CULTURE MEDIA FOR MYCOPLASMA

FIELD OF THE INVENTION

The invention relates to cell culture medium formulations. Specifically, the invention provides serum-free cell culture medium formulations that facilitate the in vitro cultivation of Mycoplasma cells.

BACKGROUND OF INVENTION

Mycoplasmas comprise a large group of species capable of colonizing a wide range of organisms, including animals, plants, insects and humans. They are characterized to lack a cell wall, which makes them naturally resistant to antibiotics that target cell wall synthesis, but also more sensitive to membrane-active agents. Mycoplasmas have also reduced genomes with few metabolic capabilities, thus relying on their host for much of their nutrition. For all these reasons, growth of mycoplasma species in axenic conditions has been historically difficult and dependent on animal serum.

Mycoplasmas are generally surface parasites. Some species act as commensal bacteria, living innocuously with their host as part of the natural flora. However, many species like M. pneumoniae act as pathogens, often causing chronic infections. Indeed, diseases associated to mycoplasmas are an important economic burden, both in human and livestock systems. Vaccination has been proven as an efficient strategy to alleviate the economic impact that some mycoplasma infections cause on milk production, weight gain and animal health. In this regard, some of the most effective vaccines available against mycoplasma infections are live-attenuated or inactivated vaccines, in which growth and production of the bacterial strain is required. The culture medium used in the production of these vaccines usually contains animal components such as BHI (brain heart infusion broth), PPLO (beef heart infusion, pancreatic digest of casein and beef extract broth) and animal serum. The use of animal components in culture medium entails several disadvantages, including: low batch-to-batch reproducibility, not defined composition, possible interference with downstream processing, and increase considerably the cost of the medium. In addition, both the use of animal serum and raw materials derived from animals have important safety concerns, since these may contain viruses, antibiotics, endotoxins and other bioactive molecules. Therefore, there is a need for a culture medium that: (1) is safe and free of animal components, (2) supports robust growth, and (3) allows serial passaging. These media would be advantageous over other media known in the art for the development of mycoplasma vaccine development or other mycoplasma-based therapies.

SUMMARY OF THE INVENTION

The authors of the invention have developed several culture media that are capable of supporting the growth of Mycoplasma cells and, in particular, of Mycoplasma pneumoniae cells, to biomass levels similar to those obtained with standard growth medium, said media being characterized in that they are free of animal serum and, in some cases, free of components of animal origin (i.e. bovine seroalbumin, BSA).

In a first aspect, the invention relates to a serum free medium suitable for the culture of Mycoplasma pneumoniae that comprises:

(i) a carbon source,

(ii) an amino acid mixture,

(iii) a vitamin mixture, said mixture comprising thioctic acid,

(iv) a lipid mixture, comprising cholesterol, sphingomyelin or ceramide and phosphatidylcholine,

(v) inorganic salts,

(vi) a nucleoside mixture,

(vii) a mixture of enzymatic cofactors,

(viii) a buffering agent,

(ix) a proteolytic digest of a microorganism or of a plant or animal tissue and

(x) a vehicle to solubilize and deliver the lipids.

In a second aspect, the invention relates to a method for the culture of a Mycoplasma strain comprising placing an inoculum of said strain into the media according to the first aspect of the invention and maintaining the inoculated media under conditions adequate for the proliferation of the Mycoplasma strain.

In a third aspect, the invention relates to a method for obtaining a biomass of Mycoplasma strain which comprises placing an inoculum of said Mycoplasma strain into the media according to the first aspect of the invention and maintaining the inoculated media under conditions adequate for the proliferation of the Mycoplasma strain and the formation of the biomass. DESCRIPTION OF THE FIGURES

Figure 1. (Left) M. pneumoniae grown in 5x CMRL; (centre) M. pneumoniae grown in 5x CMRL supplemented with 10mg/ml RNA and 30mg/ml peptone; (right) M. pneumoniae grown in Hayflick rich media. DAPI staining is used to label DNA and quantify the number of viable cells in the different conditions.

Figure 2. Layout of 96 well plate used to assay multiple growth media formulations.

Figure 3. Levels of growth (biomass increase with respect to time 0) observed in each condition after normalization with blank controls PeptJ peptoneJ TrypJ Tryptone. Black bars represent the positive controls with Hayflick and MM 16 media. Figure 4. Scheme showing the workflow during the process of medium optimization.

Figure 5. Optimization of the culture buffering system for our high-throughput screening method. (A, B, C). Growth curve analysis determined by the 430/560 absorbance ratio index comparing the performance of Hayflick rich medium (HF) and vB2 containing OmM, 50mM and 100mM HEPES. Cultures were initiated with 1.5μg (A), 3μg (B) or 6μg (C) of starting inocula. (D) Protein biomass yields at the end of growth curves shown in A, B and C growth curves. Data represents the mean ± standard deviation of three replicates.

Figure 6. Impact of thioctic acid (TA) and glycerol (Gly) on protein biomass yield using a medium free of serum.

Figure 7. Impact of phosphatidylcholine and sphingomyelin on M. pneumoniae cell growth using a medium free of serum. (A) Growth curve analysis determined by the metabolic growth index comparing cell growth after adding phosphatidylcholine (PC) and sphingomyelin (SMP) individually or in combination in a medium free of serum. (+/-) indicates presence or absence of the indicated phospholipid. (B) Protein biomass measurement at 96 h, corresponding to the end of the growth curve shown in panel A. Data represent the mean ± standard deviation of two replicates. Figure 8. Growth curve analyses of vB10 (serum-free) and vB13 (animal component-free) mediums along 96h of culture in 25crri ₂ tissue culture flasks containing 5ml of medium. Cell growth in (A, B) vB10 and (C, D) vB13 mediums was assessed by (A, C) protein and (B, D) DNA biomass quantification and compared to rich medium (Hayflick).

Figure 9. Pearson’s correlation of gene expression between M. pneumoniae samples grown in rich medium (HF) or animal component-free medium (vB13). (A) Comparative analyses of RNA levels assessed by RNA-seq experiments. (B) Comparative analyses of protein levels assessed by mass spectrometry experiments. RNAseq and proteomic datasets were obtained from two biological replicates.

Figure 10. Growth curve analyses of several M. pneumoniae chassis strains.

(A, B) Growth curve analysis determined by the metabolic growth index comparing cell growth of several M. pneumoniae strains grown in (A) rich medium (HF with 10mM HEPES) or (B) animal component-free medium (vB13).

(C) Protein biomass measurement at 89 h, corresponding to the end of the growth curve shown in panel A and B. Data represent the mean ± standard deviation of two replicates. Strain nomenclature is as follows: WT (wild-type), CV31 (WT with a deletion in mpn133 nuclease), CV1 (WT with a deletion in mpn372 CARDS toxin), CV2 (WT with a deletion in mpn133 and mpn372 ), and CV7 (CV2 strain expressing mg186 gene from M. genitalium).

Figure 11. Table showing the detailed composition of each one of the different media versions developed for M. pneumoniae cultivation.

Figure 12. Growth curve analysis determined by the metabolic growth index showing growth performance of each serum-free medium version shown in Annex 1 and compared to rich medium (Hayflick)

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a serum-free culture medium for the growing of Mycoplasma pneumoniae. Said serum-free culture medium comprises at least one carbon source, amino acids, vitamins, amino acids, nucleotides, lipids, inorganic salts enzymatic cofactors, buffering agents, a polyamine and a proteolytic digest of a microorganism, plant or animal tissue. Thus in a first aspect, the invention relates to a serum free medium suitable for the culture of Mycoplasma pneumoniae that comprises:

(i) a carbon source,

(ii) an amino acid mixture,

(iii) a vitamin mixture, said mixture comprising thioctic acid,

(iv) a lipid mixture, comprising cholesterol, sphingomyelin or ceramide and phosphatidylcholine,

(v) inorganic salts,

(vi) a nucleoside mixture,

(vii) a mixture of enzymatic cofactors,

(viii) a buffering agent,

(ix) a proteolytic digest of a microorganism or of a plant or animal tissue.

(x) a vehicle to solubilize and deliver the lipids.

As used herein, the term "serum-free medium" refers to medium which is substantially free of serum and which can maintain in vitro growth and proliferation of cells for a long period. There is no particular limitation on the serum-free medium of the present invention, which can be any serum-free culture medium suitable for cultivation or preservation of a culture of Mycoplasma pneumoniae. In some embodiments, the medium according to the present invention can maintain growth of Mycoplasma cells, preferably M. pneumoniae cells until biomass levels are reached which are at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the biomass levels that can be reached with rich medium such as, for instance, the Hayflick medium,

The term, “culture”, “cell culture” or “cultivation” refers to the maintenance or proliferation of cells in an artificial in vitro environment. The culture media of the present invention can be used for the culture of any Mycoplasma species, especially M. pneumoniae. The term “Mycoplasma” as used herein, refers to a genus of spherical to filamentous cells with no cell walls. Presently, more than 100 species have been included in the genus Mycoplasma. Mycoplasma are the smallest self-replicating organisms with the smallest genomes, comprising a total of about 500 to 1000 genes. Mycoplasma are nutritionally fastidious. Many require cholesterol, a unique property among prokaryotes. The term “Mycoplasma pneumoniae ”, as used herein, refers to a human pathogen that causes related to cold agglutinin disease. As all Mycoplasma species, also M. pneumoniae is characterized by the absence of a peptidoglycan cell wall and resulting resistance to many antibacterial agents.

The carbon source in a serum-free culture medium according to the invention is a sugar, preferably a monosaccharide such as fructose and glucose, a disaccharide such as lactose and maltose, or a mixture thereof. In an embodiment, the carbon source is selected from glucose, mannose, acetate, glucoronate or a combination thereof. In another embodiment, the carbon source is mannose or glucose.

A carbon source suitable for the serum-free culture medium according to the invention preferably is or comprises glucose. The carbon source in the serum-free culture medium according to the invention preferably is present between 0.01 and 30 % (w/w), more preferably between 0.02 and 2% (w/w).

In some embodiments, the carbon source is provided in combination with a second polyol compound, such as glycerol. When present, glucose and glycerol preferably are present between 0.2 and 25% (w/w), more preferably between 0.5 and 2% (w/w), preferably at 0,75% (w/w) for glucose; and between 0.01 and 0.5% (w/w), more preferably between 0.002 and 0.5% (w/w), more preferably between 0.005 and 0.05% (w/w), preferably at 0.025% (w/w) for glycerol. In a preferred embodiment, the glycerol concentration is of at least 0.01% (w/w), preferably of at least 0.025% (w/w) and more preferably of at least 0,05% (w/w).

In a particular embodiment, the source of carbon is D-glucose in a concentration of between 100 to 5000 mg/L. In another embodiment, the concentration of D-glucose is of about 500 mg/L. In another embodiment, the concentration of D-glucose is of about 2000 mg/L.

The chemically defined medium comprises one or more of the amino acids. The term "one or more amino acids" refers to native amino acids or their derivatives (e.g., amino acid analogs), as well as their D- and L-forms.

In a preferred embodiment, the serum-free culture medium according to the invention comprises at least the amino acids Glycine, Hydroxy L-proline, L-Alanine, L-Arginine hydrochloride, L-Aspartic acid, L-Cysteine, L-Cystine, L-Glutamic Acid, L-Glutamine, L- Histidine hydrochloride-H ₂ 0, L-lsoleucine, L-Leucine, L-Lysine hydrochloride, L- Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine.

In a preferred embodiment, the serum-free culture medium of the invention comprises the amino acids Glycine, Hydroxy L-proline, L-Alanine, L-Arginine hydrochloride, L-Cysteine, L- Cystine, L-Glutamine, L-Histidine hydrochloride-H ₂ 0, L-lsoleucine, L-Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L-Threonine, L- Tryptophan, L-Tyrosine, L-Valine but lacks L-Aspartic acid, L-Glutamic Acid and L- asparagine.

In a preferred embodiment, the serum-free culture medium of the invention comprises the amino acids Glycine, Hydroxy L-proline, L-Alanine, L-Arginine hydrochloride, L-Cysteine, L- Cystine, L-Glutamine, L-Glutamic Acid, L-Histidine hydrochloride-H ₂ 0, L-lsoleucine, L- Leucine, L-Lysine hydrochloride, L-Methionine, L-Phenylalanine, L-Proline, L-Serine, L- Threonine, L-Tryptophan, L-Tyrosine, L-Valine but lacks L-Aspartic acid.

When present in the serum-free culture medium, the amino acids are preferably contained in the concentration ranges defined below.

Glycine in a range of 0.1 mg/L to 1000 mg/L, more preferably in a range between 1 mg/L to 500mg/L, preferably between 10 to 200 mg/L. In a particular embodiment, the concentration of Glycine is of about 25 mg/L. In another embodiment, the concentration of glycine if of about 50 mg/L. In another embodiment, the concentration of glycine is of about 100 mg/L.

Hydroxy-L-proline in a range between 0.01 mg/L to 200 mg/L, more preferably between 0.1 mg/L to 100 mg/L. in a particular embodiment, the concentration of Hydroxy-L-proline is of about 1 mg/L. In another embodiment, the concentration of Hydroxy-L-proline is of about 10 mg/L. In another embodiment, the concentration of Hydroxy-L-proline is of about 20 mg/L.

L-alanine in a range of 0.1 mg/L to 1000 mg/L. more preferably between 1 mg/L to 100 mg/L. In a particular embodiment, the concentration of L-alanine is of about 12 mg/L. In another embodiment, the concentration of L-alanine is of about 25 mg/L. In another embodiment, the concentration of L-alanine is of about 50 mg/L. L-arginine hydrochloride in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 500 mg/L. In a particular embodiment, the concentration of L-arginine hydrochloride is of about 35 mg/L. In another embodiment, the concentration of L-arginine hydrochloride is of about 70 mg/L. In another embodiment, the concentration of L-arginine hydrochloride is of about 140 mg/L.

L-aspartic acid in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 100 mg/L. In a particular embodiment, the concentration of L-aspartic acid is of about 15 mg/L. In another embodiment, the concentration of L-aspartic acid is of about 30 mg/L. In another embodiment, the concentration of L-aspartic acid is of about 60 mg/L.

L-cysteine in a range of 1 mg/L to 1000 mg/L, more preferably between 50 mg/L to 500. In a particular embodiment, the concentration of L-cysteine is about 100 mg/L. In another embodiment, the concentration of L-cysteine is of about 200 mg/L. In another embodiment, the concentration of L-cysteine is of about 400 mg/L.

L-cystine in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 100 mg/L. In a particular embodiment, the concentration of L-cystine is of about 10 mg/L. In another embodiment, the concentration of L-cystine is of about 20 mg/L. In another embodiment, the concentration of L-cystine is of about 40 mg/L.

L-glutamic acid in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 500 mg/L. In a particular embodiment, the concentration of L-glutamic acid is of about 35 mg/L. In another embodiment, the concentration of L-glutamic acid is of about 15 mg/L. In another embodiment, the concentration of L-glutamic acid is of about 15 mg/L, preferably at 150 mg/L.

L-histidine hydrochloride monohydrate in a range of 0.1 mg/L to 500 mg/L, more preferably between 1 mg/L to 100 mg/L. In a particular embodiment, the concentration of L-histidine hydrochloride monohydrate is of about 10 mg/L. In another embodiment, the concentration of L-histidine hydrochloride monohydrate is of about 20 mg/L. In another embodiment, the concentration of L-histidine hydrochloride monohydrate is of about 40 mg/L. L-isoleucine in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 100 mg/L. In a particular embodiment, the concentration of L-isoleucine is of about 10 mg/L. In another embodiment, the concentration of L-isoleucine is of about 20 mg/L. In another embodiment, the concentration of L-isoleucine is of about 40 mg/L.

L-leucine in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 500 mg/L. In a particular embodiment, the concentration of L-leucine of about 30 mg/L. In another embodiment, the concentration of L-leucine is of about 60 mg/L. In another embodiment, the concentration of L-leucine is of about 120 mg/L.

L-lysine hydrochloride in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 500 mg/L. In a particular embodiment, the concentration of L-lysine hydrochloride is of about 35 mg/L. In another embodiment, the concentration of L-lysine hydrochloride is of about 70 mg/L. In another embodiment, the concentration of L-lysine hydrochloride is of about 140 mg/L.

L-methionine in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 100 mg/L. In a particular embodiment, the concentration of L-methionine is of about 7.5 mg/L. In another embodiment, the concentration of L-methionine is of about 15 mg/L. In another embodiment, the concentration of L-methionine is of about 30 mg/L.

L-phenylalanine in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 100 mg/L. In a particular embodiment, the concentration of L-phenylalanine is of about 12.5 mg/L. In another embodiment, the concentration of L-phenylalanine is of about 25 mg/L. In another embodiment, the concentration of L-phenylalanine is of about 50 mg/L.

L-proline in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 100 mg/L. In a particular embodiment, the concentration of L-proline is of about 20 mg/L. In another embodiment, the concentration of L-proline is of about 40 mg/L. In another embodiment, the concentration of L-proline is of about 80 mg/L.

L-serine in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 100 mg/L. In a particular embodiment, the concentration of L-serine is of about 12.5 mg/L. In another embodiment, the concentration of L-serine is of about 25 mg/L. In another embodiment, the concentration of L-serine is of about 50 mg/L. L-threonine in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 100 mg/L. In a particular embodiment, the concentration of L-threonine is of about 15 mg/L. In another embodiment, the concentration of L-threonine is of about 30 mg/L. In another embodiment, the concentration of L-threonine is of about 60 mg/L.

L-tryptophan in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 1000 mg/L. In a particular embodiment, the concentration of L-tryptophan is of about 5 mg/L. In another embodiment, the concentration of L-tryptophan is of about 10 mg/L. In another embodiment, the concentration of L-tryptophan is of about 20 mg/L.

L-tyrosine in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 100mg/L. In a particular embodiment, the concentration of L-tyrosine is of about 20 mg/L. In another embodiment, the concentration of L-tyrosine is of about 40 mg/L. In another embodiment, the concentration of L-tyrosine is of about 80 mg/L.

L-valine in a range of 0.1 mg/L to 1000 mg/L, more preferably between 1 mg/L to 100 mg/L. In a particular embodiment, the concentration of L-valine is of about 12.5 mg/L. In another embodiment, the concentration of L-valine is of about 25 mg/L. In another embodiment, the concentration of L-valine is of about 50 mg/L.

The serum-free culture medium comprises L-Glutamine. In a preferred embodiment, the content of L-Glutamine in the serum-free culture medium in is of at least 1 nM, more preferably of at least 2 mM.

Glutamine is known to be quite unstable in an aqueous solution. As been reported, glutamine may be stabilized with one or more divalent cations selected from the group consisting of calcium and magnesium, whereby the ration of glutamine to divalent cations is in the range of from 2:1 to 2.9:1 (US patent 5,474,931). In addition, the presence of pantothenate and sodium chloride may help stabilize glutamine and prevent its degradation into pyrrolidone carboxylic acid and ammonia. As an alternative, or in addition, unstable glutamine may be replaced with the dipeptide l-alanyl-l-glutamine or glycyl-l-glutamine. Thus in an embodiment, the content of the l-alanyl-l-glutamine or glycyl-l-glutamine in the free-serum culture medium is of at least 1 mM, more preferably, of at least 2 mM. Similarly, redox active cysteine may be replaced with N-acetyl-l-cysteine of S-sulfo-l- cysteine (Hecklau et al., 2016, J. Biotech 218: 53-63), and the amino acid derivative phosphor-l-tyrosine may replace tyrosine (Zimmer et al., 2014, J. Biotechnol 186: 110-8).

In another embodiment, the essential amino acids or the serum-free culture medium are preferentially administered in the form of peptides. Suitable peptides for the serum-free medium or the invention include without limitation, dipeptides selected from the group consisting of L-alanyl-L-glutamine, glycyl-L-glutamine and N-acetyl-L-glutamine. If the amino acids are provided in the form of peptides, then the culture medium may further comprise a reducing or antioxidant agent so as to avoid the formation of disulphide bridges betweem the Cys residues. Suitable antioxidant agents and concentrations thereof are defined below in the context of the antioxidant components. In a preferred embodiment, the antioxidant is reduced glutathione.

In a particular embodiment, the peptides are derived from the proteolytic digest of a microorganism or of a plant or animal tissue, in which case the peptide may be selected from peptone, PPLO and yeastolate, as will be specified below.

The serum-free culture medium of the invention comprises a vitamin mixture, said vitamin mixture comprising thioctic acid. In addition to the thioic acid, the vitamin mixture preferably includes ascorbic acid, biotin, Choline chloride, D-Calcium pantothenate, Folic Acid, Niacinamide, Nicotinic acid (Niacin), Para-Aminobenzoic Acid, vitamin B6, Pyridoxal hydrochloride, Pyridoxine hydrochloride, Riboflavin and Thiamine hydrochloride. These vitamins and/or essential nutrients can be obtained commercially, for example from Sigma (Sant Louis, Missouri). Said vitamins optionally may be provided by addition of a yeast extract, as it is known by a person skilled in the art.

The term “vitamin” or “essential nutrient” as used herein refers to an organic molecule (or related set of molecules) that is an essential micronutrient that an organism needs in small quantities for the proper functioning of its metabolism. Essential nutrients cannot be synthesized in the organism, either at all or not in sufficient quantities, and therefore must be obtained through the diet. The term vitamin does not include the three other groups of essential nutrients: minerals, essential fatty acids, and essential amino acids. The term “Ascorbic acid” or “vitamin C”, as used herein refers to a natural water soluble vitamin of formula C ₆ H ₈ O ₆ or HC ₆ H ₇ O ₆ . Ascorbic acid is a potent reducing and antioxidant agent that functions in fighting bacterial infections, in detoxifying reactions, and in the formation of collagen in fibrous tissue, teeth, bones, connective tissue, skin, and capillaries.

As used herein, the term “biotin”, also known as vitamin H, vitamin B7 or coenzyme R, is an enzyme cofactor of formula C ₁₀ H ₁₆ N ₂ O ₃ S. It occurs mainly bound to proteins or polypeptides and is abundant in liver, kidney, pancreas, yeast, and milk. Biotin has been recognized as an essential nutrient.

The term “Choline chloride”, also known as hepacholine, biocolina, 2-Hydroxy-N,N,N- trimethylethanaminium, refers to a quaternary ammonium salt with choline cation and chloride anion, of formula C ₅ H ₁₄ NO.CI or C ₅ H1 ₄ CINO. It has a role as an animal growth promotant. It is a chloride salt and a quaternary ammonium salt. It contains a choline.

As used herein, the term “D-Calcium pantothenate”, also known as calpanate refers to the calcium salt of the water soluble vitamin B5, ubiquitously found in plants and animal tissues with antioxidant property. Pentothenate is a component of coenzyme A (CoA) and a part of the vitamin B2 complex. Vitamin B5 is a growth factor and is essential for various metabolic functions, including the metabolism of carbohydrates, proteins, and fatty acids. This vitamin is also involved in the synthesis of cholesterol, lipids, neurotransmitters, steroid hormones, and hemoglobin.

The term “Folic Acid”, as used herein, also known as folate or vitamin M, of formula C ₁₉ H ₁₉ N ₇ O ₆ , is a collective term for pteroylglutamic acids and their oligoglutamic acid conjugates. As a natural water-soluble substance, folic acid is involved in carbon transfer reactions of amino acid metabolism, in addition to purine and pyrimidine synthesis, and is essential for hematopoiesis and red blood cell production.

The term “Nicotinamide”, also known as niacinamide or3-Pyridinecarboxamide is the active form of vitamin B3 and a component of the coenzyme nicotinamide adenine dinucleotide (NAD). It has the molecular formula C ₆ H ₆ N ₂ O.

The term “Nicotinic acid”, also known as niacin, or vitamin B3, with molecular formula C ₆ H ₅ NO ₂ or HOOC ₅ H ₄ N or C ₅ H ₄ NCOOH, is a water-soluble vitamin whose derivatives such as NADH, NAD, NAD+, and NADP play essential roles in energy metabolism in the living cell and DNA repair. The designation vitamin B3 also includes the amide form, nicotinamide or niacinamide.

In some embodiments, the medium contains nicotinic acid at a concentration of at least 0,2 μg/ml, preferably 0,5 μg/ml.

The term “Para-Aminobenzoic Acid”, also known as 4-aminobenzoic acid, or simply PABA, is an organic compound with molecular formula C7H7NO2. PABA is a white crystalline substance that is only slightly soluble in water. It consists of a benzene ring substituted with an amino group and a carboxylic acid. PABA is an essential nutrient for some bacteria and is sometimes called Vitamin Bx.

As used herein, the term “vitamin B6” refers to a refers to a group of chemically similar compounds which can be interconverted in biological systems to yield the active form which is, pyridoxal 5'-phosphat. The term includes pyridoxal hydrochloride, pyridoxine hydrochloride and pyridoxamine.

As used herein, the term “Pyridoxal hydrochloride”, also known as Pyridoxal HCI, or 3- Hydroxy-5-(hydroxymethyl)-2-methylisonicotinaldehyde hydrochloride, with molecular formula C8H10CINO3, refers to a hydrochloride obtained by combining pyridoxal with one molar equivalent of hydrochloric acid. It has a role as an Escherichia coli metabolite, a Saccharomyces cerevisiae metabolite, a cofactor, a human metabolite and a mouse metabolite. It is a hydrochloride and a pyridinium salt. It contains a pyridoxal(1+).

The term “Pyridoxine hydrochloride”, as used herein, refers to the compound of formula C8H12CINO3, which is the hydrochloride salt form of pyridoxine, a water-soluble vitamin B.

The term pyridoxamine, as used herein, refers to one form of vitamin B6 which consists of a pyridine ring structure, with hydroxyl, methyl, aminomethyl, and hydroxymethyl substituents. It differs from pyridoxine by the substituent at the 4-position, which is an aminomethyl group in the pyridoxamine and a hydroxymethyl group in pyridoxine

The term “Riboflavin”, as used herein, also known as lactoflavin or vitamin B2, refers to is an essential human nutrient that is a heat-stable and water-soluble flavin belonging to the vitamin B family. Riboflavin is a precursor of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). It has the molecular formula C17H20N4O6.

The term “Thiamine hydrochloride”, as used herein, also known as Thiamine HCI, Aneurine hydrochloride or Vitamin B1 hydrochloride, refers to the hydrochloride salt form of thiamine, a vitamin essential for aerobic metabolism, cell growth, transmission of nerve impulses and acetylcholine synthesis. Upon hydrolysis, thiamine hydrochloride is phosphorylated by thiamine diphosphokinase to form active thiamine pyrophosphate (TPP), also known as cocarboxylase. TPP is a coenzyme for many enzymatic activities involving fatty acid, amino acid and carbohydrate metabolism. It has the molecular formula HC12H17ON4SCI2 or C ₁₂ H ₁₈ CI ₂ N ₄ OS.

As used herein the term “thioctic acid” also known as “Lipoic acid (LA)” or “alpha lipoic acid (ALA)” refers to the compound of formula (R)-5-(1 ,2-Dithiolan-3-yl)pentanoic acid and is an organosulfur compound derived from octanoic acid. Thioctic acid contains two sulfur atoms (at C6 and C8) connected by a disulfide bond and is thus considered to be oxidized although either sulfur atom can exist in higher oxidation states.

In a preferred embodiment the serum-free culture medium of the invention comprises thioctic acid at a concentration in a range between 0.001 mg/L to 10 mg/L, more preferably between 0.01 to 1 mg/L, preferably at 0.1 mg/L. In another embodiment, the serum-free culture medium of the invention comprises thioctic acid at a concentration of at least 0,1 μg/mL, preferably 0,2 μg/mL.

The vitamin mixture preferably contains the following vitamins in the concentration ranges defined below:

Thioctic acid, in a range between 0.001 mg/L to 10 mg/L, more preferably between 0.01 to 1 mg/L, preferably at 0.1 mg/L. In one embodiment, the thioctic acid is present at a concentration of at least 0,1 μg/ml, preferably of at least 0,2 μg/ml,

- Ascorbic acid in a range between 0.1 to 1000 mg/L, more preferably between 1 to 100 mg/L. In a particular embodiment, the concentration of ascorbic acid is of about 25 mg/L. In another embodiment, the concentration of ascorbic acid is of about 50 mg/L. In another embodiment, the concentration of ascorbic acid is of about 100 mg/L. Biotin in a range between 0.0001 to 1 mg/ml, preferably between 0.001 to 0.1 mg/L. In a particular embodiment, the concentration of biotin is of about 0.005 mg/L. In another embodiment, the concentration of biotin is of about 0.01 mg/L. In another embodiment, the concentration of biotin is of about 0.02 mg/L.

Choline chloride in a range between 0.001 to 10 mg/L, more preferably between 0.01 to 1 mg/L. In a particular embodiment, the concentration of Choline chloride is of about 0.25 mg/L. In another embodiment, the concentration of Choline chloride is of about 0.5 mg/L. In another embodiment, the concentration of Choline chloride is of about 1 mg/L. In another embodiment, choline is present at a concentration of at least 0,2 μg/ml, preferably of at least 0,5 μg/ml.

D-Calcium pantothenate in a range between 0.0001 to 1 mg/L, preferably between 0.001 to 0.1 mg/L. In a particular embodiment, the concentration of D-Calcium pantothenate is of about 0.005 mg/L. In another embodiment, the concentration of D-Calcium pantothenate is of about 0.01 mg/L. In another embodiment, the concentration of D-Calcium pantothenate is of about 0.02 mg/L.

Folic acid in a range between 0.0001 to 1 mg/L, preferably between 0.001 to 0.1 mg/L, preferably at 0.01 mg/L. In a particular embodiment, the concentration of Folic acid is of about 0.005 mg/L. In another embodiment, the concentration of Folic acid is of about 0.01 mg/L. In another embodiment, the concentration of Folic acid is of about 0.02 mg/L.

Niacinamide in a range between 0.0001 to 1 mg/L, preferably between 0.001 to 0.1 mg/L. In a particular embodiment, the concentration of Niacinamide is of about 0.0125 mg/L. In another embodiment, the concentration of Niacinamide is of about 0.025 mg/L. In another embodiment, the concentration of Niacinamide is of about 0.05 mg/L.

Nicotinic acid (Niacin) in a range between 0.0001 to 1 mg/L, preferably between 0.001 to 0.1 mg/L. In a particular embodiment, the concentration of Nicotinic acid is of about 0.0125 mg/L. In another embodiment, the concentration of Nicotinic acid is of about 0.025 mg/L. In another embodiment, the concentration of Nicotinic acid is of about 0.05 mg/L. In one embodiment, the nicotinic acid is present at a concentration of at least 0,2 μg/ml, preferably of about 0,5 μg/ml or of at least 0,5 μg/ml.

Para-Aminobenzoic acid in a range between 0.0001 to 1 mg/L, preferably between 0.001 to 0.1 mg/L. In a particular embodiment, the concentration of Para- Aminobenzoic acid is of about 0.025 mg/L. In another embodiment, the concentration of Para-Aminobenzoic acid is of about 0.05 mg/L. In another embodiment, the concentration of Para-Aminobenzoic acid is of about 0.1 mg/L. Pyridoxal-hydrochloride in a range between 0.0001 to 1 mg/L, preferably between 0.001 to 0.1 mg/L. In a particular embodiment, the concentration of Pyridoxal- hydrochloride is of about 0.0125 mg/L. In another embodiment, the concentration of Pyridoxal-hydrochloride is of about 0.025 mg/L. In another embodiment, the concentration of P Pyridoxal-hydrochloride is of about 0.05 mg/L.

Pyridoxine hydrochloride in a range between 0.0001 to 1 mg/L, preferably between 0.001 to 0.1 mg/L. In a particular embodiment, the concentration of Pyridoxine hydrochloride is of about 0.0125 mg/L. In another embodiment, the concentration of Pyridoxine hydrochloride is of about 0.025 mg/L. In another embodiment, the concentration of Pyridoxine hydrochloride is of about 0.05 mg/L.

Pyridoxamine at a concentration of at least 0,2 μg/mL, preferably at least 0,5 μg/mL. Riboflavin in a range between 0.0001 to 1 mg/L, preferably between 0.001 to 0.1 mg/L. In a particular embodiment, the concentration of Riboflavin hydrochloride is of about 0.005 mg/L. In another embodiment, the concentration of Riboflavin is of about 0.01 mg/L. In another embodiment, the concentration of Riboflavin is of about 0.02 mg/L. In further embodiments, riboflavin is present at a concentration of at least 0,2 μg/ml, preferably 0,5 μg/ml.

Thiamine hydrochloride in a range between 0.0001 to 1 mg/L, preferably between 0.001 to 0.1 mg/L. In a particular embodiment, the concentration of Thiamine hydrochloride is of about 0.005 mg/L. In another embodiment, the concentration of Thiamine hydrochloride is of about 0.01 mg/L. In another embodiment, the concentration of Thiamine hydrochloride is of about 0.02 mg/L.

The serum-free culture medium of the present invention may additionally comprise i-inositol in a range between 0.0001 to 1 mg/L, preferably between 0.001 to 0.1 mg/L, preferably at 0.05 mg/L. In a particular embodiment, the concentration of i-inositol is of about 0.025 mg/L. In another embodiment, the concentration of i-inositol is of about 0.05 mg/L. In another embodiment, the concentration of i-inositol is of about 0.1 mg/L.

The serum-free culture medium of the invention comprises a lipid mixture comprising cholesterol, sphingomyelin or ceramide and phosphatidylcholine, wherein the ratio of sphingomyelin (SPM) to phosphatidylcholine (PC) is of between 10:1 and 1:10 (w/w). In a particular embodiments, the ratio of SPM, PC is of about 10:1, about 9:1 , about 8:1 , about 7:1 , about 6:1, about 5:1, about 4:1 , about 3:1, about 2:1, about 1.1 , about 1:2, about 1:3, about 1:4, about 1 :5, about 1:6, about 1 :7, about 1:8, about 1 :9 or about 1:10 (w/w). In a preferred embodiment, the ratio of sphingomyelin (SPM) to phosphatidylcholine (PC) is of about 1 :1.

It will be understood that the concentrations of these components, while being present at concentration ratios as defined above, should also be present at concentrations that do not result in a substantially toxicity to the cells.

The term “concentration that does not result in a substantial toxicity to the cells” as used herein, refers to the concentration of a component in a culture medium that does not substantially delay the growth of the cells in comparison with the growth in the same medium and under the same condition in the absence of the component. The concentration of a given component that does not result in a substantial toxicity to the cells prevent the cells to grow when present in a culture medium can determined by calculating the IC50 for said component, said IC50 being the concentration that causes a 50% growth inhibition. In some embodiments, the concentration that does not result in a substantial toxicity to the cells for sphingomyelin and for phosphatidylcholine corresponds to the IC50, to 90% of the IC50, to 80% of the IC50, to 70% of the IC50, to 60% of the IC50 or less.

In one embodiment, the concentration of sphingomyelin is of less than 80 μg/ml, less than 70 μg/ml, less than 60 μg/ml, less than 50 μg/ml or less than 45 μg/ml, less than 30 μg/ml.

In one embodiment, the concentration phosphatidylcholine is of less than 80 μg/ml, less than 70 μg/ml, less than 60 μg/ml, less than 50 μg/ml or less than 45 μg/ml, less than 30 μg/ml.

In another embodiment, the concentration of sphingomyelin and of phosphatidylcholine is of about 40 μg/ml.

The term cholesterol as used herein refers to the chemical compound with the molecular formula C ₂₇ H ₄₆ O. Cholesterol is an animal sterol found in the body tissues (and blood plasma) of vertebrates. It is a cholestanoid consisting of cholestane having a double bond at the 5,6-position as well as a 3beta-hydroxy group. It is a 3beta-sterol, a cholestanoid, a C27-steroid and a 3beta-hydroxy-Delta(5)-steroid

Cholesterol is present in the lipid mixture at a concentration between 0.01 and 100 mg/L (w/w), more preferably between 0.1 and 100 mg/L (w/w), preferably between 20 and 40 mg/L (w/w) preferably at about 30 mg/L (w/w), and more particularly at 33.3 mg/L (w/w).

The term “sphingomyelin” (SPM), or formula (E,2S,3R)-3-hydroxy-2-[[(Z)-tetracos-15- enoyl]amino]octadec-4-enyl] 2-(trimethylazaniumyl)ethyl phosphate), is a type of sphingolipid found in animal cell membranes, especially in the membranous myelin sheath that surrounds some nerve cell axons. It usually consists of phosphocholine and ceramide, or a phosphoethanolamine head group; therefore, sphingomyelins can also be classified as sphingophospholipids.

The term “ceramide” as used herein, refers to a family of compounds which result from esterification of sphingosine by a fatty acid. Depending on the type of fatty acid forming part of the ceramide, this can be classified as a Ceramide I, ceramide I la, ceramide lib, ceramide III, ceramide IV, ceramide V, ceramide V or ceramide VI.

The term “phosphatidylcholine”, as used herein refers to a class of phospholipids that have choline as a head group. This phospholipid is composed of a choline head group and glycerophosphoric acid, with a variety of fatty acids. Usually, one is a saturated fatty acid (in the given figure, this can be palmitic or hexadecanoic acid, H C-(CH )i -COOH; margaric acid identified by Gobley in egg yolk, or heptadecanoic acid H C-(CH )i -COOH, also belong to that class); and the other is an unsaturated fatty acid (here oleic acid, or 9Z- octadecenoic acid, as in Gobley's original egg yolk lecithin). However, there are also examples of disaturated species.

Optimal concentrations of PC and SPM in the serum-free culture medium of the invention are in a range between 0.1 and 100 mg/L (w/w), more preferably between 1 and 75 mg/L (w/w), preferably between 20 and 50 mg/L (w/w), preferably at about 40 mg/L (w/w) for PC and in a range between 0.1 and 100 μg/L (w/w), more preferably between 1 and 75 μg/L (w/w), preferably between 20 and 50 μg/L (w/w), preferably at about 40 μg/L (w/w) for SPM. In a particular embodiment, the serum-free culture medium of the invention comprises at least 20 mg/L (w/w), preferably 40 mg/L (w/w) of phosphatidylcholine.

In another embodiment, the serum-free culture medium of the invention comprises at least 30 μg/L (w/w), preferably at least 40 μg/L of sphingomyeline (w/w).

In another embodiment, the cholesterol constitutes 35-50% of the total lipids; sphingomyelin constitutes 9-15% of the total lipids and phosphatidylcholine constitutes 6-10% of the total lipids.

In another embodiment the lipid mixture additionally comprises palmitic acid and oleic acid. In particular the concentration of palmitic acid in the serum-free culture medium is in the range between 0.01 and 100 mg/L (w/w), more preferably between 0.1 and 100 mg/L (w/w), preferably between 10 and 40 mg/L (w/w) preferably at about 20 mg/L (w/w), and more particularly at 16.6 mg/L (w/w). In another embodiment, the concentration of oleic acid in the serum-free culture medium is in the range between 0.01 and 100 mg/L (w/w), more preferably between 0.1 and 100 mg/L (w/w), preferably between 10 and 40 mg/L (w/w) preferably at about 20 mg/L (w/w), and more particularly at 20 mg/L (w/w).

The lipids are preferably complexed to a vehicle or a carrier, such as bovine serum albumin (BSA), preferably delipidated BSA, cyclodextrine, polyvinyl alcohol (PVA) or lipid vesicles. In particular embodiment, the carrier to deliver cholesterol is BSA, at a concentration range of about 0,33% (w/w) or in a concentration range of 0,01 to 1%, 0,05 to 0,75%, 0,1 to 0,5 % (w/w). In another embodiment, the carrier is cyclodextrine, at a concentration in a range between 1 and 10 mg/L, more particularly between 3 and 6 mg/L, preferably of about 4-4 mg/L.

In a particular embodiment, BSA is complexed with a lipid mix containing palmitic acid, oleic acid and cholesterol at any of the preferred concentrations defined before.

The serum-free culture medium also comprises inorganic salts. In an embodiment, the inorganic salt is selected from the group comprising Calcium Chloride (CaCl ₂ -2H ₂ O), Magnesium Sulfate (MgSO ₄ -7H ₂ O), Potassium Chloride (KCI), Sodium Bicarbonate (NaHCO ₃ ), Sodium Chloride (NaCI), Sodium Phosphate monobasic (NaH ₂ PO ₄ - ₂ H ₂ O). The inorganic salts are preferably contained in the medium in the concentration ranges defined below.

Calcium Chloride (CaCl ₂ -2H ₂ O), in a range between 1 to 1000 mg/L, more particularly between 10 to 600 mg/L. In an embodiment, the concentration of Calcium Chloride is of about 130 mg/L. In another embodiment, the concentration of Calcium Chloride is about 165 mg/L. In another embodiment, the concentration of Calcium Chloride is about 530 mg/L.

Magnesium Sulfate (MgSO ₄ -7H ₂ O), in a range between 1 to 1000 mg/L, more particularly between 10 to 600 mg/L. In a particular embodiment, the concentration of Magnesium Sulfate is of about 100 mg/L. In another embodiment, the concentration of Magnesium Sulfate is of about 400 mg/L. In another embodiment, the concentration of Magnesium Sulfate is of about 200 mg/L.

Potassium Chloride (KCI), in a range between 1 and 15000 mg/L, more particularly between 100 to 1000 mg/L. In an embodiment the concentration of Potassium Chloride is of about 200 mg/L. In another embodiment the concentration of Potassium Chloride is of about 400 mg/L. In another embodiment, the concentration of Potassium Chloride is of about 800 mg/L.

Sodium Bicarbonate (NaHCCh), in a range between 100 and 10000 mg/L, preferably between 500 and 5000 mg/L. In a particular embodiment, the concentration of Sodium Bicarbonate is of about 1100 mg/L. In another embodiment, the concentration of Sodium Bicarbonate is of about 2200 mg/L, In another embodiment, the concentration of Sodium Bicarbonate is of about 4400 mg/L.

Sodium Chloride (NaCI), in a range between 100 and 20000 mg/L, preferably between 1000 and 15000 mg/L. In a particular embodiment, the concentration of Sodium Chloride is of about 3400 mg/L. In another embodiment, the concentration of Sodium Chloride is of about 6800 mg/L. In another embodiment, the concentration of Sodium Chloride is of about 13600 mg/L.

In a particular embodiment, the inorganic salts present in the serum-free culture medium of the invention include at least one salt wherein the cation is calcium, magnesium, potassium or sodium and wherein the anion is chloride, sulfate, bicarbonate or phosphate monobasic.

The serum-free culture medium of the invention comprises nucleosides or nucleotides monophosphate. Nucleosides are molecules consisting of a nitrogenous base and either ribose ordeoxyribose. Nucleotides monophosphate are molecule consisting of a nucleoside and one phosphate group.

Nucleosides include cytidine, deoxycytidine, uridine, adenosine, deoxyadenosine, guanosine, deoxyguanosine and thymidine.

Nucleotides monophosphate include adenosine monophosphate, guanosine monophosphate, cytidine monophosphate, thymidine monophosphate and uridine monophosphate. Said nucleotides preferably include adenosine ribonucleotide monophosphate, and adenosine deoxyribonucleotide monophosphate, collectively termed herein adenine; guanosine ribonucleotide monophosphate and guanosine deoxyribonucleotide monophosphate, collectively termed herein guanine; cytidine ribonucleotide monophosphate, termed cytidine; cytidine deoxyribonucleotide monophosphate, termed deoxycytidine; thymidine deoxyribonucleotide monophosphate, termed thymidine; uridine ribonucleotide monophosphate, termed uracil; adenosine ribonucleotide 3’5’-biphosphate, or a combination thereof. These nucleosides and nucleotides monophosphate may be obtained commercially, for example from Sigma (Saint Louis, Missouri).

Said nucleotides monophosphate or nucleosides as defined above are preferably present in the serum-free culture medium of the invention at a concentration between 1 mM and 1mM, more preferably at a concentration between 50 pM and 200 pM.

In an embodiment, the serum-free culture medium of the invention comprises nucleosides selected from the group comprising 2'-deoxyadenosine, 2'-deoxycytidine and 2'- deoxyguanosine but lacks thymidine and uridine.

In an embodiment, the serum-free culture medium of the invention comprises nucleosides selected from the group comprising adenosine, cytidine and guanosine but lacks thymidine and uridine.

In an embodiment, the serum-free culture medium of the invention comprises nucleotides monophosphate selected from the group comprising 2'-deoxyadenosine monophosphate, 2'-deoxycytidine monophosphate, 2'-deoxyguanosine monophosphate but lacks thymidine monophosphate and uridine-5’-monophosphate. In an embodiment, the serum-free culture medium of the invention comprises nucleotides monophosphate selected from the group comprising adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, but lacks thymidine monophosphate and uridine-5’-monophosphate.

These nucleosides and nucleotides monophosphate are preferably contained in the medium in the concentration ranges defined below. 2'-deoxyadenosine or 2'-deoxyadenosine monophosphate can be found in a range between 0.1 and 100 mg/L, preferably between 1 and 100 mg/L. In a particular embodiment, the concentration of 2'Deoxyadenosine or 2'-deoxyadenosine monophosphate is of about 5 mg/L. In another embodiment, the concentration of 2'Deoxyadenosine or 2'- deoxyadenosine monophosphate is of about 10 mg/L. In another embodiment, the concentration of 2'Deoxyadenosine or 2'-deoxyadenosine monophosphate is of about 20 mg/L.

Adenosine or adenosine monophosphate can be found in a range between 0.1 and 100 mg/L, preferably between 1 and 100 mg/L. In a particular embodiment, the concentration of adenosine or adenosine monophosphate is of about 5 mg/L. In another embodiment, the concentration of adenosine or adenosine monophosphate is of about 10 mg/L. In another embodiment, the concentration of adenosine or adenosine monophosphate is of about 20 mg/L. 2'-deoxycytidine or 2'-deoxycytidine monophosphate can be found in a range between 0.1 and 100 mg/L, preferably between 1 and 100 mg/L. In a particular embodiment, the concentration of 2'Deoxycytidine or of 2'-deoxycytidine monophosphate is of about 5 mg/L. In another embodiment, the concentration of 2'Deoxycytidine or of 2'-deoxycytidine monophosphate is of about 10 mg/L. In another embodiment, the concentration of 2'Deoxycytidine or of 2'-deoxycytidine monophosphate is of about 20 mg/L.

Deoxycytidine or cytidine monophosphate can be found in a range between 0.1 and 100 mg/L, preferably between 1 and 100 mg/L. In a particular embodiment, the concentration of cytidine or of cytidine monophosphate is of about 5 mg/L. In another embodiment, the concentration of cytidine or of cytidine monophosphate is of about 10 mg/L. In another embodiment, the concentration of cytidine or of cytidine monophosphate is of about 20 mg/L.

2'-deoxyguanosine or2'-deoxyguanosine monophosphate can be found in a range between 0.1 and 100 mg/L, preferably between 1 and 100 mg/L. In a particular embodiment, the concentration of 2'Deoxyguanosine or 2'-deoxyguanosine monophosphate is of about 5 mg/L. In another embodiment, the concentration of 2'Deoxyguanosine or 2'- deoxyguanosine monophosphate is of about 10 mg/L. In another embodiment, the concentration of 2'Deoxyguanosine or 2'-deoxyguanosine monophosphate is of about 20 mg/L.

Guanosine or guanosine monophosphate can be found in a range between 0.1 and 100 mg/L, preferably between 1 and 100 mg/L. In a particular embodiment, the concentration of guanosine or guanosine monophosphate is of about 5 mg/L. In another embodiment, the concentration of guanosine or guanosine monophosphate is of about 10 mg/L. In another embodiment, the concentration of guanosine or guanosine monophosphate is of about 20 mg/L.

When present, 5-Methyl-deoxycytidine or 5-Methyl-deoxycytidine monophosphate can be found in a range between 0.01 and 100 mg/L, preferably between 0.1 and 10 mg/L. In a particular embodiment, the concentration of 5-Methyl-deoxycytidine or 5-Methyl- deoxycytidine monophosphate is of about 0.05 mg/L. In another embodiment, the concentration of 5-Methyl-deoxycytidine or 5-Methyl-deoxycytidine monophosphate is of about 0.1 mg/L. In another embodiment, the concentration of 5-Methyl-deoxycytidine or 5- Methyl-deoxycytidine monophosphate is of about 0.2 mg/L.

Uridine or uridine monophosphate can be found in a range of between 0.01 to 100 mg/L, preferably between 0.1 and 10 mg/L. In a particular embodiment, the concentration of uridine or uridine monophosphate is of about 0.5 mg/L. In another embodiment, the concentration of uridine or uridine monophosphate is of about 1 mg/L. In another embodiment, the concentration of uridine or uridine monophosphate is of about 2 mg/L.

Thymidine or thymidine monophosphate can be found at a a concentration range between 0.01 to 100 mg/L, preferably between 0.1 to 50 mg/L. In a particular embodiment, the concentration of thymidine or thymidine monophosphate is about 5 mg/L. In another embodiment, the concentration of thymidine or thymidine monophosphate is about 11 mg/L. In another embodiment, the concentration of thymidine or thymidine monophosphate is about 20 mg/L.

The serum-free culture medium of the invention comprises a mixture of enzymatic cofactors. A used herein, the term “enzymatic cofactor” or “enzyme cofactor” refers to a compound of non-proteinaceous structure which is required by an enzyme to perform its catalytic activity. Cofactors are classified into two groups: (a) metals or metalloorganic compounds; and (b) organic molecules, or coenzymes. Coenzymes can be further divided into two subgroups. In the first of these, the coenzyme is attached to the active site and can be separated, usually reversibly, from it. Thiamine pyrophosphate and pyridoxal phosphate are good examples of such coenzymes. The coenzymes of the second group are not parts of the active site, but are specific and necessary reagents of the catalyzed reactions. Suitable enzymatic cofactors for the serum-free culture medium of the invention include, without limitation NAD, NADP, NADH, NADPH, FAD and FMN.

In another embodiment, mixture of enzymatic cofactors of the serum-free medium of the invention comprises co-carboxylase, Coenzyme A, diphosphopyridine nucleotide (NAD) and flavin adenine dinucleotide (FAD) and triphosphopyridine Nucleotide (NADP). In one embodiment, the mixture of enzymatic cofactors contains NAD but lacks NADP

The term co-carboxylase, also known as thiamine pyrophosphate co-carboxylase as used herein refers to the coenzyme form of Vitamin B1 present in many animal tissues. It is a required intermediate in the pyruvate dehydrogenase complex and the ketoglutarate dehydrogenase complex. Is has the molecular formula C ₁₂ H ₁₉ CIN4O7P2S. The concentration of co-carboxylase within the serum-free culture medium of the invention is in a range between 0.1 to 100 mg/L, more preferably between 1 to 10 mg/L. In a particular embodiment, the concentration of co-carboxylase in the culture medium of the invention is of about 0.5 mg/L. In another embodiment, the concentration of co-carboxylase in the culture medium of the invention is of about 1 mg/L. In another embodiment, the concentration of co-carboxylase in the culture medium of the invention is of about 2 mg/L.

The term Coenzyme A, as used herein, also known as S-Acetyl coenzyme A refers to acyl- CoA having acetyl as its S-acetyl component. It has a role as an effector, a coenzyme, an acyl donor and a fundamental metabolite. It derives from an acetic acid and a coenzyme A. It is a conjugate acid of an acetyl-CoA. The concentration of coenzyme A within the serum- free culture medium of the invention is in a range between 0.1 to 100 mg/L, more preferably between 1 to 10 mg/L. In a particular embodiment, the concentration of coenzyme A in the culture medium of the invention is of about 1.25 mg/L. In another embodiment, the concentration of coenzyme A in the culture medium of the invention is of about 2.5 mg/L. In another embodiment, the concentration of coenzyme A in the culture medium of the invention is of about 5 mg/L.

The term “diphosphopyridine nucleotide” (NAD), as used herein, also known as alpha-NAD, makes reference to a cofactor that is central to metabolism. Found in all living cells, NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine nucleobase and the other nicotinamide. NAD exists in two forms: an oxidized and reduced form, abbreviated as NAD+ and NADH respectively. It has the molecular formula C21H27N7O14P2. The concentration of NAD within the serum-free culture medium of the invention is in a range between 0.1 to 100 mg/L, more preferably between 1 to 20 mg/L. In a particular embodiment, the concentration of NAD in the culture medium of the invention is of about 3.5 mg/L. In another embodiment, the concentration of NAD in the culture medium of the invention is of about 7 mg/L. In another embodiment, the concentration of NAD in the culture medium of the invention is of about 14 mg/L.

The term flavin adenine dinucleotide (FAD), also known as flavin-adenine dinucleotide or flavitan, refers to a flavin adenine dinucleotide in which the substituent at position 10 of the flavin nucleus is a 5'-adenosyldiphosphoribityl group. It has a role as a human metabolite, an Escherichia coli metabolite, a mouse metabolite, a prosthetic group and a cofactor. It has the molecular formula C27H33N9O15P2. The concentration of FAD within the serum-free culture medium of the invention is in a range between 0.1 to 100 mg/L, more preferably between 1 to 10 mg/L. In a particular embodiment, the concentration of FAD in the culture medium of the invention is of about 0.5 mg/L. In another embodiment, the concentration of FAD in the culture medium of the invention is of about 1 mg/L. In another embodiment, the concentration of FAD in the culture medium of the invention is of about 2 mg/L.

As used herein, the term triphosphopyridine Nucleotide (NADP), also known as codehydrogenase II, or nicotinamide adenine dinucleotide phosphate refers to a coenzyme composed of ribosylnicotinamide 5'-phosphate (NMN) coupled by pyrophosphate linkage to the 5'-phosphate adenosine 2',5'-bisphosphate. It serves as an electron carrier in a number of reactions, being alternately oxidized (NADP+) and reduced (NADPH) .The concentration of NADP in the culture medium of the invention is in a range between 0.01 to 100 mg/L, more particularly between 0.1 to 10 mg/L, preferably of about 1 mg/L. In a particular embodiment the concentration of NADP within the serum-free culture medium is of about 0.5 mg/L. In another embodiment the concentration of NADP in the culture medium of the invention is of about 1 mg/L. In another embodiment, the concentration of NADP in the culture medium of the invention if of about 2 mg/L.

The serum-free culture medium of the invention comprises a chemical buffering system or a buffering agent. The terms “buffer,” “buffering system,” and/or “buffer solution” refer to a solution, which reduces the change of pH upon addition of small amounts of acid or base, or upon dilution. The term “buffering agent” refers to a weak acid or weak base in a buffer solution. Suitable buffering agents include, but are not limited to, N-[2-hydroxyethyl]- piperazine-N'-[2-ethanesulfonic acid] (HEPES), MOPS, MES, phosphate, bicarbonate and other buffering agents suitable for use in other cell culture applications. A suitable buffering agent is one that provides buffering capacity without substantial cytotoxicity to the cells cultured. The selection of suitable buffering agents is within the ambit of ordinary skill in the art of cell culture. The buffering agent will keep the pH of the medium between 6.8 and 7.8. In an embodiment, the buffering system is provided by sodium bicarbonate. Sodium bicarbonate reacts with the hydrogen ions generated by C02 and sequesters them to maintain pH at physiological levels. In another embodiment, the pH indicator. In an embodiment, the concentration of sodium bicarbonate in the serum-free medium of the invention is between 1 and 10000 mg/L, more preferably between 1000 to 5000 mg/L. In an embodiment the concentration of sodium bicarbonate is about 1100 mg/L. In another embodiment, the concentration of sodium bicarbonate is about 2200 mg/L. In another embodiment, the concentration of sodium bicarbonate is about 4400 mg/L.

In another embodiment, the buffering agent is HEPES (N-[2-hydroxyethyl]-piperazine-N'-[2- ethanesulfonic acid]). In a particular embodiment the concentration of HEPES in the serum- free medium of the invention is in a range between 1mM to 500 mM, more particularly between 10 mM to 100 mM, preferably about 50 mM.

In another embodiment, the serum-free culture medium of the invention comprises at least one buffering agent. In another embodiment, the serum-free culture medium of the invention comprises more than one buffering agent. In an embodiment, the serum-free culture medium of the invention comprises sodium bicarbonate and HEPES at any of the preferred concentrations of the embodiments already described. In a particular embodiment, the serum-free culture medium comprises sodium bicarbonate at a concentration in a range between 1000 mg/L to 5000 mg/L and HEPES at a concentration in a range between 10 mM to 100 mM.

The serum-free culture medium of the invention comprises a proteolytic digest of a microorganism or a plant or animal tissue. The term “proteolytic digest” refers to the product derived from the breakdown of proteins into smaller polypeptides or amino acids. Proteolysis is typically catalysed by cellular enzymes called proteases, but may also occur by intra-molecular digestion or different processes known by a skilled in the art. As mentioned above, the proteolytic digest will provide amino acids to the medium in the form of peptides. In a particular embodiment, the peptides are derived from the proteolytic digest of a microorganism or of a plant or animal tissue, in which case the peptide may be selected from peptone, PPLO and yeastolate.

It will be understood that, although not specifically excluded from the invention, the media defined herein usually contain a single type of proteolytic digest of a microorganism or a plant or animal tissue. Thus, in some embodiments, the media contains PPLO but lacks yeastolate. In other embodiments, the media contains yeastolate but lacks PPLO.

The term PPLO, refers to a beef heart infusion, pancreatic digest of casein and beef extract broth. In a particular embodiment, the culture medium of the invention comprises PPLO in a concentration in a range between 0.01 to 10 mg/ml, more particularly between 0.1 to 10 mg/ml. In preferred embodiment, the concentration of PPLO is of about 1 mg/ml. In a preferred embodiment, the concentration of PPLO in the serum-free culture medium of the invention is of 15 mg/ml.

The term “yeastolate” refers to are animal-free and water-soluble portions of autolyzed yeast or Saccharomyces cerevisiae. It comprises a mixture of peptides, amino acids, carbohydrates, simple and complex as well as vitamins. In a particular embodiment, the culture medium of the invention comprises yeastolate in a concentration in a range between 0.1 to 100 mg/ml, more particularly between 1 to 50 mg/ml. In preferred embodiment, the concentration of yeastolate is of about 10 mg/ml. In a preferred embodiment, the concentration of yeastolate in the serum-free culture medium of the invention is of 10 mg/ml.

In another embodiment, the culture medium according to the invention contains a vehicle to solubilize and deliver the lipids.

The term “vehicle for solubilizing and delivering lipids” as used herein, refers to any compound which is capable of forming a complex with lipids and of shielding their hydrophobic parts such as the complexes can be found dissolved or dispersed in an aqueous solution.

Suitable vehicles for solubilizing and delivering lipids include, without limitation, proteins (preferably a lipid-binding protein), cyclodextrins, polyvinyl alcohol (PVA) and lipid vesicles (i.e. liposomes).

In another embodiment, the vehicle to solubilize and deliver the lipids forming part of the culture medium according to the invention is a protein, particularly a lipid-binding protein. In another embodiment, the protein Is albumin. The term albumin, as used herein, refers to any polypeptide of the albumin family of proteins such as human serum albumin, including variants and derivatives thereof, such as genetically engineered or chemically modified albumin variants and fragments of albumin proteins. The albumin may also be derived from any vertebrate, especially any mammal, and includes fetal bovine serum or bovine serum albumin.

In one embodiment, the albumin is provided to the culture medium in delipidated form. In another embodiment, the albumin is delipidated bovine serum albumin, i.e. an albumin which is free of fatty acids.

In one embodiment, the albumin is used at a concentration of about 0,33% (w/w). In another embodiment, the albumin appears at concentrations of 0,01 to 1%, 0,05 to 0,75%, 0,1 to 0,5 % (w/w).

In yet another embodiment, the culture medium comprises an albumin substitute. .The term “albumin substitute” refers to any functional equivalent of albumin, which may be of proteinaceous or non-proteinaceous nature. Examples include but are not limited to bovine pituitary extract, plant hydrolysate (e.g., rice/soy hydrolysate), fetuin, egg albumin, human serum albumin (HSA), or another animal-derived albumins, chick extract, bovine embryo extract, AlbuMAX(R) I, and AlbuMAX(R) II. Further examples of albumin substitutes are polymers such as polyvinylpyrrolidone (PVP) and polyethylenglycol (PEG).

In another embodiment, the vehicle to solubilize and deliver the lipids forming part of the culture medium according to the invention comprises a cyclodextrin.

As used herein, cyclodextrins refer to a macrocyclic ring of glucose subunits joined by a- 1,4 glycosidic bonds and which consist of 5 or more a-D-glucopyranoside units linked 1->4, as in amylose (a fragment of starch). Typical cyclodextrins contain a number of glucose monomers ranging from six to eight units in a ring, creating a cone shape and include a- cyclodextrin (6 glucose subunits), b-cyclodextrin: (7 glucose subunits) and y-cyclodextrin (8 glucose subunits). Suitable cyclodextrins for use in the present invention include any of the cyclodextrins commonly used in drug delivery such as those described by Chordiya and Senthilkumaran (2012) Research and Reviews: Journal of Pharmacy and Pharmaceutical Sciences l(l):19-29 (especially Table 2 thereof as it describes different cyclodextrins in use as carriers). These include a-cyclodextrin, b-cyclodextrin, and g-cyclodextrin, or derivatives of any of these cyclodextrins. The cyclodextrin derivatives herein refers to, for example, compounds obtained by replacing hydrogen or hydroxyl group(s) of cyclodextrin with other substituent(s). Examples of such substituents include, but are not limited to, alkyl having 1 to 4 carbon atoms, alkenyl having 1 to 4 carbon atoms, and groups having a C-0 bond, such as acetyl.

In one embodiment, the cyclodextrin is 2-Hydroxypropyl^-cyclodextrin. In another embodiment, the cyclodextrin, more preferably the 2-Hydroxypropyl^-cyclodextrin, is present at a concentration of about 40 mg/ml or of about 30 mg/ml. In further embodiments, the cyclodextrin is present at a concentration of between 1 and 100 mg/ml, between 5 and 90 mg/ml, between 7 and 80 mg/ml, between 10 and 70 mg/ml, between 20 and 60 mg/ml or between 40 and 50 mg/ml. in certain embodiment, the cyclodextrin or the 2- Hydroxypropyl^-cyclodextrin is present at a concentration of at least 3 mg/ml_, at least 4 mg/ml_ or more preferably at least 5 mg/ml_

The cyclodextrin can be added to the culture medium in free form, i.e. , not complexed to any other component or, alternatively, can be added as complex with a least one lipid, such as cholesterol, fatty acids or a combination of both. In certain embodiments, a cyclodextrin and/or lipid (such as cholesterol and/or a fatty acid) is present in an amount or molar ratio disclosed with respect to a cell culture medium. In embodiments, the cyclodextrin is any cyclodextrin or combination of cyclodextrins disclosed herein. In embodiments, the lipid is any lipid (such as a fatty acid and/or cholesterol) or combination of lipids disclosed herein.

The lipophilic substances which can be complexed with cyclodextrin include unsaturated fatty acids such as linoleic acid, cholesterol and oleic acid. The linoleic acid, cholesterol and oleic acid are present in effective amounts and can be present in equal proportions such that the total amount is 0.001 to 100 mug/ml, preferably 0.1 to 10 mug/ml. The preparation of such complexes is known in the art and is described, for example, in U.S. Pat. No. 4,533,637 of Yamane et al, the entire contents of which is hereby incorporated by reference.

In certain embodiments, if a cyclodextrin is present or if 2-Hydroxypropyl^-cyclodextrin is present and if it is provided as complex with one or more fatty acids, preferably, oleic acid or palmitic acid, then no additional fatty acids are added to the medium.

In another embodiment, the vehicle to solubilize and deliver the lipids forming part of the culture medium according to the invention comprises polyvinyl alcohol. PVA is well known in the art and can be commercially purchased. PVA analogues or derivatives can also be used for the preparation of the culture medium according to the present invention but PVA as disclosed herein is preferred. The skilled person knows how to prepare and dissolve PVA to obtain a suitable PVA liquid form.

In some embodiment, if the culture medium contains PVA, then it lacks 2-Hydroxypropyl^- cyclodextrin or lacks any cyclodextrin.

In some embodiments, PVA is present in the culture medium at a concentration of at least about 0,5%, at least about 1% or at least about 2%.

In another embodiment, the vehicle to solubilize and deliver the lipids forming part of the culture medium according to the invention comprises liposomes.

The term liposomes, as used herein, refers to a closed structure comprising an outer lipid bi- or multi-layer membrane surrounding an internal aqueous space. Liposomes may be multi-laminar or unilaminar. The liposome is contemplated to range in size from 5 to 10 micro M in diameter to nanoparticle size. In certain embodiments, the liposome nanoparticle is from about 50 to 500 nm, from about 100 nm to 300 nm or from about 100 to 200 nm in diameter.

In one embodiment, the liposomes are provided at a concentration of about present 0.1% (w/w). In some embodiments, the liposomes are provided at concentrations of between 0,01 and 1%, between 0,02 and 0,9%, between 0,03 and 0,7%, between 0,04% and 0,6%, between 0,05% and 0,5%, between 0,06 and 0,4%, between 0,07 and 0,8% or between 0,09 and 0,11%.

In various embodiments, the liposomes comprise lipids, fatty acids, sterols and/or free fatty acids. In various embodiments, the liposomes comprise cholesterol and phosphatidylcholine at concentrations that provide a more physiologically relevant milieu for cell growth. In one embodiment, the liposomes contain L-alpha-phosphatidylcholine and cholesterol. In additional embodiments, the liposomes contain about 18,8 mg/ml of L-alpha- phosphatidylcholine and about 4,2 mg/ml of cholesterol. In additional embodiments, the ratio of phosphatidylcholine and cholesterol in the liposomes is of about 4:1 (w/w).

It will be understood that, although not specifically excluded from the invention, the media defined herein usually contain a single type of vehicle for solubilizing and delivering lipids. Thus, in some embodiments, the medium according to the present invention contains a lipid-binding protein but lacks cyclodextrins, polyvinyl alcohol (PVA) and liposomes. In some embodiments, the medium according to the present invention contains a cyclodextrin but lacks lipid-binding protein, polyvinyl alcohol (PVA) and liposomes. In some embodiments, the medium according to the present invention contains a polyvinyl alcohol (PVA) but lacks a lipid-binding protein, cyclodextrin and liposomes. In some embodiments, the medium according to the present invention contains a liposomes, but lacks lipid-binding protein, polyvinyl alcohol (PVA) or a cyclodextrin.

In another embodiment, the serum-free culture medium of the invention also comprises a polyamine. The term "polyamine" refers to any of a group of organic compounds composed of carbon, nitrogen, and hydrogen, and containing two or more amino groups. For example, the term encompasses molecules selected from the group consisting of cadaverine, putrescine, spermidine, spermine, agmatine, and ornithine. In a preferred embodiment, the polyamine is spermine. The term spermine, also known as musculamine, or neuridine refers to a spermidine-derived biogenic polyamine found as a polycation at all pH values. The concentration of spermine in the culture medium of the invention is in a range between 0.01 to 100 mg/L, more particularly between 0.1 to 50 mg/L, preferably of about 10 mg/L. In a preferred embodiment the concentration of spermine is of about 1 mg/ml.

In a particular embodiment, the medium of the invention further comprises one or more organic acids or a salt thereof.

As used herein “organic salts” are defined as compounds build on a carbon skeleton, containing a functional group with acidic properties, usually weaker than mineral acid. This term includes in particular carboxylic acids and sulfonic acids, containing the group -SO2OH. Carboxylic acids are characterized with the presence of a carboxyl group (-COOH) composed of two functional groups: a hydroxyl group (-OH) that is bonded to a carbonyl group (C=0). Organic acids are written in a condensed form R-COOH. Carboxylic acids include aliphatic, aromatic and cycloaliphatic carboxylic acids, depending on the structure of the carbon skeleton (R).

Suitable organic acids to be used in the culture medium according to the invention include, without limitation, one or more of lactic acid, acetic acid, glucuronic acid, succinic acid, propionic acid, butyric acid, methyl butyric acid, hydroxybutyric acid, aminobutyric acid (in particular GABA, gamma-aminobutyric acid), valeric acid, formic acid, aspartic acid, fumaric acid, oxalic acid, orotic acid, ketoglutaric acids, citric acid, glutamic acid, glyoxylic acid, glycolic acid, pyruvic acid, malic acid, sorbic acid and tartaric acid.

In another embodiment, the organic acids present in the culture medium of the invention are acetate or glucuronate. In additional embodiments, acetate is found in the culture medium at concentrations of at least about 41,5 mg/L, at least about 83 mg/L or at least about 166 mg/L. In other embodiments, acetate is found in the culture medium at concentrations of between 1 and 500 mg/L, between 10 and 400 mg/L, between 20 and 300 mg/L or between 40 and 200 mg/L. In additional embodiments, glucuronate is found in the culture medium at concentrations of about 8,40 mg/L, about 4,2 mg/L or about 2.1 mg/L. In additional embodiments, acetate is found in the culture medium at concentrations of between about 0,1 mg/L and 50 mg/L, of between 0,2 and 40 mg/L, of between 0,5 and 30 mg/L, of between 1 and 20 mg/L or of between 2 and 10 mg/L.

In another embodiment, the serum-free media of the invention may comprise RNA. RNA can be provided to the serum-free culture medium from any source. In a particular embodiment, the RNA is from yeast. In a particular embodiment, the serum-free culture medium of the invention comprises RNA at a concentration in a range between 0.01 to 100 mg/L, preferably between 0.1 to 10 mg/L, more particularly of about 1 mg/L. In a preferred embodiment, the concentration of RNA in the culture medium of the invention if of at least 0.1 mg/ml, or at least 0.5 mg/ml, of at least 1 mg/ml. In a preferred embodiment, the concentration of RNA in the serum-free culture medium of the invention is of at least 2 mg/ml.

In a particular embodiment the serum-free culture medium of the invention further comprises an antioxidant.

As used herein, an "antioxidant" can be defined as "any substance that delays, prevents or removes oxidative damage to a target molecule". A target molecule can be any oxidizable substrate, e.g., any organic molecule found in vivo. In some embodiments, an antioxidant is a substance that significantly delays or prevents oxidation of an oxidizable substrate. In some embodiments, an antioxidant is capable of significantly delaying or preventing oxidation of an oxidizable substrate even when present at low concentrations compared with those of an oxidizable substrate

As used herein, the term "antioxidant" includes compounds with a known structure as well as compositions that may be at least partially uncharacterized. An antioxidant can be a naturally occurring compound or may be a compound invented by man. Exemplary antioxidants include vitamin C, vitamin E, lipoic acid, L-sulforaphane, reduced L-glutathione, butylated hydroxyanisole, alpha tocopherol, deferoxamine, resveratrol, N-acetylcysteine, Trolox, curcumin, morin hydrate, vitamin A, a vitamin B (e.g., vitamin B I, B2, B6, and/or B12), coenzyme Q, green tea (epigallocatechin gallate-EGCG), citric acid, oxalic acid, phytic acid, chicoric acid, chlorogenic acid, cinnamic acid, ellagic acid, gallic acid, gallotannins, rosmarinic acid, silymarin, eugenol, manganese, zinc, adenosine, transferrin, lactoferrin, cysteine, histidine-containing dipeptides, pyridoxamine, carotenoids, flavonoids (e.g., kaempferol, myricetin, silymarin, quercetin), flavones and flavonols (e.g., planar flavones and flavonols with a 7-hydroxyl group), other phenolic compounds (e.g., plant- derived phenolics), melatonin, coelenterazine, nordihydroguaiaretic acid (NDGA), curcumin (diferuloylmethane), 21 -amino steroids (also called lazaroids), various antibiotics (e.g., tetracyclines), azoles (e.g., ketoconazole), thiols (e.g., mercaptopropionylglycine), metal ion chelators (e.g., iron chelators such as ICRF-187, deferasirox, deferiprone, hydroxypyridones), fullerenes, xanthine oxidase inhibitors (e.g., purine analogues such as allopurinol, oxypurinol, and tisopurine and other compounds such as febuxostat and inositols), selenium (e.g., sodium selenite, selenous acid), uric acid, bilirubin, trehalose, lipid-soluble chain-breaking antioxidants such as BHT and ethoxyquin, superoxide dismutase (SOD), catalase, superoxide dismutase (SOD).

In a preferred embodiment the antioxidant is reduced glutathione. The concentration of glutathione in the medium is of at least 20 mg/L, of at least 10 mg/L or of at least 5 mg/L. In other embodiments, reduced glutathione is present in the culture medium at a concentration of between 1 and 50 mg/L, between 2 and 40 mg/L, between 3 and 30 mg/L, between 4 and 20 g/L or between 5 and 10 mg/L.

In another embodiment, the serum-free culture medium of the invention further comprises one or more detergents. Detergents are amphipathic molecules with a polar portion and a hydrophobic portion. Detergents respond to an aqueous environment following the same principles as do membrane lipids. Suitable detergents for the culture medium of the invention include without limitation

In a particular embodiment, the detergent is selected from Tween 40 and/or Tween 80. In a particular embodiment, the detergent is Tween 80, in which case, the concentration is of at least 0.00075% (w/w). In another embodiment the detergent present in the culture medium of the invention is Tween 40, in which case, the concentration is of at least 0.0015%. In another embodiment the detergent is Tween 80, Tween 40 ora mixture thereof.

In preferred embodiments, the serum-free culture media according to the present invention comprise the vB2, vB3, vB4, vB6, vB7, vB8, vB9, vB10 or vB11 as defined in Figure 8. In another embodiment, the serum-free culture medium of the invention comprises components defined in the “Components” column in the Table 2 at concentrations indicated in the “Concentration 1” column, at the concentrations indicated in the “Concentration 2” column or at the concentrations indicated in the “Concentration 3” column.

Components Concentration 1 Concentration 2 Concentration (mg/L) (mg/L) 3 (mg/L) Components Concentration 1 Concentration 2 Concentration (mg/L) (mg/L) 3 (mg/L)

Table 2: Composition of the CMRL medium at 1x, 2x or 0,5 x.

In another embodiment the culture medium according to present invention further comprises fatty acids.

The term “fatty acid” as used herein, refers to an aliphatic carboxylic acid having the formula RCOOH wherein R is an aliphatic group having at least 4 carbons, typically between about 4 and about 28 carbon atoms. The aliphatic R group can be saturated or unsaturated, branched or unbranched. Unsaturated fatty acids" may be monounsaturated or polyunsaturated.

In one embodiment, the fatty acid is palmitic acid, oleic acid or a mixture thereof.

In one embodiment, the fatty acid is palmitic acid and is used at a concentration of about 16,6 μg/ml. In another embodiment, the fatty acid is palmitic acid and is used at concentrations of about 1 to 100 μg/ml, 5 to 75 μg/ml, 10 to 50 μg/ml or 15 to 20 μg/ml.

In one embodiment, the fatty acid is oleic acid and is used at a concentration of about 20 μg/ml. In another embodiment, the fatty acid is oleic acid and is used at concentrations of about 1 to 100 μg/ml, 5 to 75 μg/ml, 10 to 50 μg/ml or 15 to 25 μg/ml. In another embodiment, the culture medium contains yestolate but lacks a protein digest from animal sources such as PPLO broth, Preferred media having these features include the vB11, vB11.1 and vB12 as defined in Figure 8.

In other embodiments, the medium according to the invention contains yeastolate and lacks any component of animal origin, including PPLO broth and albumin. The term “albumin or an albumin substitute as defined above. The term “lacks albumin” is to be understood as that the medium contains less than 0,01%, less than 0,001%, less than 0,0001%, less than 0,00001% or less of albumin (w/w).

Preferred media having these features are the vB11, vB11.1 and vB12 media as shown in Figure 8 In another embodiment, if the medium lacks albumin, then it contains a vehicle for the solubilization and the delivery of lipids which is not from animal origin. Suitable vehicles have been defined above and include, without limitation, cyclodextrins, PVA or liposomes. Suitable media having these features include the vB13, vB14 and vB14b as defined in Figure 8.

In another embodiment, the medium according to the present invention further comprises a pH indicator.

Suitable pH indicators for use in the culture media according to the invention are those pH indicators with a transition range that extends below pH 7.0. Non-limiting examples of suitable pH indicators include halochromic compounds with a transition pH range that extends below the pH of the inoculated culture medium. A suitable pH indicator will have a transition pH range that extends far enough below the pH of the inoculated medium to detect (visually and/or by using an imaging system) a change in the pH indicator in or adjacent to a growing colony of acid-producing bacteria. Preferably, the pH indicator will have a transition pH range with a low endpoint that is not less than 0.25 pH units below the pH of the inoculated culture medium. More preferably, the pH indicator will have a transition pH range with a low endpoint that is not less than 0.5 pH units below the pH of the inoculated culture medium. Even more preferably, the pH indicator will have a transition pH range that extends not less than 1.0 pH unit below the pH of the inoculated culture medium. Most preferably, the pH indicator will have a transition pH range with a low endpoint that is about 3.5. Nonlimiting examples of suitable pH indicators include bromcresol purple, bromphenol blue, chlorophenol red, and bromcresol green.

A pH indicator such as phenol red may be added that turns yellow in acidic conditions (<6.8), and pink in basic conditions (>8.2). Incubation pH is controlled by the high CO2 levels in the incubator of 5% or more. In a particular embodiment, the serum-free medium of the invention comprises phenol red pH 7 at a concentration in a range between 0.00001% to 0.1%, more preferably between 0.0001% to 0.01%, preferably at about 0.0005%, preferably at about 0.0035%.

In another embodiment, the medium according to the present invention further comprises an antibiotic.

The term "antibiotic" is intended to mean any compound capable of preventing or slowing down the growth of a bacterium. Suitable antibiotics for use in the present invention includes antibiotics of the aminoglycoside family, of the ansamycin family, of the carbacephem family, of the cephalsporine family, of the macrolide family, of the monobactam family, of the penicillin family, of the Sulfonamide family or of the tetracycline family.

An aminoglycoside, including, but not limited to amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin, and paromomycin; an ansamycin, including, but not limited to, geldanamycin and herbimycin. A carbacephem, including, but not limited to, loracarbef. A carbapenem, including, but not limited to, ertapenem, doripenem, imipenem/cilastatin, and meropenem. First generation cephalosporins, including, but not limited to, cefadroxil, cefazolin, cefalotin, and cephalexin. Second generation cephalosporins, including, but not limited to, cefaclor, cefamandole, cefoxitin, cefprozil, and cefuroxime. Third generation cephalosporins, including, but not limited to, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, and cefdinir. Fourth generation cephalosporins, including, but not limited to cefepime. Glycopeptides, including, but not limited to, teicoplanin and vancomycin. Macrolides, including, but not limited to azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, telithromycin, and spectinomycin. Monobactams, including but not limited to aztreonam. Penicillins, including, but not limited to, amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, penicillin, piperacillin, and ticarcillin. Polypeptides, including, but not limited to, bacitracin, colistin, and polymyxin B. Quinolones, including, but not limited to, ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, ofloxacin, and trovafloxacin. Sulfonamides, including, but not limited to, mafenide, prontosil, sulfacetamide, sulfamethizole, sulfanilamide, sulfasalazine, sulfisoxazole, trimethoprim, and trimethoprim-sulfamethoxazole (co-trimoxazole (TMP- SMX). Tetracyclines, including, but not limited to demeclocycline, doxycycline, minocycline, oxytetracycline, and tetracycline. And other antibiotics, including, but not limited to, arsphenamine, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampin, and tinidazole.

In preferred embodiments, the medium of the present invention is a medium having the composition of vB1 to vB15, vB11.1, vB12.1, vB13.1 or vB13.2 as shown in figure 11. In a still more referred embodiment, the medium according to the invention is the medium defined as vB11.1 of vB13.1.

Methods for the culture of Mycoplasma In another aspect, the invention relates to a method for the culture of a Mycoplasma strain comprising placing an inoculum of said strain into the media according to the invention and maintaining the inoculated media under conditions adequate for the proliferation of the Mycoplasma strain. In another aspect, the invention relates to a method for obtaining a biomass of a Mycoplasma strain which comprises placing an inoculum of said Mycoplasma strain into the media according to the invention and maintaining the inoculated media under conditions adequate for the proliferation of the Mycoplasma strain and the formation of the biomass. In one embodiment, the Mycoplasma is Mycoplasma pneumoniae. In another embodiment, he Mycoplasma is a Mycoplasma penumoniae wild-type strain or a Mycoplasma pneumoniae variant strain. In another embodiment, the methods as defined above are carried out an the inoculum that contains about 10 ⁷ cells/ml. In some embodiments, the serum-free media can be used in methods for culturing cells.. In some embodiments, cells are cultured for at least at or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 days with the provided serum- free media. In some embodiments, cells are cultured for about 2-3 days, 3-4 days, 4-5 days, 5-6 days, 6-7 days, 7-8 days, 8-9 days, 9-10 days, 10-11 days, 11-12 days, 12-13 days, 13-14 days, 14-15 days, 15-16 days, 16-17 days, 17-18 days, 18-19 days, or 19-20 days, each inclusive. In some embodiments, the cells are cultured at about 37 degrees centigrade In some embodiments, the cells are cultured in 5 percent CO2/95 percent air atmosphere. In some embodiments, the culturing can be carried for more than or more than about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days without changing the media when culturing cells. In some embodiments, methods of perfusion, such as semi-continuous perfusion, can be employed in connection with culturing the cells.

In some embodiments, provided herein are methods for culturing cells, such as for cultivation, expansion or proliferation of cells. In some embodiments, the methods are carried out to expand the cells at least at or about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold,

50-fold, 55- fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, 100-fold or more, such as following culture in the presence of serum-free media for at or about 1, 2,

3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. In some embodiments, the cells expand at least about 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, or 50- fold or more after culture in the serum-free medium formulation for about 5 to 6 days. In some embodiments, the expansion is comparable or improved compared to culture with serum-containing media under the same conditions, such as following culture for at or about

1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days.

The invention will be described by way of the following examples which are to be considered as merely illustrative and not limitative of the scope of the invention.

EXAMPLES EXAMPLE 1 Development of a serum-free medium for mycoplasma growth.

This example shows the results of the first version of defined medium optimized to grow M. pneumoniae in absence of serum.

Four different experiments have been designed to explore different medium compositions that could promote M. pneumoniae growth in the absence of serum:

Experiment 1 : CMRL-1066 augmented with fresh yeast extracts, peptone and milks. Tested by observing colony growth via microscopy in culture media and SP4 agar plating (Table 2).

Table 2. Experiment 1. CMRL-1066 augmented with fresh yeast extracts, peptone and milk. Conditions tested in fist medium screening. Experiment 2:

CMRL-1066 augmented with either filtered, autoclaved or skimmed milk types as lipid supplements. Tested by observing colony growth via microscopy in culture media and SP4 agar plating (Table 3).

Table 3. Experiment 2. Fresh and powdered yeast extracts as lipid supplement with 0, 1 , 2, and 4x CMRL-1066 concentrations and with and without peptone supplement Experiment 3:

Fresh and powdered yeast extracts as lipid supplement with 0, 1, 2, and 4x CMRL-1066 concentrations and with and without peptone supplement. Tested by observing colony growth via microscopy in culture media and SP4 agar plating (Table 4).

Table 4. Experiment 3: Fresh and powdered yeast extracts as lipid supplement with 0, 1 , 2, and 4x CMRL-1066 concentrations and with and without peptone supplement. Experiment 4:

CMRL-1066, Minimal Media (M.M) and Hayf lick with supplements of RNA, Peptone and adiposomes of palmitic acid vs additions of PPLO and FBS (SP4 serum additions). Growth measured via DAPI staining for visual quantification (Table 5).

Table 5. Experiment 4: CMRL-1066, Minimal Media (M.M) and Hayflick with supplements of RNA, Peptone and adiposomes of palmitic acid vs additions of PPLO and FBS (SP4 serum additions). Growth measured via DAPI staining for visual quantification. Table with selected conditions is already described in section 2

Experiments 1 and 2 were addressing the idea that milk could supply the lipids that are required for growth. However, no colonies were observed after growing cells in conditions described in Tables 1 and 2 and platting them in SP4 plates. Experiments 3 and 4 were overcoming the difficulties of preparing a whole defined medium from each individual component (ca. 50) of the defined medium. Optimizing a commercial medium base CMRL-1 066 (Sigma, Cat. Nr. C0422) was started as it contains almost all of the nutrient supplements specified in Yus et al. , 20091 and it is also used in some formulations for other Mycoplasma spp. (such as SP4).

Important missing components of CMRL (and limiting factors to M. pneumoniae growth) are lipids. Their introduction was attempted, via a natural lipid substitute, in this case milk (Tables 1 and 2), via whole cell yeast extracts (Table 1) or the chemical addition of pure lipids (cholesterol, oleic, linoleic and palmitic acids; Tables 3 and 4). All of the above lipid supplements were added individually to CMRL-1066 and only pure lipids were effective based on light microscopy analysis (data not shown). The yeast extracts did not provide growth when observed under light microscopy and the milk was difficult to sterilize at concentrations where the lipids would be useful. It was also found that aside from lipids, addition of RNA and peptone (Gibco Cat. Nr. 211677; Table S4) extracts boost the growth of M. pneumoniae, as shown in Figure 1.

To systematically understand the contribution of all these components, they were added individually or in combinations in a 96-well format. Growth was monitored by pH change with a pH indicator (Yus et al., 2009) and total protein measurement.

As shown in the Figure 2 below, CMRL-1066 basal medium was supplemented with either lipids, RNA bases or a mixture of lipids and bases (composition based on our published minimal medium; Table 5 for all tested conditions) with either RNA, peptone, tryptone (Gibco Cat. Nr. 211705) or peptone + RNA. The former defined medium (MM16)1 and standard Hayflick were used as control.

Each well was inoculated with 200 pi of the indicated media and 4 mI (approx. 1 μg total protein) of M. pneumoniae cells were then added to wells in two replicates. Three 96 well plates were set in parallel, one for a 48 h protein time point, another for 120 h and another one was left without cells, as control for contaminations. The pH was measured via change of absorption of the pH indicator Phenol Red at 430 and 560 nm over 5 days (M. pneumoniae produces lactate and acetate that acidify the medium). Unfortunately, pH measurements gave no apparent growth (data not shown), indicating that i) growth was not robust enough or ii) the metabolism of the cells differs from that in Hayflick. Nevertheless, growth could indeed be detected by an increase in biomass in the defined medium with lipids, bases, RNA and peptone added. Though not as strong as that observed in the Hayflick media (Figure 3), this does show that M. pneumoniae can grow in the new chemically defined media. This growth is similar to that obtained in the published defined medium (Yus et al., 2009 and Figure 3, lower panel).

In conclusion, the first preliminary version of a medium that promotes growth of M. pneumoniae in the absence of horse serum (Table 6) was obtained. Table 6 shows the composition of the stocks used to create the growth media and their supplements.

Table 6. First version of defined medium optimized for M. pneumoniae growth in the absence of horse serum.

Table 7 summarizes the features of the different media versions developed in the present example. The composition of the CMRL-1006 medium is provided in Table 8.

Table 7: Composition of different serum-free medium versions for M. pneumoniae.

Concentration

Components Molecular Weight mM

(mg/L) Amino Acids

Glycine 75.0 50.0 0.6666667

Hydroxy L-proline 131.0 10.0 0.07633588 Concentration

Components Molecular Weight mM

(mg/L) Concentration

Components Molecular Weight mM

(mg/L)

Table 8: Composition of the CMRL-1006 minimal medium.

EXAMPLE 2 Optimization of the serum-free medium for mycoplasma growth.

Here, a further development and optimization of a serum-free and animal component-free medium capable to support and maintain growth of M. pneumoniae is presented. In principle, the composition of the medium developed can be used as a platform to grow other mycoplasmas species. 2. 1. Preparation and quantification of bacterial stocks

Frozen stocks of wild-type M. pneumoniae strain M129 were used as starting inocula in the culture tests. Bacterial stocks were prepared as follows. Culture flasks of 300cm ² containing each 75ml of modified Hayflick rich medium (Yus et., 2009) were inoculated with 200mI of M. pneumoniae cell suspension and cultured at 37°C under 5% C02. After 72 hours of culture, the culture medium was removed and cells scraped off from the flask in 10 ml of fresh medium. Then, bacteria cell suspension was stored at -80°C in aliquots of 200mI until needed. For each experiment, an aliquot was thaw to avoid repeating freezing and thawing cycles. To measure the inoculum concentration, one aliquot was used to quantify protein biomass using the Pierce TM BCA Protein Assay Kit.

2.2. Culture conditions and methods used to monitor growth

To optimize the formulation of the serum-free medium, it was established a high-throughput method to monitor growth, allowing multiple culture tests. For this, a 96-well plate format incubated at 37°C in a Tecan Spark plate reader was used. Each media was tested in duplicate wells containing 200mI of medium inoculated generally with 3μg or 1.5μg of starting inocula. Growth was monitored by growth curve analyses based on the “growth index” method previously described (Yus et al., 2009), in which growth is estimated overtime by measuring the change of absorbance (ratio 430 and 560nm). This read out is aided by a pH indicator (in this case phenol red) which is added to the medium. This method is based on the fact that M. pneumoniae acidifies the culture medium when it grows. Thus, a decrease of pH in the medium is indicative of active metabolism and therefore an indirect but straightforward method to measure growth. As the buffering capacity and/or metabolism can change depending on the media composition, w the final protein concentration at the end of the growth curve (typically 5 days) was also measured to confirm gain of cell biomass. For this, wells of the 96-well plate were washed twice with PBSxl and attached cells lysed with 100mI of lysis buffer (10mM Tris pH8, 6mM MgCh, 1mM EDTA, 100mM NaCI, 0.1%Triton-X100 plus cocktail of protease inhibitors). Then, protein biomass for each well lysate was measured using the Pierce TM BCA Protein Assay Kit.

To corroborate the results of the 96-well plate format assays, scaling up experiments of certain media formulations were performed in tissue culture flasks of 25cm ² containing 5ml of medium. Cultures were incubated at 37°C under 5% CO2, and protein and DNA biomass were measured at different time points (Oh, 24h, 48h, 72, 96h) as follows. For each time point, cells were scraped off from the flasks in the culture medium and 1ml of cell suspension harvested by centrifugation (14000rpm, 10min). This was performed per duplicate to obtain samples for both, protein and DNA measurements. For protein biomass quantification, the cell pellet was washed twice with PBSxl and lysed with 10OmI of lysis buffer (10mM Tris pH8, 6mM MgCI ₂ , 1mM EDTA, 100mM NaCI, 0.1%Triton-X100 plus cocktail of protease inhibitors) prior to duplicate protein measurements using the Pierce TM BCA Protein Assay Kit. For DNA biomass quantification, the cell pellet was directly lysed and the DNA extracted using the MasterPure DNA purification Kit (Epicentre) following the recommendations of the Kit manufacturer. Finally, extracted DNA for each time point was measured using a fluorometric method (Qubit dsDNA HS assay Kit, Invitrogen).

To test whether certain media formulations could support growth after consecutive passages, passaging experiments were performed as follows. Tissue culture flasks of 25crri ₂ containing 5ml of medium were grown for 3 to 5 days, and then mycoplasma cells scraped off from the flasks in the same culture medium. For the next passage, 200mI of cell suspension was diluted in 5ml of fresh medium to start a new culture. This process was repeated up to 10 consecutive passages.

2.3 Development of a first version of a serum- free medium

A first version of a serum-free medium was established based on the formulation of a chemically defined medium previously published by Yus et al., (2009). To facilitate medium preparation and reproducibility, amino acids, bases, vitamins and inorganic salts were replaced by RNA and the commercially available CMRL-1066 medium (Invitrogen). As a source of lipids, this medium contained phosphatidylcholine (PC), and a lipid mix consisting in palmitic acid, oleic acid and cholesterol complexed to non-lipidated bovine serum albumin (BSA). This formulation (named vB2,) supported a slightly increase in protein biomass, but growth was not robust enough to produce sufficient biomass and support serial passaging, suggesting rapid consumption of nutrients and loss of viability.

2.4. Medium optimization workflow

To optimize the medium composition, a workflow method to systematically test different media formulations was stablished (Fig. 4). For this, we developed a 96-well plate culture format in which growth was monitored by combining growth curve analyses based on the metabolic “growth index” and quantification of protein biomass gain at the end of the growth curve (for details see method section entitled “culture conditions and methods used to monitor growth”). Using this experimental set up, it was possible to assess the contribution of different concentrations of the original components in the performance of vB2 medium. Then, the contribution of new components that could improve performance based on the review of the literature and the analysis of the metabolic map of M. pneumoniae (Yus et al., 2009) was tested. When an improvement was found, it was fixed, and then the influence of different concentrations of the other components was tested again on a systematic manner, as it was found that small variations could affect the optimal range of the other components. As a result, this methodology was repeated until we it was not possible to find a significant improvement, performing a total of more than 300 experiments.

To implement this method, the buffering capacity of vB2 was optimized, as this medium was not robust enough to change the medium pH when using 100mM of HEPES, the buffering conditions used in Hayflick.

For this, decreased concentrations of HEPES (OmM and 50mM) combined with several inoculum concentrations were tested. As shown in Fig. 5, a measurable change in pH in vB2 with no HEPES or supplemented with 50mM HEPES was detected. Based on these results, 50mM HEPES was used for future optimization experiments. To compare yield performance to rich media, Hayflick containing 50mM HEPES was used as a reference medium.

2. 5 Major improvements found during medium optimization

A culture medium for mycoplasma likely requires a carbon source, amino acids, nucleosides, lipids, vitamins and co-factors, essential metals and minerals. In the current available mycoplasma rich mediums, many of these components are generally provided by animal serums and animal mixtures such as peptones or animal extracts. The chemically defined medium published by Yus et al in 2009 contains most of these nutrients but the performance of this medium was far to support robust growth, which is required in industry production. This suggests that optimized concentrations of media components is crucial, and that this medium probably lacks important growth factors likely present in the animal serum.

In this invention, two culture media that were optimized for M. pneumoniae cultivation: (1) a serum-free medium containing BSA as a lipid carrier (see vB10 and vB11 medium versions in Annex 1), and (2) an animal component-free medium in which BSA and complex animal extracts have been completely removed (vB13, see in Annex 1) were presented. Below, a summary of the major improvements found during our optimization process aimed to replace animal serum and other animal components is described. The composition details and performance of intermediate media versions is summarized in Figures 11 y 12

Carbon source

Glucose was found essential as a carbon source. In addition, it was found that glycerol improved significantly the growth performance with an optimal concentration of 0.025% in the context of the medium composition tested.

Amino acids and broth extracts

All the essential amino acids were provided by the commercially available CMRL-1066 medium (Invitrogen). Only glutamine was added separately as this amino acid is absent in CMRL. Increasing amino acids amounts did not improve growth. In fact, M. pneumoniae encodes a low number of amino acid permeases and transporters, but encodes a peptide importer (oppB-F cluster) as well as aminopeptidases, suggesting a requirement for peptides in the medium. Indeed, this was confirmed experimentally by Yus et al., in 2009. Thus, the presence of peptones or similar hydrolyzed extracts is critical. We found that PPLO and Yeastolate were superior to peptone, probably because these broth extracts also contains other beneficial growth factors such as vitamins, nucleosides and lipid sources, the later especially derived from PPLO. Yeastolate and PPLO supported similar growths at their respective optimal concentrations, with the advantage that Yeastolate is not derived from animal materials.

Bases and nucleosides

Essential nucleosides were mostly provided by the CMRL-1066 medium, and broth extracts like Yeastolate and PPLO depending on the version of the medium. Addition of RNA improved slightly the growth performance.

Vitamins

Essential vitamins were mostly provided by the CMRL-1066 medium, except for thioctic acid. Accordingly, supplementation of this vitamin improved significantly the growth performance. Figure 6 illustrates the role of thioctic acid and glycerol in increasing the medium performance. Moreover, addition of extra amounts of vitamins such as pyridoxamine, nicotinic acid, riboflavin and choline slightly enhanced growth, suggesting that CMRL and broth extracts did not provide enough. CMRL as a nutrient substitute

As described above, the commercially available CMRL-1066 medium was used to facilitate media preparation and substitute several components that should be otherwise supplemented separately, including for example amino acids, nucleosides, essential minerals, anti-oxidants, vitamins and other co-factors. The presence of CMRL in the medium is essential to support growth and we found particularly important the adjustment of the amount of CMRL supplemented, being 0.5X the optimal concentration. It was noted that higher concentrations had inhibitory effects. Although the use of CMRL extremely simplify the preparation of the medium, it is also possible to add separately the components, since the formulation of CMRL is publically available.

Lipids and lipid carriers

The delivery of lipids was critical for medium development, as variations in the lipid content resulted in the major improvements achieved. By one hand, the cholesterol amount present in CMRL was not enough to support growth, demanding additional supplementation with an optimal concentration nearby 30μg/ml. In addition, cholesterol had to be delivered by a carrier protein such as BSA. Absence of cholesterol or BSA resulted in growth failure. It was found that BSA complexed with a lipid mix containing palmitic acid, oleic acid and cholesterol resulted in the most efficient medium (see vB10 or vB11 in Annex 1). BSA concentrations around 0.3% appeared to perform well, although increasing concentrations could also support efficient growth. To obtain a full animal component-free medium, BSA was also replaced by cyclodextrin or PVA. Although growth was decreased compared to BSA-based media, cyclodextrin was capable to support robust growth, especially at concentrations ranging from 4 to 5 mg/ml. PVA also showed a positive effect, but under performing compared to cyclodextrin. Of note, it was found that addition of palmitic and oleic acid resulted to have toxic effects when cyclodextrin was used as a lipid delivery system. Thus, the vB13 version, which is free of animal components (including BSA), only contains cholesterol and phospholipids as lipid sources.

As hinted above, addition of phospholipids were also found fundamental to improve growth performance. In particular, it was found that both, phosphatidylcholine (PC) and sphingomyelin (SPM) improved growth separately, but there was a synergistic effect when both were combined (Fig. 7). Optimal concentrations for both, PC and SPM were found to be 40μg/mI, although 20μg/ml of PC could also perform well. Addition of other phospholipids such as cardiolipin did not result in significant improvements, nor could replace PC or SPM.

2.6. Scaling up and passaging experiments to test medium performance

After the process of medium optimization in the 96-well plate format, the size of the cultures was scaled up to confirm growth performance. For this, growth over time in 5ml cultures was monitored by measuring protein and DNA biomass as described above (for details see method section entitled “culture conditions and methods used to monitor growth”). As shown in Fig8 , both vB10 serum-free medium and vB13 animal component-free medium supported growth by reaching in 72h of culture at 60-70% of biomass as compared to rich medium. Importantly, both mediums also allowed up to 10 serial passages, indicating that serum residuals in the starting inocula were not influencing the growth performance. It is therefore concluded that vB10 or vB13 compositions can be used for bacterial production upon the process of industrial scaling up.

7.2 Quality control of cellular behavior under culture conditions using animal component- free medium.

Vaccines or other bacterial based therapies produced in animal component-free medium should have preferably similar cellular characteristics to those produced in rich medium. To test this, comparative transcriptomic and proteomic analyses of M. pneumoniae grown in rich medium or animal component free medium (vB13) was performed. RNA-seq and mass spectrometry (MS) analyses were performed respectively at the Genomics and Proteomics core facilities of the CRG, as previously described (Yus et al., 2019). As shown in Fig. 9, it was found high correlations between the overall gene expression at RNA (r=0.95) and protein levels (r=0.89), thus indicating a similar cell response in both media.

8.2 Growth assessment of other M. pneumoniae strains

Next, the growth of several M. pneumoniae chassis strains was assessed, in which several virulence factors were removed by genetic engineering. Growth curve analysis determined by the metabolic growth index were performed in a 96-well plate format and protein biomass quantified at the end of the growth curve (Fig. 10). As with the wild-type strain, it was found that all the tested strains could be grown in vB13 medium. The differences observed in the growth curves between strains could be probably explained by differences in the quality of the inoculum as these strains exhibited similar growth profiles in rich medium. Thus, it was concluded that the developed serum-free medium is suitable to grow modified strains derived from wild-type.