US4767704A
Expired / Lapsed
US
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Protein-free culture medium
Assignee: Marcor Development Corp
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Filing: 1983-10-07
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Granted: 1988-08-30
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Expires: 2003-10-07
Abstract
Abstract A trace element mixture suitable for use in a protein-free tissue culture medium, which comprises water-soluble compounds selected from acids, bases, and salts containing copper, iron, zinc, manganese, silicon, molybdenum, vanadium, nickel, tin, aluminum, silver, barium, bromine, cadmium, cobalt, chromium, fluorine, germanium, iodine, rubidium, zirconium, or selenium, the compounds being devoid of any additional metals other than those present as positive ions selected from groups IA and IIA of the periodic table of elements, wherein the compounds produce a solution containing specified minimum concentrations of the listed elements when dissolved in an amount of water sufficient to produce a concentration for one of the elements equal to the corresponding minimum concentration of the one element while maintaining each remaining element at a concentration equal to or greater than the minimum concentration for the remaining element is disclosed along with cell culture media containing these trace elements and methods of culturing cells using these media.
Inventors
- 👤 William L. Cleveland
- 👤 Bernard F. Erlanger
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-04 15:50
CPC Classifications
C; C12; C12N; C12N5/00; C12N5/0018; C12N5/005; C12N2500/00; C12N2500/05; C12N2500/10; C12N2500/12; C12N2500/20; C12N2500/30; C12N2500/32; C12N2500/36; C12N2501/00; C12N2501/30; C12N2501/38; C12N2501/39; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/948
Claims Analysis
Total Claims: 25
Claim 1
Independent Claim
116 words
In a protein-free chemically defined tissue culture medium wherein the improvement comprises a combination of a basal medium and a trace element mixture, said trace element mixture comprising a bromine salt, a fluorine salt, and an iodine salt, and, as metal salt other than positive metal ions selected from Group IA and IIA of the Periodic Table of the Elements, said mixture containing only at least one species selected from each member of the group consisting of copper salts, iron salts, zinc salts, manganese salts, silicon salts, molybdenum salts, vanadium salts, nickel salts, tin salts, aluminum salts, silver salts, barium salts, cadminum salts, cobalt salts, chromium salts, germanium salts, rubidium salts, zirconium salts and selenium salts.
Claim 2
Dependent Claim
116 words
In a protein-free chemically defined tissue culture medium wherein the improvement comprises a combination of a basal medium and a trace element mixture, said trace element mixture comprising a bromine salt, a fluorine salt, and an iodine salt, and, as metal salt other than positive metal ions selected from Group IA and IIA of the Periodic Table of the Elements, said mixture containing only at least one species selected from each member of the group consisting of copper salts, iron salts, zinc salts, manganese salts, silicon salts, molybdenum salts, vanadium salts, nickel salts, tin salts, aluminum salts, silver salts, barium salts, cadminum salts, cobalt salts, chromium salts, germanium salts, rubidium salts, zirconium salts and selenium salts.
Claim 3
Dependent Claim
157 words
The tissue culture medium of claim 1, wherein said trace element mixture produces a solution containing 3.1×10 -5 -3.1×10 -3 mg/L copper, 8.4×10 -3 -8.4×10 -1 mg/L iron, 9.9×10 -3 -9.9×10 -1 mg/L zinc, 2.8×10 -6 -2.8×10 -4 mg/L manganese, 7.0×10 -4 -7.0×10 -2 mg/L silicon, 4.8×10 -6 -4.8×10 -4 mg/L molybdenum, 1.3×10 -5 -1.3×10 -3 mg/L vanadium, 1.4×10 -6 -1.4×10 -4 mg/L nickel, 3.1×10 -6 -3.1×10 -4 mg/L tin, 1.3×10 -5 -1.3×10 -3 mg/L aluminum, 1.1×10 -5 -1.1×10 -3 mg/L silver, 1.4×10 -4 -1.4×10 -2 mg/L barium, 4.0×10 -6 -4.0× 10 -4 mg/L bromine, 1.1×10 -4 -1.1×10 -2 mg/L cadmium, 6.0×10 -5 -6.0×10 -3 mg/L cobalt, 5.3×10 -6 -5.3×10 -4 mg/L chromium, 2.3×10 -4 -2.3×10 -2 mg/L fluorine, 3.7×10 -5 -3.7×10 -3 mg/L germanium, 4.1×10 -6 -4.1×10 -4 mg/L iodine, 8.5×10 -5 -8.5×10 -3 mg/L rubidium, 9.0×10 -5 -9.0×10 -3 mg/L zirconium, and 2.6×10 -4 -2.6×10 -2 mg/L selenium when dissolved in said amount of water.
Claim 4
Dependent Claim
164 words
The tissue culture medium of claim 1, wherein said trace element mixture produces a solution containing 1.6×10 -4 -6.4×10 -4 mg/L copper, 4.6×10 -2 -1.8×10 -1 mg/L iron, 4.9×10 -2 -1.9×10 -1 mg/L zinc, 1.4×10 -5 -5.6×10 -5 mg/L manganese, 3.5×10 -3 -1.4 ×10 -2 mg/L silicon, 2.4×10 -5-1 .0×10 -4 mg/L silicon, 2.4×10 -5 -1.0×10 -4 mg/L molybdenum, 6.5×10 -5 -2.6×10 -4 mg/L vanadium, 7.5×10 -6 -3.0×10 -5 mg/L nickel, 1.5×10 -5 -6.0×10 -5 mg/L tin, 6.5×10 -5 -2.6×10 -4 mg/L aluminum, 5.0×10 -5 -2.2×10 -4 mg/L silver, 7.0×10 -4 -2.8×10 -5 mg/L barium, 2.0×10 -5 -8.0×10 -5 mg/L bromine, 5.5×10 -4 -2.2×10 -3 mg/L cadminum, 3.0×10 -4 -1.2×10 -3 mg/L cobalt, 2.5×10 -5 -1.1×10 -4 mg/L chromium, 1.2×10 -3 -4.6×10 -3 mg/L fluorine, 1.8×10 -4 -7.4×10 -4 mg/L germanium, 2.0×10 -5 -8.2× 10 -5 mg/L iodine, 4.2×10 -4 -1.7×10 -3 mg/L rubidium, 4.5×10 -4 -1.8×10 -3 mg/l zirconium, and 1.3×10 -3 -5.1×10 -3 mg/L selenium when dissolved in said amount of water.
Claim 5
Dependent Claim
36 words
The tissue culture medium of claim 1, wherein said basal culture medium is capable of supporting long term growth of a hybridoma cell line without said trace element mixture when supplemented with 5% fetal calf serum.
Claim 6
Dependent Claim
36 words
The tissue culture medium of claim 1, wherein said basal medium contains an amino acid, an inorganic salt, a vitamin, an antibotic, a fatty acid, an amine, a sulfur source, or a non-proteinaceous growth providing hormone.
Claim 7
Dependent Claim
136 words
The tissue culture medium of claim 5, wherein said tissue culture medium contains a component selected from the group consisting of L-alanine, minimum concentration 0.2 mg/L; L-arginine, minimum concentration 1.5 mg/L; L-asparagine, minimum concentration 0.2 mg/L; L-aspartic acid, minimum concentration 0.2 mg/L; L-cystine, minimum concentration 0.1 mg/L; L-cysteine, minimum concentration 0.5 mg/L; L-glumatic acid, minimum concentration 0.4 mg/L; L-glutamine, minimum concentration 3.7 mg/L; glycine, minimum concentration 0.1 mg/L; L-histidine, minimum concentration 0.3 mg/L; L-isoleucine, minimum concentration 0.5 mg/L; L-leucine, minimum concentration 0.6 mg/L; L-lysine, minimum concentration 0.9 mg/L; L-methionine, minimum concentration 0.2 mg/L; L-phenylalanine, minimum concentration 0.4 mg/L; L-proline; minimum concentration 0.4 mg/L; :L-serrine, minimum concentration 0.3 mg/L; L-threonine, minimum concentration 0.5 mg/L; L-tryptophan, minimum concentration 0.09 mg/L; L-tyrosine, minimum concentration 0.5 mg/L; and L-valine, minimum concentration 0.5 mg/L, when dissolved in said amount of water.
Claim 8
Dependent Claim
96 words
The tissue culture medium of claim 5, wherein said tissue culture medium contains a component selected from the group consisting of 1.7-170 mg/L L-alanine, 14.7-1470 mg/L L-arginine, 2.0-200 mg/L-asparagine, 2.2-220 mg/L L-aspartic acid, 1.1-110 mg/L L-cystine, 4.9-490 mg/L L-cysteine, 4.5-450 mg/L L-glutamic acid, 36.5-3650 mg/L L-glutamine, 1.1 -110 mg/L glycine, 3.2-320 mg/L L-histidine, 5.4-540 mg/ L L-isoleucine, 5.9-590 mg/L L-leucine, 9.1-910 mg/L L-lysine, 1.7-170 mg/L L-methionine, 3.5-350 mg/L L-phenylalanine 3.7-370 mg/L L-proline, 2.6-260 mg/L L-serine, 5.3-530 mg/L L-threonine, 0.9-90 mg/ L L-tryptophan, 5.2-520 mg/L L-tyrosine, and 5.3-530 mg/L L-valine, when dissolved in said amount of water.
Claim 9
Dependent Claim
94 words
The tissue culture medium of claim 5, wherein said tissue culture medium contains a component selected from the group consisting of 8.5-34 mg/L L-alanine, 74-295 mg/L L-arginine, 10-40 mg/L L-asparagine, 11-43 mg/L L-aspartic acid, 5.5-22 mg/L L-cystine, 25-98 mg/L L-cysteine, 22-90 mg/L L-glutamic acid, 192-730 mg/L L-glutamine, 5.5-23 mg/L glycine, 16-63 mg/L L-histidine, 27-109 mg/L L-isoleucine, 29-118 mg/L L-leucine, 46-183 mg/L L-lysine, 8.5-34 mg/L L-methionine, 18-71 mg/L L-phenylalanine, 19-75 mg/L L-proline, 13-53 mg/L L-serine, 27-106 mg/L L-threonine, 4.5-18 mg/L L-tryptophan, 26-104 mg/L L-tyrosine, and 26-106 mg/L L-valine, when dissolved in said amount of water.
Claim 10
Dependent Claim
69 words
The tissue culture medium of claim 5, wherein said tissue culture medium contains a component selected from the group consisting of 0.36-3600 mg/L Ca 2+ , 1.76-17,300 mg/L K + , 0.17-1720 mg/L Mg 2+ , 30-×10 5 mg/L Na + , 39-3.9×10 5 mg/L Cl - , 0.96-10,000 mg/L PO 4 3- , and 15-1.5×10 5 mg/L HCO 3 - , when dissolved in said amount of water.
Claim 11
Dependent Claim
66 words
The tissue culture medium of claim 5, wherein said tissue culture medium contains a component selected from the group consisting of 9.0-1.44 mg/L Ca 2+ , 43-690 mg/L K + , 4.3-69 mg/L Mg 2+ , 750-12,000 mg/L Na + , 975-15,600 mg/L CL - , 24-400 mg/L PO 4 3 -, and 380-6100 mg/L HCO 3 - , when dissolved in said amount of water.
Claim 12
Dependent Claim
65 words
The tissue culture medium of claim 5, wherein said tissue culture medium contains a component selected from the group consisting of 18-72 mg/L Ca 2+ , 86-345 mg/L K + , 8.6-35 mg/L Mg 2+ , 1500-6000 mg/L Na + , 1950-7800 mg/L Cl - , 48-192 mg/L PO 4 3- , and 760-3050 mg/L HCO 3 , when dissolved in said amount of water.
Claim 13
Dependent Claim
124 words
The tissue culture medium of claim 5, wherein said tissue culture medium contains a component selected from the group consisting of 1.0×10 -4 -1.0 mg/L biotin, 0.022-225 mg/L pantothenate, 0.090-900 mg/L choline, 0.027-275 mg/L folic acid, 0.12-1250 mg/L inositol, 0.020-200 mg/L nicotinamide, 0.020-200 mg/L pyridoxal, 3.1×10 -4 -3.1 mg/L pyridoxine, 2.2×10 -3 -22 mg/L riboflavin, 0.022-220 mg/L thiamine, 6.9×10 -3 -69 mg/L vitamin B 12 , 1.1-11,000 mg/L pyruvate, 14-1.4×10 5 mg/L glucose, 0.1-1000 mg/L phenol red, 8.1×10 -4 -8.1 mg/L putrescine, 0.020-200 mg/L hypoxanthine, 4.1×10 -4 -4.1 mg/L linoleic acid, 1.0×10 -3 -12 mg/L lipoic acid, 3.6×10 -3 -37 mg/L thymidine, 1-10,000 U/ml penicillin, 1-10,000 μg/ml streptomycin, 0.063-625 μL/L α-thioglycerol, and 1.0×10 -4 -1.0 mg/L progesterone, when dissolved in said amount of water.
Claim 14
Dependent Claim
116 words
The tissue culture medium of claim 5, wherein said tissue culture medium contains a component selected from the group consisting of 2.5×10 -3 -0.040 mg/L biotin, 0.56-9.0 mg/L pantothenate, 2.3-36 mg/L choline, 0.66-10.6 mg/L folic acid, 3.1-50 mg/L inositol, 0.50-8.0 mg/L nicotinamide, 0.50-8.0 mg/L pyridoxal, 7.8×10 -3 -0.12 mg/L pyridoxal, 7.8×10 -3 -0.12 mg/L pyridoxine, 0.055-0.88 mg/L riboflavin, 0.55-8.8 mg/L thiamine, 0.17-2.7 mg/L vitamin B 12 , 28-450 mg/L pyruvate, 350-5600 mg/L glucose, 2.6-43 mg/L phenol red, 0.020-0.32 mg/L putrescine, 0.50-8.0 mg/L hypoxanthine, 0.010-0.16 mg/L linoleic acid, 0.025-0.40 mg/L lipoic acid, 0.091-1.5 mg/L thymidine, 25-400 U/ml penicillin, 25-400 μg/ml streptomycin, 1.6-25 μL/L α-thioglycerol, and 2.5×10 -3 -0.040 mg/L progesterone, when dissolved in said amount of water.
Claim 15
Dependent Claim
110 words
The tissue culture medium of claim 5, wherein said tissue culture medium contains a component selected from the group consisting of 5.0×10 -3 -0.020 mg/L biotin, 1.1-4.5 mg/L pantothenate, 4.6 -18 mg/L choline, 1.3-5.3 mg/L folic acid, 6.2-25 mg/L inositol, 1.0-4.0 mg/L nicotinamide, 1.0-4.0 mg/L pyridotal, 0.015-0.0060 mg/L pyridoxine, 0.11-0.44 mg/L riboflavin, 1.1-4.4 mg/L thiamine, 0.34-1.4 mg/L vitamin B 12 , 56-225 mg/L pyruvate, 700-6300 mg/L glucose, 5.2-22 mg/L phenol red, 0.040-0.16 mg/L putrescine, 1.0-4.0 mg/L hypoxanthine, 0.020-0.080 mg/L linoleic acid, 0.050-0.20 mg/L lipoic acid, 0.18-0.75 mg/L thymidine, 50-200 U/ml penicillin, 50-200 μg/ml streptomycin, 3.2-13 μL/L α-thioglycerol, and 5.0×10 -3 -0.020 mg/L progesterone, when dissolved in said amount of water.
Claim 16
Dependent Claim
11 words
The tissue culture medium of claim 1, comprising a buffering agent.
Claim 17
Dependent Claim
19 words
The tissue culture medium of claim 1, wherein said basal medium has a pH of from 6.5 to 8.2.
Claim 18
Dependent Claim
51 words
In a method of cultivating hybridoma cells in a protein free basal medium, the improvement comprising adding to the basal medium a trace element mixture consisting essentially of copper, iron, zinc, manganese, silicon, molybdenum, banadium, nickel, tin, aluminum, silver, barium, bromine, cadmium, cobalt, chromium, fluorine, germanium, iodine, rubidium, ziroconium, and selenium.
Claim 19
Dependent Claim
51 words
In a method of cultivating hybridoma cells in a protein free basal medium, the improvement comprising adding to the basal medium a trace element mixture consisting essentially of copper, iron, zinc, manganese, silicon, molybdenum, banadium, nickel, tin, aluminum, silver, barium, bromine, cadmium, cobalt, chromium, fluorine, germanium, iodine, rubidium, ziroconium, and selenium.
Claim 20
Dependent Claim
118 words
The method of claim 17 wherein the basal medium contains 8.5 to 34 mg/L L-alanine, 74 to 295 mg/L L-arginine, 10 to 40 mg/L L-asparagine, 11 to 43 mg/L L-aspartic acid, 5.5 to 22 mg/L L-cystine, 24 to 98 mg/L L-cysteine, 22 to 90 mg/L L-glutamic acid, 192 to 730 mg/L L-glutamine, 5.5 to 23 mg/L glycine, 16 to 63 mg/L L-histidine, 27 to 109 mg/L L-isoleucine, 29 to 118 mg/L L-leucine, 46 to 183 mg/L L-lysine, 8.5 to 34 mg/L L-methionine, 18 to 71 mg/L L-phenylalanine, 19 to 75 mg/L L-proline, 13 to 53 mg/L L-serine, 27 to 106 mg/L L-threonine, 4.5 to 18 mg/L L-tryptophan, 26 to 104 mg/L L-tyrosine, and 26 to 106 mg/L valine.
Claim 21
Dependent Claim
61 words
The method of claim 18, wherein the basal medium contains 18 to 72 mg/L Ca 2+ 86 to 345 mg/L K + , 8.6 to 35 mg/L Mg 2+ , 1500 to 6000 mg/L Na + , 1950 to 7800 mg/L Cl - , 48 to 192 mg/L PO 4 3- , and 760 to 3050 mg/L HCO 3 - .
Claim 22
Dependent Claim
157 words
The method of claim 18, wherein the basal medium contains 5.0×10 -3 to 0.020 mg/L biotin, 1.1 to 4.5 mg/L of pantothenate, 4.6 to 18 mg/L of choline, 1.3 to 5.3 mg/L of folic acid, 6.2 to 25 mg/L of inositol, 1.0 to 4.0 mg/L of nicotinamide, 1.0 to 4.0 mg/L of pyridoxal, 0.015 to 0.060 mg/L of pyridoxine, 0.11 to 0.44 mg/L of riboflavin, 1.1 to 4.4 mg/L of thiamine, 0.34 to 1.4 mg/L of vitamin B 12 , 56 to 225 mg/L of pyruvate, 700 to 6300 mg/L of glucose, 5.2 to 22 mg/L of phenol red, 0.040 to 0.16 mg/L of putrescine, 1.0 to 4.0 mg/L of hypoxanthine, 0.020 to 0.080 mg/L of linoleic acid, 0.050 to 0.20 mg/L of lipoic acid, 0.18 to 0.75 mg/L of thymidine, 50 to 200 U/ml of penicillin, 50 to 200 μg/ml of streptomycin, 3.2 to 13 μL/L of α-thioglycerol, and 5.0×10 -3 to 0.020 mg/L of progesterone.
Claim 23
Dependent Claim
217 words
The method of claim 18, comprising: contacting the said cells with a medium containing 5.0×10 -3 to 0.0020 mg/L biotin; 1.1 to 4.5 mg/L of pentothenate; 4.6 to 18 mg/L of choline; 1.3 to 5.3 mg/L of folic acid; 6.2 to 25 mg/L of inositol; 1.0 to 4.0 mg/L of nicotinamide; 1.0 to 4.0 mg/L of pyridoxal; 0.015 to 0.060 mg/L of pyridoxine; 0.11 to 0.44 mg/L of riboflavin; 1.1 to 4.4 mg/L of thiamine; 0.34 to 1.4 mg/L of vitamine B 12 ; 56 to 225 mg/L of pyruvate; 700 to 6300 mg/L of glucose; 5.2 to 22 mg/L of phenol red; 0.040 to 0.16 mg/L of putrescine; 1.0 to 4.0 mg/L of hypoxanthine; 0.020 to 0.080 mg/L of linoleic acid; 0.050 to 0.20 mg/L of lipoic acid; 0.18 to 0.75 mg/L of thymidine; 50 to 200 U/ml of penicillin; 50 to 200 g/ml of streptomycin; 3.2 to 13 L/L of a-thioglycerol; 5.0×10 -3 to 0.020 mg/L of progesterone; 18 to 72 mg/L Ca 2+ ; 86 to 345 mg/L K + ; 8.6 to 35 mg/L Mg 2+ ; 500 to 6000 mg/L Na + ; 1950 to 7800 mg/L Cl - ; 48 to 192 mg/L PO 4 3- ; and 760 to 3050 mg/L HCO 3 - ; and growing the said cells.
Claim 24
Dependent Claim
62 words
The method of claim 17 wherein the basal medium contains 18 to 72 mg/L Ca 2+ , 86 to 345 mg/L K + , 8.6 to 35 mg/L Mg 2+ , 1500 to 6000 mg/L Na + , 1950 to 7800 mg/L Cl - , 48 to 192 mg/L PO 4 3- , and 760 to 3050 mg/L HCO 3 - .
Claim 25
Dependent Claim
157 words
The method of claim 17, wherein the basal medium contains 5.0×10 -3 to 0.020 mg/L biotin, 1.1 to 4.5 mg/L of pantothenate, 4.6 to 18 mg/L of choline, 1.3 to 5.3 mg/L of folic acid, 6.2 to 25 mg/L of inositol, 1.0 to 4.0 mg/L of nicotinamide, 1.0 to 4.0 mg/L of pyridoxal, 0.015 to 0.060 mg/L of pyridoxine, 0.11 to 0.44% mg/L of riboflavin, 1.1 to 4.4 mg/L of thiamine, 0.34 to 1.4 mg/L of vitamin B 12 , 56 to 225 mg/L of pyruvate, 700 to 6300 mg/L of glucose, 5.2 to 22 mg/L of phenol red, 0.040 to 0.16 mg/L of putrescine, 1.0 to 4.0 mg/L of hypoxanthine, 0.020 to 0.080 mg/L of linoleic acid, 0.050 to 0.20 mg/L of lipoic acid, 0.18 to 0.75 mg/L of thymidine, 50 to 200 U/ml of penicillin, 50 to 200 μg/ml of streptomycin, 3.2 to 13 μL/L of α-thioglycerol, and 5.0×10 -3 to 0.020 mg/L of progesterone.
References Cited (Backward Citations)
No citation records indexed.
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