Serum-free cell culture media

Claim 2

An MEM zinc option medium wherein the amino acid profile is about as follows: ______________________________________ Item Amount (milligrams per liter) ______________________________________ L alanine 8.9 L asparagine 75.0 L aspartic acid 13.0 L glutamic acid 14.7 L glycine 7.5 L proline 11.5 L serine 52.5 L arginine HCl 196.52 L cystine 37.2 L glutamine 584.0 L histidine HCl 65.0 L isoleucine 80.88 L leucine 160.88 L lysine 112.38 L methionine 23.30 L phenyl-alanine 50.15 L threonine 74.18 L tryptophan 15.61 L valine 71.4 L tyrosine 45.9 ______________________________________

Claim 3

An MEM zinc option medium, as defined in claim 1, wherein said medium is augmented with cell growth promoting quantities of biotin and folic acid.

Claim 4

A culture media as defined in claim 1 further comprising ______________________________________ Thyroxine 0.01 to 0.03 micrograms per liter Insulin 4 milligrams per liter Hydrocortisone 0.075 to 0.3 milligrams per liter Essential Fatty Acids 2.5 to 15 milligrams per liter Vitamin A 0.1 to 0.4 milligrams per liter Vitamin E 7.5 to 30 milligrams per liter Surfactants Sufficient quantity to disperse vitamins and fatty acids. ______________________________________

Claim 5

A culture media as defined in claim 2 further comprising ______________________________________ Thyroxine 0.01 to 0.03 micrograms per liter Insulin 4 milligrams per liter Hydrocortisone 0.075 to 0.3 milligrams per liter Essential Fatty Acids 2.5 to 15 milligrams per liter Vitamin A 0.1 to 0.4 milligrams per liter Vitamin E 7.5 to 30 milligrams per liter Surfactants Sufficient quantity to disperse vitamins and fatty acids. ______________________________________

Claim 6

A culture media as defined in claim 3 wherein said surfactant is a polysorbate 20.

Claim 7

A culture media as defined in claim 1 further comprising ______________________________________ Item Amount ______________________________________ thyroxine 0.02 micrograms per liter insulin 4 milligrams per liter hydrocortisone 1.5 milligrams per liter linoleic acid 5.0 milligrams per liter linolenic acid 5.0 milligrams per liter arachidonic acid 5.0 milligrams per liter surfactant, emulsifier 110 milligrams per liter Vitamin E 20 milligrams per liter Vitamin A 0.25 milligrams per liter ______________________________________

Claim 8

An aqueous culture media for use in cell growth having about the following formula: ______________________________________ Item Amount (milligrams per liter) ______________________________________ L alanine 8.9 L asparagine 75.0 L aspartic acid 13.0 L glutamic acid 14.7 L glycine 7.5 L proline 11.5 L serine 52.5 L arginine HCl 196.52 l cystine 37.2 L glutamine 584.0 L histidine HCl 65.0 L isoleucine 80.88 L leucine 160.88 L lysine 112.38 L methionine 23.30 L phenyl-alanine 50.15 L threonine 74.18 L tryptophan 15.61 L valine 71.4 L tyrosine 45.9 CaCl 200 KCl 400 MgCl6H.sub.2 O 183 MgSO.sub.4 7H.sub.2 O 12 NaCl 6800 NaH.sub.2 PO.sub.4 H.sub.2 O 150 phenol red 10 glucose 2000 Na pyruvate 110 biotin 1.0 D-calcium pantothenate 1.0 choline Cl 56.0 folic acid 1.0 inositol 36.0 nicotinamide 1.0 pyridoxal HCl 1.0 riboflavin 0.1 thiamine 1.0 B.sub.12 1.36 folinic acid 1.0 DL thioctic acid 0.20 putrescine HCl 0.16 linoleic 0.084 FeCl.sub.3 6H.sub.2 O 0.54 ZnSO.sub.4 7H.sub.2 O 0.14 insulin 4.00 HEPES buffer solution 2380.0 NaHCO.sub.3 sufficient to adjust to a pH of about 7.4 ______________________________________ Distilled water to make a total of 1 liter

Claim 9

An aqueous culture media for use in cell growth having about the following formula: ______________________________________ Item Amount (milligrams per liter) ______________________________________ L alanine 8.9 L asparagine 75.0 L aspartic acid 13.0 L glutamic acid 14.7 L glycine 7.5 L proline 11.5 L serine 52.5 L arginine HCl 196.52 l cystine 37.2 L glutamine 584.0 L histidine HCl 65.0 L isoleucine 80.88 L leucine 160.88 L lysine 112.38 L methionine 23.30 L phenyl-alanine 50.15 L threonine 74.18 L tryptophan 15.61 L valine 71.4 L tyrosine 45.9 CaCl 200 KCl 400 MgCl6H.sub.2 O 183 MgSO.sub.4 7H.sub.2 O 12 NaCl 6800 NaH.sub.2 PO.sub.4 H.sub.2 O 150 phenol red 10 glucose 2000 Na pyruvate 110 biotin 1.0 D-calcium pantothenate 1.0 choline Cl 56.0 folic acid 1.0 inositol 36.0 nicotinamide 1.0 pyridoxal HCl 1.0 riboflavin 0.1 thiamine 1.0 B.sub.12 1.36 folinic acid 1.0 DL thioctic acid 0.20 putrescine HCl 0.16 linoleic 0.084 FeCl.sub.3 6H.sub.2 O 0.54 ZnSO.sub.4 7H.sub.2 O 0.14 insulin 4.00 HEPES buffer solution 2380.0 NaHCO.sub.3 sufficient to adjust to a pH of about 7.4 ______________________________________ Distilled water to make a total of 1 liter

Claim 10

A culture media as defined in claim 7 further comprising ______________________________________ Thyroxine 0.01 to 0.03 micrograms per liter Insulin 4 milligrams per liter Hydrocortisone 0.075 to 0.3 milligrams per liter Essential Fatty Acids 2.5 to 15 milligrams per liter Vitamin A 0.1 to 0.4 milligrams per liter Vitamin E 7.5 to 30 milligrams per liter Surfactants Sufficient quantity to disperse vitamins and fatty acids. ______________________________________

Claim 13

An improved process for growing lines of living cells comprising the steps of growing said cells in a growth medium, through at least two passages, said medium an MEM-zinc-option type medium having about the following amino acid profile: ______________________________________ Item Amount (milligrams per liter) ______________________________________ L alanine 8.9 L asparagine 75.0 L aspartic acid 13.0 L glutamic acid 14.7 L glycine 7.5 L proline 11.5 L serine 52.5 L arginine HCl 196.52 L cystine 37.2 L glutamine 584.0 L histidine HCl 65.0 L isoleucine 80.88 L leucine 160.88 L lysine 112.38 L methionine 23.30 L phenyl-alanine 50.15 L threonine 74.18 L tryptophan 15.61 L valine 71.4 L tyrosine 45.9 ______________________________________

Claim 14

An improved process for growing lines of living cells comprising the steps of growing said cells in a growth medium, through at least two passages, said medium an MEM-zinc-option type medium having about the following amino acid profile: ______________________________________ Item Amount (milligrams per liter) ______________________________________ L alanine 8.9 L asparagine 75.0 L aspartic acid 13.0 L glutamic acid 14.7 L glycine 7.5 L proline 11.5 L serine 52.5 L arginine HCl 196.52 L cystine 37.2 L glutamine 584.0 L histidine HCl 65.0 L isoleucine 80.88 L leucine 160.88 L lysine 112.38 L methionine 23.30 L phenyl-alanine 50.15 L threonine 74.18 L tryptophan 15.61 L valine 71.4 L tyrosine 45.9 ______________________________________

Claim 15

A process as defined in claim 11 wherein said medium comprises effective amounts of additional nutrients as follows: biotin folic acid Thyroxine Insulin Hydrocortisone Essential Fatty Acids Vitamin A Vitamin E Surfactants

Claim 16

A process as defined in claims 11 or 12, wherein transfer of cells from one passage to another is primarily effected by use of physical means and without use of cell-harming quantities of proteolytic enzymes.