CN100482786C - Human embryo kidney 293 cell amplifying protein-free medium - Google Patents

Human embryo kidney 293 cell amplifying protein-free medium Download PDF

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Publication number
CN100482786C
CN100482786C CNB2007103070612A CN200710307061A CN100482786C CN 100482786 C CN100482786 C CN 100482786C CN B2007103070612 A CNB2007103070612 A CN B2007103070612A CN 200710307061 A CN200710307061 A CN 200710307061A CN 100482786 C CN100482786 C CN 100482786C
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sodium
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hydrochloride
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CN101220347A (en
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黄宗堂
唐贺文
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TIANJIN BAINUOKE MEDICINE BIOTECHNOLOGY CO Ltd
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TIANJIN BAINUOKE MEDICINE BIOTECHNOLOGY CO Ltd
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Abstract

The invention relates to a culture medium and the usage thereof, which particularly relates to a human embryonic kidney 293 cell amplification culture medium and the usage thereof. The invention pertains to the field of biological products. The human embryonic kidney 293 cell amplification protein-free culture medium includes a basic culture medium and further includes a phosphatidylcholine and trace elements. The human embryonic kidney 293 cell protein-free culture medium does not contain any animal serum or protein, and consists of amino acids, vitamins, inorganic salts, sugars, phosphatidyl choline and trace elements. The human embryonic kidney 293 cell serum-free culture medium can directly replace the conventional culture medium which contains serum, and the human embryonic kidney 293 cell does not need an adaptation process.

Description

A kind of human embryo kidney 293 cell amplifying protein-free medium
Technical field
Invention relates to a kind of substratum, more specifically to a kind of human embryo kidney (HEK) 293 cell amplification culture mediums.Belong to field of biological product.
Background technology
Cell culture medium is material commonly used in the field of biological product, and is very necessary in bacterium and cell cultures.Human embryo kidney (HEK) 293 cells are people's renal epithelial cell systems of transfection adenovirus e1a gene.The shortcoming of the substratum of cultivator embryonic kidney 293 cells is at present: at present, animal serum is widely used as the key component of mammalian cell substratum, based on bovine serum.But the animal serum shortcoming is: cost an arm and a leg, composition is indeterminate, and the product differences between batches are big, contains cell growth-inhibiting composition, and even more serious be possible contain bacterium, virus etc., cause the potential source biomolecule contact scar.
Summary of the invention
[problem that will solve]
Purpose of the present invention provides a kind of human embryo kidney (HEK) 293 cell amplification culture mediums and uses thereof.
[technical scheme]
A kind of human embryo kidney 293 cell amplifying protein-free medium of the present invention wherein, comprises minimum medium, also comprises: phosphatidylcholine and trace element.
Component that above-mentioned minimum medium comprises and content are:
Composition Mg/L
Glycine 30-120
L-L-Ala 5-50
L-arginine 100-800
One water L-amino-succinamic acid 3-60
L-aspartic acid 5-80
One water L-cysteine hydrochloride 10-120
L-cystinic acid dihydrochloride 10-120
L-L-glutamic acid 10-100
L-glutaminate 200-3000
One water L-L-Histidine hydrochloride 3-50
L-oxyproline 5-50
L-Isoleucine 30-150
L-leucine 30-300
L-Methionin 5-50
L-methionine(Met) 10-100
L-phenylalanine 2-50
L-proline(Pro) 5-100
L-Serine 10-300
L-Threonine 10-150
L-tryptophane 10-150
L-tyrosine sodium salt 50-500
L-Xie Ansuan 5-50
L-ornithine hydrochloride 50-400
L-citrulline 40-350
D-vitamin H 0.001-0.8
Choline chloride 60 0.1-3
D-pantothenic acid 0.001-0.8
Folic acid 0.001-0.8
Inositol 0.001-0.2
Niacinamide 0.001-0.5
P-benzaminic acid 0.02-2
Pyridoxal hydrochloride 0.01-1
Pyridoxine hydrochloride hydrochloride 0.01-2
Riboflavin 0.001-0.3
Thiamine salt hydrochlorate 0.001-0.4
Vitamins B 120.02-0.6
Racemization tocopherol acetate 0.01-0.2
Nicotinic acid 0.001-0.5
Vitamin A acetic acid 0.001-0.6
Vitamin d 0.001-0.1
VITAMIN B4 0.01-1
Cholesterol 0.01-0.2
Citric acid monohydrate Food grade 0.0001-0.01
2-deoxy-D-ribose 3.5-15
Calcium dichloride dihydrate 0.5-6.5
Magnesium chloride hexahydrate 0.5-7
Magnesium sulfate heptahydrate 6.5-25
Repone K 0.8-4.5
Sodium-chlor 2500-10000
Sodium bicarbonate 500-4500
Glucose 1500-6000
Phenol red sodium 0.8-25
Sodium.alpha.-ketopropionate 45-250
Xanthine 0.9-15
Linolic acid 0.01-0.8
DL-Thioctic Acid 0.1-6.0
Putrescine 0.01-1.2
Gsh 0.01-0.98
Thymidine 0.2-3.0
MES (2-(N-morpholine) ethyl sulfonic acid) 1500-6500
D-ribose 0.05-3.0
Sodium ethylene diamine tetracetate 1.5-15
Guanine hydrochloride 0.01-0.9
Oxalic acid acetate 0.5-15
Cholesterol 0.001-0.08
Sodium nitroprusside 0.5-15
Taurine 10.0-45
Polysorbate 80 (be Tween 80,
Sigma-Aldrich company, article No.
P1754) 0.5-6.5
Iron vitriol 0.5-3.5
Nine water Iron nitrate 0.1-0.85
Zinc Sulphate Heptahydrate 0.5-3.5
Sodium phosphate dibasic 45-300
Above-mentioned preferred ingredients and content are
Composition Mg/L
Glycine 65
L-L-Ala 20
L-arginine 300
One water L-amino-succinamic acid 15
L-aspartic acid 20
One water L-cysteine hydrochloride 50
L-cystinic acid dihydrochloride 50
L-L-glutamic acid 40
L-glutaminate 1000
One water L-L-Histidine hydrochloride 15
L-oxyproline 20
L-Isoleucine 52
L-leucine 72.5
L-Methionin 15
L-methionine(Met) 32
L-phenylalanine 11.5
L-proline 30
L-Serine 66
L-Threonine 40
L-tryptophane 42
L-tyrosine sodium salt 95
L-Xie Ansuan 16
L-ornithine hydrochloride 104
L-citrulline 94
D-vitamin H 0.01
Choline chloride 60 0.5
D-pantothenic acid 0.01
Folic acid 0.01
Inositol 0.05
Niacinamide 0.025
P-benzaminic acid 0.05
Pyridoxal hydrochloride 0.025
Pyridoxine hydrochloride hydrochloride 0.025
Riboflavin 0.01
Thiamine salt hydrochlorate 0.01
Vitamins B 120.1
Racemization tocopherol acetate 0.05
Nicotinic acid 0.01
Vitamin A acetic acid 0.01
Vitamin d 0.025
VITAMIN B4 0.1
Cholesterol 0.09
Citric acid monohydrate Food grade 0.0035
2-deoxy-D-ribose 8.98
Calcium dichloride dihydrate 2.24
Magnesium chloride hexahydrate 2.65
Magnesium sulfate heptahydrate 12.6
Repone K 2.02
Sodium-chlor 6900
Sodium bicarbonate 2250
Glucose 3000
Phenol red sodium 7.3776
Sodium.alpha.-ketopropionate 98.6
Xanthine 4.756
Linolic acid 0.09744
DL-Thioctic Acid 0.754
Putrescine 0.09338
Gsh 0.0348
Thymidine 0.87
MES (2-(N-morpholine) ethyl sulfonic acid) 3654
D-ribose 0.232
Sodium ethylene diamine tetracetate 4.872
Guanine hydrochloride 0.116
Oxalic acid acetate 3.48
Cholesterol 0.00464
Sodium nitroprusside 3.48
Taurine 23.2
Polysorbate 80 (be Tween 80,
Sigma-Aldrich company, article No.
P1754) 2.9
Iron vitriol 1.2504
Nine water Iron nitrates 0.3126
Zinc Sulphate Heptahydrate 1.563
Sodium phosphate dibasic 125
Component that above-mentioned phosphatidylcholine comprises and content are:
Composition Mg/L
Dilinoleoylphosphatidylcholine (Dilineoyl-phosphatidylcholin) 0.5-3.5
Distearoyl phosphatidylcholine (Distearoyl-phosphatidylcholin) 0.1-1.8
Component that above-mentioned phosphatidylcholine comprises and content are
Composition Mg/L
Dilinoleoylphosphatidylcholine (Dilineoyl-phosphatidylcholin) 1.2504
Distearoyl phosphatidylcholine (Distearoyl-phosphatidylcholin) 0.9378
Component that above-mentioned trace element comprises and content are:
Composition Mg/L
Cupric sulfate pentahydrate 0.001-0.08
Sodium selenite (Na2SeO3) pentahydrate 0.001-0.05
Sodium Fluoride 0.001-0.08
Nine water Sodium Selenite 0.001-0.5
CoCL2 0.001-0.045
Barium chloride 0.001-0.08
Lithium chloride 0.0031-0.015
Sodium Molybdate Dihydrate 0.00001-0.0007
Ammonium meta-vanadate 0.0001-0.006
Nickelous chloride 0.000008-0.0002
Nickelous nitrate 0.00002-0.002
Rubidium chloride 0.000003-0.0005
Tin protochloride 0.0006-0.03
Titanium dioxide 0.001-0.0085
Silver chloride 0.000001-0.00009
Six water aluminum chloride 0.0005-0.035
Potassium chromium sulfate 0.0006-0.036
Germanium oxide 0.0001-0.0096
Potassium Bromide 0.00001-0.00025
Potassiumiodide 0.00006-0.0032
Manganous chloride tetrahydrate 0.00006-0.0033
Component that above-mentioned trace element comprises and content are:
Composition Mg/L
Cupric sulfate pentahydrate 0.007815
Sodium selenite (Na2SeO3) pentahydrate 0.007127
Sodium Fluoride 0.009378
Nine water Sodium Selenites 0.012504
CoCL2 0.003126
Barium chloride 0.00719
Lithium chloride 0.007815
Sodium Molybdate Dihydrate 0.000156
Ammonium meta-vanadate 0.000625
Nickelous chloride 0.000016
Nickelous nitrate 0.000313
Rubidium chloride 0.000013
Tin protochloride 0.002188
Titanium dioxide 0.002501
Silver chloride 0.000009
Six water aluminum chlorides 0.002501
Potassium chromium sulfate 0.002188
Germanium oxide 0.000938
Potassium Bromide 0.000078
Potassiumiodide 0.00025
Manganous chloride tetrahydrate 0.00025
Above-mentioned substratum has the purposes in cultivator embryonic kidney 293 cells, and described substratum can directly be replaced and contains the conventional substratum of serum, and human embryo kidney (HEK) 293 cells do not need adaptive process.
The condition that substratum uses is as follows: culture condition: 37 ℃ of temperature, CO 2Concentration 5%, relative humidity〉95%.
[advantage of invention product]
These human embryo kidney (HEK) 293 cell protein-free mediums do not contain any animal serum, do not contain protein, with amino acid, and VITAMIN, inorganic salt, carbohydrate, phosphatidylcholine and trace element are formed.These human embryo kidney (HEK) 293 cell non-serum culture mediums can directly be replaced and contain the conventional substratum of serum, and human embryo kidney (HEK) 293 cells do not need adaptive process.
Description of drawings
Fig. 1 is the cultivation figure of serum free medium human embryo kidney (HEK) 293 cell amplification culture medium embodiment 5;
Fig. 2 is the cultivation figure of serum free medium human embryo kidney (HEK) 293 cell amplification culture medium embodiment 4.
Embodiment
The following embodiment content in the book that is to explain is the embodiment of content, and is not intended to the protection domain of restriction invention.
Embodiment 1
Substratum is made up of minimum medium, phosphatidylcholine and trace element, and wherein, the composition and the composition of minimum medium are as follows:
Composition Mg/L
Glycine 65
L-L-Ala 20
L-arginine 300
One water L-amino-succinamic acid 15
L-aspartic acid 20
One water L-cysteine hydrochloride 50
L-cystinic acid dihydrochloride 50
L-L-glutamic acid 40
L-glutaminate 1000
One water L-L-Histidine hydrochloride 15
L-oxyproline 20
L-Isoleucine 52
L-leucine 72.5
L-Methionin 15
L-methionine(Met) 32
L-phenylalanine 11.5
L-proline 30
L-Serine 66
L-Threonine 40
L-tryptophane 42
L-tyrosine sodium salt 95
L-Xie Ansuan 16
L-ornithine hydrochloride 104
L-citrulline 94
D-vitamin H 0.01
Choline chloride 60 0.5
D-pantothenic acid 0.01
Folic acid 0.01
Inositol 0.05
Niacinamide 0.025
P-benzaminic acid 0.05
Pyridoxal hydrochloride 0.025
Pyridoxine hydrochloride hydrochloride 0.025
Riboflavin 0.01
Thiamine salt hydrochlorate 0.01
Vitamins B 120.1
Racemization tocopherol acetate 0.05
Nicotinic acid 0.01
Vitamin A acetic acid 0.01
Vitamin d 0.025
VITAMIN B4 0.1
Cholesterol 0.09
Citric acid monohydrate Food grade 0.0035
2-deoxy-D-ribose 8.98
Calcium dichloride dihydrate 2.24
Magnesium chloride hexahydrate 2.65
Magnesium sulfate heptahydrate 12.6
Repone K 2.02
Sodium-chlor 6900
Sodium bicarbonate 2250
Glucose 3000
Phenol red sodium 7.3776
Sodium.alpha.-ketopropionate 98.6
Xanthine 4.756
Linolic acid 0.09744
DL-Thioctic Acid 0.754
Putrescine 0.09338
Gsh 0.0348
Thymidine 0.87
MES (2-(N-morpholine) ethyl sulfonic acid) 3654
D-ribose 0.232
Sodium ethylene diamine tetracetate 4.872
Guanine hydrochloride 0.116
Oxalic acid acetate 3.48
Cholesterol 0.00464
Sodium nitroprusside 3.48
Taurine 23.2
Polysorbate 80 (be Tween 80,
Sigma-Aldrich company, article No.
P1754) 2.9
Iron vitriol 1.2504
Nine water Iron nitrates 0.3126
Zinc Sulphate Heptahydrate 1.563
Sodium phosphate dibasic 125
Component that phosphatidylcholine comprises and content are
Composition Mg/L
Dilinoleoylphosphatidylcholine (Dilineoyl-phosphatidylcholin) 1.2504
Distearoyl phosphatidylcholine (Distearoyl-phosphatidylcholin) 0.9378
Component that trace element comprises and content are:
Composition Mg/L
Cupric sulfate pentahydrate 0.007815
Sodium selenite (Na2SeO3) pentahydrate 0.007127
Sodium Fluoride 0.009378
Nine water Sodium Selenites 0.012504
CoCL2 0.003126
Barium chloride 0.00719
Lithium chloride 0.007815
Sodium Molybdate Dihydrate 0.000156
Ammonium meta-vanadate 0.000625
Nickelous chloride 0.000016
Nickelous nitrate 0.000313
Rubidium chloride 0.000013
Tin protochloride 0.002188
Titanium dioxide 0.002501
Silver chloride 0.000009
Six water aluminum chlorides 0.002501
Potassium chromium sulfate 0.002188
Germanium oxide 0.000938
Potassium Bromide 0.000078
Potassiumiodide 0.00025
Manganous chloride tetrahydrate 0.00025
Embodiment 2
Substratum is made up of minimum medium, phosphatidylcholine and trace element, and wherein, the composition and the composition of minimum medium are as follows:
Composition Mg/L
Glycine 30
L-L-Ala 50
L-arginine 100
One water L-amino-succinamic acid 60
L-aspartic acid 5
One water L-cysteine hydrochloride 120
L-cystinic acid dihydrochloride 10
L-L-glutamic acid 100
L-glutaminate 200
One water L-L-Histidine hydrochloride 50
L-oxyproline 5
L-Isoleucine 150
L-leucine 30
L-Methionin 50
L-methionine(Met) 10
L-phenylalanine 50
L-proline(Pro) 5
L-Serine 300
L-Threonine 10
L-tryptophane 150
L-tyrosine sodium salt 50
L-Xie Ansuan 50
L-ornithine hydrochloride 50
L-citrulline 350
D-vitamin H 0.001
Choline chloride 60 3
D-pantothenic acid 0.001
Folic acid 0.8
Inositol 0.001
Niacinamide 0.5
P-benzaminic acid 0.02
Pyridoxal hydrochloride 1
Pyridoxine hydrochloride hydrochloride 0.01
Riboflavin 0.3
Thiamine salt hydrochlorate 0.001
Vitamins B 120.6
Racemization tocopherol acetate 0.01
Nicotinic acid 0.5
Vitamin A acetic acid 0.001
Vitamin d 0.1
VITAMIN B4 0.01
Cholesterol 0.2
Citric acid monohydrate Food grade 0.0001
2-deoxy-D-ribose 15
Calcium dichloride dihydrate 0.5
Magnesium chloride hexahydrate 7
Magnesium sulfate heptahydrate 6.5
Repone K 4.5
Sodium-chlor 2500
Sodium bicarbonate 4500
Glucose 1500
Phenol red sodium 25
Sodium.alpha.-ketopropionate 45
Xanthine 15
Linolic acid 0.01
DL-Thioctic Acid 6.0
Putrescine 0.01
Gsh 0.98
Thymidine 0.2
MES (2-(N-morpholine) ethyl sulfonic acid) 6500
D-ribose 0.05
Sodium ethylene diamine tetracetate 15
Guanine hydrochloride 0.01
Oxalic acid acetate 15
Cholesterol 0.001
Sodium nitroprusside 15
Taurine 10
Polysorbate 80 (be Tween 80,
Sigma-Aldrich company, article No.
P1754) 6.5
Iron vitriol 0.5
Nine water Iron nitrates 0.85
Zinc Sulphate Heptahydrate 0.5
Sodium phosphate dibasic 300
Component that phosphatidylcholine comprises and content are
Composition Mg/L
Dilinoleoylphosphatidylcholine (Dilineoyl-phosphatidylcholin) 0.5
Distearoyl phosphatidylcholine (Distearoyl-phosphatidylcholin) 1.8
Component that trace element comprises and content are:
Composition Mg/L
Cupric sulfate pentahydrate 0.001
Sodium selenite (Na2SeO3) pentahydrate 0.05
Sodium Fluoride 0.001
Nine water Sodium Selenites 0.5
CoCL2 0.001
Barium chloride 0.08
Lithium chloride 0.0031
Sodium Molybdate Dihydrate 0.0007
Ammonium meta-vanadate 0.0001
Nickelous chloride 0.0002
Nickelous nitrate 0.00002
Rubidium chloride 0.0005
Tin protochloride 0.0006
Titanium dioxide 0.0085
Silver chloride 0.000001
Six water aluminum chlorides 0.035
Potassium chromium sulfate 0.0006
Germanium oxide 0.0096
Potassium Bromide 0.00001
Potassiumiodide 0.0032
Manganous chloride tetrahydrate 0.00006
Embodiment 3
Substratum is made up of minimum medium, phosphatidylcholine and trace element, and wherein, the composition and the composition of minimum medium are as follows:
Composition Mg/L
Glycine 120
L-L-Ala 5
L-arginine 800
One water L-amino-succinamic acid 3
L-aspartic acid 80
One water L-cysteine hydrochloride 10
L-cystinic acid dihydrochloride 120
L-L-glutamic acid 10
L-glutaminate 3000
One water L-L-Histidine hydrochloride 3
L-oxyproline 50
L-Isoleucine 30
L-leucine 300
L-Methionin 5
L-methionine(Met) 100
L-phenylalanine 2
L-proline(Pro) 100
L-Serine 10
L-Threonine 150
L-tryptophane 10
L-tyrosine sodium salt 500
L-Xie Ansuan 5
L-ornithine hydrochloride 400
L-citrulline 40
D-vitamin H 0.8
Choline chloride 60 0.1
D-pantothenic acid 0.8
Folic acid 0.001
Inositol 0.2
Niacinamide 0.001
P-benzaminic acid 2
Pyridoxal hydrochloride 0.01
Pyridoxine hydrochloride hydrochloride 2
Riboflavin 0.001
Thiamine salt hydrochlorate 0.4
Vitamins B 120.02
Racemization tocopherol acetate 0.2
Nicotinic acid 0.001
Vitamin A acetic acid 0.6
Vitamin d 0.001
VITAMIN B4 1
Cholesterol 0.01
Citric acid monohydrate Food grade 0.01
2-deoxy-D-ribose 3.5
Calcium dichloride dihydrate 6.5
Magnesium chloride hexahydrate 0.5
Magnesium sulfate heptahydrate 25
Repone K 0.8
Sodium-chlor 10000
Sodium bicarbonate 500
Glucose 6000
Phenol red sodium 0.8
Sodium.alpha.-ketopropionate 250
Xanthine 0.9
Linolic acid 0.8
DL-Thioctic Acid 0.1
Putrescine 1.2
Gsh 0.01
Thymidine 3.0
MES (2-(N-morpholine) ethyl sulfonic acid) 1500
D-ribose 3.0
Sodium ethylene diamine tetracetate 1.5
Guanine hydrochloride 0.9
Oxalic acid acetate 0.5
Cholesterol 0.08
Sodium nitroprusside 0.5
Taurine 45
Polysorbate 80 (be Tween 80,
Sigma-Aldrich company, article No.
P1754) 0.5
Iron vitriol 3.5
Nine water Iron nitrates 0.1
Zinc Sulphate Heptahydrate 3.5
Sodium phosphate dibasic 45
Component that phosphatidylcholine comprises and content are
Composition Mg/L
Dilinoleoylphosphatidylcholine (Dilineoyl-phosphatidylcholin) 3.5
Distearoyl phosphatidylcholine (Distearoyl-phosphatidylcholin) 0.1
Component that trace element comprises and content are:
Composition Mg/L
Cupric sulfate pentahydrate 0.08
Sodium selenite (Na2SeO3) pentahydrate 0.001
Sodium Fluoride 0.08
Nine water Sodium Selenites 0.001
CoCL2 0.045
Barium chloride 0.001
Lithium chloride 0.015
Sodium Molybdate Dihydrate 0.00001
Ammonium meta-vanadate 0.006
Nickelous chloride 0.000008
Nickelous nitrate 0.002
Rubidium chloride 0.000003
Tin protochloride 0.03
Titanium dioxide 0.001
Silver chloride 0.00009
Six water aluminum chlorides 0.0005
Potassium chromium sulfate 0.036
Germanium oxide 0.0001
Potassium Bromide 0.00025
Potassiumiodide 0.00006
Manganous chloride tetrahydrate 0.0033
Embodiment 4
Substratum is made up of minimum medium, phosphatidylcholine and trace element, and wherein, the composition and the composition of minimum medium are as follows:
Composition Mg/L
Glycine 30
L-L-Ala 5
L-arginine 100
One water L-amino-succinamic acid 3
L-aspartic acid 5
One water L-cysteine hydrochloride 10
L-cystinic acid dihydrochloride 10
L-L-glutamic acid 10
L-glutaminate 200
One water L-L-Histidine hydrochloride 3
L-oxyproline 5
L-Isoleucine 30
L-leucine 30
L-Methionin 5
L-methionine(Met) 10
L-phenylalanine 2
L-proline(Pro) 5
L-Serine 10
L-Threonine 10
L-tryptophane 10
L-tyrosine sodium salt 50
L-Xie Ansuan 5
L-ornithine hydrochloride 50
L-citrulline 40
D-vitamin H 0.001
Choline chloride 60 0.1
D-pantothenic acid 0.001
Folic acid 0.001
Inositol 0.001
Niacinamide 0.001
P-benzaminic acid 0.02
Pyridoxal hydrochloride 0.01
Pyridoxine hydrochloride hydrochloride 0.01
Riboflavin 0.001
Thiamine salt hydrochlorate 0.001
Vitamins B 120.02
Racemization tocopherol acetate 0.01
Nicotinic acid 0.001
Vitamin A acetic acid 0.001
Vitamin d 0.001
VITAMIN B4 0.01
Cholesterol 0.01
Citric acid monohydrate Food grade 0.0001
2-deoxy-D-ribose 3.5
Calcium dichloride dihydrate 0.5
Magnesium chloride hexahydrate 0.5
Magnesium sulfate heptahydrate 6.5
Repone K 0.8
Sodium-chlor 2500
Sodium bicarbonate 500
Glucose 1500
Phenol red sodium 0.8
Sodium.alpha.-ketopropionate 45
Xanthine 0.9
Linolic acid 0.01
DL-Thioctic Acid 0.1
Putrescine 0.01
Gsh 0.01
Thymidine 0.2
MES (2-(N-morpholine) ethyl sulfonic acid) 1500
D-ribose 0.05
Sodium ethylene diamine tetracetate 1.5
Guanine hydrochloride 0.01
Oxalic acid acetate 0.5
Cholesterol 0.001
Sodium nitroprusside 0.5
Taurine 10.0
Polysorbate 80 (be Tween 80,
Sigma-Aldrich company, article No.
P1754) 0.5
Iron vitriol 0.5
Nine water Iron nitrates 0.1
Zinc Sulphate Heptahydrate 0.5
Sodium phosphate dibasic 45
Component that phosphatidylcholine comprises and content are
Composition Mg/L
Dilinoleoylphosphatidylcholine (Dilineoyl-phosphatidylcholin) 0.5
Distearoyl phosphatidylcholine (Distearoyl-phosphatidylcholin) 0.1
Component that trace element comprises and content are:
Composition Mg/L
Cupric sulfate pentahydrate 0.001
Sodium selenite (Na2SeO3) pentahydrate 0.001
Sodium Fluoride 0.001
Nine water Sodium Selenites 0.001
CoCL2 0.001
Barium chloride 0.001
Lithium chloride 0.0031
Sodium Molybdate Dihydrate 0.00001
Ammonium meta-vanadate 0.0001
Nickelous chloride 0.000008
Nickelous nitrate 0.00002
Rubidium chloride 0.000003
Tin protochloride 0.0006
Titanium dioxide 0.001
Silver chloride 0.000001
Six water aluminum chlorides 0.0005
Potassium chromium sulfate 0.0006
Germanium oxide 0.0001
Potassium Bromide 0.00001
Potassiumiodide 0.00006
Manganous chloride tetrahydrate 0.00006
Embodiment 5
Substratum is made up of minimum medium, phosphatidylcholine and trace element, and wherein, the composition and the composition of minimum medium are as follows:
Composition Mg/L
Glycine 120
L-L-Ala 50
L-arginine 800
One water L-amino-succinamic acid 60
L-aspartic acid 80
One water L-cysteine hydrochloride 120
L-cystinic acid dihydrochloride 120
L-L-glutamic acid 100
L-glutaminate 3000
One water L-L-Histidine hydrochloride 50
L-oxyproline 50
L-Isoleucine 150
L-leucine 300
L-Methionin 50
L-methionine(Met) 100
L-phenylalanine 50
L-proline(Pro) 100
L-Serine 300
L-Threonine 150
L-tryptophane 150
L-tyrosine sodium salt 500
L-Xie Ansuan 50
L-ornithine hydrochloride 400
L-citrulline 350
D-vitamin H 0.8
Choline chloride 60 3
D-pantothenic acid 0.8
Folic acid 0.8
Inositol 0.2
Niacinamide 0.5
P-benzaminic acid 2
Pyridoxal hydrochloride 1
Pyridoxine hydrochloride hydrochloride 2
Riboflavin 0.3
Thiamine salt hydrochlorate 0.4
Vitamins B 120.6
Racemization tocopherol acetate 0.2
Nicotinic acid 0.5
Vitamin A acetic acid 0.6
Vitamin d 0.1
VITAMIN B4 1
Cholesterol 0.2
Citric acid monohydrate Food grade 0.01
2-deoxy-D-ribose 15
Calcium dichloride dihydrate 6.5
Magnesium chloride hexahydrate 7
Magnesium sulfate heptahydrate 25
Repone K 4.5
Sodium-chlor 10000
Sodium bicarbonate 4500
Glucose 6000
Phenol red sodium 25
Sodium.alpha.-ketopropionate 250
Xanthine 15
Linolic acid 0.8
DL-Thioctic Acid 6.0
Putrescine 1.2
Gsh 0.98
Thymidine 3.0
MES (2-(N-morpholine) ethyl sulfonic acid) 6500
D-ribose 3.0
Sodium ethylene diamine tetracetate 15
Guanine hydrochloride 0.9
Oxalic acid acetate 15
Cholesterol 0.08
Sodium nitroprusside 15
Taurine 45
Polysorbate 80 (be Tween 80,
Sigma-Aldrich company, article No. P1754) 6.5
Iron vitriol 3.5
Nine water Iron nitrates 0.85
Zinc Sulphate Heptahydrate 3.5
Sodium phosphate dibasic 300
Component that phosphatidylcholine comprises and content are
Composition Mg/L
Dilinoleoylphosphatidylcholine (Dilineoyl-phosphatidylcholin) 3.5
Distearoyl phosphatidylcholine (Distearoyl-phosphatidylcholin) 1.8
Component that trace element comprises and content are:
Composition Mg/L
Cupric sulfate pentahydrate 0.08
Sodium selenite (Na2SeO3) pentahydrate 0.05
Sodium Fluoride 0.08
Nine water Sodium Selenites 0.5
CoCL2 0.045
Barium chloride 0.08
Lithium chloride 0.015
Sodium Molybdate Dihydrate 0.0007
Ammonium meta-vanadate 0.006
Nickelous chloride 0.0002
Nickelous nitrate 0.002
Rubidium chloride 0.0005
Tin protochloride 0.03
Titanium dioxide 0.0085
Silver chloride 0.00009
Six water aluminum chlorides 0.035
Potassium chromium sulfate 0.036
Germanium oxide 0.0096
Potassium Bromide 0.00025
Potassiumiodide 0.0032
Manganous chloride tetrahydrate 0.0033

Claims (6)

1. a human embryo kidney 293 cell amplifying protein-free medium is characterized in that: comprise minimum medium, also comprise: phosphatidylcholine and trace element;
The component of wherein said minimum medium and content are:
Composition Mg/L
Glycine 30-120
L-L-Ala 5-50
L-arginine 100-800
One water L-amino-succinamic acid 3-60
L-aspartic acid 5-80
One water L-cysteine hydrochloride 10-120
L-cystinic acid dihydrochloride 10-120
L-L-glutamic acid 10-100
L-glutaminate 200-3000
One water L-L-Histidine hydrochloride 3-50
L-oxyproline 5-50
L-Isoleucine 30-150
L-leucine 30-300
L-Methionin 5-50
L-methionine(Met) 10-100
L-phenylalanine 2-50
L-proline(Pro) 5-100
L-Serine 10-300
L-Threonine 10-150
L-tryptophane 10-150
L-tyrosine sodium salt 50-500
L-Xie Ansuan 5-50
L-ornithine hydrochloride 50-400
L-citrulline 40-350
D-vitamin H 0.001-0.8
Choline chloride 60 0.1-3
D-pantothenic acid 0.001-0.8
Folic acid 0.001-0.8
Inositol 0.001-0.2
Niacinamide 0.001-0.5
P-benzaminic acid 0.02-2
Pyridoxal hydrochloride 0.01-1
Pyridoxine hydrochloride hydrochloride 0.01-2
Riboflavin 0.001-0.3
Thiamine salt hydrochlorate 0.001-0.4
Vitamins B 120.02-0.6
Racemization tocopherol acetate 0.01-0.2
Nicotinic acid 0.001-0.5
Vitamin A acetic acid 0.001-0.6
Vitamin d 0.001-0.1
VITAMIN B4 0.01-1
Cholesterol 0.01-0.2
Citric acid monohydrate Food grade 0.0001-0.01
2-deoxy-D-ribose 3.5-15
Calcium dichloride dihydrate 0.5-6.5
Magnesium chloride hexahydrate 0.5-7
Magnesium sulfate heptahydrate 6.5-25
Repone K 0.8-4.5
Sodium-chlor 2500-10000
Sodium bicarbonate 500-4500
Glucose 1500-6000
Phenol red sodium 0.8-25
Sodium.alpha.-ketopropionate 45-250
Xanthine 0.9-15
Linolic acid 0.01-0.8
DL-Thioctic Acid 0.1-6.0
Putrescine 0.01-1.2
Gsh 0.01-0.98
Thymidine 0.2-3.0
MES (2-(N-morpholine) ethyl sulfonic acid) 1500-6500
D-ribose 0.05-3.0
Sodium ethylene diamine tetracetate 1.5-15
Guanine hydrochloride 0.01-0.9
Oxalic acid acetate 0.5-15
Cholesterol 0.001-0.08
Sodium nitroprusside 0.5-15
Taurine 10.0-45
Polysorbate 80 0.5-6.5
Iron vitriol 0.5-3.5
Nine water Iron nitrate 0.1-0.85
Zinc Sulphate Heptahydrate 0.5-3.5
Sodium phosphate dibasic 45-300;
Wherein, the component of described trace element and content are:
Composition Mg/L
Cupric sulfate pentahydrate 0.001-0.08
Sodium selenite (Na2SeO3) pentahydrate 0.001-0.05
Sodium Fluoride 0.001-0.08
Nine water Sodium Selenite 0.001-0.5
CoCL2 0.001-0.045
Barium chloride 0.001-0.08
Lithium chloride 0.0031-0.015
Sodium Molybdate Dihydrate 0.00001-0.0007
Ammonium meta-vanadate 0.0001-0.006
Nickelous chloride 0.000008-0.0002
Nickelous nitrate 0.00002-0.002
Rubidium chloride 0.000003-0.0005
Tin protochloride 0.0006-0.03
Titanium dioxide 0.001-0.0085
Silver chloride 0.000001-0.00009
Six water aluminum chloride 0.0005-0.035
Potassium chromium sulfate 0.0006-0.036
Germanium oxide 0.0001-0.0096
Potassium Bromide 0.00001-0.00025
Potassiumiodide 0.00006-0.0032
Manganous chloride tetrahydrate 0.00006-0.0033;
Wherein, the component of described phosphatidylcholine and content are:
Composition Mg/L
Dilinoleoylphosphatidylcholine (Dilineoyl-phosphatidylcholin) 0.5-3.5
Distearoyl phosphatidylcholine (Distearoyl-phosphatidylcholin) 0.1-1.8.
2. substratum as claimed in claim 1 is characterized in that: the component of described minimum medium and content are:
Composition Mg/L
Glycine 65
L-L-Ala 20
L-arginine 300
One water L-amino-succinamic acid 15
L-aspartic acid 20
One water L-cysteine hydrochloride 50
L-cystinic acid dihydrochloride 50
L-L-glutamic acid 40
L-glutaminate 1000
One water L-L-Histidine hydrochloride 15
L-oxyproline 20
L-Isoleucine 52
L-leucine 72.5
L-Methionin 15
L-methionine(Met) 32
L-phenylalanine 11.5
L-proline 30
L-Serine 66
L-Threonine 40
L-tryptophane 42
L-tyrosine sodium salt 95
L-Xie Ansuan 16
L-ornithine hydrochloride 104
L-citrulline 94
D-vitamin H 0.01
Choline chloride 60 0.5
D-pantothenic acid 0.01
Folic acid 0.01
Inositol 0.05
Niacinamide 0.025
P-benzaminic acid 0.05
Pyridoxal hydrochloride 0.025
Pyridoxine hydrochloride hydrochloride 0.025
Riboflavin 0.01
Thiamine salt hydrochlorate 0.01
Vitamins B 120.1
Racemization tocopherol acetate 0.05
Nicotinic acid 0.01
Vitamin A acetic acid 0.01
Vitamin d 0.025
VITAMIN B4 0.1
Cholesterol 0.09
Citric acid monohydrate Food grade 0.0035
2-deoxy-D-ribose 8.98
Calcium dichloride dihydrate 2.24
Magnesium chloride hexahydrate 2.65
Magnesium sulfate heptahydrate 12.6
Repone K 2.02
Sodium-chlor 6900
Sodium bicarbonate 2250
Glucose 3000
Phenol red sodium 7.3776
Sodium.alpha.-ketopropionate 98.6
Xanthine 4.756
Linolic acid 0.09744
DL-Thioctic Acid 0.754
Putrescine 0.09338
Gsh 0.0348
Thymidine 0.87
MES (2-(N-morpholine) ethyl sulfonic acid) 3654
D-ribose 0.232
Sodium ethylene diamine tetracetate 4.872
Guanine hydrochloride 0.116
Oxalic acid acetate 3.48
Cholesterol 0.00464
Sodium nitroprusside 3.48
Taurine 23.2
Polysorbate 80 2.9
Iron vitriol 1.2504
Nine water Iron nitrates 0.3126
Zinc Sulphate Heptahydrate 1.563
Sodium phosphate dibasic 125.
3. substratum as claimed in claim 1 is characterized in that: the component of described trace element and content are:
Composition Mg/L
Cupric sulfate pentahydrate 0.007815
Sodium selenite (Na2SeO3) pentahydrate 0.007127
Sodium Fluoride 0.009378
Nine water Sodium Selenites 0.012504
CoCL2 0.003126
Barium chloride 0.00719
Lithium chloride 0.007815
Sodium Molybdate Dihydrate 0.000156
Ammonium meta-vanadate 0.000625
Nickelous chloride 0.000016
Nickelous nitrate 0.000313
Rubidium chloride 0.000013
Tin protochloride 0.002188
Titanium dioxide 0.002501
Silver chloride 0.000009
Six water aluminum chlorides 0.002501
Potassium chromium sulfate 0.002188
Germanium oxide 0.000938
Potassium Bromide 0.000078
Potassiumiodide 0.00025
Manganous chloride tetrahydrate 0.00025.
4. substratum as claimed in claim 1 is characterized in that: the component of described phosphatidylcholine and content are
Composition Mg/L
Dilinoleoylphosphatidylcholine (Dilineoyl-phosphatidylcholin) 1.2504
Distearoyl phosphatidylcholine (Distearoyl-phosphatidylcholin) 0.9378.
5. as the purposes of any described substratum in cultivator embryonic kidney 293 cells among the claim 1-4.
6. purposes as claimed in claim 5 is characterized in that: the condition of use is as follows: culture condition: 37 ℃ of temperature, CO 2Concentration 5%, relative humidity〉95%.
CNB2007103070612A 2007-12-28 2007-12-28 Human embryo kidney 293 cell amplifying protein-free medium Expired - Fee Related CN100482786C (en)

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