CN100482783C - 一种用于Vero细胞的培养基及其培养方法 - Google Patents

一种用于Vero细胞的培养基及其培养方法 Download PDF

Info

Publication number
CN100482783C
CN100482783C CNB2007101874667A CN200710187466A CN100482783C CN 100482783 C CN100482783 C CN 100482783C CN B2007101874667 A CNB2007101874667 A CN B2007101874667A CN 200710187466 A CN200710187466 A CN 200710187466A CN 100482783 C CN100482783 C CN 100482783C
Authority
CN
China
Prior art keywords
acid
sodium
hydrochloride
water
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.)
Expired - Fee Related
Application number
CNB2007101874667A
Other languages
English (en)
Other versions
CN101182490A (zh
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/zh
Publication of CN101182490A publication Critical patent/CN101182490A/zh
Application granted granted Critical
Publication of CN100482783C publication Critical patent/CN100482783C/zh
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

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

Abstract

本发明涉及一种培养基的配制和使用方法，更具体地说涉及一种用于Vero细胞的培养基及其培养方法。属于生物制品领域。本发明产品的培养方法如下：将贴壁生长的Vero细胞，Vero细胞悬浮适应SFM无血清培养基驯化适应培养，Vero生长方式可由贴壁生长转化为悬浮生长；进一步使Vero细胞可以在Vero扩增CHEMICALLY DEFINED MEDIUM培养基中扩增生长。本发明方法具有良好的效果，因为使Vero细胞生长模式，从贴壁生长转化为悬浮生长，改变细胞的生长模式，可以极大地增加细胞产量。

Description

一种用于Vero细胞的培养基及其培养方法
技术领域
发明涉及一种培养基的配制和使用方法，更具体地说涉及一种用于Vero细胞的培养基及其培养方法。属于生物制品领域。
背景技术
动物血清是培养基是生物制品领域中常用的物质，在细菌和细胞培养中十分必要，但是血清具有明显且无法客服的缺点：成分不明确，产品批间差异大，容易传播细菌，病毒等疾病，价格昂贵，使用血清培养的细胞所产生的重组蛋白，其下游纯化工艺复杂，成本高昂。重组蛋白工业生产中所用的Vero细胞特性为贴壁生长，其培养体系所能提供的贴附底物面积有限，使其增殖扩增数量难以大幅度提升。
发明内容
【要解决的问题】
本发明的目的，是提供一种可以使Vero细胞经适应性培养，将其生长模式从贴壁生长转化为悬浮生长的方法，本发明描述的培养基不含有任何动物血清成分，即提供一种用于Vero细胞的培养基及其培养方法。
【技术方案】
本发明的一种Vero细胞悬浮适应SFM无血清培养基，其中，培养基的组成成份及浓度为：
成份                                                  mg/L
甘氨酸(Glycine)                                       10-50
L-丙氨酸(L-Alanine)                                   5-30
L-精氨酸(L-Arginine)                                  30-200
L-门冬酰胺(L-Asparagine)                              10-60
L-天冬氨酸(L-Aspartic acid)                           1-15
L-半胱氨酸盐酸盐-一水(L-Cysteine hydrochloride-H2O)    0.01-0.6
L-胱胺酸二盐酸(L-Cystine 2HCl)                     15-100
L-谷氨酸(L-Glutamic Acid)                          10-100
L-谷氨酰胺(L-Glutamine)                            50-200
L-组氨酸(L-Histidine)                              5-100
L-羟脯氨酸(L-Hydroxyproline)                       2-20
L-异亮氨酸(L-Isoleucine)                           10-100
L-亮氨酸(L-Leucine)                                10-100
L-赖氨酸盐酸盐(L-Lysine hydrochloride)             15-120
L-甲硫氨酸(L-Methionine)                           2-30
L-苯丙氨酸(L-Phenylalanine)                        5-50
L-脯氨酸(L-Proline)                                10-100
L-丝氨酸(L-Serine)                                 5-60
L-苏氨酸(L-Threonine)                              5-50
L-色氨酸(L-Tryptophan)                             2-50
二水酪氨酸钠盐(L-Tyrosine disodium salt dihydrate) 5-100
L-缬氨酸(L-Valine)                                 5-100
生物素(Biotin)                                     0.00001-0.5
氯化胆碱(Choline chloride)                         0.5-20.
泛酸钙(D-Calcium pantothenated)                    0.001-0.9
叶酸(Folic Acid)                                   0.001-1.5
肌醇(i-Inositol)                                   2.-30
烟酰胺(Niacinamide)                                0.0001-0.98
氨基乙酸(Para-Aminobenzoic Acid)                   0.05-3.
盐酸吡哆醛(Pyridoxal hydrochloride)                0.002-0.7
盐酸吡哆辛(Pyridoxine hydrochloride)               0.006-3.5
核黄素(Riboflavin)                                 0.0001-6.
盐酸硫胺(Thiamine hydrochloride)                   0.003-0.15
维生素B12(Vitamin B12)                             0.002-1.8
抗坏血酸(Ascorbic Acid)                            0.01-1.2
阿尔法-生育酚磷酸酯(Alpha-tocopherol Phosphate)   0.001-3
(四水)硝酸钙(Calcium nitrate(Ca(NO3)24H2O)        50-200
(无水)氯化镁(Magnesium Chloride(anhydrous))       3-40
(无水)硫酸镁                                      25-150
(Magnesium Sulfate(MgSO4)(anhyd.))
氯化钾(Potassium Chloride(KCl))                   150-600
氯化钠(Sodium Chloride(NaCl))                     3000-10000
一水磷酸氢二钠                                    5-200
(Sodium Phosphate monobasic(NaH2PO4-H2O))
五水硫酸铜(Cupric sulfate(CuS4-5H2O))              0.0001-0.03
七水硫酸亚铁(Ferric sulfate(FeSO4-7H2O))           0.002-0.8
九水硝酸铁(Ferric Nitrate(Fe(NO3)3-9H2O))          0.02-3.5
七水硫酸锌(Zinc sulfate(ZnS4-7H2O))                0.003-0.86
无水磷酸氢二钠                                    50-300
(Sodium Phosphate dibasic(Na2HPO4)anhydrous)
D-葡萄糖(D-Glucose(Dextrose))                    800-2000
酚红(Phenol Red)                                 1-30
丙酮酸钠(Sodium Pyruvate)                        2-50
次黄嘌呤钠(Hypoxanthine Na)                      0.008-8
亚油酸(Linoleic Acid)                            0.0003-1.2
硫辛酸(Lipoic Acid)                              0.003-0.8
腐胺(Putrescine 2HCl)                            0.0001-0.5
谷胱甘肽(Glutathione(reduced))                   0.05-3
胸苷(Thymidine)                                  0.02-6
缓冲液(HEPES)                                    2000-6500
白蛋白(Albumin)                                  300-800
转铁蛋白(Holo Transferrin)                       1-20
胰岛素(Insulin)                                  1-20
上述培养基的组成成份及浓度优选为：
成份                                               mg/L
甘氨酸(Glycine)                                    33.5
L-丙氨酸(L-Alanine)                                16.78
L-精氨酸(L-Arginine)                               124.2
L-门冬酰胺(L-Asparagine)                           31.002
L-天冬氨酸(L-Aspartic acid)                        6.66
L-半胱氨酸盐酸盐-水(L-Cysteine hydrochloride-H2O)  0.06
L-胱胺酸二盐酸(L-Cystine 2HCl)                     35.624
L-谷氨酸(L-Glutamic Acid)                          51.94
L-谷氨酰胺(L-Glutamine)                            149.2
L-组氨酸(L-Histidine)                              20.328
L-羟脯氨酸(L-Hydroxyproline)                       10
L-异亮氨酸(L-Isoleucine)                           34.8
L-亮氨酸(L-Leucine)                                48.62
L-赖氨酸盐酸盐(L-Lysine hydrochloride)             57.3
L-甲硫氨酸(L-Methionine)                           12.9
L-苯丙氨酸(L-Phenylalanine)                        19
L-脯氨酸(L-Proline)                                34.9
L-丝氨酸(L-Serine)                                 23.1
L-苏氨酸(L-Threonine)                              24.38
L-色氨酸(L-Tryptophan)                             7.408
二水酪氨酸钠盐(L-Tyrosine disodium salt dihydrate) 42.162
L-缬氨酸(L-Valine)                                 21.34
生物素(Biotin)                                     0.04746
氯化胆碱(Choline chloride)                         3.7
泛酸钙(D-Calcium pantothenated)                    0.156
叶酸(Folic Acid)                                   0.466
肌醇(i-Inositol)                                   10.63
烟酰胺(Niacinamide)                                0.2222
氨基乙酸(Para-Aminobenzoic Acid)                        0.23
盐酸吡哆醛(Pyr idoxal hydrochloride)                    0.015
盐酸吡哆辛(Pyridoxine hydrochloride)                    0.227
核黄素(Riboflavin)                                      0.0534
盐酸硫胺(Thiamine hydrochloride)                        0.266
维生素B12(Vitamin B12)                                  0.281
抗坏血酸(Ascorbic Acid)                                0.03
阿尔法-生育酚磷酸酯(Alpha-tocopherol Phosphate)        0.006
(四水)硝酸钙(Calcium nitrate(Ca(NO3)24H2O)             146.644
(无水)氯化镁(Magnesium Chloride(anhydrous))            11.444
(无水)硫酸镁                                           68.37
(Magnesium Sulfate(MgSO4)(anhyd.))
氯化钾(Potassium Chloride(KCl))                        364.72
氯化钠(Sodium Chloride(NaCl))                          6799.8
一水磷酸氢二钠                                         84
(Sodium Phosphate monobasic(NaH2PO4-H2O))
五水硫酸铜(Cupric sulfate(CuSO4-5H2O))                 0.0005
七水硫酸亚铁(Ferric sulfate(FeSO4-7H2O))               0.1668
九水硝酸铁(Ferric Nitrate(Fe(NO3)3-9H2O))              0.42
七水硫酸锌(Zinc sulfate(ZnSO4-7H2O))                   0.1726
无水磷酸氢二钠                                         188.4
(Sodium Phosphate dibasic(Na2HPO4)anhydrous)
D-葡萄糖(D-Glucose(Dextrose))                          1360.4
酚红(Phenol Red)                                       8
丙酮酸钠(Sodium Pyruvate)                              22
次黄嘌呤钠(Hypoxanthine Na)                            1.194
亚油酸(Linoleic Acid)                                  0.0168
硫辛酸(Lipoic Acid)                                    0.042
腐胺(Putrescine 2HCl)                                  0.0322
谷胱甘肽(Glutathione(reduced))             0.23
胸苷(Thymidine)                            0.14
缓冲液(HEPES)                              4500
白蛋白(Albumin)                            500
转铁蛋白(Holo Transferrin)                 5
胰岛素(Insulin)                            5
上述的培养基，其中还包含如下成份及其重量比例：
成份                                       mg/L
乙醇胺(Ethanolamine)                       2-20
亚硒酸钠(sodium Selenite)                  0.001-0.3
胆固醇(Cholesterol)                        0.1-4.5
2-巯基乙醇(2-MercaptOethanol)              2-20
亚油酸(linoleic acid)                      0.5-10
脂酸(palmitic acid)                        0.5-10
肝素(Heparin)                             5-40
右旋糖酐(Dextran)                         3-50
上述还包含的成份及其浓度为：
成份                                       mg/L
乙醇胺(Ethanolamine)                       8
亚硒酸钠(sodium Selenite)                  0.003
胆固醇(Cholesterol)                        1.5
2-巯基乙醇(2-MercaptOethanol)              6
脂酸(palmitic acid)                        1
肝素(Heparin)                             10
右旋糖酐(Dextran)                         15
本发明一种Vero扩增CHEMICALLY DEFINED MEDIUM(化学成份明确的合成培养基)培养基，其中，培养基的组成成份及浓度为：
成份                                               mg/L
甘氨酸(Glycine)                                    10-50
L-丙氨酸(L-Alanine)                                5.2-40.2
L-精氨酸(L-Arginine)                               60.2-200
L-左旋门冬酰胺(L-Asparagine)                       5-60
L-天冬氨酸(L-Aspartic acid)                        2.011-36
L-半胱氨酸盐酸盐-水(L-Cysteine hydrochloride-H2O)   0.5-15
胱胺酸二盐酸盐(L-Cystine 2HCl)                     2-50.6
L-谷氨酸(L-Glutamic Acid)                          5-100
L-谷氨酰胺(L-Glutamine)                            50-200
L-组氨酸(L-Histidine)                              6.003-50.005
L-羟脯氨酸(L-Hydroxyproline)                       0.05-30.
L-异亮氨酸(L-Isoleucine)                           5-60
L-亮氨酸(L-Leucine)                                10.05-80
L-赖氨酸盐酸盐(L-Lysine hydrochloride)             20.05-100
L-甲硫氨酸(L-Methionine)                           2-30.5
L-苯丙氨酸(L-Phenylalanine)                        5-50
L-脯氨酸(L-Proline)                                20-150
L-丝氨酸(L-Serine)                                 5-50
L-苏氨酸(L-Threonine)                              5-50
L-色氨酸(L-Tryptophan)                             5-30
二水酪氨酸钠盐(L-Tyrosine disodium salt dihydrate)  5-50
L-缬氨酸(L-Valine)                                 5-50
生物素(Biotin)                                    0.0003-0.9
氯化胆碱(Choline chloride)                         2-30
泛酸钙(D-Calcium pantothenated)                    0.01-3
叶酸(Folic Acid)                                   0.5-15
肌醇(i-Inositol)                                   5-40
烟酰胺(Niacinamide)                                0.01-15
氨基乙酸(Para-Aminobenzoic Acid)                   0.01-3
盐酸吡哆醛(Pyridoxal hydrochloride)                0.01-2
盐酸吡哆辛(Pyridoxine hydrochloride)               0.01-0.8
核黄素(Riboflavin)                                 0.02-5
盐酸硫胺(Thiamine hydrochloride)                   0.05-3
维生素B12(Vitamin B12)                              0.05-4.5
抗坏血酸(Ascorbic Acid)                            0.05-2
阿尔法-生育酚磷酸酯(Alpha-tocopherol Phosphate)     0.001-0.5
维生素K3(Menadione(Vitamin K3))                    0.001-0.5
萘醌(Niacin)                                      0.001-0.8
维生素A(Vitamin A)                                 0.01-3
维生素D2(Vitamin D2)                               0.01-4
氯化钙(CaCl2)                                      2-30
四水硝酸钙(Ca(NO3)24H2O)                           15-200
氯化镁(MgCl2)                                     5-60
硫酸镁(MgSO4)                                     20-100
氯化钾(KCl)                                       100-500
氯化钠(NaCl)                                      5000-10000
一水磷酸二氢钠(NaH2PO4-H2O)                        50-250
磷酸氢二钠(Na2HPO4)                               20-120
硝酸钾(KNO3)                                      0.1-5
亚硒酸钠(Na2SeO3-5H2O)                            0-20
碳酸氢钠(NaHCO3)                                 50-300
D-葡萄糖(D-Glucose(Dextrose))                   1000-3000
酚红(Phenol Red)                                2-30
丙酮酸钠(Sodium Pyruvate)                       20-200
谷胱甘肽(Glutathione(reduced))                  0.001-0.25
胸苷(Thymidine)                                 0.05-3.0
缓冲液(HEPES)                                   1500-4000
硫酸腺嘌呤(Adenine sulfate)                       3-40
胆固醇(Cholesterol)                               0.3-4.5
2-脱氧D-核糖(2-deoxy-D-Ribose)                    0.02-0.40
D-核糖(D-Ribose)                                  0.05-3
谷胱甘肽(Glutathione(reduced))                    0.3-4.5
盐酸鸟嘌呤(Guanine.HCL)                           5-40
次黄嘌呤钠(Hypoxanthine Na)                       0.05-3
亚油酸(Linoleic Acid)                             0.01-3
硫辛酸(Lipoic Acid)                               0.05-0.9
酚红(Phenol Red-Na)                               5-30
腐胺(Putrescine-2HCl)                             0.8-4.5
丙酮酸钠(Pyruvic Acid Na)                         5-100
胸腺嘧啶(Thymine)                                 0.05-0.8
尿嘧啶(uracil)                                    0.01-2
乙醇胺(Ethanolamine)                              0.5-10
上述的培养基的组成成份及浓度优选为：
成份                                               mg/L
甘氨酸(Glycine)                                    28.75
L-丙氨酸(L-Alanine)                                17.45
L-精氨酸(L-Arginine)                               123.61
L-左旋门冬酰胺(L-Asparagine)                       25.504
L-天冬氨酸(L-Aspartic acid)                        7.317
L-半胱氨酸盐酸盐-水(L-Cysteine hydrochloride-H2O)   3.2
胱胺酸二盐酸盐(L-Cystine 2HCl)                     27.048
L-谷氨酸(L-Glutamic Acid)                          45.59
L-谷氨酰胺(L-Glutamine)                            130.32
L-组氨酸(L-Histidine)                              21.436
L-羟脯氨酸(L-Hydroxyproline)                       6.97
L-异亮氨酸(L-Isoleucine)                           25.536
L-亮氨酸(L-Leucine)                              39.176
L-赖氨酸盐酸盐(L-Lysine hydrochloride)            53.25
L-甲硫氨酸(L-Methionine)                         10.79
L-苯丙氨酸(L-Phenylalanine)                      16.15
L-脯氨酸(L-Proline)                              35.53
L-丝氨酸(L-Serine)                               19.33
L-苏氨酸(L-Threonine)                            21.55
L-色氨酸(L-Tryptophan)                           6.126
二水酪氨酸钠盐(L-Tyrosine disodium salt dihydrate)33.934
L-缬氨酸(L-Valine)                               18.94
生物素(Biotin)                                   0.02792
氯化胆碱(Choline chloride)                       6.35
泛酸钙(D-Calcium pantothenated)                  0.225
叶酸(Folic Acid)                                 1.525
肌醇(i-Inositol)                                 10.825
烟酰胺(Niacinamide)                              0.0769
氨基乙酸(Para-Aminobenzoic Acid)                 0.125
盐酸吡哆醛(Pyridoxal hydrochloride)              0.0625
盐酸吡哆辛(Pyridoxine hydrochloride)             0.0865
核黄素(Riboflavin)                               0.0398
盐酸硫胺(Thiamine hydrochloride)                 0.145
维生素B12(Vitamin B12)                           0.76
抗坏血酸(Ascorbic Acid)                         0.075
阿尔法-生育酚磷酸酯(Alpha-tocopherol Phosphate)  0.005
维生素K3(Menadione(Vitamin K3))                 0.005
萘醌(Niacin)                                    0.0625
维生素A(Vitamin A)                              0.05
维生素D2(Vitamin D2)                            0.05
氯化钙(CaCl2)                                   10
四水硝酸钙(Ca(NO3)24H2O)               113.288
氯化镁(MgCl2)                         22.888
硫酸镁(MgSO4)                         68.835
氯化钾(KCl)                           329.44
氯化钠(NaCl)                          7085.6
一水磷酸二氢钠(NaH2PO4-H2O)            128
磷酸氢二钠(Na2HPO4)                   56.8
硝酸钾(KNO3)                          0
亚硒酸钠(Na2SeO3-5H2O)                 0
碳酸氢钠(NaHCO3)                      220
D-葡萄糖(D-Glucose(Dextrose))         1520.8
酚红(Phenol Red)                      11.48
丙酮酸钠(Sodium Pyruvate)             44
谷胱甘肽(Glutathione(reduced))        0.075
胸苷(Thymidine)                       0.28
缓冲液(HEPES)                         2600
硫酸腺嘌呤(Adenine sulfate)           15
胆固醇(Cholesterol)                   1.5
2-脱氧D-核糖(2-deoxy-D-Ribose)        0.16
D-核糖(D-Ribose)                      0.22
谷胱甘肽(Glutathione(reduced))        1.5
盐酸鸟嘌呤(Guanine.HCL)               22
次黄嘌呤(Hypoxanthine)                0.45
亚油酸(Linoleic Acid)                 0.04
硫辛酸(Lipoic Acid)                   0.105
酚红(Phenol Red-Na)                   10.6
腐胺(Putrescine-2HCl)                 1.6905
丙酮酸钠(Pyruvic Acid Na)              55
胸腺嘧啶(Thymine)                     0.15
尿嘧啶(uracil)                                 0.15
乙醇胺(Ethanolamine)                           2
Vero扩增CHEMICALLY DEFINED MEDIUM培养基还包含如下成份及其重量比例：
成份                                                     mg/L
半乳糖(Galactose)                                        1000-3000
表皮生长因子(Epidermal Growth Factor(recombinant human))  0.05-2
可的松(Hydrocortisone)                                   0.001-0.8
胰岛素(Insulin(recombinant human，Zn Full Chain))         2-30
前列腺素(Prostaglandin E1)                                0.0001-0.56
三碘甲状腺氨酸(Triiodothyronine(T3))                     0.0001-0.4
上述的Vero扩增CHEMICALLY DEFINED MEDIUM培养基成份及其浓度为：
成份                                                    mg/L
半乳糖(Galactose)                                       2000
表皮生长因子(Epidermal Growth Factor(recombinant human)) 0.33
可的松(Hydrocortisone)                                  0.01
胰岛素(Insulin(recombinant human，Zn Full Chain))        12
前列腺素(Prostaglandin E1)                              0.001
三碘甲状腺氨酸(Triiodothyroninc(T3))                    0.0005
本发明的一种Vero细胞的无血清培养方法，其中，将贴壁生长的Vero细胞，经上述的Vero细胞悬浮适应SFM无血清培养基驯化适应培养，Vero生长方式可由贴壁生长转化为悬浮生长；进一步使Vero细胞可以在Vero扩增CHEMICALLYDEFINED MEDIUM(化学成份明确的合成培养基)培养基中扩增生长。
将贴壁生长的Vero细胞驯化为悬浮生长的Vero细胞悬浮适应SFM无血清培养基不含任何动物血清。
培养的条件是本领域中常规技术中使用的条件，优选的培养条件是：37摄氏度，5％CO2，湿度大于95％的培养环境。
将贴壁生长的Vero细胞驯化为悬浮生长的Vero细胞悬浮适应SFM无血清培养基不含任何动物血清；Vero细胞悬浮生长适应后；扩增生长所用的培养基不含蛋白质。
本发明中培养基的配制和使用方法，其用化学成分明确的物质替代血清，使Vero细胞生长模式，从贴壁生长转化为悬浮生长，改变细胞的生长模式，可以极大地增加细胞产量。
更为重要的是本发明描述的扩增培养基，不含有任何蛋白成分，极大地简化了重组蛋白的纯化工艺过程，使重组蛋白的产量和纯度明显提高。
本发明方法具有良好的效果，因为使Vero细胞生长模式，从贴壁生长转化为悬浮生长，改变细胞的生长模式，可以极大地增加细胞产量。
更为重要的是本发明描述的扩增培养基，不含有任何蛋白成分，极大地简化了重组蛋白的纯化工艺过程，使重组蛋白的产量和纯度明显提高。
本发明方法及其得到的产品具有良好的效果，用悬浮适应SFM无血清培养基使Vero细胞转化为悬浮生长模式；
进一步Vero细胞在Vero扩增CHEMICALLY DEFINED(即：化学成份明确的合成培养基)无蛋白培养基中扩增生长，Vero扩增CHEMICALLY DEFINED无蛋白培养基中不含有任何蛋白质组分，极大地简化了由Vero细胞生产的重组蛋白的下游纯化工艺，提高了产量和纯度。
发明效果：
从如下生长曲线可以看出细胞生长速度很快
 
培养天数 细胞密度(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
附图说明
图1为细胞培养的生长曲线
具体实施方式
实施例1
Vero细胞的无血清培养方法，其中，将贴壁生长的Vero细胞，经Vero细胞悬浮适应SFM无血清培养基驯化适应培养，Vero生长方式可由贴壁生长转化为悬浮生长；进一步使Vero细胞可以在Vero扩增CHEMICALLY DEFINED MEDIUM(化学成份明确的合成培养基)培养基中扩增生长。培养的条件如下：37摄氏度，5％CO2，湿度大于95％的培养环境。
实施例2
培养方法如实施例1所述，其中，Vero细胞悬浮适应SFM无血清培养基和Vero扩增CHEMICALLY DEFINED MEDIUM培养基的组成成分及其比例分别为：
Vero细胞悬浮适应SFM无血清培养基成份及配比：
成份                                mg/L
甘氨酸(Glycine)                     33.5
L-丙氨酸(L-Alanine)                 16.78
L-精氨酸(L-Arginine)                124.2
L-门冬酰胺(L-Asparagine)                           31.002
L-天冬氨酸(L-Aspartic acid)                        6.66
L-半胱氨酸盐酸盐-水(L-Cysteine hydrochloride-H2O)   0.06
L-胱胺酸二盐酸(L-Cystine 2HCl)                     35.624
L-谷氨酸(L-Glutamic Acid)                          51.94
L-谷氨酰胺(L-Glutamine)                            149.2
L-组氨酸(L-Histidine)                              20.328
L-羟脯氨酸(L-Hydroxyproline)                       10
L-异亮氨酸(L-Isoleucine)                           34.8
L-亮氨酸(L-Leucine)                                48.62
L-赖氨酸盐酸盐(L-Lysine hydrochloride)              57.3
L-甲硫氨酸(L-Methionine)                           12.9
L-苯丙氨酸(L-Phenylalanine)                        19
L-脯氨酸(L-Proline)                                34.9
L-丝氨酸(L-Serine)                                 23.1
L-苏氨酸(L-Threonine)                              24.38
L-色氨酸(L-Tryptophan)                             7.408
二水酪氨酸钠盐(L-Tyrosine disodium salt dihydrate)  42.162
L-缬氨酸(L-Valine)                                 21.34
生物素(Biotin)                                     0.04746
氯化胆碱(Choline chloride)                         3.7
泛酸钙(D-Calcium pantothenated)                    0.156
叶酸(Folic Acid)                                   0.466
肌醇(i-Inositol)                                   10.63
烟酰胺(Niacinamide)                                0.2222
氨基乙酸(Para-Aminobenzoic Acid)                   0.23
盐酸吡哆醛(Pyridoxal hydrochloride)                0.015
盐酸吡哆辛(Pyridoxine hydrochloride)               0.227
核黄素(Riboflavin)                                 0.0534
盐酸硫胺(Thiamine hydrochloride)                0.266
维生素B12(Vitamin B12)                          0.281
抗坏血酸(Ascorbic Acid)                        0.03
阿尔法-生育酚磷酸酯(Alpha-tocopherol Phosphate) 0.006
(四水)硝酸钙(Calcium nitrate(Ca(NO3)24H2O)      146.644
(无水)氯化镁(Magnesium Chloride(anhydrous))    11.444
(无水)硫酸镁
                                              68.37
(Magnesium Sulfate(MgSO4)(anhyd.))
氯化钾(Potassium Chloride(KCl))               364.72
氯化钠(Sodium Chloride(NaCl))                 6799.8
一水磷酸氢二钠
                                             84
(Sodium Phosphate monobasic(NaH2PO4-H2O))
五水硫酸铜(Cupric sulfate(CuSO4-5H2O))        0.0005
七水硫酸亚铁(Ferric sulfate(FeSO4-7H2O))      0.1668
九水硝酸铁(Ferric Nitrate(Fe(NO3)3-9H2O))     0.42
七水硫酸锌(Zinc sulfate(ZnS4-7H2O))           0.1726
无水磷酸氢二钠
                                            188.4
(Sodium Phosphate dibasic(Na2HPO4)anhydrous)
D-葡萄糖(D-Glucose(Dextrose))                1360.4
酚红(Phenol Red)                             8
丙酮酸钠(Sodium Pyruvate)                    22
次黄嘌呤钠(Hypoxanthine Na)                  1.194
亚油酸(Linoleic Acid)                        0.0168
硫辛酸(Lipoic Acid)                          0.042
腐胺(Putrescine 2HCl)                       0.0322
谷胱甘肽(Glutathione(reduced))              0.23
胸苷(Thymidine)                             0.14
缓冲液(HEPES)                               4500
白蛋白(Albumin)                             500
转铁蛋白(Holo Transferrin)                         5
胰岛素(Insulin)                                    5
Vero扩增CHEMICALLY DEFINED MEDIUM培养基
成份                                               mg/L
甘氨酸(Glycine)                                    28.75
L-丙氨酸(L-Alanine)                                17.45
L-精氨酸(L-Arginine)                               123.61
L-左旋门冬酰胺(L-Asparagine)                       25.504
L-天冬氨酸(L-Aspartic acid)                        7.317
L-半胱氨酸盐酸盐-水(L-Cysteine hydrochloride-H2O)   3.2
胱胺酸二盐酸盐(L-Cystine 2HCl)                     27.048
L-谷氨酸(L-Glutamic Acid)                          45.59
L-谷氨酰胺(L-Glutamine)                            130.32
L-组氨酸(L-Histidine)                              21.436
L-羟脯氨酸(L-Hydroxyproline)                       6.97
L-异亮氨酸(L-Isoleucine)                           25.536
L-亮氨酸(L-Leucine)                                39.176
L-赖氨酸盐酸盐(L-Lysine hydrochloride)             53.25
L-甲硫氨酸(L-Methionine)                           10.79
L-苯丙氨酸(L-Phenylalanine)                        16.15
L-脯氨酸(L-Proline)                                35.53
L-丝氨酸(L-Serine)                                 19.33
L-苏氨酸(L-Threonine)                              21.55
L-色氨酸(L-Tryptophan)                             6.126
二水酪氨酸钠盐(L-Tyrosine disodium salt dihydrate)  33.934
L-缬氨酸(L-Valine)                                 18.94
生物素(Biotin)                                     0.02792
氯化胆碱(Choline chloride)                         6.35
泛酸钙(D-Calcium pantothenated)                    0.225
叶酸(Folic Acid)                                1.525
肌醇(i-Inositol)                                10.825
烟酰胺(Niacinamide)                             0.0769
氨基乙酸(Para-Aminobenzoic Acid)                0.125
盐酸吡哆醛(Pyridoxal hydrochloride)             0.0625
盐酸吡哆辛(Pyridoxine hydrochloride)            0.0865
核黄素(Riboflavin)                              0.0398
盐酸硫胺(Thiamine hydrochloride)                0.145
维生素B12(Vitamin B12)                           0.76
抗坏血酸(Ascorbic Acid)                         0.075
阿尔法-生育酚磷酸酯(Alpha-tocopherol Phosphate)  0.005
维生素K3(Menadione(Vitamin K3))                  0.005
萘醌(Niacin)                                    0.0625
维生素A(Vitamin A)                              0.05
维生素D2(Vitamin D2)                             0.05
氯化钙(CaCl2)                                   10
四水硝酸钙(Ca(NO3)24H2O)                         113.288
氯化镁(MgCl2)                                   22.888
硫酸镁(MgSO4)                                   68.835
氯化钾(KCl)                                     329.44
氯化钠(NaCl)                                    7085.6
一水磷酸二氢钠(NaH2PO4-H2O)                      128
磷酸氢二钠(Na2HPO4)                             56.8
硝酸钾(KNO3)                                    0
亚硒酸钠(Na2SeO3-5H2O)                          0
碳酸氢钠(NaHCO3)                               220
D-葡萄糖(D-Glucose(Dextrose))                  1520.8
酚红(Phenol Red)                               11.48
丙酮酸钠(Sodium Pyruvate)                      44
谷胱甘肽(Glutathione(reduced))               0.075
胸苷(Thymidine)                              0.28
缓冲液(HEPES)                                2600
硫酸腺嘌呤(Adenine sulfate)                  15
胆固醇(Cholesterol)                          1.5
2-脱氧D-核糖(2-deoxy-D-Ribose)               0.16
D-核糖(D-Ribose)                            0.22
谷胱甘肽(Glutathione(reduced))              1.5
盐酸鸟嘌呤(Guanine.HCL)                     22
次黄嘌呤(Hypoxanthine)                      0.45
亚油酸(Linoleic Acid)                       0.04
硫辛酸(Lipoic Acid)                         0.105
酚红(Phenol Red-Na)                         10.6
腐胺(Putrescine-2HCl)                       1.6905
丙酮酸钠(Pyruvic Acid Na)                   55
胸腺嘧啶(Thymine)                           0.15
尿嘧啶(uracil)                             0.15
乙醇胺(Ethanolamine)                        2
实施例3
培养方法如实施例1所述，其中，Vero细胞悬浮适应SFM无血清培养基和Vero扩增CHEMICALLY DEFINED MEDIUM培养基的组成成分及其比例分别为：
Vero细胞悬浮适应SFM无血清培养基成份及配比：
成份                                                mg/L
甘氨酸(Glycine)                                     50
L-丙氨酸(L-Alanine)                                 5
L-精氨酸(L-Arginine)                                200
L-门冬酰胺(L-Asparagine)                            10
L-天冬氨酸(L-Aspartic acid)                         15
L-半胱氨酸盐酸盐-一水(L-Cysteine hydrochloride-H2O)  0.01
L-胱胺酸二盐酸(L-Cystine 2HCl)                   100
L-谷氨酸(L-Glutamic Acid)                        10
L-谷氨酰胺(L-Glutamine)                          200
L-组氨酸(L-Histidine)                            5
L-羟脯氨酸(L-Hydroxyproline)                     20
L-异亮氨酸(L-Isoleucine)                         10
L-亮氨酸(L-Leucine)                              100
L-赖氨酸盐酸盐(L-Lysine hydrochloride)            15
L-甲硫氨酸(L-Methionine)                         30
L-苯丙氨酸(L-Phenylalanine)                      5
L-脯氨酸(L-Proline)                              100
L-丝氨酸(L-Serine)                               5
L-苏氨酸(L-Threonine)                            50
L-色氨酸(L-Tryptophan)                           2
二水酪氨酸钠盐(L-Tyrosine disodium salt dihydrate)100
L-缬氨酸(L-Valine)                               5
生物素(Biotin)                                   0.5
氯化胆碱(Choline chloride)                       0.5
泛酸钙(D-Calcium pantothenated)                  0.9
叶酸(Folic Acid)                                 0.001
肌醇(i-Inositol)                                 30
烟酰胺(Niacinamide)                              0.0001
氨基乙酸(Para-Aminobenzoic Acid)                 3.
盐酸吡哆醛(Pyridoxal hydrochloride)              0.002
盐酸吡哆辛(Pyridoxine hydrochloride)             3.5
核黄素(Riboflavin)                               0.0001
盐酸硫胺(Thiamine hydrochloride)                 0.15
维生素B12(Vitamin B12)                            0.002
抗坏血酸(Ascorbic Acid)                          1.2
阿尔法-生育酚磷酸酯(Alpha-tocopherol Phosphate) 0.001
(四水)硝酸钙(Calcium nitrate(Ca(NO3)24H2O)        200
(无水)氯化镁(Magnesium Chloride(anhydrous))      3
(无水)硫酸镁
                                                150
(Magnesium Sulfate(MgSO4)(anhyd.))
氯化钾(Potassium Chloride(KCl))                  150
氯化钠(Sodium Chloride(NaCl))                    10000
一水磷酸氢二钠
                                                5
(Sodium Phosphate monobasic(NaH2PO4-H2O))
五水硫酸铜(Cupric suifate(CuSO4-5H2O))            0.03
七水硫酸亚铁(Ferric sulfate(FeSO4-7H2O))          0.002
九水硝酸铁(Ferric Nitrate(Fe(NO3)3-9H2O))         3.5
七水硫酸锌(Zinc sulfate(ZnSO4-7H2O))              0.003
无水磷酸氢二钠
                                                 300
(Sodium Phosphate dibasic(Na2HPO4)anhydrous)
D-葡萄糖(D-Glucose(Dextrose))                    800
酚红(Phenol Red)                                 30
丙酮酸钠(Sodium Pyruvate)                        2
次黄嘌呤钠(Hypoxanthine Na)                      8
亚油酸(Linoleic Acid)                            0.0003
硫辛酸(Lipoic Acid)                              0.8
腐胺(Putrescine 2HCl)                            0.0001
谷胱甘肽(Glutathione(reduced))                   0.05
胸苷(Thymidine)                                  6
缓冲液(HEPES)                                    2000
白蛋白(Albumin)                                  800
转铁蛋白(Holo Transferrin)                       1
胰岛素(Insulin)                                  20
Vero扩增CHEMICALLY DEFINED MEDIUM培养基
成份                                             mg/L
甘氨酸(Glycine)                                  10
L-丙氨酸(L-Alanine)                              40.2
L-精氨酸(L-Arginine)                             60.2
L-左旋门冬酰胺(L-Asparagine)                     60
L-天冬氨酸(L-Aspartic acid)                      2.011
L-半胱氨酸盐酸盐-水(L-Cysteine hydrochloride-H2O)15
胱胺酸二盐酸盐(L-Cystine 2HCl)                   2
L-谷氨酸(L-Glutamic Acid)                        100
L-谷氨酰胺(L-Glutamine)                          50
L-组氨酸(L-Histidine)                            50.005
L-羟脯氨酸(L-Hydroxyproline)                     0.05
L-异亮氨酸(L-Isoleucine)                         60
L-亮氨酸(L-Leucine)                              10.05
L-赖氨酸盐酸盐(L-Lysine hydrochloride)            100
L-甲硫氨酸(L-Methionine)                         2
L-苯丙氨酸(L-Phenylalanine)                      50
L-脯氨酸(L-Proline)                              20
L-丝氨酸(L-Serine)                               50
L-苏氨酸(L-Threonine)                            5
L-色氨酸(L-Tryptophan)                           30
二水酪氨酸钠盐(L-Tyrosine disodium salt dihydrate)5
L-缬氨酸(L-Valine)                               50
生物素(Biotin)                                   0.0003
氯化胆碱(Choline chloride)                       30
泛酸钙(D-Calcium pantothenated)                  0.01
叶酸(Folic Acid)                                 15
肌醇(i-Inositol)                                 5
烟酰胺(Niacinamide)                              15
氨基乙酸(Para-Aminobenzoic Acid)                     0.01
盐酸吡哆醛(Pyridoxal hydrochloride)                  2
盐酸吡哆辛(Pyridoxine hydrochloride)                 0.01
核黄素(Riboflavin)                                   5
盐酸硫胺(Thiamine hydrochloride)                     0.05
维生素B12(Vitamin B12)                               4.5
抗坏血酸(Ascorbic Acid)                              0.05
阿尔法-生育酚磷酸酯(Alpha-tocopherol Phosphate)       0.5
维生素K3(Menadione(Vitamin K3))                      0.001
萘醌(Niacin)                                         0.8
维生素A(Vitamin A)                                   0.01
维生素D2(Vitamin D2)                                 4
氯化钙(CaCl2)                                        2
四水硝酸钙(Ca(NO3)24H2O)                              200
氯化镁(MgCl2)                                        5
硫酸镁(MgSO4)                                        100
氯化钾(KCl)                                          100
氯化钠(NaCl)                                         10000
一水磷酸二氢钠(NaH2PO4-H2O)                            50
磷酸氢二钠(Na2HPO4)                                   120
硝酸钾(KNO3)                                          0.1
亚硒酸钠(Na2SeO3-5H2O)                                20
碳酸氢钠(NaHCO3)                                      50
D-葡萄糖(D-Glucose(Dextrose))                        3000
酚红(Phenol Red)                                     2
丙酮酸钠(Sodium Pyruvate)                            200
谷胱甘肽(Glutathione(reduced))                       0.001
胸苷(Thymidine)                                      3.0
缓冲液(HEPES)                                        1500
硫酸腺嘌呤(Adenine sulfate)                            40
胆固醇(Cholesterol)                                    0.3
2-脱氧D-核糖(2-deoxy-D-Ribose)                         0.40
D-核糖(D-Ribose)                                       0.05
谷胱甘肽(Glutathione(reduced))                         4.5
盐酸鸟嘌呤(Guanine.HCL)                                5
次黄嘌呤钠(Hypoxanthine Na)                            3
亚油酸(Linoleic Acid)                                  0.01
硫辛酸(Lipoic Acid)                                    0.9
酚红(Phenol Red-Na)                                    5
腐胺(Putrescine-2HCl)                                  4.5
丙酮酸钠(Pyruvic Acid Na)                              5
胸腺嘧啶(Thymine)                                      0.8
尿嘧啶(uracil)                                         0.01
乙醇胺(Ethanolamine)                                   10
实施例4
培养方法如实施例1所述，其中，Vero细胞悬浮适应SFM无血清培养基和Vero扩增CHEMICALLY DEFINED MEDIUM培养基的组成成分及其比例分别为：
Vero细胞悬浮适应SFM无血清培养基成份及配比：
成份                                                 mg/L
甘氨酸(Glycine)                                      10
L-丙氨酸(L-Alanine)                                  30
L-精氨酸(L-Arginine)                                 30
L-门冬酰胺(L-Asparagine)                             60
L-天冬氨酸(L-Aspartic acid)                          1
L-半胱氨酸盐酸盐-一水(L-Cysteine hydrochloride-H2O)    0.6
L-胱胺酸二盐酸(L-Cystine 2HCl)                       15
L-谷氨酸(L-Glutamic Acid)                            100
L-谷氨酰胺(L-Glutamine)                              50
L-组氨酸(L-Histidine)                               100
L-羟脯氨酸(L-Hydroxyproline)                        2
L-异亮氨酸(L-Isoleucine)                            100
L-亮氨酸(L-Leucine)                                 10
L-赖氨酸盐酸盐(L-Lysine hydrochloride)              120
L-甲硫氨酸(L-Methionine)                            2
L-苯丙氨酸(L-Phenylalanine)                         50
L-脯氨酸(L-Proline)                                 10
L-丝氨酸(L-Serine)                                  60
L-苏氨酸(L-Threonine)                               5
L-色氨酸(L-Tryptophan)                              50
二水酪氨酸钠盐(L-Tyrosine disodium salt dihydrate)   5
L-缬氨酸(L-Valine)                                  100
生物素(Biotin)                                      0.00001
氯化胆碱(Choline chloride)                          20
泛酸钙(D-Calcium pantothenated)                     0.001
叶酸(Folic Acid)                                    1.5
肌醇(i-Inositol)                                    2
烟酰胺(Niacinamide)                                 0.98
氨基乙酸(Para-Aminobenzoic Acid)                    0.05
盐酸吡哆醛(Pyridoxal hydrochloride)                 0.7
盐酸吡哆辛(Pyridoxine hydrochloride)                0.006
核黄素(Riboflavin)                                  6
盐酸硫胺(Thiamine hydrochloride)                    0.003
维生素B12(Vitamin B12)                              1.8
抗坏血酸(Ascorbic Acid)                             0.01
阿尔法-生育酚磷酸酯(Alpha-tocopherol Phosphate)     3
(四水)硝酸钙(Calcium nitrate(Ca(NO3)24H2O)          50
(无水)氯化镁(Magnesium Chloride(anhydrous))        40
(无水)硫酸镁
                                              25
(Magnes ium Sul fate(MgSO4)(anhyd.))
氯化钾(Potassium Chloride(KCl))                600
氯化钠(Sodium Chloride(NaCl))                  3000
一水磷酸氢二钠
                                              200
(Sodium Phosphate monobasic(NaH2PO4-H2O))
五水硫酸铜(Cupric sulfate(CuS4-5H2O))          0.0001
七水硫酸亚铁(Ferric sulfate(FeSO4-7H2O))       0.8
九水硝酸铁(Ferric Nitrate(Fe(NO3)3-9H2O))      0.02
七水硫酸锌(Zinc suifate(ZnSO4-7H2O))           0.86
无水磷酸氢二钠
                                              50
(Sodium Phosphate dibasic(Na2HPO4)anhydrous)
D-葡萄糖(D-Glucose(Dextrose))                 2000
酚红(Phenol Red)                              1
丙酮酸钠(Sodium Pyruvate)                     50
次黄嘌呤钠(Hypoxanthine Na)                   0.008
亚油酸(Linoleic Acid)                         1.2
硫辛酸(Lipoic Acid)                           0.003
腐胺(Putrescine 2HCl)                         0.5
谷胱甘肽(Glutathione(reduced))                0.05
胸苷(Thymidine)                               6
缓冲液(HEPES)                                 2000
白蛋白(Albumin)                               800
转铁蛋白(Holo Transferr in)                   1
胰岛素(Insulin)                               20
Vero扩增CHEMICALLY DEFINED MEDIUM培养基
成份mg/L
甘氨酸(Glycine)                               10
L-丙氨酸(L-Alanine)                           40.2
L-精氨酸(L-Arginine)                             60.2
L-左旋门冬酰胺(L-Asparagine)                     60
L-天冬氨酸(L-Aspartic acid)                      2.011
L-半胱氨酸盐酸盐-水(L-Cysteine hydrochloride-H2O)15
胱胺酸二盐酸盐(L-Cystine 2HCl)                   2
L-谷氨酸(L-Glutamic Acid)                        100
L-谷氨酰胺(L-Glutamine)                          50
L-组氨酸(L-Histidine)                            50.005
L-羟脯氨酸(L-Hydroxyproline)                     0.05
L-异亮氨酸(L-Isoleucine)                         60
L-亮氨酸(L-Leucine)                              10.05
L-赖氨酸盐酸盐(L-Lysine hydrochloride)           100
L-甲硫氨酸(L-Methionine)                         2
L-苯丙氨酸(L-Phenylalanine)                      50
L-脯氨酸(L-Proline)                              20
L-丝氨酸(L-Serine)                               50
L-苏氨酸(L-Threonine)                            5
L-色氨酸(L-Tryptophan)                           30
二水酪氨酸钠盐(L-Tyrosine disodium salt dihydrate)5
L-缬氨酸(L-Valine)                               50
生物素(Biotin)                                   0.0003
氯化胆碱(Choline chloride)                       30
泛酸钙(D-Calcium pantothenated)                  0.01
叶酸(Folic Acid)                                 15
肌醇(i-Inositol)                                 5
烟酰胺(Niacinamide)                              15
氨基乙酸(Para-Aminobenzoic Acid)                 0.01
盐酸吡哆醛(Pyridoxal hydrochloride)              2
盐酸吡哆辛(Pyridoxine hydrochloride)             0.01
核黄素(Riboflavin)                            5
盐酸硫胺(Thiamine hydrochloride)              0.05
维生素B12(Vitamin B12)                         4.5
抗坏血酸(Ascorbic Acid)                       0.05
阿尔法-生育酚磷酸酯(Alpha-tocopherol Phosphate)0.5
维生素K3(Menadione(Vitamin K3))                0.001
萘醌(Niacin)                                  0.8
维生素A(Vitamin A)                            0.01
维生素D2(Vitamin D2)                          4
氯化钙(CaCl2)                                 2
四水硝酸钙(Ca(NO3)24H2O)                       200
氯化镁(MgCl2)                                 5
硫酸镁(MgSO4)                                 100
氯化钾(KCl)                                   100
氯化钠(NaCl)                                  10000
一水磷酸二氢钠(NaH2PO4-H2O)                    50
磷酸氢二钠(Na2HPO4)                           120
硝酸钾(KNO3)                                  0.1
亚硒酸钠(Na2SeO3-5H2O)                        0
碳酸氢钠(NaHCO3)                              300
D-葡萄糖(D-Glucose(Dextrose))                 1000
酚红(Phenol Red)                              30
丙酮酸钠(Sodium Pyruvate)                     20
谷胱甘肽(Glutathione(reduced))                0.25
胸苷(Thymidine)                               3.0
缓冲液(HEPES)                                 1500
硫酸腺嘌呤(Adenine sulfate)                    40
胆固醇(Cholesterol)                            0.3
2-脱氧D-核糖(2-deoxy-D-Ribose)                 0.40
D-核糖(D-Ribose)                                 0.05
谷胱甘肽(Glutathione(reduced))                   4.5
盐酸鸟嘌呤(Guanine.HCL)                          5
次黄嘌呤钠(Hypoxanthine Na)                      3
亚油酸(Linoleic Acid)                            0.01
硫辛酸(Lipoic Acid)                              0.9
酚红(Phenol Red-Na)                              5
腐胺(Putrescine-2HCl)                            4.5
丙酮酸钠(Pyruvic Acid Na)                        5
胸腺嘧啶(Thymine)                                0.8
尿嘧啶(uracil)                                   0.01
乙醇胺(Ethanolamine)                             10
实施例5
培养方法如实施例3或4所述，其中，在实施例3或4所做的实验中采用的Vero细胞悬浮适应SFM无血清培养基和Vero扩增CHEMICALLY DEFINED MEDIUM培养基的组成成分及其比例分别增加的组分和配比为：
Vero细胞悬浮适应SFM无血清培养基中可增加的成份及配比：
成份                                          mg/L
乙醇胺(Ethanolamine)                          8
亚硒酸钠(sodium Selenite)                     0.003
胆固醇(Cholesterol)                           1.5
2-巯基乙醇(2-MercaptOethanol)                 6
亚油酸(linoleic acid)                         1
脂酸(palmitic acid)                           1
肝素(Heparin)                                 10
右旋糖酐(Dextran)                             15
Vero扩增CHEMICALLY DEFINED MEDIUM培养基中可增加的成份及配比：
成份                                         mg/L
半乳糖(Galactose)                            2000
表皮生长因子(Epidermal Growth Factor(recombinant human))   0.33
可的松(Hydrocortisone)                                    0.01
胰岛素(Insulin(recombinant human，Zn Full Chain))           12
前列腺素(Prostaglandin E1)                                 0.001
三碘甲状腺氨酸(Triiodothyronine(T3))                      0.0005
实施例6
培养方法如实施例3或4所述，其中，在实施例3或4所做的实验中采用的Vero细胞悬浮适应SFM无血清培养基和Vero扩增CHEMICALLY DEFINED MEDIUM培养基的组成成分及其比例分别增加的组分和配比为：
Vero细胞悬浮适应SFM无血清培养基中可增加的成份及配比：
成份                           mg/L
乙醇胺(Ethanolamine)           20
亚硒酸钠(sodium Selenite)      0.001
胆固醇(Cholesterol)            4.5
2-巯基乙醇(2-MercaptOethanol)  2
亚油酸(linoleic acid)          10
脂酸(palmitic acid)            0.5
肝素(Heparin)                 40
右旋糖酐(Dextran)             3
Vero扩增CHEMICALLY DEFINED MEDIUM培养基中可增加的成份及配比：
成份                                                    mg/L
半乳糖(Galactose)                                       3000
表皮生长因子(Epidermal Growth Factor(recombinant human)) 0.05
可的松(Hydrocortisone)                                  0.8
胰岛素(Insulin(recombinant human，Zn Full Chain))         2
前列腺素(Prostaglandin E1)                               0.56
三碘甲状腺氨酸(Triiodothyronine(T3))                    0.0001

Claims (4)

1.一种Vero细胞悬浮适应SFM无血清培养基，其特征在于：培养基的组成成份及浓度为：
成份                  mg/L
甘氨酸                10-50
L-丙氨酸              5-30
L-精氨酸              30-200
L-门冬酰胺            10-60
L-天冬氨酸            1-15
L-一水半胱氨酸盐酸盐   0.01-0.6
L-胱氨酸二盐酸        15-100
L-谷氨酸              10-100
L-谷氨酰胺            50-200
L-组氨酸              5-100
L-羟脯氨酸            2-20
L-异亮氨酸            10-100
L-亮氨酸              10-100
L-赖氨酸盐酸盐        15-120
L-甲硫氨酸            2-30
L-苯丙氨酸            5-50
L-脯氨酸              10-100
L-丝氨酸                            5-60
L-苏氨酸                            5-50
L-色氨酸                            2-50
二水酪氨酸钠盐                      5-100
L-缬氨酸                            5-100
生物素                              0.00001-0.5
氯化胆碱                            0.5-20
泛酸钙                              0.001-0.9
叶酸                                0.001-1.5
肌醇                                2-30
烟酰胺                              0.0001-0.98
氨基乙酸                            0.05-3
盐酸吡哆醛                          0.002-0.7
盐酸吡哆辛                          0.006-3.5
核黄素                              0.0001-6
盐酸硫胺                            0.003-0.15
维生素B12                            0.002-1.8
抗坏血酸                            0.01-1.2
阿尔法-生育酚磷酸酯                 0.001-3
四水硝酸钙                          50-200
氯化镁                              3-40
硫酸镁                              25-150
氯化钾                                150-600
氯化钠                                3000-10000
一水磷酸氢二钠                        5-200
五水硫酸铜                            0.0001-0.03
七水硫酸亚铁                          0.002-0.8
九水硝酸铁                            0.02-3.5
七水硫酸锌                            0.003-0.86
无水磷酸氢二钠                        50-300
D-葡萄糖                              800-2000
酚红                                  1-30
丙酮酸钠                              2-50
次黄嘌呤钠(hypoxanthine Na)           0.008-8
亚油酸                                0.0003-1.2
硫辛酸                                0.003-0.8
腐胺                                  0.0001-0.5
谷胱甘肽                              0.05-3
胸苷                                  0.02-6
HEPES缓冲液                           2000-6500
白蛋白                                300-800
转铁蛋白                              1-20
胰岛素                                1-20。
2.如权利要求1所述的Vero细胞悬浮适应SFM无血清培养基，其特征在于：培养基的组成成份及浓度优选为：
成份                               mg/L
甘氨酸                             33.5
L-丙氨酸                           16.78
L-精氨酸                           124.2
L-门冬酰胺                         31.002
L-天冬氨酸                         6.66
L-一水半胱氨酸盐酸盐               0.06
L-胱氨酸二盐酸                     35.624
L-谷氨酸                           51.94
L-谷氨酰胺                         149.2
L-组氨酸                           20.328
L-羟脯氨酸                         10
L-异亮氨酸                         34.8
L-亮氨酸                           48.62
L-赖氨酸盐酸盐                     57.3
L-甲硫氨酸                         12.9
L-苯丙氨酸                         19
L-脯氨酸                           34.9
L-丝氨酸                           23.1
L-苏氨酸                           24.38
L-色氨酸                           7.408
二水酪氨酸钠盐                         42.162
L-缬氨酸                               21.34
生物素                                 0.04746
氯化胆碱                               3.7
泛酸钙                                 0.156
叶酸                                   0.466
肌醇                                   10.63
烟酰胺                                 0.2222
氨基乙酸                               0.23
盐酸吡哆醛                             0.015
盐酸吡哆辛                             0.227
核黄素                                 0.0534
盐酸硫胺                               0.266
维生素B12                              0.281
抗坏血酸                               0.03
阿尔法-生育酚磷酸酯                    0.006
四水硝酸钙                             146.644
氯化镁                                 11.444
硫酸镁                                 68.37
氯化钾                                 364.72
氯化钠                                 6799.8
一水磷酸氢二钠                         84
五水硫酸铜                        0.0005
七水硫酸亚铁                      0.1668
九水硝酸铁                        0.42
七水硫酸锌                        0.1726
无水磷酸氢二钠                    188.4
D-葡萄糖                          1360.4
酚红                              8
丙酮酸钠                          22
次黄嘌呤钠(hypoxanthine Na)       1.194
亚油酸                            0.0168
硫辛酸                            0.042
腐胺                              0.0322
谷胱甘肽                          0.23
胸苷                              0.14
HEPES缓冲液                       4500
白蛋白                            500
转铁蛋白                          5
胰岛素                            5。
3.如权利要求1或2所述的培养基，其特征在于：其中还包含如下成份及其浓度：
成份                           mg/L
乙醇胺                         2-20
亚硒酸钠         0.001-0.3
胆固醇           0.1-4.5
2-巯基乙醇       2-20
脂酸             0.5-10
肝素             5-40
右旋糖酐         3-50。
4.如权利要求3所述的培养基，其特征在于：其中所述的成份及其浓度为：
成份                 mg/L
乙醇胺               8
亚硒酸钠             0.003
胆固醇               1.5
2-巯基乙醇           6
脂酸                 1
肝素                 10
右旋糖酐             15。
CNB2007101874667A 2007-11-27 2007-11-27 一种用于Vero细胞的培养基及其培养方法 Expired - Fee Related CN100482783C (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CNB2007101874667A CN100482783C (zh) 2007-11-27 2007-11-27 一种用于Vero细胞的培养基及其培养方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CNB2007101874667A CN100482783C (zh) 2007-11-27 2007-11-27 一种用于Vero细胞的培养基及其培养方法

Publications (2)

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

Family

ID=39447915

Family Applications (1)

Application Number Title Priority Date Filing Date
CNB2007101874667A Expired - Fee Related CN100482783C (zh) 2007-11-27 2007-11-27 一种用于Vero细胞的培养基及其培养方法

Country Status (1)

Country Link
CN (1) CN100482783C (zh)

Families Citing this family (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102827804B (zh) * 2012-08-30 2019-02-12 苏州市沃美生物技术有限公司 适用于Vero细胞微载体悬浮放大培养的培养基及方法
US20140170748A1 (en) * 2012-12-14 2014-06-19 DePuy Synthes Products, LLC Nutrient Enriched Media for hUTC Growth
CN104099392B (zh) * 2014-07-08 2017-01-25 苏州康聚生物科技有限公司 高糖基化修饰的重组人红细胞生长刺激蛋白的工业化生产方法
CN105441378A (zh) * 2015-12-22 2016-03-30 肇庆大华农生物药品有限公司 一种培养Vero细胞用的无血清培养基及其制备方法
CN105462911B (zh) * 2015-12-22 2018-04-13 肇庆大华农生物药品有限公司 用于培养病毒的无血清培养液及其制备方法
CN105647851A (zh) * 2016-03-01 2016-06-08 赛百慷（上海）生物技术股份有限公司 一种内皮培养体系
CN106834420A (zh) * 2016-12-30 2017-06-13 河北德路通生物科技有限公司 具有靶细胞样本保护功能的细胞清洗液、其制备方法及应用
CN106754634A (zh) * 2016-12-31 2017-05-31 山东金周生物科技有限公司 一种无血清培养基及其制备方法
CN106676053B (zh) * 2016-12-31 2020-08-25 山东甲骨文生物科技有限公司 一种具有广泛适应性的低血清细胞培养基及其制备方法
CN106754648A (zh) * 2017-01-19 2017-05-31 弎西艾斯（苏州）生物科技有限公司 一种无动物源培养基
CN106834229B (zh) * 2017-01-25 2021-08-17 华东理工大学 用于人免疫杀伤细胞体外扩增的无血清培养基
CN108690825B (zh) * 2017-04-06 2021-08-17 上海倍谙基生物科技有限公司 保护动物细胞培养中抗体二硫键完整性的无血清培养基
CN107236708A (zh) * 2017-06-23 2017-10-10 王晓柯 一种支持HeLa细胞贴壁培养的无血清培养基
CN107267443B (zh) * 2017-07-27 2020-06-05 郑州爱科生物科技有限公司 一种适应全悬浮培养的vero E6细胞株及其应用
CN107801718B (zh) * 2017-10-30 2019-09-10 华南农业大学 一种免耕旱直播水稻抗草甘膦浸种剂
CN107858174B (zh) * 2017-11-23 2020-04-28 成都艾伟孚生物科技有限公司 一种体外受精专用胚胎培养矿物油及其制备方法
CN108486061B (zh) * 2018-03-16 2021-08-20 李军涛 一种贴壁细胞的悬浮培养化方法及其应用
CN110272864B (zh) * 2019-07-25 2021-07-27 北京鼎持生物技术有限公司 一种适应无血清悬浮培养的Vero33细胞株及其驯化方法和应用
CN110616183A (zh) * 2019-09-23 2019-12-27 山东甲骨文生物科技有限公司 一种用于Vero细胞培养及相应病毒生产的低血清培养基
CN110628697A (zh) * 2019-09-23 2019-12-31 山东甲骨文生物科技有限公司 一种用于vero无血清细胞培养及相应病毒生产的无血清培养基
CN112094802B (zh) * 2020-09-28 2022-10-11 成都柏奥特克生物科技股份有限公司 一种用于培养Vero细胞的无血清培养基
CN114107178B (zh) * 2021-12-16 2024-04-16 无锡多宁生物科技有限公司 一种Vero细胞无血清基础培养基及其制备方法
CN114540277A (zh) * 2022-03-18 2022-05-27 杭州荣泽生物科技集团有限公司 一种用于培养Vero细胞的无血清培养基及其制备方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
利用生物反应器和Vero细胞培养鸡传染性法氏囊病病毒的研究. 石岗等.中国农业科学，第35卷第9期. 2002
利用生物反应器和Vero细胞培养鸡传染性法氏囊病病毒的研究. 石岗等.中国农业科学，第35卷第9期. 2002 *

Also Published As

Publication number Publication date
CN101182490A (zh) 2008-05-21

Similar Documents

Publication Publication Date Title
CN100482783C (zh) 一种用于Vero细胞的培养基及其培养方法
CN100362098C (zh) 适用于多种动物细胞大规模培养的无血清培养基
KR101264940B1 (ko) 배아 줄기 세포용 배지 및 이의 배양물
CN101760442A (zh) 适于mdck细胞大规模贴壁培养和单细胞悬浮培养的无血清培养基
US4657866A (en) Serum-free, synthetic, completely chemically defined tissue culture media
US5474931A (en) Media concentrate technology
US5712163A (en) Chemically defined cell culture media and system and methods for use, particularly for culturing epithelial cells
CN106834229B (zh) 用于人免疫杀伤细胞体外扩增的无血清培养基
CN109337861B (zh) 一种支持产物高表达的cho细胞无血清培养基
EP0501435B1 (en) A serum-free medium for culturing animal cells
KR20070058584A (ko) 인간 배아 줄기 세포의 배양
CN101418330B (zh) 适于nso细胞大规模培养及抗体生产的无蛋白培养基
CN103045533B (zh) 适于大规模生产流感疫苗的无血清培养基
CN100506977C (zh) 一种中国仓鼠卵巢细胞扩增培养基及其用途
CN101195817A (zh) 一种杂交瘤细胞扩增培养基及其用途
CN102827804A (zh) 适用于Vero细胞微载体悬浮放大培养的培养基及方法
RU2018112452A (ru) Обогащенная питательная среда для выращивания клеток, извлеченных из ткани пуповины человека
JP6845218B2 (ja) 細胞におけるグルタチオンレベルを増加させる方法
KR20100108424A (ko) 개선된 배양 배지 첨가제 및 이를 이용한 방법
CA2552229A1 (en) Basal medium for es cell culturing
CN107435037A (zh) 一种用于bhk细胞的无血清培养基
AU662491B2 (en) Medium for culture of mammalian cells
CN105087460A (zh) 一种st细胞培养基
JPH0249579A (ja) 培地
Hanss et al. Studies of culture media for the growth of human tumor cells

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