Serum free cell culture medium

Claim 2

A serum-free culture composition containing less than 10 μg/ml of protein comprising a basal medium supplemented with a solution of 2-aminoethanol to a concentration of at least 100 μM, a solution of 2-mercaptoethanol, a solution of transferrin, a solution of insulin and an added solution of essential amino acids.

Claim 3

The composition as set forth in claim 1 wherein the basal media comprises a 2:1:1 ratio mixture of RPMI 1640:Dulbecco's modified Eagle's Medium:Ham's F12.

Claim 4

The composition as set forth in claim 1 wherein the 2-aminoethanol concentration is 600 μM.

Claim 5

The composition as set forth in claim 1 wherein the 2-mercaptoethanol concentration is 50 μM.

Claim 6

The composition as set forth in claim 1 wherein the transferrin concentration is 1 to 5 μg/ml of one-third iron saturated transferrin.

Claim 7

The composition as set forth in claim 1 wherein the insulin concentration is 5 μg/ml.

Claim 8

The composition as set forth in claim 6 wherein the trace element solution comprises a trace element mixture of compounds of Mn, Mo, Ni, Se, Si, Sn, V and Cd.

Claim 9

The composition as set forth in claim 7 wherein the selenium comprises 2×10 -8 M Na 2 SeO 3 .

Claim 10

The composition as set forth in claim 1 wherein 40 ml/L of minimum essential medium essential amino acids is added.

Claim 11

A serum free cell culture composition containing less than 10 ug/ml protein comprising the following compounds in the following proportions: ______________________________________ mg/liter ______________________________________ Salts Ca(No.sub.3).sub.2.4H.sub.2 O 50. CaCl.sub.2 58.3 Fe(No.sub.3).sub.3.9H.sub.2 O 0.25 KCl 355.9 MgSO.sub.4 (Anhydrous) 48.84 NaCl 6497.975 NaHCO.sub.3 1600. NaH.sub.2 PO.sub.4 (Anhydrous ) 31.25 CuSO.sub.4.5H.sub.2 O 0.00065 FeSO.sub.4.7H.sub.2 O 0.2085 MgCl.sub.2 14.32 Na.sub.2 HPO.sub.4 435.51 ZnSO.sub.4.7H.sub.2 O 0.216 Other Components Sodium Hypoxanthine 1.195 Linoleic Acid 0.021 D-glucose 2575.5 Lipoic Acid 0.0525 Putrescine.2HCl 0.0405 Thymidine 0.1825 Phenol red 6.55 DL-Thioetic acid 0.525 Glutathione (reduced) 0.5 HEPES 1787.25 Sodium Pyruvate 27.5 Amino Acids L-Alanine 2.225 L-Asparagine 25. L-Asparagine.H.sub.2 O 3.75 L-Arginine free base 100. L-Arginine.HCl 326.55 L-Aspartic Acid 13.325 L-Cysteine.HCl.H.sub.2 O 8.78 L-Cystine 48. L-Cystine.2HCl 48.22 L-Glutamic Acid 13.675 Glycine 14.375 L-Histidine (free base) 7.5 L-Histidine.HCl.H.sub.2 O 99.74 L-Hydroxyproline 10. L-Isoleucine 157.235 L-Leucine 159.325 L-Lysine.HCl 210.625 L-Methionine 46.325 L-Phenylalanine 91.24 L-Proline 18.625 L-Serine 28.125 L-Threonine 131.925 L-Tryptophan 27.41 L-Tyrosine 72. L-Tyrosine.2Na 14.415 L-Tyrosine.2Na.2H.sub.2 O 27.895 L-Valine 130.025 Vitamins Biotin 0.10175 D-calcium pantothenate 1.245 Choline Chloride 5.99 Folic Acid 1.825 i-Inositol 23.8 Nicotinamide 0.5 Para-aminobenzoic acid 0.5 Pyridoxine.HCl 0.5155 Riboflavin 0.2095 Thiamine.HCl 1.585 Vitamin B12 0.3425 Niacinamide 1.01 Pyridoxa1.HCl 1.0 Trace Elements MnCl.sub.2 0.5 nM (NH.sub.4).sub.6 Mo.sub.7 O.sub.24 0.5 nM NiSO.sub.4 0.25 nM H.sub.2 SeO.sub.3 15. nM Na.sub.2 SeO.sub.3 50. nM Na.sub.2 SiO.sub.3 250. nM SnCl.sub.2 0.25 nM NaVO.sub.3 2.5 nM CdSO.sub.4 50. nM Added at Time of Use - Unstable Components L-Glutamine 332.5 mg/L 2-mercaptoethanol 5 × 10.sup.-5 μM 2-aminoethanol (ethanolamine) 600 uM Transferrin l to 5 mg/L Insulin 5 mg/L ______________________________________

Claim 12

A serum free cell culture composition containing less than 10 ug/ml protein comprising the following compounds in the following proportions: ______________________________________ mg/liter ______________________________________ Salts Ca(No.sub.3).sub.2.4H.sub.2 O 50. CaCl.sub.2 58.3 Fe(No.sub.3).sub.3.9H.sub.2 O 0.25 KCl 355.9 MgSO.sub.4 (Anhydrous) 48.84 NaCl 6497.975 NaHCO.sub.3 1600. NaH.sub.2 PO.sub.4 (Anhydrous ) 31.25 CuSO.sub.4.5H.sub.2 O 0.00065 FeSO.sub.4.7H.sub.2 O 0.2085 MgCl.sub.2 14.32 Na.sub.2 HPO.sub.4 435.51 ZnSO.sub.4.7H.sub.2 O 0.216 Other Components Sodium Hypoxanthine 1.195 Linoleic Acid 0.021 D-glucose 2575.5 Lipoic Acid 0.0525 Putrescine.2HCl 0.0405 Thymidine 0.1825 Phenol red 6.55 DL-Thioetic acid 0.525 Glutathione (reduced) 0.5 HEPES 1787.25 Sodium Pyruvate 27.5 Amino Acids L-Alanine 2.225 L-Asparagine 25. L-Asparagine.H.sub.2 O 3.75 L-Arginine free base 100. L-Arginine.HCl 326.55 L-Aspartic Acid 13.325 L-Cysteine.HCl.H.sub.2 O 8.78 L-Cystine 48. L-Cystine.2HCl 48.22 L-Glutamic Acid 13.675 Glycine 14.375 L-Histidine (free base) 7.5 L-Histidine.HCl.H.sub.2 O 99.74 L-Hydroxyproline 10. L-Isoleucine 157.235 L-Leucine 159.325 L-Lysine.HCl 210.625 L-Methionine 46.325 L-Phenylalanine 91.24 L-Proline 18.625 L-Serine 28.125 L-Threonine 131.925 L-Tryptophan 27.41 L-Tyrosine 72. L-Tyrosine.2Na 14.415 L-Tyrosine.2Na.2H.sub.2 O 27.895 L-Valine 130.025 Vitamins Biotin 0.10175 D-calcium pantothenate 1.245 Choline Chloride 5.99 Folic Acid 1.825 i-Inositol 23.8 Nicotinamide 0.5 Para-aminobenzoic acid 0.5 Pyridoxine.HCl 0.5155 Riboflavin 0.2095 Thiamine.HCl 1.585 Vitamin B12 0.3425 Niacinamide 1.01 Pyridoxa1.HCl 1.0 Trace Elements MnCl.sub.2 0.5 nM (NH.sub.4).sub.6 Mo.sub.7 O.sub.24 0.5 nM NiSO.sub.4 0.25 nM H.sub.2 SeO.sub.3 15. nM Na.sub.2 SeO.sub.3 50. nM Na.sub.2 SiO.sub.3 250. nM SnCl.sub.2 0.25 nM NaVO.sub.3 2.5 nM CdSO.sub.4 50. nM Added at Time of Use - Unstable Components L-Glutamine 332.5 mg/L 2-mercaptoethanol 5 × 10.sup.-5 μM 2-aminoethanol (ethanolamine) 600 uM Transferrin l to 5 mg/L Insulin 5 mg/L ______________________________________

Claim 13

A composition as set forth in claim 1 wherein the concentration of 2-aminoethanol is 400 to 800 μm.

Claim 14

A composition as set forth in claim 1 which is also supplemented with a selenium-containing solution of trace elements or with a solution of selenium.