CN114540277A - 一种用于培养Vero细胞的无血清培养基及其制备方法 - Google Patents
一种用于培养Vero细胞的无血清培养基及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于培养Vero细胞的无血清培养基及其制备方法,包括基础培养基,所述基础培养基包括葡萄糖和谷氨酰胺,还包括主要添加因子和次要添加因子,所述主要添加因子包括蛋白质类、激素、生长因子、促贴壁因子、微量元素和维生素,所述次要添加因子包括还原剂、胰蛋白酶抑制剂和剪切力保护剂。本发明中,通过本发明提供的培养基培养Vero细胞,不仅能维持Vero细胞的正常生长,还能提供Vero细胞的扩增效率,有利于加快新冠病毒疫苗的研发和生产,其次,培养基化学成分明确,不需要血清,成本低廉,避免了因使用血清带来的生物安全风险,有利于提高生物制品生产工艺和成品质量的稳定性。
Description
技术领域
本发明涉及Vero细胞培养基技术领域,尤其涉及一种用于培养Vero细胞的无血清培养基及其制备方法。
背景技术
Vero细胞世界卫生组织和我国生物制品规程认可的疫苗生产细胞系,目前已用于狂犬病毒、流感病毒等多种疫苗的生产,同时,对于通过获得灭活毒株或弱毒株新冠病毒进行疫苗研发途径的,Vero细胞也是极其优异的宿主细胞。
在疫苗生产中,为实现细胞的高密度培养,通常会在基础培养基中添加5%-10%的血清,血清可以提供细胞生长所需的多种营养物质,如激素、微量元素、贴壁因子等,但使用血清存在很多不足,因此,发展无血清培养基是细胞走向临床应用的重要条件,首先,目前现有部分市售的无血清培养基存在细胞贴壁性差、细胞增殖速率不够理想、成分不明等缺陷,因此,本发明提供一种用于培养Vero细胞的无血清培养基及其制备方法。
发明内容
为了解决上述背景技术中所提到的技术问题,而提出的一种用于培养Vero细胞的无血清培养基及其制备方法。
为了实现上述目的,本发明采用了如下技术方案:
一种用于培养Vero细胞的无血清培养基,包括基础培养基,所述基础培养基包括葡萄糖和谷氨酰胺,还包括主要添加因子和次要添加因子,所述主要添加因子包括蛋白质类、激素、生长因子、促贴壁因子、微量元素和维生素,所述次要添加因子包括还原剂、胰蛋白酶抑制剂和剪切力保护剂;
所述蛋白质类包括350~500mg/L血清白蛋白或者800~1000mg/L植物水解蛋白、5~15mg/L转铁蛋白或者8~10mg/L铁螯合剂,所述激素包括2~5mg/L胰岛素、1.5~2.0mg/L多肽类激素、1.6~1.8mg/L甾类激素和2.2~2.8mg/L皮质醇;
所述生长因子包括0.01~0.03mg/L表皮生长因子EGF、0.04~0.6mg/L转化生长因子TGF、0.05~0.15mg/L血小板生长因子PDGF和1.8~2.2mg/L成纤维细胞生长因子FGF,所述促贴壁因子包括1.8~2.5mg/L纤粘连蛋白、1.0~1.4mg/L胶原蛋白、0.3~0.8mg/L层粘连蛋白、1.1~1.4mg/L多聚-L赖氨酸或者多聚-D赖氨酸和0.4~0.7mg/L鸟氨酸;
所述微量元素包括0.01~0.03mg/L亚硒酸钠、0.03~0.04mg/L金精三羧酸和0.008~0.015mg/L Zn2+,所述维生素包括0.05~0.07mg/L核黄素、0.22~0.47mg/L叶酸、0.17~0.28mg/L钴胺素、0.1~0.226mg/L泛酸钙、7.68~10.63mg/L肌醇、0.2~0.3mg/L烟酰胺、2.1~3.5mg/L氯化胆碱、0.04~0.06mg/L硫辛酸、0.02~0.07mg/L盐酸吡哆醛、0.25~0.27mg/L维生素C、0.01~0.03mg/L维生素E、0.20~0.23mg/L盐酸吡哆醇和0.04~0.06mg/L生物素;
所述还原剂包括10~12mg/L乙醇胺、3~5mg/L丙酮酸钠和1~2mg/Lβ-疏基乙醇,所述剪切力保护剂包括0.1~0.5mg/L聚醚F-68。
作为上述技术方案的进一步描述:
所述主要添加因子还包括无机盐、抗生素和脂类,所述次要添加因子还包括缓冲液和泡沫稳定剂;
所述无机盐包括301.44~329.44mg/L氯化钾、10~13mg/L氯化钙、118~128mg/L磷酸二氢钠、6801~7085.6mg/L氯化钠和54.178~68.835mg/L硫酸镁,所述抗生素包括1.2~1.4mg/L两性霉素B、2.1~3.1mg/L喷他霉素和1.0~1.4mg/L抗真菌抗生素,所述脂类包括0.15~0.17mg/L亚油酸、0.22~0.23mg/L谷胱甘肽和0.2~0.5mg/L精胺;
所述缓冲液包括4000~4500mg/L HEPES和碳酸氢钠缓冲液,所述泡沫稳定剂包括3.5~4.2mg/L泊洛沙姆188。
作为上述技术方案的进一步描述:
所述铁螯合剂包括2.8~4.2mg/L Fe3+-硫酸酯-亚氨基二乙酸复合物和5.8~7.2mg/L Fe3+-硫酸酯-甘氨酰甘氨酸复合物。
作为上述技术方案的进一步描述:
所述多肽类激素包括1.1~1.28mg/L生长素、0.1~0.2mg/L胰高血糖素和0.02~0.03mg/L甲状腺素,所述甾类激素包括0.06~1.1mg/L孕酮、0.7~1.0mg/L氢化可的松和0.8~1.2mg/L雌二醇。
作为上述技术方案的进一步描述:
所述生物素包括0.001~0.005mg/L羧酸酯酶、0.015~0.018mg/L乙酰辅酶A羧化酶、0.015~0.017mg/L二氢吡啶二羧酸合成酶和0.008~0.015mg/Lβ-羟基羧酸酯羟基氧化酶。
作为上述技术方案的进一步描述:
一种用于培养Vero细胞的无血清培养基的制备方法,包括以下步骤:
S1、制备基础培养基溶解液:制备葡萄糖和谷氨酰胺的溶解液;
S2、分别制备主要添加因子的溶解液、次要添加因子的溶解液,将两组溶解液混合后过滤,得到混合溶解液;
S3、对混合溶解液进行预冻、升华和干燥处理,获得冻干粉剂的添加剂;
S31、对于预冻处理,是在-35~45℃的温度下,保持3~5h;
S32、对于升华处理,首先,将温度降低到-45~55℃,然后对整个工作系统抽真空,当真空度控制到<10Pa后,在19~25℃的温度下,升华干燥24h;
S33、对于干燥处理,是在7~9℃的温度下,干燥3~5h;
S34、干燥处理结束后,向工作系统中输入空气,使得工作系统的压力达到0.9~1.1个标准大气压,得到冻干粉剂的添加剂;
S4、对冻干粉剂的添加剂进行再次溶解,获得无血清培养的添加剂的溶解液,将基础培养基的溶解液和无血清培养的添加剂的溶解液混匀后过滤,得到无血清培养基。
综上所述,由于采用了上述技术方案,本发明的有益效果是:
1、本发明中,通过本发明提供的培养基培养Vero细胞,不仅能维持Vero细胞的正常生长,还能提供Vero细胞的扩增效率,有利于加快新冠病毒疫苗的研发和生产,其次,培养基化学成分明确,不需要血清,成本低廉,避免了因使用血清带来的生物安全风险,有利于提高生物制品生产工艺和成品质量的稳定性。
2、本发明中,本发明制备的无血清培养基细胞接种密度大,病毒滴度高,批间差以高度可控,质量均一,有利于新冠病毒的高效生产。
具体实施方式
下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例,都属于本发明保护的范围。
实施例
本发明提供一种技术方案:一种用于培养Vero细胞的无血清培养基,包括基础培养基,基础培养基包括葡萄糖和谷氨酰胺,还包括主要添加因子和次要添加因子,主要添加因子包括蛋白质类、激素、生长因子、促贴壁因子、微量元素、维生素、无机盐、抗生素和脂类,次要添加因子包括还原剂、胰蛋白酶抑制剂、剪切力保护剂、缓冲液和泡沫稳定剂;
蛋白质类包括350~500mg/L血清白蛋白或者800~1000mg/L植物水解蛋白、5~15mg/L转铁蛋白或者8~10mg/L铁螯合剂,铁螯合剂包括2.8~4.2mg/L Fe3+-硫酸酯-亚氨基二乙酸复合物和5.8~7.2mg/L Fe3+-硫酸酯-甘氨酰甘氨酸复合物,激素包括2~5mg/L胰岛素、1.5~2.0mg/L多肽类激素、1.6~1.8mg/L甾类激素和2.2~2.8mg/L皮质醇,多肽类激素包括1.1~1.28mg/L生长素、0.1~0.2mg/L胰高血糖素和0.02~0.03mg/L甲状腺素,甾类激素包括0.06~1.1mg/L孕酮、0.7~1.0mg/L氢化可的松和0.8~1.2mg/L雌二醇;
生长因子包括0.01~0.03mg/L表皮生长因子EGF、0.04~0.6mg/L转化生长因子TGF、0.05~0.15mg/L血小板生长因子PDGF和1.8~2.2mg/L成纤维细胞生长因子FGF,促贴壁因子包括1.8~2.5mg/L纤粘连蛋白、1.0~1.4mg/L胶原蛋白、0.3~0.8mg/L层粘连蛋白、1.1~1.4mg/L多聚-L赖氨酸或者多聚-D赖氨酸和0.4~0.7mg/L鸟氨酸;
微量元素包括0.01~0.03mg/L亚硒酸钠、0.03~0.04mg/L金精三羧酸和0.008~0.015mg/L Zn2+,维生素包括0.05~0.07mg/L核黄素、0.22~0.47mg/L叶酸、0.17~0.28mg/L钴胺素、0.1~0.226mg/L泛酸钙、7.68~10.63mg/L肌醇、0.2~0.3mg/L烟酰胺、2.1~3.5mg/L氯化胆碱、0.04~0.06mg/L硫辛酸、0.02~0.07mg/L盐酸吡哆醛、0.25~0.27mg/L维生素C、0.01~0.03mg/L维生素E、0.20~0.23mg/L盐酸吡哆醇和0.04~0.06mg/L生物素,生物素包括0.001~0.005mg/L羧酸酯酶、0.015~0.018mg/L乙酰辅酶A羧化酶、0.015~0.017mg/L二氢吡啶二羧酸合成酶和0.008~0.015mg/Lβ-羟基羧酸酯羟基氧化酶;
无机盐包括301.44~329.44mg/L氯化钾、10~13mg/L氯化钙、118~128mg/L磷酸二氢钠、6801~7085.6mg/L氯化钠和54.178~68.835mg/L硫酸镁,抗生素包括1.2~1.4mg/L两性霉素B、2.1~3.1mg/L喷他霉素和1.0~1.4mg/L抗真菌抗生素,脂类包括0.15~0.17mg/L亚油酸、0.22~0.23mg/L谷胱甘肽和0.2~0.5mg/L精胺;
还原剂包括10~12mg/L乙醇胺、3~5mg/L丙酮酸钠和1~2mg/Lβ-疏基乙醇,剪切力保护剂包括0.1~0.5mg/L聚醚F-68,缓冲液包括4000~4500mg/LHEPES和碳酸氢钠缓冲液。
具体的,一种用于培养Vero细胞的无血清培养基的制备方法,包括以下步骤:
S1、制备基础培养基溶解液:制备葡萄糖和谷氨酰胺的溶解液;
S2、分别制备主要添加因子的溶解液、次要添加因子的溶解液,将两组溶解液混合后过滤,得到混合溶解液;
S3、对混合溶解液进行预冻、升华和干燥处理,获得冻干粉剂的添加剂;
S31、对于预冻处理,是在-35~45℃的温度下,保持3~5h;
S32、对于升华处理,首先,将温度降低到-45~55℃,然后对整个工作系统抽真空,当真空度控制到<10Pa后,在19~25℃的温度下,升华干燥24h;
S33、对于干燥处理,是在7~9℃的温度下,干燥3~5h;
S34、干燥处理结束后,向工作系统中输入空气,使得工作系统的压力达到0.9~1.1个标准大气压,得到冻干粉剂的添加剂;
S4、对冻干粉剂的添加剂进行再次溶解,获得无血清培养的添加剂的溶解液,将基础培养基的溶解液和无血清培养的添加剂的溶解液混匀后过滤,得到无血清培养基。
首先,通过本发明提供的培养基培养Vero细胞,不仅能维持Vero细胞的正常生长,还能提供Vero细胞的扩增效率,有利于加快新冠病毒疫苗的研发和生产,其次,培养基化学成分明确,不需要血清,成本低廉,避免了因使用血清带来的生物安全风险,有利于提高生物制品生产工艺和成品质量的稳定性。
下面通过实施例和对比例来验证本发明培养基相较于传统培养基在细胞增殖和新冠疫苗生产方面的差异表现:
实施例1:培养基1:基础培养基、800mg/L植物水解蛋白、2.8mg/L Fe3+-硫酸酯-亚氨基二乙酸复合物、7.2mg/L Fe3+-硫酸酯-甘氨酰甘氨酸复合物、5mg/L胰岛素、1.28mg/L生长素、1.0mg/L氢化可的松、2.2mg/L皮质醇、0.01mg/L表皮生长因子EGF、0.6mg/L转化生长因子TGF、0.15mg/L血小板生长因子PDGF、1.8mg/L成纤维细胞生长因子FGF、2.5mg/L纤粘连蛋白、1.4mg/L胶原蛋白、0.3mg/L层粘连蛋白、1.1mg/L多聚-L赖氨酸、0.4mg/L鸟氨酸、0.01mg/L亚硒酸钠、0.03mg/L金精三羧酸、0.008mg/L Zn2+、0.07mg/L核黄素、0.22mg/L叶酸、0.17mg/L钴胺素、0.1mg/L泛酸钙、7.68mg/L肌醇、0.3mg/L烟酰胺、3.5mg/L氯化胆碱、0.04mg/L硫辛酸、0.02mg/L盐酸吡哆醛、0.27mg/L维生素C、0.01mg/L维生素E、0.23mg/L盐酸吡哆醇、0.005mg/L羧酸酯酶、301.44mg/L氯化钾、13mg/L氯化钙、118mg/L磷酸二氢钠、6801mg/L氯化钠、68.835mg/L硫酸镁、1.2mg/L两性霉素B、3.1mg/L喷他霉素、1.4mg/L抗真菌抗生素、0.15mg/L亚油酸、0.22mg/L谷胱甘肽、0.2mg/L精胺、10mg/L乙醇胺、3mg/L丙酮酸钠、2mg/Lβ-疏基乙醇、0.5mg/L聚醚F-68、4000mg/L HEPES和碳酸氢钠缓冲液;
实施例2:培养基2:基础培养基、500mg/L血清白蛋白、15mg/L转铁蛋白、5mg/L胰岛素、1.28mg/L生长素、1.0mg/L氢化可的松、2.2mg/L皮质醇、0.01mg/L表皮生长因子EGF、0.6mg/L转化生长因子TGF、0.15mg/L血小板生长因子PDGF、1.8mg/L成纤维细胞生长因子FGF、2.5mg/L纤粘连蛋白、1.4mg/L胶原蛋白、0.3mg/L层粘连蛋白、1.1mg/L多聚-L赖氨酸、0.4mg/L鸟氨酸、0.01mg/L亚硒酸钠、0.03mg/L金精三羧酸、0.008mg/L Zn2+、0.07mg/L核黄素、0.22mg/L叶酸、0.17mg/L钴胺素、0.1mg/L泛酸钙、7.68mg/L肌醇、0.3mg/L烟酰胺、3.5mg/L氯化胆碱、0.04mg/L硫辛酸、0.02mg/L盐酸吡哆醛、0.27mg/L维生素C、0.01mg/L维生素E、0.23mg/L盐酸吡哆醇、0.005mg/L羧酸酯酶、301.44mg/L氯化钾、13mg/L氯化钙、118mg/L磷酸二氢钠、6801mg/L氯化钠、68.835mg/L硫酸镁、0.15mg/L亚油酸、0.22mg/L谷胱甘肽、0.2mg/L精胺、10mg/L乙醇胺、3mg/L丙酮酸钠、2mg/Lβ-疏基乙醇、0.5mg/L聚醚F-68、4000mg/L HEPES和碳酸氢钠缓冲液;
实施例3:培养基3:基础培养基、500mg/L血清白蛋白、15mg/L转铁蛋白、5mg/L胰岛素、1.28mg/L生长素、1.0mg/L氢化可的松、2.2mg/L皮质醇、0.01mg/L表皮生长因子EGF、0.6mg/L转化生长因子TGF、0.15mg/L血小板生长因子PDGF、1.8mg/L成纤维细胞生长因子FGF、2.5mg/L纤粘连蛋白、1.4mg/L胶原蛋白、0.3mg/L层粘连蛋白、1.1mg/L多聚-L赖氨酸、0.4mg/L鸟氨酸、0.01mg/L亚硒酸钠、0.03mg/L金精三羧酸、0.008mg/L Zn2+、0.07mg/L核黄素、0.22mg/L叶酸、0.17mg/L钴胺素、0.1mg/L泛酸钙、7.68mg/L肌醇、0.3mg/L烟酰胺、3.5mg/L氯化胆碱、0.04mg/L硫辛酸、0.02mg/L盐酸吡哆醛、0.27mg/L维生素C、0.01mg/L维生素E、0.23mg/L盐酸吡哆醇、0.005mg/L羧酸酯酶、301.44mg/L氯化钾、13mg/L氯化钙、118mg/L磷酸二氢钠、6801mg/L氯化钠、68.835mg/L硫酸镁、1.2mg/L两性霉素B、3.1mg/L喷他霉素、1.4mg/L抗真菌抗生素、0.15mg/L亚油酸、0.22mg/L谷胱甘肽、0.2mg/L精胺、10mg/L乙醇胺、3mg/L丙酮酸钠、2mg/Lβ-疏基乙醇、0.5mg/L聚醚F-68、4000mg/L HEPES和碳酸氢钠缓冲液;
对比例1:培养基4:商品化培养基MEM添加8~12%(V/V)胎牛血清;
对比例2:培养基5:商品化培养基VP-SFM添加2~8mM/L谷氨酰胺;
对比例3:培养基6:商品化培养基OptiPRO添加2~8mM/L谷氨酰胺;
Vero细胞培养方法:用培养基1-6分别在35mm培养皿中培养Vero细胞,细胞接种密度0.5~2×104/cm2,培养条件:37℃,5~10%CO2,培养40~80h,将细胞消化,1~2mL胰酶终止液终止消化并收集细胞计数后,以相同方法继续传代培养两次。
将Vero细胞株利用上述培养方法分别接种于培养基1-6中,培养基1-6的细胞连续传代三次的活细胞密度如表1所示:
表1
将Vero细胞株利用上述培养方法分别接种于培养基1-6中,培养基的细胞连续传代三次的活细胞总数如表2所示:
表2
通过表1和表2可以看出,实施例1-3中,培养基3有利于Vero细胞的高效生长,对比例1-3中,培养基5较有利于Vero细胞的获得,进一步,利用培养基3、5进行新型冠状病毒的培养,培养方法如下:首先,分别利用培养基3、5培养Vero细胞,培养至6~8天时,进行新型冠状病毒SARS-CoV-2的接种,三个批次病毒MOI分别为0.005、0.01、0.3,种毒后于37±1℃进行通气搅拌培养,培养转速50~150rpm,溶氧不低于40%,培养期间控制pH值7.2~7.6之间,以下表3为培养基3中的新型冠状病毒在不同培养时间条件下的病毒滴度,表4为培养基5中的新型冠状病毒在不同培养时间条件下的病毒滴度:
表3
表4
通过表1可以看出,新冠病毒在培养基3的培养阶段,病毒接种48h后病毒滴度明显升高,达到7.0LgCCID50/mL以上,在培养基5中,病毒接种在48h后病毒滴度达到6.0LgCCID50/mL以上,则自病毒接种开始,培养48~96h内进行病毒液收获,具体培养基3、5中病毒收获液病毒滴度如以下表5所示:
表5
通过表1-5可以看出,本发明制备的无血清培养基细胞接种密度大,病毒滴度高,批间差以高度可控,质量均一,有利于新冠病毒的高效生产。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
Claims (6)
1.一种用于培养Vero细胞的无血清培养基,包括基础培养基,所述基础培养基包括葡萄糖和谷氨酰胺,其特征在于,还包括主要添加因子和次要添加因子,所述主要添加因子包括蛋白质类、激素、生长因子、促贴壁因子、微量元素和维生素,所述次要添加因子包括还原剂、胰蛋白酶抑制剂和剪切力保护剂;
所述蛋白质类包括350~500mg/L血清白蛋白或者800~1000mg/L植物水解蛋白、5~15mg/L转铁蛋白或者8~10mg/L铁螯合剂,所述激素包括2~5mg/L胰岛素、1.5~2.0mg/L多肽类激素、1.6~1.8mg/L甾类激素和2.2~2.8mg/L皮质醇;
所述生长因子包括0.01~0.03mg/L表皮生长因子EGF、0.04~0.6mg/L转化生长因子TGF、0.05~0.15mg/L血小板生长因子PDGF和1.8~2.2mg/L成纤维细胞生长因子FGF,所述促贴壁因子包括1.8~2.5mg/L纤粘连蛋白、1.0~1.4mg/L胶原蛋白、0.3~0.8mg/L层粘连蛋白、1.1~1.4mg/L多聚-L赖氨酸或者多聚-D赖氨酸和0.4~0.7mg/L鸟氨酸;
所述微量元素包括0.01~0.03mg/L亚硒酸钠、0.03~0.04mg/L金精三羧酸和0.008~0.015mg/L Zn2+,所述维生素包括0.05~0.07mg/L核黄素、0.22~0.47mg/L叶酸、0.17~0.28mg/L钴胺素、0.1~0.226mg/L泛酸钙、7.68~10.63mg/L肌醇、0.2~0.3mg/L烟酰胺、2.1~3.5mg/L氯化胆碱、0.04~0.06mg/L硫辛酸、0.02~0.07mg/L盐酸吡哆醛、0.25~0.27mg/L维生素C、0.01~0.03mg/L维生素E、0.20~0.23mg/L盐酸吡哆醇和0.04~0.06mg/L生物素;
所述还原剂包括10~12mg/L乙醇胺、3~5mg/L丙酮酸钠和1~2mg/Lβ-疏基乙醇,所述剪切力保护剂包括0.1~0.5mg/L聚醚F-68。
2.根据权利要求1所述的一种用于培养Vero细胞的无血清培养基,其特征在于,所述主要添加因子还包括无机盐、抗生素和脂类,所述次要添加因子还包括缓冲液和泡沫稳定剂;
所述无机盐包括301.44~329.44mg/L氯化钾、10~13mg/L氯化钙、118~128mg/L磷酸二氢钠、6801~7085.6mg/L氯化钠和54.178~68.835mg/L硫酸镁,所述抗生素包括1.2~1.4mg/L两性霉素B、2.1~3.1mg/L喷他霉素和1.0~1.4mg/L抗真菌抗生素,所述脂类包括0.15~0.17mg/L亚油酸、0.22~0.23mg/L谷胱甘肽和0.2~0.5mg/L精胺;
所述缓冲液包括4000~4500mg/L HEPES和碳酸氢钠缓冲液,所述泡沫稳定剂包括3.5~4.2mg/L泊洛沙姆188。
3.根据权利要求1所述的一种用于培养Vero细胞的无血清培养基,其特征在于,所述铁螯合剂包括2.8~4.2mg/L Fe3+-硫酸酯-亚氨基二乙酸复合物和5.8~7.2mg/L Fe3+-硫酸酯-甘氨酰甘氨酸复合物。
4.根据权利要求1所述的一种用于培养Vero细胞的无血清培养基,其特征在于,所述多肽类激素包括1.1~1.28mg/L生长素、0.1~0.2mg/L胰高血糖素和0.02~0.03mg/L甲状腺素,所述甾类激素包括0.06~1.1mg/L孕酮、0.7~1.0mg/L氢化可的松和0.8~1.2mg/L雌二醇。
5.根据权利要求1所述的一种用于培养Vero细胞的无血清培养基,其特征在于,所述生物素包括0.001~0.005mg/L羧酸酯酶、0.015~0.018mg/L乙酰辅酶A羧化酶、0.015~0.017mg/L二氢吡啶二羧酸合成酶和0.008~0.015mg/Lβ-羟基羧酸酯羟基氧化酶。
6.一种用于培养Vero细胞的无血清培养基的制备方法,其特征在于,包括以下步骤:
S1、制备基础培养基溶解液:制备葡萄糖和谷氨酰胺的溶解液;
S2、分别制备主要添加因子的溶解液、次要添加因子的溶解液,将两组溶解液混合后过滤,得到混合溶解液;
S3、对混合溶解液进行预冻、升华和干燥处理,获得冻干粉剂的添加剂;
S31、对于预冻处理,是在-35~45℃的温度下,保持3~5h;
S32、对于升华处理,首先,将温度降低到-45~55℃,然后对整个工作系统抽真空,当真空度控制到<10Pa后,在19~25℃的温度下,升华干燥24h;
S33、对于干燥处理,是在7~9℃的温度下,干燥3~5h;
S34、干燥处理结束后,向工作系统中输入空气,使得工作系统的压力达到0.9~1.1个标准大气压,得到冻干粉剂的添加剂;
S4、对冻干粉剂的添加剂进行再次溶解,获得无血清培养的添加剂的溶解液,将基础培养基的溶解液和无血清培养的添加剂的溶解液混匀后过滤,得到无血清培养基。
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