 2002, Placenta, 23 (10), 716-26. MOV: Archer F et al., 2004, J. Virol., 78 (1), 482-90. Hybridomas: G16, K9, F17, and R29: Haridon et al., 1991 ,. Hybridoma, 10, (1), 35-47 (g): The cells of these 5 lines have a propensity for autoagglutination when cultured in synthetic medium in polystyrene containers. As shown in Table 20, of these 25 lines 4 are not able to propagate indefinitely in the medium H-MSM-2: a hybridoma clone (R29), a line of avian lymphoma (LSCC-RP9) , a trout fibroblast line (RTG-2), and a hamster kidney fibroblast line (BHK21). The CaCo2 line has low cloning efficiency and is growing slowly.

Example 3: Cryopreservation The cells can be stored in liquid nitrogen in a HMSM + 5% DMSO (0.7M) mixture, resulting osmolarity of the order of 1000 mOsM.

Example 4: Production of monoclonal antibodies by hybridomas. Four F17, G16, K9, and R29 hybridomas (Haridon et al., 1991, Hybridoma, 10, (1), 35-47) were cultured in H-MSM medium. Hybridoma R29, difficult to culture in RPMI 1640 medium + 10% SFB, is not cultured in the absence of serum.

The F17 hybridoma ceased secreting a few months after a first culture. So he had to be subcloned again. After this second subcloning, the clone was perfectly stable. The G16 and K9 hybridomas were maintained in culture for nearly 2 years, and the F17 hybridoma for 8 months in H-MSM medium without modification of their ability to secrete their monoclonal antibody. Hybridoma K9 secretes a high proportion of Fab fragments. • Culture in H-MSM-1 medium Productions of monoclonal antibodies of hybridoma G16 grown in traditional medium (RPMI 1640 + 5% SFB) or in H-MSM-1 medium, until cultured senescence were compared . The production in H-MSM-1 medium is 2 times more important than in RPMI 1640 + 5% SFB medium, in terms of the amount of immunoglobulins purified on a protein A-sepharose column, and 3 times higher in term of neutralizing activity secreted in culture supernatants (Table 21). Table 21: Production of monoclonal antibody by hybridoma G16 medium RPMI1640 medium + medium H-MSM-1 + RPMI1640 + 10% 10% SFB hybridoma G16 SFB alone + G16 hybridoma (without hybridomaL total proteins 7,500 μg / ml 7,500 μg / ml ml 65 μg / ml (Bradford Method) Neutralizing Activity C) 15,000 50,000 (UL / ml) Neutralizing Activity C) 2,800 (UL / μg Protein) Purified Protein 60 μg / 50ml 370 μg / 50m1 800 μg / 50m1 chromatography Either 1.2 μg / ml Either 7.4 μg / ml Or 16 μg / ml protein A affinity (mg / I) Neutralizing activity ND 400 000 800 000 purified / protein Neutralizing activity ND 106 106 (UL / mg purified protein) The neutralizing activity is indicated as the reciprocal of the dilution of an antibody solution which incubated in the presence of an interferon solution induces a 75% reduction in antiviral titer (ie two successive / 2). Culture in H-MSM-2 Medium Hybridomas F17 and G16 were seeded at 104 cells / ml in H-MSM-2 medium and in RPMI 1640 + 10% SFB medium and cultured for 7 days.

The number of cells was determined on days 3, 4, 5, 6, and 7. The culture supernatants at day 7 were tested. The graphs indicate that the growths and antibody productions are of the same order that the hybridomas are grown in RPMI 1640 + 10% SFB or in H-MSM-2 (Figures 1, 2 and 3). The antibodies produced in H-MSM medium are very clean (Figure 4).

The difference which is noted between the antibody activity produced in H-MSM-1 medium and in H-MSM-2 medium can be explained by a slower cell growth in H-MSM-1 medium. which promotes the accumulation of antibodies in the culture medium. Virus production Pseudorasal herpes virus (PRV, or Aujesky's disease virus) was produced on PK15 cells previously cultured in EMEM + 5% SFB medium or in H-MSM medium. PK15 cells grown in H-MSM medium or in EMEM + 5% SFB medium produce the same amount of virus titrated in lysis bands. Bovine rotavirus (strain RF: Haridon, R., and Scherrer, R. 1976.

In vitro culture of rotavirus associated with neonatal diarrhea in the calf. Ann. Veterinary Research, 7: 373-381) was produced in MA104 cells grown in EMEM medium or in H-MSM-2 medium. MA104 cells cultured in H-MSM-2 medium produce more virus than MA104 cells grown in EMEM. • Production of recombinant protein in mammalian cells There are many recombinant protein expression systems in mammalian cells. The most commonly used are in particular cloned CV1 cells COS7, CHO cells, and RK13 cells.

The model of expression of porcine interferon gamma in RK13 cells (Cencic et al., 1999, Anim Biotechnol 10 (1-2): 63-79) available in our Research Unit was kindly provided by the Dr. Claude La Bonnardière 25 (Unit 893 - Virology and Immunology - INRA). The cells of clone Al2RK13 carry the gamma interferon gamma gene placed under the control of a promoter modulated by tetracycline. The synthesis of the protein of interest is inhibited by tetracycline. They are therefore likely to secrete the protein of interest when cultured in the absence of tetracycline.

One batch of these cells was cultured in the usual medium (EMEM + 10% SFB + tetracycline) and another batch was cultured in synthetic medium (HMSM-2 + tetracycline). These cells after three passages were subcultured in these same media but in the absence of tetracycline. An aliquot of supernatant from each culture was taken after 4, 7, 10, and 14 days of culture. The presence of interferon 2910024 in these supernatants was detected by an immunochemical method: -ELISA- (Lefevre F. et al., 1998, Biol Reprod 58: 1026-1031) and by the biological inhibition method of the cytopathic effect caused by VSV virus infecting MDBK cells. The results are shown in Figures 5 and 6.

The present application therefore proposes a chemically defined synthetic medium formulation, completely free of proteins. There are many works of the prior art describing the performance of semi-synthetic media, that is to say that these so-called serum-free media, sometimes without protein (but in fact natural protein free). ). These media have been developed by researching serum substitutes. They contain, in most cases, one or more recombinant proteins (insulin for example) intended to partially replace the serum. There is only a few works Hamilton W., G., & Ham R., G., 1977, Vitro 13, (9): 537-547, Barnes D., and Sato G., 1980, Analyt. Biochem. 102, 255-270; Schneider Y., 1989, J. Immunol Methods, 116 (1): 65-77; Darfler FJ, 1990, Vitro Cell Dev Biol .; 26 (8): 769-78; and US Pat. No. 5,045,468) which describe media completely free of proteins or peptides. On the other hand, these formulations contain substances that are presumed toxic or pharmacologically active (EDTA, steroid hormones, nitroprusside). The H-MSM medium, unlike the previous ones, was developed for the long-term cultivation of different cell lines in strict aprotic medium, without looking for substitutes for the serum. It is therefore considered better balanced than existing formulations. It allows the production of secure virus for the preparation of vaccines. For the production of recombinant proteins or monoclonal antibodies, it leaves the possibility of the addition of one (or more) specific factor (s) likely to increase the production yields (for example TGF, insulin, IRTKA, IGF, ...).

Claims (23)

claims   A synthetic and chemically defined cell culture medium comprising: a) a mixture of natural amino acids; b) at least one mineral salt; (c) at least one sugar; d) at least one organic acid; e) at least one nitrogenous nutrient; and f) at least one vitamin and / or antioxidant; wherein the percentage (in molarity / total molarity of the mixture) of each amino acid in the natural amino acid mixture is:   The cell culture medium according to claim 1, wherein the concentration of each of the amino acids in said culture medium is equal to the following concentrations multiplied by a factor of between 0.75 and 3.75 (inclusive): Ala Arg Asn Asp (Cys-Cys) Eventually Gln Glu Gly His lle Leu Lys Met Phe Pro Ser r Trp Tyr Val 6.20 1.24 4.58 0.92 96 0.39 3.71 0.74 5.61 1.12 0 , 81 0.16 1, 59 0.32 9.52 1,90 1.12 0.22 3.74 0.75 5 10 2910024 39 Ala 1.345 0.269 x10-3 M Arg 0.75 0.150 x103 M Asn 0, 0.138 x 10-3 M Asp 0.23 0.046 x 10 -3 M (Cys-Cys) 0.56 0.112 x 10 -3 M 2, 215 Possibly Gln 0.443 x 10-3 M Glu 0.31 0.062 x 10-3 M Gly 0, Mg 0.62 0.124 x10-3 M d 0.265 0.053 xlo-3 M Leu 0.502 0.100 x10-3 M Lys 0.76 + 0.152 xl0m Met 0.11 0.022 x103 M Phe 0.215 0.043 x10 -3 M Pro 1.29 0.258 xl M Ser 1.35 0.270 x10-3 M Thr 0.7 7 0.154x10-3M Trp 0.07 0.014 x10-3 M x10-3 Val 0.15 0.030 M 0.507 0.101 x10-3 Mr.   3. A cell culture medium according to claim 1 or 2, comprising sodium, potassium, calcium, magnesium, phosphate, carbonate, iron, zinc, cobalt, molybdenum, selenium, iodine, boron, manganese, tin and lanthanum salts.   4. A cell culture medium according to any one of claims 1 to 3, comprising iron in the form of iron phosphate (Fe4 (P2O7) 3).   Cell culture medium according to claim 3 or 4, comprising: NaCl 11.5-115 x 10 -3M KCl 0.537-5.37 x 10 3M CaCl 2 O 2 H 2 O 0.18-1.8 xlo-3m MgSO 4 .7H 2 O 1.8 x 10 3M NaH 2 PO 4 .H 2 O 0.2-2 x 10- 3M NaHCO 3 2.62-26.2 x 10 3m Fe 4 O 2 P 6 (Fe 4 (P 2 O 7) 3) 0.1-1 x 10 -5 Mn Zn SO 4 .7H 2 O , 1-1 x10 m * CoCl2.6H2O 0.1-1 x10 m * MoO4Na2.H2O 0.1-1 x10-8M * SeO3Na2 0.1-1 x10-M 2910024 40 * IK 0.1-1 x10- 8M BO3H3 0.1-1 x10-8M MnCl2.4H2O 0.1-1 x1 0 "9M * SnCl2.2H2O 0.1-1 x1 0" 9M * LaCI3.7H2O 0.1-1 x10-9M.   A cell culture medium according to any one of claims 1 to 5, comprising glucose, fructose, galactose, mannose, inositol, sodium pyruvate, sodium butyrate, choline, ethanolamine and optionally alaninol.   The cell culture medium of claim 6, comprising: Glucose 2.5-10 x 10-3M Fructose 0.79-3.16 x10-3M Galactose 10-3-10-10 2M Mannose 10-3 5-10-2,5M Inositol Io-4,5-1 0 3,5M Sodium Pyruvate 10-3.5-10 "2M Sodium Butyrate 10" 6-10 ~ M Choline HCI 10 "5-10-4M Ethanolamine 10-5 , 5-10-4.5M Possibly Alaninol 10-6-10 4M.   8. A cell culture medium according to any one of claims 1 to 7, comprising biotin, vitamin B12 and retinyl acetate, the ratio of biotin: vitamin 12 concentrations being between 80: 1 and 120: 1, and the ratio of biotin: retinyl acetate concentrations between 8: 1 and 12: 1.   Cell culture medium according to any one of claims 1 to 7, comprising biotin, D-Ca-pantothenate, nicotinamide, pyridoxine, thiamine, thioctic acid, folic acid and optionally its precursor P-amino benzoic acid, retinylacetate, riboflavin, ethyl linoleate, vitamin B12, and possibly ascorbic acid.   Cell culture medium according to claim 8 or 9, comprising: Biotin 10-b-10-4M D-Ca-pantothenate 10b-10M Nicotinamide 10-6-10MM Pyridoxine 10b-10 4M Thiamine 10-b-10-4M Thioctic acid 10-6-10-4M 5 2910024 41 Folic acid 10-63-10 "5.5M Retinylacetate 10-6.5-10" 5'5M Possibly Ac. amino benzoic acid 10 "'- 10-5M Riboflavin 10' '10" 6M Ethyl linoleate 10-7-lem Vitamin B12 10-6-10 "6M Possibly Ascorbic acid 10610" 3M.   The cell culture medium of any one of claims 1 to 10, comprising: NaCl 11.5-115x10 -3M KCl 0.537-5.37 x 10 -3-M CaCl2.2H2O 0.18-1.8 x10- 3M MgSO 4 .7H 2 O 0.18-1.8 x 10-3-M NaH 2 PO 4 .H 2 O 0.2-2 x 10 -2 NaHCO 3 2.62-26.2 x 10-3M Fe 4 O 2 P 6 (Fe 4 (P2O 7) 3) 0, 1-1 x10-5'5M _ * Zn SO4.7H2O 0.1-1 x10-6'5M _ 0.1-1 x10 "i'5M * CoCl2.6H2O _ * MoO4Na.H 0 _ 0.1- 1 x 10 -M * SeO3Na2 0.1-1 x10- $ M _ * IK 0.1-1 x10 8M _ BO3H3 0.1-1 x10- M MnCl2.4H2O 0.1-1 x10- SnCl2. 2H2O 0.1-1 x10 "M * LaCI3.7H2O 0.1-1 x10" Ï Glucose 2.5-10 x10 "3M Fructose 0.79-3.16 x10" 3M Galactose 10-3.5-10- 2'5M Mannose 10 "3'5-10" 2'5M Inositol Io-4,5-1 o3'5M Sodium Pyruvate 10 "3'5-10" 2M Sodium Butyrate 10-10 MM _ Choline HCI 10 5-10 "4M Ethanolamine 10" 5.5-10 4.5M Possibly Alaninol 10 "-10 M Alanine 1.01-5.04x10" 3M Arginine 0.56-2.81 x10 "3M Asparagine 0.52-2.59 x10 -3M Aspartic Acid 0.17-0.86 x10-3M Cystine 0.42-2.10 x1 0-3-M Glutamic Acid 0.23-1.16 x10-3 M Possibly Glutamine 1.66-8 , 31 x10-3-Ni 29100 24 42 Glycine 0.63-3.15 x10-3M Histidine 0.47-2.33 x10-3M Isoleucine 0.20-0.99 x1 0 "3M Leucine 0.38-1.88 x10-3M Lysine 0, 57-2.85 × 10 3 M Methionine 0.08-0.41 x 10 -3 M Phenylalanine 0.16-0.81 x 10 -3 M Proline 0.97-4.84 x 1.0 M 3 Serine 1.01-5, 06 x10-3M Threonine 0.58-2.89 x10-3M Tryptophan 0.05-0.26 x10-3M Tyrosine 0.11-0.56 x10-3M Valine 0.38-1.90 x10-3M Biotin 10 "b-10" 4M D-Ca-Pantothenate 10-6-10 "4M Nicotinamide 10-b1 0" 4M Pyridoxine 10 "6-10" 4M Thiamine 10-6-10 "4M Thioctic Acid 10-6-104M Folic Acid 10 "6; 5-10" 5.5M Retinylacetate 10- ~ 5-10-5'5M Possibly Ac. P.-amino 10 "7-10" 5M Benzoic Riboflavin 10 "'- 10-5M Ethyl Linoleate 10"' - 10 6M Vitamin B12 10 "6-10-5M Possibly Acid 10" 6-10 "3M Ascorbic   The cell culture medium of any one of claims 1 to 11, comprising: NaCl 11.5-115x10-3M KCl 0.537-5.37 x10-3M CaCl222H2O 0.18-1.8 x10-3M MgSO4.7H2O 0.18-1.8 x 10 -3 M NaH 2 PO 4 .H 2 O 0.2-2 x 10 -3 M NaHCO 3 2.62-26.2 x 1 0 -3 M Fe 4 O 2 P 6 (Fe 4 (P 2 O) 3) 0.1-1 x 10 -5 ' 5M * Zn SO4.7H20 0.1-1 x10- "m * CoCl2.6H2O 0.1-1 x10- ', 5M * MoO4Na2.H2O 0.1-1 x10-6M * SeO3Na2 0.1-1 x10- 6M * IK 0.1-1 x1 0 "$ M B03H3 0.1-1 x10-6M MnCl2.4H20 0.1-1 x10-9M 2910024 43 * SnCl2.2H2O 0.1-1 x10-9M * LaCI3. 7H2O 0.1-1 x10-9M Glucose 2.5-10 x10-3M Fructose 0.79-3.16 x10-3M Galactose 10 "3.5-10" 2'5M Mannose 10-3.5-10 " 2.5M Inositol 1 0 4'5-10 3.5M Sodium Pyruvate 10-3'5-10 "2M Sodium Butyrate 10" 6-10M Choline HCI 10 "5-10-4M Ethanolamine 10" 5.5-10 4.5M Possibly Alaninol 10 "b-104M Alanine 1.345 0.269 x1 0" 3M Arginine 0.75 0.150 x10 "3M Asparagine 0.69 0, 138 x10-3M Aspartic Acid 0.23 0.046 x10-3M Cystine 0.56 0.112 x10 -3M Glutamic Acid 2,215 0,443 x10-3M Possibly Glutamine 0.31 0.062 x10 -3M Glycine 0.84 0.168 x10-3M Histidine 0.62 0.124 x10 "3M Isoleucine 0.265 0.053 x10-3M Leucine 0.502 0.100 x10-3M Lysine 0.76 0.152 x10-3M Methionine 0.11 0.022 x10-3M Phenylalanine 0.215 0.043 x10 -3M Proline 1.29 0.258 x10-3M Serine 1.35 0.270 x10-3M Threonine 0.77 0.154 x10-3M Tryptophan 0.07 0.014 x10 "3M Tyrosine 0.15 0.030 x1 0" 3M Valine 0.507 0.101 x10-3M Biotin 10-b-10-4M D-Ca-Pantothenate 10 "6-10-4M Nicotinamide 10-6-10-4M Pyridoxine 10" 6-10 "4M Thiamine 10-b-10" 4M Thioctic Acid 10k-10 4M Acid folic 10 "6'5-10-5'5M Retinylacetate 10" ~ 5-10 5 5M Possibly Ac. P. amino 10 "'- 10-5M benzoic Riboflavin 10"' - 10-'M 2910024 44 Ethyl linoleate 10 "- 10" bM Vitamin B12 10 "8-10-6M Possibly 10-b-10" 3M acid ascorbic   Cell culture medium according to any one of claims 1 to 11, comprising: NaCl 11.5-115x1 0-3M KCl 0.537-5.37 x10-3M CaCl2.2H2O 0.18-1.8 x10-3M MgSO4.7H2O 0.18-1.8 x10-3M NaH2PO4.H2O 0.2-2 x10-3M NaHCO3 2.62-26.2 x1 0-3M Fe4O21 P6 (Fe4 (P2O7) 3) 0.1-1 x10-5'5M * Zn SO4.7H2O 0.1-1 x1 0 "6.5M * CoCl2.6H2O 0.1-1 x1 0" ', 5M * MoO4Na2.H2O 0.1-1 x10-8M * SeO3Na2 0.1-1 x10-8M * IK 0.1-1 x10-8M BO3H3 0.1-1 x10-8M MnCl2.4H2O 0.1-1 x10-9M * SnCl2.2H2O 0.1-1 x10-9M * LaCI3.7H2O 0.1-1 x10-9M Glucose 2.5-10 x1 0 "3M Fructose 0.79-3.16 x1 0" 3M Galactose 10-3'S-10-2'5M Mannose 10 "3'5 -10 '5M Inositol I 0 4'5-10 3'5M Sodium Pyruvate 10 "3.5-10" 2M Sodium Butyrate 10-6-10 "5M Choline HCI 10" 5-10 "4M Ethanolamine 10" 5'5 -10 4'5M Possibly Alaninol 10-b-10 "4M Alanine 2.69 0.54 x10" M Arginine 1.5 0.30 x10-3M Asparagine 1.38 0.28 x10 "3M Aspartic Acid 0.46 0 , 09 x10-3M Cystine 1.12 0.22 x1 0 "3M Glutamic acid 4.43 0.89 x1 0" 3M Possibly Glutamine 0.62 0.12 x10-3M Glycine 1, 68 0.34 x10-3M Histidine 1.24 0.25 x10-3M Isoleucine 0.53 0.11 x10-3M 2910024 45 Leucine 1.04 0.20 x10-3M Lysine 1.52 0.30 x10-3M Methionine 0.22 0.04 x 1.07 M Phenylalanine 0.43 0.09 x 10 -3 M Proline 2.58 0.52 x 10-3 M Serine 2.7 0.54 x 10 -3 M Threonine 1, 54 0.31 x 10 -3M Tryptophan 0.14 0.03 x10-3M Tyrosine 0.306 x10-3M Valine 1.14 0.20 x 10-3M Biotin 10-6-10-4M D-Ca-pantothenate 10-b- 10 "4M Nicotinamide 10" b-10-4M Pyridoxine 10 "b-10" 4M Thiamine 10 "6-10" 4M Thioctic acid 10 "b-10" 4M Folic acid 10e, 5-15M Retinyl acetate 10-6 5- 10-5 5M Possibly Ac. P. amino 10 "'- 10-5M benzoic Riboflavin 10"' - 10 "6M Ethyl linoleate 10" '- 10 "bM Vitamin B12 10" 6-10-bM Possibly 10-b-10 "3M Ascorbic acid   14. A cell culture medium according to any one of claims 1 to 13, said medium being serum-free and / or aprotic. 5   15. A cell culture medium according to any one of claims 1 to 14, said medium being intended for the culture of vertebrate cells.   16. A cell multiplication method comprising the steps of: a) contacting the culture medium of any one of claims 1 to 15 with at least one cell; and b) cultivating said at least one cell for a time and under conditions allowing the multiplication of said cell.   The method of claim 16, wherein said at least one cell is an adherent cell or a suspended cell. 2910024 46   The method of claim 16 or 17, wherein said at least one cell expresses a protein of interest or produces a virus.   19. The method according to claim 18, wherein said cell expressing a protein of interest is a hybridoma expressing a monoclonal antibody or a cell transformed with a nucleic acid comprising a sequence encoding said protein of interest.   20. A method of producing a protein of interest comprising the steps of: a) contacting the culture medium of any one of claims 1 to 15 with at least one cell expressing a protein of interest; b) cultivating said at least one cell for a time and under conditions allowing the expression of the protein of interest; and c) optionally harvesting the expressed protein of interest.   The method of claim 20, wherein said cell expressing a protein of interest is a cell that has been transformed with a nucleic acid comprising a sequence encoding said protein of interest.   The method of claim 20, wherein said cell expressing a protein of interest is a hybridoma expressing a monoclonal antibody.   23. A method of producing a virus comprising the steps of: a) contacting the culture medium of any one of claims 1 to 15 with at least one cell infected with a virus; b) cultivating said at least one cell for a time and under conditions enabling the replicative cycle of said virus to be carried out; and (c) possibly harvest the viruses produced.