CN101182490A - Culture medium used for Vero cell and cultivation method thereof - Google Patents

Culture medium used for Vero cell and cultivation method thereof Download PDF

Info

Publication number
CN101182490A
CN101182490A CNA2007101874667A CN200710187466A CN101182490A CN 101182490 A CN101182490 A CN 101182490A CN A2007101874667 A CNA2007101874667 A CN A2007101874667A CN 200710187466 A CN200710187466 A CN 200710187466A CN 101182490 A CN101182490 A CN 101182490A
Authority
CN
China
Prior art keywords
acid
sodium
hydrochloride
vitamin
composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CNA2007101874667A
Other languages
Chinese (zh)
Other versions
CN100482783C (en
Inventor
黄宗堂
唐贺文
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
TIANJIN BAINUOKE MEDICINE BIOTECHNOLOGY CO Ltd
Original Assignee
TIANJIN BAINUOKE MEDICINE BIOTECHNOLOGY CO Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by TIANJIN BAINUOKE MEDICINE BIOTECHNOLOGY CO Ltd filed Critical TIANJIN BAINUOKE MEDICINE BIOTECHNOLOGY CO Ltd
Priority to CNB2007101874667A priority Critical patent/CN100482783C/en
Publication of CN101182490A publication Critical patent/CN101182490A/en
Application granted granted Critical
Publication of CN100482783C publication Critical patent/CN100482783C/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Cosmetics (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention relates to the preparation and application method of a medium, which is specifically related to a medium which is used for Vero cell and a culture method of the Vero cell. The invention belongs to the biological product area. The culture method of the invention is that the adhering Vero cells are suspend and cultured in SFM serum-free medium for the domestication and adaptation; the growing pattern of the Vero cell changes from adherence growth to suspension growth; the Vero cells are further amplified in Vero amplification CHEMICALLY DEFINED MEDIUM. The method of the invention has good effects; the growing pattern of the Vero cell is transformed from adherence growth to suspension growth, which changes the growth pattern of the cell and greatly increases the yield of the cell.

Description

A kind of substratum and cultural method thereof that is used for the Vero cell
Technical field
Invention relates to a kind of culture medium preparation and using method, more specifically to a kind of substratum and cultural method thereof that is used for the Vero cell.Belong to field of biological product.
Background technology
Animal serum is that substratum is a material commonly used in the field of biological product, very necessary in bacterium and cell cultures, the shortcoming that but serum has obviously and can't customer service: composition is indeterminate, the product differences between batches are big, spread germs diseases such as virus easily, cost an arm and a leg, the recombinant protein that uses the serum cultured cells to be produced, its downstream purification complex process, with high costs.Used Vero cell characteristics is an adherent growth in the recombinant protein industrial production, and the attaching substrate area that its culture system can provide is limited, makes its propagation amplification quantity be difficult to promote significantly.
Summary of the invention
[problem that will solve]
Purpose of the present invention, providing a kind of Vero cell that can make cultivates through adaptability, its growth pattern is converted into the method for suspension growth from adherent growth, and the substratum that the present invention describes does not contain any animal serum composition, and a kind of substratum and cultural method thereof of the Vero of being used for cell promptly is provided.
[technical scheme]
A kind of Vero cell suspension of the present invention adapts to the SFM serum free medium, and wherein, the composition of substratum and weight part are:
Composition mg/L
Glycine (Glycine) 10-50
L-L-Ala (L-Alanine) 5-30
L-arginine (L-Arginine) 30-200
L-amino-succinamic acid (L-Asparagine) 10-60
L-aspartic acid (L-Aspartic acid) 1-15
L-cysteine hydrochloride (L-Cysteine hydrochloride-H 2O) 0.01-0.6
L-cystinic acid dihydrochloride (L-Cystine 2HCl) 15-100
L-L-glutamic acid (L-Glutamic Acid) 10-100
L-glutaminate (L-Glutamine) 50-200
L-Histidine (L-Histidine) 5-100
L-oxyproline (L-Hydroxyproline) 2-20
L-Isoleucine (L-Isoleucine) 10-100
L-leucine (L-Leucine) 10-100
L lysine HCL (L-Lysine hydrochloride) 15-120
L-methionine(Met) (L-Methionine) 2-30
L-phenylalanine (L-Phenyla lanine) 5-50
L-proline(Pro) (L-Proline) 10-100
L-Serine (L-Serine) 5-60
L-Threonine (L-Threonine) 5-50
L-tryptophane (L-Tryptophan) 2-50
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 5-100
L-Xie Ansuan (L-Valine) 5-100
Vitamin H (Biotin) 0.00001-0.5
Choline chloride 60 (Choline chloride) 0.5-20.
Calcium pantothenate (D-Calcium pantothenated) 0.001-0.9
Folic acid (Folic Acid) 0.001-1.5
Inositol (i-Inositol) 2.-30
Niacinamide (Niacinamide) 0.0001-0.98
Padil (Para-Aminobenzoic Acid) 0.05-3.
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.002-0.7
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.006-3.5
Riboflavin (Riboflavin) 0.0001-6.
Thiamine hydrochloride (Thiamine hydrochloride) 0.003-0.15
Vitamins B 12(Vitamin B 12) 0.002-1.8
Xitix (Ascorbic Acid) 0.01-1.2
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.001-3
(four water) nitrocalcite (Calcium nitrate (Ca (NO 3) 24H 2O) 50-200
(anhydrous) magnesium chloride (Magnesium Chloride (anhydrous)) 3-40
(anhydrous) sal epsom
25-150
(Magnesium Sulfate(MgSO 4)(anhyd.))
Repone K (Potassium Chloride (KCl)) 150-600
Sodium-chlor (Sodium Chloride (NaCl)) 3000-10000
One water Sodium phosphate dibasic
5-200
(Sodium Phosphate monobasic(NaH 2PO 4-H 2O))
Cupric sulfate pentahydrate (Cupric sulfate (CuSO 4-5H 2O)) 0.0001-0.03
Iron vitriol (Ferric sulfate (FeSO 4-7H 2O)) 0.002-0.8
Nine water iron nitrates (Ferric Nitrate (Fe (NO 3) 3-9H 2O)) 0.02-3.5
Zinc Sulphate Heptahydrate (Zinc sulfate (ZnSO 4-7H 2O)) 0.003-0.86
Disodium hydrogen phosphate,anhydrous
50-300
(Sodium Phosphate dibasic(Na 2HPO 4)anhydrous)
Glucose (D-Glucose (Dextrose)) 800-2000
Phenol red (Phenol Red) 1-30
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 2-50
Xanthine sodium salt (Hypoxanthine Na) 0.008-8
Linolic acid (Linoleic Acid) 0.0003-1.2
Thioctic Acid (Lipoic Acid) 0.003-0.8
Putrescine (Putrescine 2HCl) 0.0001-0.5
Gsh (Glutathione (reduced)) 0.05-3
Thymidine (Thymidine) 0.02-6
Damping fluid (HEPES) 2000-6500
Albumin (Albumin) 300-800
Transferrins,iron complexes (Holo Transferrin) 1-20
Regular Insulin (Insulin) 1-20
The composition and the weight part of above-mentioned substratum are preferably:
Composition mg/L
Glycine (Glycine) 33.5
L-L-Ala (L-Alanine) 16.78
L-arginine (L-Arginine) 124.2
L-amino-succinamic acid (L-Asparagine) 31.002
L-aspartic acid (L-Aspartic acid) 6.66
Cysteine hydrochloride-water (L-Cysteine hydrochloride-H 2O) 0.06
L-cystinic acid dihydrochloride (L-Cystine 2HCl) 35.624
L-L-glutamic acid (L-Glutamic Acid) 51.94
L-glutaminate (L-Glutamine) 149.2
L-Histidine (L-Histidine) 20.328
L-oxyproline (L-Hydroxyproline) 10
L-Isoleucine (L-Isoleucine) 34.8
L-leucine (L-Leucine) 48.62
L lysine HCL (L-Lysine hydrochloride) 57.3
L-methionine(Met) (L-Methionine) 12.9
L-phenylalanine (L-Phenylalanine) 19
L-proline(Pro) (L-Proline) 34.9
L-Serine (L-Serine) 23.1
L-Threonine (L-Threonine) 24.38
L-tryptophane (L-Tryptophan) 7.408
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 42.162
L-Xie Ansuan (L-Valine) 21.34
Vitamin H (Biotin) 0.04746
Choline chloride 60 (Choline chloride) 3.7
Calcium pantothenate (D-Calcium pantothenated) 0.156
Folic acid (Folic Acid) 0.466
Inositol (i-Inositol) 10.63
Niacinamide (Niacinamide) 0.2222
Padil (Para-Aminobenzoic Acid) 0.23
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.015
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.227
Riboflavin (Riboflavin) 0.0534
Thiamine hydrochloride (Thiamine hydrochloride) 0.266
Vitamins B 12(Vitamin B 12) 0.281
Xitix (Ascorbic Acid) 0.03
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.006
(four water) nitrocalcite (Calcium nitrate (Ca (NO 3) 24H 2O) 146.644
(anhydrous) magnesium chloride (Magnesium Chloride (anhydrous)) 11.444
(anhydrous) sal epsom
68.37
(Magnesium Sulfate(MgSO 4)(anhyd.))
Repone K (Potassium Chloride (KCl)) 364.72
Sodium-chlor (Sodium Chloride (NaCl)) 6799.8
One water Sodium phosphate dibasic
84
(Sodium Phosphate monobasic(NaH 2PO 4-H 2O))
Cupric sulfate pentahydrate (Cupric sulfate (CuSO 4-5H 2O)) 0.0005
Iron vitriol (Ferric sulfate (FeSO 4-7H 2O)) 0.1668
Nine water iron nitrates (Ferric Nitrate (Fe (NO 3) 3-9H 2O)) 0.42
Zinc Sulphate Heptahydrate (Zinc sulfate (ZnSO 4-7H 2O)) 0.1726
Disodium hydrogen phosphate,anhydrous
188.4
(Sodium Phosphate dibasic(Na 2HPO 4)anhydrous)
Glucose (D-Glucose (Dextrose)) 1360.4
Phenol red (Phenol Red) 8
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 22
Xanthoglobulin (Hypoxanthine Na) 1.194
Linolic acid (Linoleic Acid) 0.0168
Thioctic Acid (Lipoic Acid) 0.042
Putrescine (Putrescine 2HCl) 0.0322
Gsh (Glutathione (reduced)) 0.23
Thymidine (Thymidine) 0.14
Damping fluid (HEPES) 4500
Albumin (Albumin) 500
Transferrins,iron complexes (Holo Transferrin) 5
Regular Insulin (Insulin) 5
Above-mentioned substratum wherein also comprises following composition and part by weight thereof:
Composition mg/L
Thanomin (Ethanolamine) 2-20
Sodium Selenite (sodium Selenite) 0.001-0.3
Cholesterol (Cholesterol) 0.1-4.5
2 mercapto ethanol (2-MercaptOethanol) 2-20
Linolic acid (linoleic acid) 0.5-10
Resin acid (palmitic acid) 0.5-10
Heparin (Heparin) 5-40
Dextran (Dextran) 3-50
The preferred proportion of above-mentioned composition that also comprises and weight thereof is:
Composition mg/L
Thanomin (Ethanolamine) 8
Sodium Selenite (sodium Selenite) 0.003
Cholesterol (Cholesterol) 1.5
2 mercapto ethanol (2-MercaptOethanol) 6
Linolic acid (linoleic acid) 1
Resin acid (palmitic acid) 1
Heparin (Heparin) 10
Dextran (Dextran) 15
A kind of Vero amplification of the present invention CHEMICALLY DEFINED MEDIUM (synthetic medium that Chemical Composition is clear and definite) substratum, wherein, the composition of substratum and weight part are:
Composition mg/L
Glycine (Glycine) 10-50
L-L-Ala (L-Alanine) 5.2-40.2
L-arginine (L-Arginine) 60.2-200
The left-handed amino-succinamic acid of L-(L-Asparagine) 5-60
L-aspartic acid (L-Aspartic acid) 2.011-36
Cysteine hydrochloride-water (L-Cysteine hydrochloride-H 2O) 0.5-15
Cystinic acid dihydrochloride (L-Cystine 2HCl) 2-50.6
L-L-glutamic acid (L-Glutamic Acid) 5-100
L-glutaminate (L-Glutamine) 50-200
L-Histidine (L-Histidine) 6.003-50.005
L-oxyproline (L-Hydroxyproline) 0.05-30.
L-Isoleucine (L-Isoleucine) 5-60
L-leucine (L-Leucine) 10.05-80
L lysine HCL (L-Lysine hydrochloride) 20.05-100
L-methionine(Met) (L-Methionine) 2-30.5
L-phenylalanine (L-Phenylalanine) 5-50
L-proline(Pro) (L-Proline) 20-150
L-Serine (L-Serine) 5-50
L-Threonine (L-Threonine) 5-50
L-tryptophane (L-Tryptophan) 5-30
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 5-50
L-Xie Ansuan (L-Valine) 5-50
Vitamin H (Biotin) 0.0003-0.9
Choline chloride 60 (Choline chloride) 2-30
Calcium pantothenate (D-Calcium pantothenated) 0.01-3
Folic acid (Folic Acid) 0.5-15
Inositol (i-Inositol) 5-40
Niacinamide (Niacinamide) 0.01-15
Padil (Para-Aminobenzoic Acid) 0.01-3
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.01-2
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.01-0.8
Riboflavin (Riboflavin) 0.02-5
Thiamine hydrochloride (Thiamine hydrochloride) 0.05-3
Vitamins B 12(Vitamin B 12) 0.05-4.5
Xitix (Ascorbic Acid) 0.05-2
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.001-0.5
Vitamin K 3(Menadione (Vitamin K 3)) 0.001-0.5
Naphthoquinones (Niacin) 0.001-0.8
Vitamin A (Vitamin A) 0.01-3
Vitamins D 2(Vitamin D 2) 0.01-4
Calcium chloride (CaCl 2) 2-30
Four water-calcium nitrate (Ca (NO 3) 24H 2O) 15-200
Magnesium chloride (MgCl 2) 5-60
Sal epsom (MgSO 4) 20-100
Repone K (KCl) 100-500
Sodium-chlor (NaCl) 5000-10000
Sodium dihydrogen phosphate-water (NaH 2PO 4-H 2O) 50-250
Sodium phosphate dibasic (Na 2HPO 4) 20-120
Saltpetre (KNO 3) 0.1-5
Sodium Selenite (Na 2SeO 3-5H 2O) 0-20
Sodium bicarbonate (NaHCO 3) 50-300
Glucose (D-Glucose (Dextrose)) 1000-3000
Phenol red (Phenol Red) 2-30
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 20-200
Gsh (Glutathione (reduced)) 0.001-0.25
Thymidine (Thymidine) 0.05-3.0
Damping fluid (HEPES) 1500-4000
Adenine sulfate (Adenine sulfate) 3-40
Cholesterol (Cholesterol) 0.3-4.5
2-deoxidation D-ribose (2-deoxy-D-Ribose) 0.02-0.40
D-ribose (D-Ribose) 0.05-3
Gsh (Glutathione (reduced)) 0.3-4.5
Guanine hydrochloride (Guanine.HCL) 5-40
Xanthoglobulin (Hypoxanthine.Na) 0.05-3
Linolic acid (Linoleic Acid) 0.01-3
Thioctic Acid (Lipoic Acid) 0.05-0.9
Phenol red (Phenol Red-Na) 5-30
Putrescine (Putrescine-2HCl) 0.8-4.5
Sodium.alpha.-ketopropionate (Pyruvic Acid Na) 5-100
Thymus pyrimidine (Thymine) 0.05-0.8
Uridylic (uracil) 0.01-2
Thanomin (Ethanolamine) 0.5-10
The composition and the weight part of above-mentioned substratum are preferably:
Composition mg/L
Glycine (Glycine) 28.75
L-L-Ala (L-Alanine) 17.45
L-arginine (L-Arginine) 123.61
The left-handed amino-succinamic acid of L-(L-Asparagine) 25.504
L-aspartic acid (L-Aspartic acid) 7.317
Cysteine hydrochloride-water (L-Cysteine hydrochloride-H 2O) 3.2
Cystinic acid dihydrochloride (L-Cystine 2HCl) 27.048
L-L-glutamic acid (L-Glutamic Acid) 45.59
L-glutaminate (L-Glutamine) 130.32
L-Histidine (L-Histidine) 21.436
L-oxyproline (L-Hydroxyproline) 6.97
L-Isoleucine (L-Isoleucine) 25.536
L-leucine (L-Leucine) 39.176
L lysine HCL (L-Lysine hydrochloride) 53.25
L-methionine(Met) (L-Methionine) 10.79
L-phenylalanine (L-Phenylalanine) 16.15
L-proline(Pro) (L-Proline) 35.53
L-Serine (L-Serine) 19.33
L-Threonine (L-Threonine) 21.55
L-tryptophane (L-Tryptophan) 6.126
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 33.934
L-Xie Ansuan (L-Valine) 18.94
Vitamin H (Biotin) 0.02792
Choline chloride 60 (Choline chloride) 6.35
Calcium pantothenate (D-Calcium pantothenated) 0.225
Folic acid (Folic Acid) 1.525
Inositol (i-Inositol) 10.825
Niacinamide (Niacinamide) 0.0769
Padil (Para-Aminobenzoic Acid) 0.125
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.0625
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.0865
Riboflavin (Riboflavin) 0.0398
Thiamine hydrochloride (Thiamine hydrochloride) 0.145
Vitamins B 12(Vitamin B 12) 0.76
Xitix (Ascorbic Acid) 0.075
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.005
Vitamin K 3(Menadione (Vitamin K 3)) 0.005
Naphthoquinones (Niacin) 0.0625
Vitamin A (Vitamin A) 0.05
Vitamins D 2(Vitamin D 2) 0.05
Calcium chloride (CaCl 2) 10
Four water-calcium nitrate (Ca (NO 3) 24H 2O) 113.288
Magnesium chloride (MgCl 2) 22.888
Sal epsom (MgSO 4) 68.835
Repone K (KCl) 329.44
Sodium-chlor (NaCl) 7085.6
Sodium dihydrogen phosphate-water (NaH 2PO 4-H 2O) 128
Sodium phosphate dibasic (Na 2HPO 4) 56.8
Saltpetre (KNO 3) 0
Sodium Selenite (Na 2SeO 3-5H 2O) 0
Sodium bicarbonate (NaHCO 3) 220
Glucose (D-Glucose (Dextrose)) 1520.8
Phenol red (Phenol Red) 11.48
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 44
Gsh (Glutathione (reduced)) 0.075
Thymidine (Thymidine) 0.28
Damping fluid (HEPES) 2600
Adenine sulfate (Adenine sulfate) 15
Cholesterol (Cholesterol) 1.5
2-deoxidation D-ribose (2-deoxy-D-Ribose) 0.16
D-ribose (D-Ribose) 0.22
Gsh (Glutathione (reduced)) 1.5
Guanine hydrochloride (Guanine.HCL) 22
Xanthoglobulin (Hypoxanthine) 0.45
Linolic acid (Linoleic Acid) 0.04
Thioctic Acid (Lipoic Acid) 0.105
Phenol red (Phenol Red-Na) 10.6
Putrescine (Putrescine-2HCl) 1.6905
Sodium.alpha.-ketopropionate (Pyruvic Acid Na) 55
Thymus pyrimidine (Thymine) 0.15
Uridylic (uracil) 0.15
Thanomin (Ethanolamine) 2
Vero amplification CHEMICALLY DEFINED MEDIUM substratum also comprises following composition and part by weight thereof:
Composition mg/L
Semi-lactosi (Galactose) 1000-3000
Urogastron (Epidermal Growth Factor (recombinant human)) 0.05-2
Cortisone (Hydrocortisone) 0.001-0.8
Regular Insulin (Insulin (recombinant human, Zn Full Chain)) 2-30
Prostaglandin(PG) (Prostaglandin E1) 0.0001-0.56
Triiodothyronine (Triiodothyronine (T3)) 0.0001-0.4
The above-mentioned Vero amplification CHEMICALLY DEFINED MEDIUM substratum composition and the preferred proportion of weight thereof are:
Composition mg/L
Semi-lactosi (Galactose) 2000
Urogastron (Epidermal Growth Factor (recombinant human)) 0.33
Cortisone (Hydrocortisone) 0.01
Regular Insulin (Insulin (recombinant human, Zn Full Chain)) 12
Prostaglandin(PG) (Prostaglandin E1) 0.001
Triiodothyronine (Triiodothyronine (T3)) 0.0005
The serum-free culture method of a kind of Vero cell of the present invention wherein, with the Vero cell of adherent growth, adapts to the domestication of SFM serum free medium through above-mentioned Vero cell suspension and adapts to cultivation, and the Vero growth pattern can be converted into suspension growth by adherent growth; Further make the growth of can in Vero increases CHEMICALLYDEFINED MEDIUM (synthetic medium that Chemical Composition is clear and definite) substratum, increasing of Vero cell.
The Vero cell suspension that the Vero cell of adherent growth is domesticated for suspension growth adapts to the SFM serum free medium and does not contain any animal serum.
Culture condition is the condition of using in the routine techniques in this area, and preferred culture condition is: 37 degrees centigrade, and 5%CO 2, humidity is greater than 95% culture environment.
The Vero cell suspension that the Vero cell of adherent growth is domesticated for suspension growth adapts to the SFM serum free medium and does not contain any animal serum; Behind the Vero cell suspension growth adaptation; The used substratum of amplification growth does not contain protein.
Culture medium preparation and using method among the present invention, it makes Vero cell growth pattern with the material alternative serum of specific chemical components, is converted into suspension growth from adherent growth, changes the growth pattern of cell, can greatly increase cell yield.
The amplification culture medium that what is more important the present invention describes does not contain any protein ingredient, has greatly simplified the purifying process process of recombinant protein, and the output of recombinant protein and purity are obviously improved.
The inventive method has good effect, because make Vero cell growth pattern, is converted into suspension growth from adherent growth, changes the growth pattern of cell, can greatly increase cell yield.
The amplification culture medium that what is more important the present invention describes does not contain any protein ingredient, has greatly simplified the purifying process process of recombinant protein, and the output of recombinant protein and purity are obviously improved.
The inventive method and the product that obtains thereof have good effect, and making the Vero cell transformation with the adaptation SFM serum free medium that suspends is the suspension growth pattern;
Further Vero cell amplification growth in Vero amplification CHEMICALLY DEFINED (that is: the clear and definite synthetic medium of Chemical Composition) protein-free medium, do not contain any protein component in the Vero amplification CHEMICALLY DEFINED protein-free medium, greatly simplified downstream purification technology, improved output and purity by the recombinant protein of Vero cells produce.
The invention effect:
The cell speed of growth is very fast as can be seen from following growth curve
Cultivate fate Cell density (cell/ml)
0 3.00E+05
1 2.30E+05
2 3.50E+05
3 4.40E+05
4 8.60E+05
5 2.10E+06
Description of drawings
Fig. 1 is the growth curve of cell cultures
Embodiment
Embodiment 1
The serum-free culture method of Vero cell wherein, with the Vero cell of adherent growth, adapts to the domestication of SFM serum free medium through the Vero cell suspension and adapts to cultivation, and the Vero growth pattern can be converted into suspension growth by adherent growth; Further make the growth of can in Vero increases CHEMICALLY DEFINED MEDIUM (synthetic medium that Chemical Composition is clear and definite) substratum, increasing of Vero cell.Culture condition is as follows: 37 degrees centigrade, and 5%CO 2, humidity is greater than 95% culture environment.
Embodiment 2
Cultural method is as described in the embodiment 1, and wherein, moiety and ratio thereof that the Vero cell suspension adapts to SFM serum free medium and Vero amplification CHEMICALLY DEFINED MEDIUM substratum are respectively:
The Vero cell suspension adapts to SFM serum free medium composition and proportioning:
Composition mg/L
Glycine (Glycine) 33.5
L-L-Ala (L-Alanine) 16.78
L-arginine (L-Arginine) 124.2
L-amino-succinamic acid (L-Asparagine) 31.002
L-aspartic acid (L-Aspartic acid) 6.66
Cysteine hydrochloride-water (L-Cysteine hydrochloride-H 2O) 0.06
L-cystinic acid dihydrochloride (L-Cystine 2HCl) 35.624
L-L-glutamic acid (L-Glutamic Acid) 51.94
L-glutaminate (L-Glutamine) 149.2
L-Histidine (L-Histidine) 20.328
L-oxyproline (L-Hydroxyproline) 10
L-Isoleucine (L-Isoleucine) 34.8
L-leucine (L-Leucine) 48.62
L lysine HCL (L-Lysine hydrochloride) 57.3
L-methionine(Met) (L-Methionine) 12.9
L-phenylalanine (L-Phenylalanine) 19
L-proline(Pro) (L-Proline) 34.9
L-Serine (L-Serine) 23.1
L-Threonine (L-Threonine) 24.38
L-tryptophane (L-Tryptophan) 7.408
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 42.162
L-Xie Ansuan (L-Valine) 21.34
Vitamin H (Biotin) 0.04746
Choline chloride 60 (Choline chloride) 3.7
Calcium pantothenate (D-Calcium pantothenated) 0.156
Folic acid (Folic Acid) 0.466
Inositol (i-Inositol) 10.63
Niacinamide (Niacinamide) 0.2222
Padil (Para-Aminobenzoic Acid) 0.23
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.015
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.227
Riboflavin (Riboflavin) 0.0534
Thiamine hydrochloride (Thiamine hydrochloride) 0.266
Vitamins B 12(Vitamin B 12) 0.281
Xitix (Ascorbic Acid) 0.03
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.006
(four water) nitrocalcite (Calcium nitra te (Ca (NO 3) 24H 2O) 146.644
(anhydrous) magnesium chloride (Magnesium Chlorde (anhydrous)) 11.444
(anhydrous) sal epsom
68.37
(Magnesium Sulfate(MgSO 4)(anhyd.))
Repone K (Potassium Chloride (KCl)) 364.72
Sodium-chlor (Sodium Chloride (NaCl)) 6799.8
One water Sodium phosphate dibasic
84
(Sodium Phosphate monobasic(NaH 2PO 4-H 2O))
Cupric sulfate pentahydrate (Cupric sulfate (CuSO 4-5H 2O)) 0.0005
Iron vitriol (Ferric sulfate (FeSO 4-7H 2O)) 0.1668
Nine water iron nitrates (Ferric Nitrate (Fe (NO 3) 3-9H 2O)) 0.42
Zinc Sulphate Heptahydrate (Zinc sulfate (ZnSO 4-7H 2O)) 0.1726
Disodium hydrogen phosphate,anhydrous
188.4
(Sodium Phosphate dibasic(Na 2HPO 4)anhydrous)
Glucose (D-Glucose (Dextrose)) 1360.4
Phenol red (Phenol Red) 8
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 22
Xanthoglobulin (Hypoxanthine Na) 1.194
Linolic acid (Linoleic Acid) 0.0168
Thioctic Acid (Lipoic Acid) 0.042
Putrescine (Putrescine 2HCl) 0.0322
Gsh (Glutathione (reduced)) 0.23
Thymidine (Thymidine) 0.14
Damping fluid (HEPES) 4500
Albumin (Albumin) 500
Transferrins,iron complexes (Holo Transferrin) 5
Regular Insulin (Insulin) 5
Vero amplification CHEMICALLY DEFINED MEDIUM substratum
Composition mg/L
Glycine (Glycine) 28.75
L-L-Ala (L-Alanine) 17.45
L-arginine (L-Arginine) 123.61
The left-handed amino-succinamic acid of L-(L-Asparagine) 25.504
L-aspartic acid (L-Aspartic acid) 7.317
Cysteine hydrochloride-water (L-Cysteine hydrochloride-H 2O) 3.2
Cystinic acid dihydrochloride (L-Cystine 2HCl) 27.048
L-L-glutamic acid (L-Glutamic Acid) 45.59
L-glutaminate (L-Glutamine) 130.32
L-Histidine (L-Histidine) 21.436
L-oxyproline (L-Hydroxyproline) 6.97
L-Isoleucine (L-Isoleucine) 25.536
L-leucine (L-Leucine) 39.176
L lysine HCL (L-Lysine hydrochloride) 53.25
L-methionine(Met) (L-Methionine) 10.79
L-phenylalanine (L-Phenylalanine) 16.15
L-proline(Pro) (L-Proline) 35.53
L-Serine (L-Serine) 19.33
L-Threonine (L-Threonine) 21.55
L-tryptophane (L-Tryptophan) 6.126
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 33.934
L-Xie Ansuan (L-Valine) 18.94
Vitamin H (Biotin) 0.02792
Choline chloride 60 (Choline chloride) 6.35
Calcium pantothenate (D-Calcium pantothenated) 0.225
Folic acid (Folic Acid) 1.525
Inositol (i-Inositol) 10.825
Niacinamide (Niacinamide) 0.0769
Padil (Para-Aminobenzoic Acid) 0.125
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.0625
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.0865
Riboflavin (Riboflavin) 0.0398
Thiamine hydrochloride (Thiamine hydrochloride) 0.145
Vitamins B 12(Vitamin B 12) 0.76
Xitix (Ascorbic Acid) 0.075
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.005
Vitamin K 3(Menadione (Vitamin K 3)) 0.005
Naphthoquinones (Niacin) 0.0625
Vitamin A (Vitamin A) 0.05
Vitamins D 2(Vitamin D 2) 0.05
Calcium chloride (CaCl 2) 10
Four water-calcium nitrate (Ca (NO 3) 24H 2O) 113.288
Magnesium chloride (MgCl 2) 22.888
Sal epsom (MgSO 4) 68.835
Repone K (KCl) 329.44
Sodium-chlor (NaCl) 7085.6
Sodium dihydrogen phosphate-water (NaH 2PO 4-H 2O) 128
Sodium phosphate dibasic (Na 2HPO 4) 56.8
Saltpetre (KNO 3) 0
Sodium Selenite (Na 2SeO 3-5H 2O) 0
Sodium bicarbonate (NaHCO 3) 220
Glucose (D-Glucose (Dextrose)) 1520.8
Phenol red (Phenol Red) 11.48
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 44
Gsh (Glutathione (reduced)) 0.075
Thymidine (Thymidine) 0.28
Damping fluid (HEPES) 2600
Adenine sulfate (Adenine sulfate) 15
Cholesterol (Cholesterol) 1.5
2-deoxidation D-ribose (2-deoxy-D-Ribose) 0.16
D-ribose (D-Ribose) 0.22
Gsh (Glutathione (reduced)) 1.5
Guanine hydrochloride (Guanine.HCL) 22
Xanthoglobulin (Hypoxanthine) 0.45
Linolic acid (Linoleic Acid) 0.04
Thioctic Acid (Lipoic Acid) 0.105
Phenol red (Phenol Red-Na) 10.6
Putrescine (Putrescine-2HCl) 1.6905
Sodium.alpha.-ketopropionate (Pyruvic Acid Na) 55
Thymus pyrimidine (Thymine) 0.15
Uridylic (uracil) 0.15
Thanomin (Ethanolamine) 2
Embodiment 3
Cultural method is as described in the embodiment 1, and wherein, moiety and ratio thereof that the Vero cell suspension adapts to SFM serum free medium and Vero amplification CHEMICALLY DEFINED MEDIUM substratum are respectively:
The Vero cell suspension adapts to SFM serum free medium composition and proportioning:
Composition mg/L
Glycine (Glycine) 50
L-L-Ala (L-Alanine) 5
L-arginine (L-Arginine) 200
L-amino-succinamic acid (L-Asparagine) 10
L-aspartic acid (L-Aspartic acid) 15
L-cysteine hydrochloride (L-Cysteine hydrochloride-H 2O) 0.01
L-cystinic acid dihydrochloride (L-Cystine 2HCl) 100
L-L-glutamic acid (L-Glutamic Acid) 10
L-glutaminate (L-Glutamine) 200
L-Histidine (L-Histidine) 5
L-oxyproline (L-Hydroxyproline) 20
L-Isoleucine (L-Isoleucine) 10
L-leucine (L-Leucine) 100
L lysine HCL (L-Lysine hydrochloride) 15
L-methionine(Met) (L-Methionine) 30
L-phenylalanine (L-Phenylalanine) 5
L-proline(Pro) (L-Proline) 100
L-Serine (L-Serine) 5
L-Threonine (L-Threonine) 50
L-tryptophane (L-Tryptophan) 2
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 100
L-Xie Ansuan (L-Valine) 5
Vitamin H (Biotin) 0.5
Choline chloride 60 (Choline chloride) 0.5
Calcium pantothenate (D-Calcium pantothenated) 0.9
Folic acid (Folic Acid) 0.001
Inositol (i-Inositol) 30
Niacinamide (Niacinamide) 0.0001
Padil (Para-Aminobenzoic Acid) 3.
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.002
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 3.5
Riboflavin (Riboflavin) 0.0001
Thiamine hydrochloride (Thiamine hydrochloride) 0.15
Vitamins B 12(Vitamin B 12) 0.002
Xitix (Ascorbic Acid) 1.2
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.001
(four water) nitrocalcite (Calcium nitrate (Ca (NO 3) 24H 2O) 200
(anhydrous) magnesium chloride (Magnesium Chloride (anhydrous)) 3
(anhydrous) sal epsom
150
(Magnesium Sulfate(MgSO 4)(anhyd.))
Repone K (Potassium Chloride (KCl)) 150
Sodium-chlor (Sodium Chloride (NaCl)) 10000
One water Sodium phosphate dibasic
5
(Sodium Phosphate monobasic(NaH 2PO 4-H 2O))
Cupric sulfate pentahydrate (Cupric sulfate (CuSO 4-5H 2O)) 0.03
Iron vitriol (Ferric sulfate (FeSO 4-7H 2O)) 0.002
Nine water iron nitrates (Ferric Nitrate (Fe (NO 3) 3-9H 2O)) 3.5
Zinc Sulphate Heptahydrate (Zinc sulfate (ZnSO 4-7H 2O)) 0.003
Disodium hydrogen phosphate,anhydrous
300
(Sodium Phosphate dibasic(Na 2HPO 4)anhydrous)
Glucose (D-Glucose (Dextrose)) 800
Phenol red (Phenol Red) 30
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 2
Xanthine sodium salt (Hypoxanthine Na) 8
Linolic acid (Linoleic Acid) 0.0003
Thioctic Acid (Lipoic Acid) 0.8
Putrescine (Putrescine 2HCl) 0.0001
Gsh (Glutathione (reduced)) 0.05
Thymidine (Thymidine) 6
Damping fluid (HEPES) 2000
Albumin (Albumin) 800
Transferrins,iron complexes (Holo Transferrin) 1
Regular Insulin (Insulin) 20
Vero amplification CHEMICALLY DEFINED MEDIuM substratum
Composition mg/L
Glycine (Glycine) 10
L-L-Ala (L-Alanine) 40.2
L-arginine (L-Arginine) 60.2
The left-handed amino-succinamic acid of L-(L-Asparagine) 60
L-aspartic acid (L-Aspartic acid) 2.011
Cysteine hydrochloride-water (L-Cysteine hydrochloride-H 2O) 15
Cystinic acid dihydrochloride (L-Cystine 2HCl) 2
L-L-glutamic acid (L-Glutamic Acid) 100
L-glutaminate (L-Glutamine) 50
L-Histidine (L-Histidine) 50.005
L-oxyproline (L-Hydroxyproline) 0.05
L-Isoleucine (L-Isoleucine) 60
L-leucine (L-Leucine) 10.05
L lysine HCL (L-Lysine hydrochloride) 100
L-methionine(Met) (L-Methionine) 2
L-phenylalanine (L-Phenylalanine) 50
L-proline(Pro) (L-Proline) 20
L-Serine (L-Serine) 50
L-Threonine (L-Threonine) 5
L-tryptophane (L-Tryptophan) 30
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 5
L-Xie Ansuan (L-Valine) 50
Vitamin H (Biotin) 0.0003
Choline chloride 60 (Choline chloride) 30
Calcium pantothenate (D-Calcium pantothenated) 0.01
Folic acid (Folic Acid) 15
Inositol (i-Inositol) 5
Niacinamide (Niacinamide) 15
Padil (Para-Aminobenzoic Acid) 0.01
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 2
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.01
Riboflavin (Riboflavin) 5
Thiamine hydrochloride (Thiamine hydrochloride) 0.05
Vitamins B 12(Vitamin B 12) 4.5
Xitix (Ascorbic Acid) 0.05
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.5
Vitamin K 3(Menadione (Vitamin K 3)) 0.001
Naphthoquinones (Niacin) 0.8
Vitamin A (Vitamin A) 0.01
Vitamins D 2(Vitamin D 2) 4
Calcium chloride (CaCl 2) 2
Four water-calcium nitrate (Ca (NO 3) 24H 2O) 200
Magnesium chloride (MgCl 2) 5
Sal epsom (MgSO 4) 100
Repone K (KCl) 100
Sodium-chlor (NaCl) 10000
Sodium dihydrogen phosphate-water (NaH 2PO 4-H 2O) 50
Sodium phosphate dibasic (Na 2HPO 4) 120
Saltpetre (KNO 3) 0.1
Sodium Selenite (Na 2SeO 3-5H 2O) 20
Sodium bicarbonate (NaHCO 3) 50
Glucose (D-Glucose (Dextrose)) 3000
Phenol red (Phenol Red) 2
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 200
Gsh (Glutathione (reduced)) 0.001
Thymidine (Thymidine) 3.0
Damping fluid (HEPES) 1500
Adenine sulfate (Adenine sulfate) 40
Cholesterol (Cholesterol) 0.3
2-deoxidation D-ribose (2-deoxy-D-Ribose) 0.40
D-ribose (D-Ribose) 0.05
Gsh (Glutathione (reduced)) 4.5
Guanine hydrochloride (Guanine.HCL) 5
Xanthoglobulin (Hypoxanthine.Na) 3
Linolic acid (Linoleic Acid) 0.01
Thioctic Acid (Lipoic Acid) 0.9
Phenol red (Phenol Red-Na) 5
Putrescine (Putrescine-2HCl) 4.5
Sodium.alpha.-ketopropionate (Pyruvic Acid Na) 5
Thymus pyrimidine (Thymine) 0.8
Uridylic (uracil) 0.01
Thanomin (Ethanolamine) 10
Embodiment 4
Cultural method is as described in the embodiment 1, and wherein, moiety and ratio thereof that the Vero cell suspension adapts to SFM serum free medium and Vero amplification CHEMICALLY DEFINED MEDIUM substratum are respectively:
The Vero cell suspension adapts to SFM serum free medium composition and proportioning:
Composition mg/L
Glycine (Glycine) 10
L-L-Ala (L-Alanine) 30
L-arginine (L-Arginine) 30
L-amino-succinamic acid (L-Asparagine) 60
L-aspartic acid (L-Aspartic acid) 1
L-cysteine hydrochloride (L-Cysteine hydrochloride-H 2O) 0.6
L-cystinic acid dihydrochloride (L-Cystine 2HCl) 15
L-L-glutamic acid (L-Glutamic Acid) 100
L-glutaminate (L-Glutamine) 50
L-Histidine (L-Histidine) 100
L-oxyproline (L-Hydroxyproline) 2
L-Isoleucine (L-Isoleucine) 100
L-leucine (L-Leucine) 10
L lysine HCL (L-Lysine hydrochloride) 120
L-methionine(Met) (L-Methionine) 2
L-phenylalanine (L-Phenylalanine) 50
L-proline(Pro) (L-Proline) 10
L-Serine (L-Serine) 60
L-Threonine (L-Threonine) 5
L-tryptophane (L-Tryptophan) 50
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 5
L-Xie Ansuan (L-Valine) 100
Vitamin H (Biotin) 0.00001
Choline chloride 60 (Choline chloride) 20
Calcium pantothenate (D-Calcium pantothenated) 0.001
Folic acid (Folic Acid) 1.5
Inositol (i-Inositol) 2
Niacinamide (Niacinamide) 0.98
Padil (Para-Aminobenzoic Acid) 0.05
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.7
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.006
Riboflavin (Riboflavin) 6
Thiamine hydrochloride (Thiamine hydrochloride) 0.003
Vitamins B 12(Vitamin B 12) 1.8
Xitix (Ascorbic Acid) 0.01
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 3
(four water) nitrocalcite (Calcium nitrate (Ca (NO 3) 24H 2O) 50
(anhydrous) magnesium chloride (Magnesium Chloride (anhydrous)) 40
(anhydrous) sal epsom
25
(Magnesium Sulfate(MgSO 4)(anhyd.))
Repone K (Potassium Chloride (KCl)) 600
Sodium-chlor (Sodium Chloride (NaCl)) 3000
One water Sodium phosphate dibasic
200
(Sodium Phosphate monobasic(NaH 2PO 4-H 2O))
Cupric sulfate pentahydrate (Cupric sulfate (CuSO 4-5H 2O)) 0.0001
Iron vitriol (Ferric sulfate (FeSO 4-7H 2O)) 0.8
Nine water iron nitrates (Ferric Nitrate (Fe (NO 3) 3-9H 2O)) 0.02
Zinc Sulphate Heptahydrate (Zinc sulfate (ZnSO 4-7H 2O)) 0.86
Disodium hydrogen phosphate,anhydrous
50
(Sodium Phosphate dibasic(Na 2HPO 4)anhydrous)
Glucose (D-Glucose (Dextrose)) 2000
Phenol red (Phenol Red) 1
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 50
Xanthine sodium salt (Hypoxanthine Na) 0.008
Linolic acid (Linoleic Acid) 1.2
Thioctic Acid (Lipoic Acid) 0.003
Putrescine (Putrescine 2HCl) 0.5
Gsh (Glutathione (reduced)) 0.05
Thymidine (Thymidine) 6
Damping fluid (HEPES) 2000
Albumin (Albumin) 800
Transferrins,iron complexes (Holo Transferrin) 1
Regular Insulin (Insulin) 20
Vero expands glass CHEMICALLY DEFINED MEDIUM substratum
Composition mg/L
Glycine (Glycine) 10
L-L-Ala (L-Alanine) 40.2
L-arginine (L-Arginine) 60.2
The left-handed amino-succinamic acid of L-(L-Asparagine) 60
L-aspartic acid (L-Aspartic acid) 2.011
Cysteine hydrochloride-water (L-Cysteine hydrochloride-H 2O) 15
Cystinic acid dihydrochloride (L-Cystine 2HCl) 2
L-L-glutamic acid (L-Glutamic Acid) 100
L-glutaminate (L-Glutamine) 50
L-Histidine (L-Histidine) 50.005
L-oxyproline (L-Hydroxyproline) 0.05
L-Isoleucine (L-Isoleucine) 60
L-leucine (L-Leucine) 10.05
L lysine HCL (L-Lysine hydrochloride) 100
L-methionine(Met) (L-Methionine) 2
L-phenylalanine (L-Phenylalanine) 50
L-proline(Pro) (L-Proline) 20
L-Serine (L-Serine) 50
L-Threonine (L-Threonine) 5
L-tryptophane (L-Tryptophan) 30
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 5
L-Xie Ansuan (L-Valine) 50
Vitamin H (Biotin) 0.0003
Choline chloride 60 (Choline chloride) 30
Calcium pantothenate (D-Calcium pantothenated) 0.01
Folic acid (Folic Acid) 15
Inositol (i-Inositol) 5
Niacinamide (Niacinamide) 15
Padil (Para-Aminobenzoic Acid) 0.01
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 2
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.01
Riboflavin (Riboflavin) 5
Thiamine hydrochloride (Thiamine hydrochloride) 0.05
Vitamins B 12(Vitamin B 12) 4.5
Xitix (Ascorbic Acid) 0.05
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.5
Vitamin K 3(Menadione (Vitamin K 3)) 0.001
Naphthoquinones (Niacin) 0.8
Vitamin A (Vitamin A) 0.01
Vitamins D 2(Vitamin D 2) 4
Calcium chloride (CaCl 2) 2
Four water-calcium nitrate (Ca (NO 3) 24H 2O) 200
Magnesium chloride (MgCl 2) 5
Sal epsom (MgSO 4) 100
Repone K (KCl) 100
Sodium-chlor (NaCl) 10000
Sodium dihydrogen phosphate-water (NaH 2PO 4-H 2O) 50
Sodium phosphate dibasic (Na 2HPO 4) 120
Saltpetre (KNO 3) 0.1
Sodium Selenite (Na 2SeO 3-5H 2O) 0
Sodium bicarbonate (NaHCO 3) 300
Glucose (D-Glucose (Dextrose)) 1000
Phenol red (Phenol Red) 30
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 20
Gsh (Glutathione (reduced)) 0.25
Thymidine (Thymidine) 3.0
Damping fluid (HEPES) 1500
Adenine sulfate (Adenine sulfate) 40
Cholesterol (Cholesterol) 0.3
2-deoxidation D-ribose (2-deoxy-D-Ribose) 0.40
D-ribose (D-Ribose) 0.05
Gsh (Glutathione (reduced)) 4.5
Guanine hydrochloride (Guanine.HCL) 5
Xanthoglobulin (Hypoxanthine.Na) 3
Linolic acid (Linoleic Acid) 0.01
Thioctic Acid (Lipoic Acid) 0.9
Phenol red (Phenol Red-Na) 5
Putrescine (Putrescine-2HCl) 4.5
Sodium.alpha.-ketopropionate (Pyruvic Acid Na) 5
Thymus pyrimidine (Thymine) 0.8
Uridylic (uracil) 0.01
Thanomin (Ethanolamine) 10
Embodiment 5
Cultural method is as described in embodiment 3 or 4, component and proportioning that the moiety of Vero cell suspension adaptation SFM serum free medium that wherein, adopts in embodiment 3 or 4 experiments of being done and Vero amplification CHEMICALLY DEFINED MEDIUM substratum and ratio thereof increase respectively are:
The Vero cell suspension adapts to increasable composition and proportioning in the SFM serum free medium:
Composition mg/L
Thanomin (Ethanolamine) 8
Sodium Selenite (sodium Selenite) 0.003
Cholesterol (Cholesterol) 1.5
2 mercapto ethanol (2-MercaptOethanol) 6
Linolic acid (linoleic acid) 1
Resin acid (palmitic acid) 1
Heparin (Heparin) 10
Dextran (Dextran) 15
Increasable composition and proportioning in the Vero amplification CHEMICALLY DEFINED MEDIUM substratum:
Composition mg/L
Semi-lactosi (Galactose) 2000
Urogastron (Epidermal Growth Factor (recombinant human)) 0.33
Cortisone (Hydrocortisone) 0.01
Regular Insulin (Insulin (recombinant human, Zn Full Chain)) 12
Prostaglandin(PG) (Prostaglandin E1) 0.001
Triiodothyronine (Triiodothyronine (T3)) 0.0005
Embodiment 6
Cultural method is as described in embodiment 3 or 4, component and proportioning that the moiety of Vero cell suspension adaptation SFM serum free medium that wherein, adopts in embodiment 3 or 4 experiments of being done and Vero amplification CHEMICALLY DEFINED MEDIUM substratum and ratio thereof increase respectively are:
The Vero cell suspension adapts to increasable composition and proportioning in the SFM serum free medium:
Composition mg/L
Thanomin (Ethanolamine) 20
Sodium Selenite (sodium Selenite) 0.001
Cholesterol (Cholesterol) 4.5
2 mercapto ethanol (2-MercaptOethanol) 2
Linolic acid (linoleic acid) 10
Resin acid (palmitic acid) 0.5
Heparin (Heparin) 40
Dextran (Dextran) 3
Increasable composition and proportioning in the Vero amplification CHEMICALLY DEFINED MEDIUM substratum:
Composition mg/L
Semi-lactosi (Galactose) 3000
Urogastron (Epidermal Growth Factor (recombinant human)) 0.05
Cortisone (Hydrocortisone) 0.8
Regular Insulin (Insulin (recombinant human, Zn Full Chain)) 2
Prostaglandin(PG) (Prostaglandin E1) 0.56
Triiodothyronine (Triiodothyronine (T3)) 0.0001

Claims (10)

1. a Vero cell suspension adapts to the SFM serum free medium, and it is characterized in that: the composition of substratum and weight part are:
Composition mg/L
Glycine (Glycine) 10-50
L-L-Ala (L-Alanine) 5-30
L-arginine (L-Arginine) 30-200
L-amino-succinamic acid (L-Asparagine) 10-60
L-aspartic acid (L-Aspartic acid) 1-15
L-cysteine hydrochloride (L-Cysteine hydrochloride-H 2O) 0.01-0.6
L-cystinic acid dihydrochloride (L-Cystine 2HCl) 15-100
L-L-glutamic acid (L-Glutamic Acid) 10-100
L-glutaminate (L-Glutamine) 50-200
L-Histidine (L-Histidine) 5-100
L-oxyproline (L-Hydroxyproline) 2-20
L-Isoleucine (L-Isoleucine) 10-100
L-leucine (L-Leucine) 10-100
L lysine HCL (L-Lysine hydrochloride) 15-120
L-methionine(Met) (L-Methionine) 2-30
L-phenylalanine (L-Phenylalanine) 5-50
L-proline(Pro) (L-Proline) 10-100
L-Serine (L-Serine) 5-60
L-Threonine (L-Threonine) 5-50
L-tryptophane (L-Tryptophan) 2-50
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 5-100
L-Xie Ansuan (L-Valine) 5-100
Vitamin H (Biotin) 0.00001-0.5
Choline chloride 60 (Choline chloride) 0.5-20.
Calcium pantothenate (D-Calcium pantothenated) 0.001-0.9
Folic acid (Folic Acid) 0.001-1.5
Inositol (i-Inositol) 2.-30
Niacinamide (Niacinamide) 0.0001-0.98
Padil (Para-Aminobenzoic Acid) 0.05-3.
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.002-0.7
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.006-3.5
Riboflavin (Riboflavin) 0.0001-6.
Thiamine hydrochloride (Thiamine hydrochloride) 0.003-0.15
Vitamins B 12(Vitamin B 12) 0.002-1.8
Xitix (Ascorbic Acid) 0.01-1.2
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.001-3
(four water) nitrocalcite (Calcium nitrate (Ca (NO 3) 24H 2O) 50-200
(anhydrous) magnesium chloride (Magnesium Chloride (anhydrous)) 3-40
(anhydrous) sal epsom 25-150
(Magnesium Sulfate(MgSO 4)(anhyd.))
Repone K (Potassium Chloride (KCl)) 150-600
Sodium-chlor (Sodium Chloride (NaCl)) 3000-10000
One water Sodium phosphate dibasic
5-200
(Sodium Phosphate monobasic(NaH 2PO 4-H 2O))
Cupric sulfate pentahydrate (Cupric sulfate (CuSO 4-5H 2O)) 0.0001-0.03
Iron vitriol (Ferric sulfate (FeSO 4-7H 2O)) 0.002-0.8
Nine water iron nitrates (Ferric Nitrate (Fe (NO 3) 3-9H 2O)) 0.02-3.5
Zinc Sulphate Heptahydrate (Zinc sulfate (ZnSO 4-7H 2O)) 0.003-0.86
Disodium hydrogen phosphate,anhydrous
50-300
(Sodium Phosphate dibasic(Na 2HPO 4)anhydrous)
Glucose (D-Glucose (Dextrose)) 800-2000
Phenol red (Phenol Red) 1-30
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 2-50
Xanthine sodium salt (Hypoxanthine Na) 0.008-8
Linolic acid (Linoleic Acid) 0.0003-1.2
Thioctic Acid (Lipoic Acid) 0.003-0.8
Putrescine (Putrescine 2HCl) 0.0001-0.5
Gsh (Glutathione (reduced)) 0.05-3
Thymidine (Thymidine) 0.02-6
Damping fluid (HEPES) 2000-6500
Albumin (Albumin) 300-800
Transferrins,iron complexes (Holo Transferrin) 1-20
Regular Insulin (Insulin) 1-20
2. Vero cell suspension as claimed in claim 1 adapts to the SFM serum free medium, and it is characterized in that: the composition and the weight part of substratum are preferably:
Composition mg/L
Glycine (Glycine) 33.5
L-L-Ala (L-Alanine) 16.78
L-arginine (L-Arginine) 124.2
L-amino-succinamic acid (L-Asparagine) 31.002
L-aspartic acid (L-Aspartic acid) 6.66
Cysteine hydrochloride-water (L-Cysteine hydrochloride-H 2O) 0.06
L-cystinic acid dihydrochloride (L-Cystine 2HCl) 35.624
L-L-glutamic acid (L-Glutamic Acid) 51.94
L-glutaminate (L-Glutamine) 149.2
L-Histidine (L-Histidine) 20.328
L-oxyproline (L-Hydroxyproline) 10
L-Isoleucine (L-Isoleucine) 34.8
L-leucine (L-Leucine) 48.62
L lysine HCL (L-Lysine hydrochloride) 57.3
L-methionine(Met) (L-Methionine) 12.9
L-phenylalanine (L-Phenylalanine) 19
L-proline(Pro) (L-Proline) 34.9
L-Serine (L-Serine) 23.1
L-Threonine (L-Threonine) 24.38
L-tryptophane (L-Tryptophan) 7.408
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 42.162
L-Xie Ansuan (L-Valine) 21.34
Vitamin H (Biotin) 0.04746
Choline chloride 60 (Choline chloride) 3.7
Calcium pantothenate (D-Calcium pantothenated) 0.156
Folic acid (Folic Acid) 0.466
Inositol (i-Inositol) 10.63
Niacinamide (Niacinamide) 0.2222
Padil (Para-Aminobenzoic Acid) 0.23
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.015
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.227
Riboflavin (Riboflavin) 0.0534
Thiamine hydrochloride (Thiamine hydrochloride) 0.266
Vitamins B 12(Vitamin B 12) 0.281
Xitix (Ascorbic Acid) 0.03
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.006
(four water) nitrocalcite (Calcium nitrate (Ca (NO 3) 24H 2O) 146.644
(anhydrous) magnesium chloride (Magnesium Chloride (anhydrous)) 11.444
(anhydrous) sal epsom 68.37
(Magnesium Sulfate(MgSO 4)(anhyd.))
Repone K (Potassium Chloride (KCl)) 364.72
Sodium-chlor (Sodium Chloride (NaCl)) 6799.8
One water Sodium phosphate dibasic
84
(Sodium Phosphate monobasic(NaH 2PO 4-H 2O))
Cupric sulfate pentahydrate (Cupric sulfate (CuSO 4-5H 2O)) 0.0005
Iron vitriol (Ferric sulfate (FeSO 4-7H 2O)) 0.1668
Nine water iron nitrates (Ferric Nitrate (Fe (NO 3) 3-9H 2O)) 0.42
Zinc Sulphate Heptahydrate (Zinc sulfate (ZnSO 4-7H 2O)) 0.1726
Disodium hydrogen phosphate,anhydrous
188.4
(Sodium Phosphate dibasic(Na 2HPO 4)anhydrous)
Glucose (D-Glucose (Dextrose)) 1360.4
Phenol red (Phenol Red) 8
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 22
Xanthoglobulin (Hypoxanthine Na) 1.194
Linolic acid (Linoleic Acid) 0.0168
Thioctic Acid (Lipoic Acid) 0.042
Putrescine (Putrescine 2HCl) 0.0322
Gsh (Glutathione (reduced)) 0.23
Thymidine (Thymidine) 0.14
Damping fluid (HEPES) 4500
Albumin (Albumin) 500
Transferrins,iron complexes (Holo Transferrin) 5
Regular Insulin (Insulin) 5
3. substratum as claimed in claim 1 or 2 is characterized in that: wherein also comprise following composition and part by weight thereof:
Composition mg/L
Thanomin (Ethanolamine) 2-20
Sodium Selenite (sodium Selenite) 0.001-0.3
Cholesterol (Cholesterol) 0.1-4.5
2 mercapto ethanol (2-MercaptOethanol) 2-20
Linolic acid (linoleic acid) 0.5-10
Resin acid (palmitic acid) 0.5-10
Heparin (Heparin) 5-40
Dextran (Dextran) 3-50
4. substratum as claimed in claim 3 is characterized in that: the preferred proportion of wherein said composition and weight thereof is:
Composition mg/L
Thanomin (Ethanolamine) 8
Sodium Selenite (sodium Selenite) 0.003
Cholesterol (Cholesterol) 1.5
2 mercapto ethanol (2-MercaptOethanol) 6
Linolic acid (linoleic acid) 1
Resin acid (palmitic acid) 1
Heparin (Heparin) 10
Dextran (Dextran) 15
5. Vero amplification CHEMICALLY DEFINED MEDIUM (synthetic medium that Chemical Composition is clear and definite) substratum, it is characterized in that: the composition of substratum and weight part are:
Composition mg/L
Glycine (Glycine) 10-50
L-L-Ala (L-Alanine) 5.2-40.2
L-arginine (L-Arginine) 60.2-200
The left-handed amino-succinamic acid of L-(L-Asparagine) 5-60
L-aspartic acid (L-Aspartic acid) 2.011-36
Cysteine hydrochloride-water (L-Cysteine hydrochloride-H 2O) 0.5-15
Cystinic acid dihydrochloride (L-Cystine 2HCl) 2-50.6
L-L-glutamic acid (L-Glutamic Acid) 5-100
L-glutaminate (L-Glutamine) 50-200
L-Histidine (L-Histidine) 6.003-50.005
L-oxyproline (L-Hydroxyproline) 0.05-30.
L-Isoleucine (L-Isoleucine) 5-60
L-leucine (L-Leucine) 10.05-80
L lysine HCL (L-Lysine hydrochloride) 20.05-100
L-methionine(Met) (L-Methionine) 2-30.5
L-phenylalanine (L-Phenylalanine) 5-50
L-proline(Pro) (L-Proline) 20-150
L-Serine (L-Serine) 5-50
L-Threonine (L-Threonine) 5-50
L-tryptophane (L-Tryptophan) 5-30
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 5-50
L-Xie Ansuan (L-Valine) 5-50
Vitamin H (Biotin) 0.0003-0.9
Choline chloride 60 (Choline chloride) 2-30
Calcium pantothenate (D-Calcium pantothenated) 0.01-3
Folic acid (Folic Acid) 0.5-15
Inositol (i-Inositol) 5-40
Niacinamide (Niacinamide) 0.01-15
Padil (Para-Aminobenzoic Acid) 0.01-3
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.01-2
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.01-0.8
Riboflavin (Riboflavin) 0.02-5
Thiamine hydrochloride (Thiamine hydrochloride) 0.05-3
Vitamins B 12(Vitamin B 12) 0.05-4.5
Xitix (Ascorbic Acid) 0.05-2
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.001-0.5
Vitamin K 3(Menadione (Vitamin K 3)) 0.001-0.5
Naphthoquinones (Niacin) 0.001-0.8
Vitamin A (Vitamin A) 0.01-3
Vitamins D 2(Vitamin D 2) 0.01-4
Calcium chloride (CaCl 2) 2-30
Four water-calcium nitrate (Ca (NO 3) 24H 2O) 15-200
Magnesium chloride (MgCl 2) 5-60
Sal epsom (MgSO 4) 20-100
Repone K (KCl) 100-500
Sodium-chlor (NaCl) 5000-10000
Sodium dihydrogen phosphate-water (NaH 2PO 4-H 2O) 50-250
Sodium phosphate dibasic (Na 2HPO 4) 20-120
Saltpetre (KNO 3) 0.1-5
Sodium Selenite (Na 2SeO 3-5H 2O) 0-20
Sodium bicarbonate (NaHCO 3) 50-300
Glucose (D-Glucose (Dextrose)) 1000-3000
Phenol red (Phenol Red) 2-30
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 20-200
Gsh (Glutathione (reduced)) 0.001-0.25
Thymidine (Thymidine) 0.05-3.0
Damping fluid (HEPES) 1500-4000
Adenine sulfate (Adenine sulfate) 3-40
Cholesterol (Cholesterol) 0.3-4.5
2-deoxidation D-ribose (2-deoxy-D-Ribose) 0.02-0.40
D-ribose (D-Ribose) 0.05-3
Gsh (Glutathione (reduced)) 0.3-4.5
Guanine hydrochloride (Guanine.HCL) 5-40
Xanthoglobulin (Hypoxanthine.Na) 0.05-3
Linolic acid (Linoleic Acid) 0.01-3
Thioctic Acid (Lipoic Acid) 0.05-0.9
Phenol red (Phenol Red-Na) 5-30
Putrescine (Putrescine-2HCl) 0.8-4.5
Sodium.alpha.-ketopropionate (Pyruvic Acid Na) 5-100
Thymus pyrimidine (Thymine) 0.05-0.8
Uridylic (uracil) 0.01-2
Thanomin (Ethanolamine) 0.5-10
6. substratum as claimed in claim 5 is characterized in that: the composition and the weight part of substratum are preferably:
Composition mg/L
Glycine (Glycine) 28.75
L-L-Ala (L-Alanine) 17.45
L-arginine (L-Arginine) 123.61
The left-handed amino-succinamic acid of L-(L-Asparagine) 25.504
L-aspartic acid (L-Aspartic acid) 7.317
Cysteine hydrochloride-water (L-Cysteine hydrochloride-H 2O) 3.2
Cystinic acid dihydrochloride (L-Cystine 2HCl) 27.048
L-L-glutamic acid (L-Glutamic Acid) 45.59
L-glutaminate (L-Glutamine) 130.32
L-Histidine (L-Histidine) 21.436
L-oxyproline (L-Hydroxyproline) 6.97
L-Isoleucine (L-Isoleucine) 25.536
L-leucine (L-Leucine) 39.176
L lysine HCL (L-Lysine hydrochloride) 53.25
L-methionine(Met) (L-Methionine) 10.79
L-phenylalanine (L-Phenylalanine) 16.15
L-proline(Pro) (L-Proline) 35.53
L-Serine (L-Serine) 19.33
L-Threonine (L-Threonine) 21.55
L-tryptophane (L-Tryptophan) 6.126
Tyrosine salt sodium monocalcium salt (L-Tyrosine disodium salt dihydrate) 33.934
L-Xie Ansuan (L-Valine) 18.94
Vitamin H (Biotin) 0.02792
Choline chloride 60 (Choline chloride) 6.35
Calcium pantothenate (D-Calcium pantothenated) 0.225
Folic acid (Folic Acid) 1.525
Inositol (i-Inositol) 10.825
Niacinamide (Niacinamide) 0.0769
Padil (Para-Aminobenzoic Acid) 0.125
Pyridoxal hydrochloride (Pyridoxal hydrochloride) 0.0625
Hydrochloric acid pyrrole (Pyridoxine hydrochloride) 0.0865
Riboflavin (Riboflavin) 0.0398
Thiamine hydrochloride (Thiamine hydrochloride) 0.145
Vitamins B 12(Vitamin B 12) 0.76
Xitix (Ascorbic Acid) 0.075
Alpha-tocopherol phosphoric acid ester (Alpha-tocopherol Phosphate) 0.005
Vitamin K 3(Menadione (Vitamin K 3)) 0.005
Naphthoquinones (Niacin) 0.0625
Vitamin A (Vitamin A) 0.05
Vitamins D 2(Vitamin D 2) 0.05
Calcium chloride (CaCl 2) 10
Four water-calcium nitrate (Ca (NO 3) 24H 2O) 113.288
Magnesium chloride (MgCl 2) 22.888
Sal epsom (MgSO 4) 68.835
Repone K (KCl) 329.44
Sodium-chlor (NaCl) 7085.6
Sodium dihydrogen phosphate-water (NaH 2PO 4-H 2O) 128
Sodium phosphate dibasic (Na 2HPO 4) 56.8
Saltpetre (KNO 3) 0
Sodium Selenite (Na 2SeO 3-5H 2O) 0
Sodium bicarbonate (NaHCO 3) 220
Glucose (D-Glucose (Dextrose)) 1520.8
Phenol red (Phenol Red) 11.48
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 44
Gsh (Glutathione (reduced)) 0.075
Thymidine (Thymidine) 0.28
Damping fluid (HEPES) 2600
Adenine sulfate (Adenine sulfate) 15
Cholesterol (Cholesterol) 1.5
2-deoxidation D-ribose (2-deoxy-D-Ribose) 0.16
D-ribose (D-Ribose) 0.22
Gsh (Glutathione (reduced)) 1.5
Guanine hydrochloride (Guanine.HCL) 22
Xanthoglobulin (Hypoxanthine) 0.45
Linolic acid (Linoleic Acid) 0.04
Thioctic Acid (Lipoic Acid) 0.105
Phenol red (Phenol Red-Na) 10.6
Putrescine (Putrescine-2HCl) 1.6905
Sodium.alpha.-ketopropionate (Pyruvic Acid Na) 55
Thymus pyrimidine (Thymine) 0.15
Uridylic (uracil) 0.15
Thanomin (Ethanolamine) 2
7. as claim 5 or 6 described substratum, it is characterized in that: wherein also comprise following composition and part by weight thereof:
Composition mg/L
Semi-lactosi (Galactose) 1000-3000
Urogastron (Epidermal Growth Factor (recombinant human)) 0.05-2
Cortisone (Hydrocortisone) 0.001-0.8
Regular Insulin (Insulin (recombinant human, Zn Full Chain)) 2-30
Prostaglandin(PG) (Prostaglandin E1) 0.0001-0.56
Triiodothyronine (Triiodothyronine (T3)) 0.0001-0.4
8. substratum as claimed in claim 7 is characterized in that: the preferred proportion of wherein said composition and weight thereof is:
Composition mg/L
Semi-lactosi (Galactose) 2000
Urogastron (Epidermal Growth Factor (recombinant human)) 0.33
Cortisone (Hydrocortisone) 0.01
Regular Insulin (Insulin (recombinant human, Zn Full Chain)) 12
Prostaglandin(PG) (Prostaglandin E1) 0.001
Triiodothyronine (Triiodothyronine (T3)) 0.0005
9. the serum-free culture method of a Vero cell, it is characterized in that: with the Vero cell of adherent growth, adapt to SFM serum free medium domestication adaptation cultivation through claim 1 or 2 described Vero cell suspensions, the Vero growth pattern can be converted into suspension growth by adherent growth; Further make the growth of can in Vero increases CHEMICALLY DEFINED MEDIUM (synthetic medium that Chemical Composition is clear and definite) substratum, increasing of Vero cell.
10. as method as described in the claim 9, it is characterized in that: the Vero cell suspension that the Vero cell of adherent growth is domesticated for suspension growth adapts to the SFM serum free medium and does not contain any animal serum; Behind the Vero cell suspension growth adaptation; The used substratum of amplification growth does not contain protein.
CNB2007101874667A 2007-11-27 2007-11-27 Culture medium used for Vero cell and cultivation method thereof Expired - Fee Related CN100482783C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CNB2007101874667A CN100482783C (en) 2007-11-27 2007-11-27 Culture medium used for Vero cell and cultivation method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNB2007101874667A CN100482783C (en) 2007-11-27 2007-11-27 Culture medium used for Vero cell and cultivation method thereof

Publications (2)

Publication Number Publication Date
CN101182490A true CN101182490A (en) 2008-05-21
CN100482783C CN100482783C (en) 2009-04-29

Family

ID=39447915

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB2007101874667A Expired - Fee Related CN100482783C (en) 2007-11-27 2007-11-27 Culture medium used for Vero cell and cultivation method thereof

Country Status (1)

Country Link
CN (1) CN100482783C (en)

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102827804A (en) * 2012-08-30 2012-12-19 苏州市沃美生物技术有限公司 Culture medium applicable to suspension and magnification cultivation of Vero cell microcarriers and method for suspension magnification cultivation of Vero cell microcarriers
CN104099392A (en) * 2014-07-08 2014-10-15 苏州康聚生物科技有限公司 Industrialized production method for high galactosylated modification recombination human erythrocyte growth stimulation protein
CN105026551A (en) * 2012-12-14 2015-11-04 德普伊新特斯产品公司 Nutrient enriched media for hUTC growth
CN105441378A (en) * 2015-12-22 2016-03-30 肇庆大华农生物药品有限公司 Serum-free medium used for culturing Vero cells, and preparation method thereof
CN105462911A (en) * 2015-12-22 2016-04-06 肇庆大华农生物药品有限公司 Serum-free medium for culturing viruses and preparation method thereof
CN105647851A (en) * 2016-03-01 2016-06-08 赛百慷（上海）生物技术股份有限公司 Endothelium culture system
CN106676053A (en) * 2016-12-31 2017-05-17 山东金周生物科技有限公司 Low-serum cell culture medium with universal adaptability and preparation method thereof
CN106754648A (en) * 2017-01-19 2017-05-31 弎西艾斯（苏州）生物科技有限公司 A kind of non-animal derived culture medium
CN106754634A (en) * 2016-12-31 2017-05-31 山东金周生物科技有限公司 A kind of serum free medium and preparation method thereof
CN106834420A (en) * 2016-12-30 2017-06-13 河北德路通生物科技有限公司 Cell cleaning fluid, its preparation method and application with target cell sample defencive function
CN106834229A (en) * 2017-01-25 2017-06-13 华东理工大学 For the serum free medium of people's immunologic cytotoxicity cell expansion ex vivo
CN107236708A (en) * 2017-06-23 2017-10-10 王晓柯 A kind of serum free medium for supporting HeLa cell attachment cultures
CN107267443A (en) * 2017-07-27 2017-10-20 郑州爱科生物科技有限公司 A kind of vero E6 cell lines for adapting to the full culture that suspends and its application
CN107858174A (en) * 2017-11-23 2018-03-30 成都艾伟孚生物科技有限公司 A kind of special Embryo Culture mineral oil in vitro fertilization and preparation method thereof
CN108486061A (en) * 2018-03-16 2018-09-04 李军涛 A kind of suspension culture method and its application of attached cell
CN108690825A (en) * 2017-04-06 2018-10-23 上海倍谙基生物科技有限公司 Protect the serum free medium of antibody disulfide bond integrality in animal cell culture
WO2019085271A1 (en) * 2017-10-30 2019-05-09 华南农业大学 Glyphosate resistant seed soaking agent for no-tillage dry direct-seeding rice
CN110272864A (en) * 2019-07-25 2019-09-24 北京鼎持生物技术有限公司 A kind of Vero33 cell strain adapting to serum free suspension culture and its acclimation method and application
CN110616183A (en) * 2019-09-23 2019-12-27 山东甲骨文生物科技有限公司 Low-serum culture medium for Vero cell culture and corresponding virus production
CN110628697A (en) * 2019-09-23 2019-12-31 山东甲骨文生物科技有限公司 Serum-free culture medium for VERO serum-free cell culture and corresponding virus production
CN112094802A (en) * 2020-09-28 2020-12-18 成都柏奥特克生物科技股份有限公司 Serum-free culture medium for culturing Vero cells
CN114107178A (en) * 2021-12-16 2022-03-01 无锡多宁生物科技有限公司 Serum-free Vero cell basal medium and preparation method thereof
CN114540277A (en) * 2022-03-18 2022-05-27 杭州荣泽生物科技集团有限公司 Serum-free medium for culturing Vero cells and preparation method thereof

Cited By (33)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102827804A (en) * 2012-08-30 2012-12-19 苏州市沃美生物技术有限公司 Culture medium applicable to suspension and magnification cultivation of Vero cell microcarriers and method for suspension magnification cultivation of Vero cell microcarriers
CN105026551A (en) * 2012-12-14 2015-11-04 德普伊新特斯产品公司 Nutrient enriched media for hUTC growth
TWI620818B (en) * 2012-12-14 2018-04-11 德派信迪思公司 Nutrient enriched media for hutc growth
CN105026551B (en) * 2012-12-14 2020-07-03 德普伊新特斯产品公司 Nutrient enrichment medium for hUTC growth
CN111690602A (en) * 2012-12-14 2020-09-22 德普伊新特斯产品公司 Nutrient enrichment medium for hUTC growth
CN104099392A (en) * 2014-07-08 2014-10-15 苏州康聚生物科技有限公司 Industrialized production method for high galactosylated modification recombination human erythrocyte growth stimulation protein
CN104099392B (en) * 2014-07-08 2017-01-25 苏州康聚生物科技有限公司 Industrialized production method for high galactosylated modification recombination human erythrocyte growth stimulation protein
CN105441378A (en) * 2015-12-22 2016-03-30 肇庆大华农生物药品有限公司 Serum-free medium used for culturing Vero cells, and preparation method thereof
CN105462911A (en) * 2015-12-22 2016-04-06 肇庆大华农生物药品有限公司 Serum-free medium for culturing viruses and preparation method thereof
CN105647851A (en) * 2016-03-01 2016-06-08 赛百慷（上海）生物技术股份有限公司 Endothelium culture system
CN106834420A (en) * 2016-12-30 2017-06-13 河北德路通生物科技有限公司 Cell cleaning fluid, its preparation method and application with target cell sample defencive function
CN106676053A (en) * 2016-12-31 2017-05-17 山东金周生物科技有限公司 Low-serum cell culture medium with universal adaptability and preparation method thereof
CN106754634A (en) * 2016-12-31 2017-05-31 山东金周生物科技有限公司 A kind of serum free medium and preparation method thereof
CN106676053B (en) * 2016-12-31 2020-08-25 山东甲骨文生物科技有限公司 Low-serum cell culture medium with wide adaptability and preparation method thereof
CN106754648A (en) * 2017-01-19 2017-05-31 弎西艾斯（苏州）生物科技有限公司 A kind of non-animal derived culture medium
CN106834229B (en) * 2017-01-25 2021-08-17 华东理工大学 Serum-free culture medium for in vitro amplification of human immune killer cells
CN106834229A (en) * 2017-01-25 2017-06-13 华东理工大学 For the serum free medium of people's immunologic cytotoxicity cell expansion ex vivo
CN108690825A (en) * 2017-04-06 2018-10-23 上海倍谙基生物科技有限公司 Protect the serum free medium of antibody disulfide bond integrality in animal cell culture
CN108690825B (en) * 2017-04-06 2021-08-17 上海倍谙基生物科技有限公司 Serum-free medium for protecting integrity of antibody disulfide bonds in animal cell culture
CN107236708A (en) * 2017-06-23 2017-10-10 王晓柯 A kind of serum free medium for supporting HeLa cell attachment cultures
CN107267443A (en) * 2017-07-27 2017-10-20 郑州爱科生物科技有限公司 A kind of vero E6 cell lines for adapting to the full culture that suspends and its application
CN107267443B (en) * 2017-07-27 2020-06-05 郑州爱科生物科技有限公司 Vero E6 cell strain suitable for full suspension culture and application thereof
WO2019085271A1 (en) * 2017-10-30 2019-05-09 华南农业大学 Glyphosate resistant seed soaking agent for no-tillage dry direct-seeding rice
CN107858174A (en) * 2017-11-23 2018-03-30 成都艾伟孚生物科技有限公司 A kind of special Embryo Culture mineral oil in vitro fertilization and preparation method thereof
CN108486061A (en) * 2018-03-16 2018-09-04 李军涛 A kind of suspension culture method and its application of attached cell
CN108486061B (en) * 2018-03-16 2021-08-20 李军涛 Suspension culture method of adherent cells and application thereof
CN110272864A (en) * 2019-07-25 2019-09-24 北京鼎持生物技术有限公司 A kind of Vero33 cell strain adapting to serum free suspension culture and its acclimation method and application
CN110628697A (en) * 2019-09-23 2019-12-31 山东甲骨文生物科技有限公司 Serum-free culture medium for VERO serum-free cell culture and corresponding virus production
CN110616183A (en) * 2019-09-23 2019-12-27 山东甲骨文生物科技有限公司 Low-serum culture medium for Vero cell culture and corresponding virus production
CN112094802A (en) * 2020-09-28 2020-12-18 成都柏奥特克生物科技股份有限公司 Serum-free culture medium for culturing Vero cells
CN114107178A (en) * 2021-12-16 2022-03-01 无锡多宁生物科技有限公司 Serum-free Vero cell basal medium and preparation method thereof
CN114107178B (en) * 2021-12-16 2024-04-16 无锡多宁生物科技有限公司 Vero cell serum-free basal medium and preparation method thereof
CN114540277A (en) * 2022-03-18 2022-05-27 杭州荣泽生物科技集团有限公司 Serum-free medium for culturing Vero cells and preparation method thereof

Also Published As

Publication number Publication date
CN100482783C (en) 2009-04-29

Similar Documents

Publication Publication Date Title
CN100482783C (en) Culture medium used for Vero cell and cultivation method thereof
CN101760442B (en) Serum-free medium for MDCK cell large-scale adherent culture and single-cell suspension culture
KR101264940B1 (en) Medium and culture of embryonic stem cells
CN100362098C (en) Non-serum culture medium for multiple animal cell large-scale culture
US5474931A (en) Media concentrate technology
US4657866A (en) Serum-free, synthetic, completely chemically defined tissue culture media
CN106834229B (en) Serum-free culture medium for in vitro amplification of human immune killer cells
NZ591651A (en) High titer antibody production and culture media comprising glucose, soy or wheat hydrolsyate, amino acids, and other chemical compounds
EP0501435B1 (en) A serum-free medium for culturing animal cells
KR20070058584A (en) Culturing human embryonic stem cells
EP2167644B1 (en) Culture media for developmental cells containing elevated concentrations of lipoic acid
CN101418330B (en) Non protein culture medium adapted to large-scale culture of NSO cell and production of antibody
CN100506977C (en) Gonad cell amplification culture medium of Chinese hamster and uses thereof
CN102827804A (en) Culture medium applicable to suspension and magnification cultivation of Vero cell microcarriers and method for suspension magnification cultivation of Vero cell microcarriers
CN101195817A (en) Hybrid tumor cell amplification culture medium and uses thereof
US8637312B2 (en) Mammalian culture media with polyamine and iron
AU2004217807A1 (en) Animal-free cell culture method
JP6845218B2 (en) How to increase glutathione levels in cells
CN107435037A (en) A kind of serum free medium for bhk cell
AU662491B2 (en) Medium for culture of mammalian cells
CN105087460A (en) ST cell culture medium
JPH0249579A (en) Culture medium
CN113943694B (en) Universal serum-free culture medium supporting adherence or suspension culture of various vaccine cells and preparation method thereof
JPH03180176A (en) Nutrient medium for cell culture
JPH0678759A (en) Serum-free cell culture medium

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
DD01 Delivery of document by public notice

Addressee: Tianjin Bainuoke Medicine Biotechnology Co., Ltd.

Document name: Notification to Pay the Fees

DD01 Delivery of document by public notice

Addressee: Tang Hewen

Document name: Notification of Termination of Patent Right

C56 Change in the name or address of the patentee
CP02 Change in the address of a patent holder

Address after: 300308 Tianjin Airport Logistics Processing Zone Tianbao Industrial Park No. 4 Building 3 layer Tianjin bairuoke biotechnology limited liability company

Patentee after: Tianjin Bainuoke Medicine Biotechnology Co., Ltd.

Address before: 300450, Tianjin Tanggu Free Trade Zone, Haibin 1 road

Patentee before: Tianjin Bainuoke Medicine Biotechnology Co., Ltd.

DD01 Delivery of document by public notice

Addressee: Xiao Jingmin

Document name: Notification to Pay the Fees

DD01 Delivery of document by public notice

Addressee: Tianjin Bainuoke Medicine Biotechnology Co., Ltd.

Document name: Notification to Pay the Fees

DD01 Delivery of document by public notice

Addressee: Tianjin Bainuoke Medicine Biotechnology Co., Ltd.

Document name: Notification of Termination of Patent Right

CF01 Termination of patent right due to non-payment of annual fee

Granted publication date: 20090429

Termination date: 20141127

EXPY Termination of patent right or utility model