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

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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
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CN101182490A (zh
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黄宗堂
唐贺文
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TIANJIN BAINUOKE MEDICINE BIOTECHNOLOGY CO Ltd
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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。
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