CN110951677B - 一种Vero细胞无血清培养基及其用途 - Google Patents
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Abstract
本发明涉及一种Vero细胞无血清培养基及其用途,所述Vero细胞无血清培养基包括基础培养基DMEM/F12和添加组分,添加组分为:乙二胺四乙酸二钠1~10mg/L、二水合乙醇胺盐酸盐5~30mg/L、乙二胺四乙酸铁纳2~25mg/L、重组表皮生长因子(rhEGF)0.005~0.02mg/L、重组胰岛素1~10mg/L、重组纤连蛋白0.1~5mg/L、亚油酸钠0.05~0.5mg/L、肉豆蔻酸0.05~0.5mg/L、硬脂酸0.05~0.8mg/L、肌醇6~80mg/L、维生素B6 1~10mg/L、维生素B7 0.1~0.8mg/L。本发明还包括所述培养基在培养Vero细胞中的应用。
Description
技术领域
本发明涉及细胞培养技术领域,具体地说,是一种Vero细胞无血清培养基及其用途。
背景技术
Vero细胞(African green kidney cell,Vero cell)是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。目前已用于多种疫苗的生产。与用作疫苗生产的原代细胞、二倍体细胞和其它一些传代细胞基质相比,Vero细胞具有以下特点:①来源方便,可连续传代,生长速度快;②对多种病毒的感染敏感、病毒增殖滴度高;③遗传性状稳定,恶性转化程度低,生物安全性较高;④培养条件要求不苛刻,易于在生物反应器实施大规模培养。
培养基是影响体外培养细胞的形态、生长、代谢、增殖乃至生存的最直接和最重要的环境因素。由于Vero细胞的培养需要在适宜的介质表面贴附、铺展才开始有丝分裂、增殖,病毒的有效感染和增殖也有赖于细胞的贴附生长状态,适用于Vero细胞微载体培养的无血清培养基应能有效地支持细胞在微载体表面的贴附、铺展。由此,不仅提高了Vero细胞无血清培养基配方成分的复杂程度,也加大了Vero细胞无血清培养基的成本控制压力。
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:①血清批间存在差异,影响疫苗生产工艺的稳定性和质量;②使用血清容易引起微生物污染;③血清成分复杂,同时存在促进细胞生长的物质和抑制细胞生长的物质,对细胞生长及后续下游纯化造成影响;④血清价格昂贵;⑤政策法规和行业指导原则不断严格,生物制品去血清化生产是未来的趋势。
中国专利申请:CN101864393B公开了一种用于Vero细胞微载体培养的无动物来源成分无血清培养基。该无血清培养基由DMEM/F12(1∶1)培养基和表皮生长因子、胰岛素、血清胺、金精三羧酸、生物素、维生素C、氨基酸、果糖、海藻糖、微量元素、羟丙基-β-环状糊精等培养基添加成分组成。中国专利申请:CN108103007A公开了一种适合Vero细胞生长的低血清培养基,将常规培养小牛血清用量由10%(V组分)降至2%,并且将常规添加的动物源成分替换成植物源成分和重组蛋白。但是关于本发明目前一种Vero细胞无血清培养基及其用途还未见报道。
本发明开发了一种适用于Vero细胞的无血清培养基,使用本发明所提供的培养基培养细胞,能够在不添加血清的情况下,显著提高细胞的扩增效率。
发明内容
本发明的第一个目的是针对现有技术的不足,提供一种Vero细胞无血清培养基。
本发明的第二个目的是针对现有技术的不足,提供如上所述Vero细胞无血清培养基的用途。
本发明的第三个目的是针对现有技术的不足,提供如上所述Vero细胞无血清培养基培养乙脑病毒或脊髓灰质炎病毒或狂犬病毒的方法。
为实现上述第一个目的,本发明采取的技术方案是:
一种Vero细胞无血清培养基,包括基础培养基和添加组分,所述基础培养基是:DMEM/F12,所述添加组分由以下成分组成:乙二胺四乙酸二钠、二水合乙醇胺盐酸盐、乙二胺四乙酸铁纳、rhEGF、重组胰岛素、重组纤连蛋白、亚油酸钠、肉豆蔻酸、硬脂酸、肌醇、维生素B6、维生素B7。
作为本发明的一个优选实施方案,所述各成分的终浓度分别为:乙二胺四乙酸二钠1~10mg/L、二水合乙醇胺盐酸盐5~30mg/L、乙二胺四乙酸铁纳2~25mg/L、rhEGF 0.005~0.02mg/L、重组胰岛素1~10mg/L、重组纤连蛋白0.1~5mg/L、亚油酸钠0.05~0.5mg/L、肉豆蔻酸0.05~0.5mg/L、硬脂酸0.05~0.8mg/L、肌醇6~80mg/L、维生素B61~10mg/L、维生素B70.1~0.8mg/L。
作为本发明的一个优选实施方案,所述各成分的终浓度分别为:乙二胺四乙酸二钠3~8mg/L、二水合乙醇胺盐酸盐15~20mg/L、乙二胺四乙酸铁纳12~15mg/L、rhEGF0.01~0.015mg/L、重组胰岛素3~8mg/L、重组纤连蛋白2.1~3mg/L、亚油酸钠0.15~0.4mg/L、肉豆蔻酸0.15~0.4mg/L、硬脂酸0.35~0.5mg/L、肌醇36~50mg/L、维生素B63~8mg/L、维生素B70.4~0.5mg/L。
作为本发明的一个优选实施方案,所述各成分的终浓度分别为:乙二胺四乙酸二钠5.5mg/L、二水合乙醇胺盐酸盐17.5mg/L、乙二胺四乙酸铁纳13.5mg/L、rhEGF 0.0125mg/L、重组胰岛素5.5mg/L、重组纤连蛋白2.55mg/L、亚油酸钠0.05~0.5mg/L、肉豆蔻酸0.275mg/L、硬脂酸0.425mg/L、肌醇43mg/L、维生素B65.5mg/L、维生素B70.45mg/L。
为实现上述第二个目的,本发明采取的技术方案是:
如上任一所述的Vero细胞无血清培养基在培养Vero细胞中的应用。
如上任一所述的Vero细胞无血清培养基在制备疫苗中的应用。
作为本发明的一个优选实施方案,所述疫苗包括乙型脑炎疫苗、脊髓灰质炎疫苗、狂犬病疫苗。
优选地,所述培养基在培养Vero细胞的培养条件是:37℃,5%CO2的培养环境。
为实现上述第三个目的,本发明采取的技术方案是:
如上任一所述的Vero细胞无血清培养基培养乙脑病毒或脊髓灰质炎病毒或狂犬病毒的方法,包括如下步骤:
(1)Vero细胞的复苏;
(2)Vero细胞的传代扩增;
(3)在Vero细胞上接种乙脑病毒毒株或脊髓灰质炎病毒毒株或狂犬病毒毒株;
(4)收获感染细胞培养上清液,病毒液进行澄清过滤,即得到所述的乙脑病毒液或脊髓灰质炎病毒液或狂犬病毒液;
其中步骤(2)中的传代扩增和步骤(3)中病毒的增殖过程均采用如上任一所述的无血清培养基。
本发明优点在于:
1、本发明开发了一种适用于Vero细胞的无血清培养基,细胞在培养基中生长良好、能够明显增强细胞的扩增能力、连续传代三次培养可收获的活细胞总数多,有利于提高生物制品生产的稳定性,降低后期纯化工艺复杂程度,降低生产成本。
2、本发明通过在基础培养基中添加乙二胺四乙酸二钠、二水合乙醇胺盐酸盐、乙二胺四乙酸铁纳、rhEGF、重组胰岛素、重组纤连蛋白、亚油酸钠、肉豆蔻酸、硬脂酸、肌醇、维生素B6、维生素B7多种营养因子替代血清功能,并确定了其各成分的之间的浓度,成分明确、稳定,排除了血清培养的不稳定性,同时各因子之间协同作用,能够高效的扩增Vero细胞,使得细胞汇合度更优、收获的活细胞总数更多、显著提高细胞的扩增效率。
附图说明
附图1是细胞连续传代三次的结果。
附图2是细胞连续传代三次后理论收获的活细胞总数结果。
具体实施方式
下面结合具体实施方式,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。此外应理解,在阅读了本发明记载的内容之后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
以下各实施例中所述的添加组分中所有成分的原料和基础培养基DMEM/F12均采购自Sigma;商业化培养基MEM采购自Gibco;细胞株Vero采购自ATCC。
实施例1Vero细胞无血清培养基(一)
包括基础培养基和添加组分:
添加组分:乙二胺四乙酸二钠1mg/L、二水合乙醇胺盐酸盐5mg/L、乙二胺四乙酸铁纳2mg/L、rhEGF 0.005mg/L、重组胰岛素1mg/L、重组纤连蛋白0.1mg/L、亚油酸钠0.05mg/L、肉豆蔻酸0.05mg/L、硬脂酸0.05mg/L、肌醇6mg/L、维生素B61mg/L、维生素B70.1mg/L;
基础培养基:DMEM/F12。
实施例2Vero细胞无血清培养基(二)
包括基础培养基和添加组分:
添加组分:乙二胺四乙酸二钠10mg/L、二水合乙醇胺盐酸盐30mg/L、乙二胺四乙酸铁纳25mg/L、rhEGF 0.02mg/L、重组胰岛素10mg/L、重组纤连蛋白5mg/L、亚油酸钠0.5mg/L、肉豆蔻酸0.5mg/L、硬脂酸0.8mg/L、肌醇80mg/L、维生素B610mg/L、维生素B70.8mg/L;
基础培养基:DMEM/F12。
实施例3Vero细胞无血清培养基(三)
包括基础培养基和添加组分:
添加组分:乙二胺四乙酸二钠1mg/L、二水合乙醇胺盐酸盐30mg/L、乙二胺四乙酸铁纳2mg/L、rhEGF 0.02mg/L、重组胰岛素1mg/L、重组纤连蛋白5mg/L、亚油酸钠0.05mg/L、肉豆蔻酸0.5mg/L、硬脂酸0.05mg/L、肌醇80mg/L、维生素B61mg/L、维生素B70.8mg/L;
基础培养基:DMEM/F12。
实施例4Vero细胞无血清培养基(四)
包括基础培养基和添加组分:
添加组分:乙二胺四乙酸二钠10mg/L、二水合乙醇胺盐酸盐5mg/L、乙二胺四乙酸铁纳25mg/L、rhEGF 0.005mg/L、重组胰岛素10mg/L、重组纤连蛋白0.1mg/L、亚油酸钠0.5mg/L、肉豆蔻酸0.05mg/L、硬脂酸0.8mg/L、肌醇6mg/L、维生素B610mg/L、维生素B70.1mg/L;
基础培养基:DMEM/F12。
实施例5Vero细胞无血清培养基(五)
包括基础培养基和添加组分:
添加组分:乙二胺四乙酸二钠1mg/L、二水合乙醇胺盐酸盐5mg/L、乙二胺四乙酸铁纳2mg/L、rhEGF 0.005mg/L、重组胰岛素1mg/L、重组纤连蛋白0.1mg/L、亚油酸钠0.5mg/L、肉豆蔻酸0.5mg/L、硬脂酸0.8mg/L、肌醇80mg/L、维生素B610mg/L、维生素B70.8mg/L;
基础培养基:DMEM/F12。
实施例6Vero细胞无血清培养基(六)
包括基础培养基和添加组分:
添加组分:乙二胺四乙酸二钠10mg/L、二水合乙醇胺盐酸盐30mg/L、乙二胺四乙酸铁纳25mg/L、rhEGF 0.02mg/L、重组胰岛素10mg/L、重组纤连蛋白5mg/L、亚油酸钠0.05mg/L、肉豆蔻酸0.05mg/L、硬脂酸0.05mg/L、肌醇6mg/L、维生素B61mg/L、维生素B70.1mg/L;
基础培养基:DMEM/F12。
实施例7Vero细胞无血清培养基(七)
包括基础培养基和添加组分:
添加组分:乙二胺四乙酸二钠3mg/L、二水合乙醇胺盐酸盐15mg/L、乙二胺四乙酸铁纳12mg/L、rhEGF 0.01mg/L、重组胰岛素3mg/L、重组纤连蛋白2.1mg/L、亚油酸钠0.15mg/L、肉豆蔻酸0.15mg/L、硬脂酸0.35mg/L、肌醇36mg/L、维生素B63mg/L、维生素B70.4mg/L;
基础培养基:DMEM/F12。
实施例8Vero细胞无血清培养基(八)
包括基础培养基和添加组分:
添加组分:乙二胺四乙酸二钠8mg/L、二水合乙醇胺盐酸盐20mg/L、乙二胺四乙酸铁纳15mg/L、rhEGF 0.015mg/L、重组胰岛素8mg/L、重组纤连蛋白3mg/L、亚油酸钠0.4mg/L、肉豆蔻酸0.4mg/L、硬脂酸0.5mg/L、肌醇50mg/L、维生素B68mg/L、维生素B70.5mg/L;
基础培养基:DMEM/F12。
实施例9Vero细胞无血清培养基(九)
包括基础培养基和添加组分:
添加组分:乙二胺四乙酸二钠3mg/L、二水合乙醇胺盐酸盐20mg/L、乙二胺四乙酸铁纳12mg/L、rhEGF 0.015mg/L、重组胰岛素3mg/L、重组纤连蛋白3mg/L、亚油酸钠0.15mg/L、肉豆蔻酸0.4mg/L、硬脂酸0.35mg/L、肌醇50mg/L、维生素B63mg/L、维生素B70.5mg/L;
基础培养基:DMEM/F12。
实施例10Vero细胞无血清培养基(十)
包括基础培养基和添加组分:
添加组分:乙二胺四乙酸二钠8mg/L、二水合乙醇胺盐酸盐15mg/L、乙二胺四乙酸铁纳15mg/L、rhEGF 0.01mg/L、重组胰岛素8mg/L、重组纤连蛋白2.1mg/L、亚油酸钠0.4mg/L、肉豆蔻酸0.15mg/L、硬脂酸0.5mg/L、肌醇36mg/L、维生素B68mg/L、维生素B70.4mg/L;
基础培养基:DMEM/F12。
实施例11Vero细胞无血清培养基(十一)
包括基础培养基和添加组分:
添加组分:乙二胺四乙酸二钠3mg/L、二水合乙醇胺盐酸盐15mg/L、乙二胺四乙酸铁纳12mg/L、rhEGF 0.01mg/L、重组胰岛素3mg/L、重组纤连蛋白2.1mg/L、亚油酸钠0.4mg/L、肉豆蔻酸0.4mg/L、硬脂酸0.5mg/L、肌醇50mg/L、维生素B68mg/L、维生素B70.5mg/L;
基础培养基:DMEM/F12。
实施例12Vero细胞无血清培养基(十二)
包括基础培养基和添加组分:
添加组分:乙二胺四乙酸二钠5.5mg/L、二水合乙醇胺盐酸盐17.5mg/L、乙二胺四乙酸铁纳13.5mg/L、rhEGF 0.0125mg/L、重组胰岛素5.5mg/L、重组纤连蛋白2.55mg/L、亚油酸钠0.05~0.5mg/L、肉豆蔻酸0.275mg/L、硬脂酸0.425mg/L、肌醇43mg/L、维生素B65.5mg/L、维生素B70.45mg/L;
基础培养基:DMEM/F12。
实施例13效果验证
1、细胞培养基的制备
培养基1:由基础培养基DMEM/F12+添加组分组成,所述添加组分由以下各成分组成:乙二胺四乙酸二钠1mg/L、二水合乙醇胺盐酸盐5mg/L、乙二胺四乙酸铁纳2mg/L、rhEGF0.005mg/L、重组胰岛素1mg/L、重组纤连蛋白0.1mg/L、亚油酸钠0.05mg/L、肉豆蔻酸0.05mg/L、硬脂酸0.05mg/L、肌醇6mg/L、维生素B61mg/L、维生素B70.1mg/L,将添加组分加入基础培养基DMEM/F12中。
培养基2:由基础培养基DMEM/F12+添加组分组成,所述添加组分由以下各成分组成:乙二胺四乙酸二钠5.5mg/L、二水合乙醇胺盐酸盐17.5mg/L、乙二胺四乙酸铁纳13.5mg/L、rhEGF 0.0125mg/L、重组胰岛素5.5mg/L、重组纤连蛋白2.55mg/L、亚油酸钠0.05~0.5mg/L、肉豆蔻酸0.275mg/L、硬脂酸0.425mg/L、肌醇43mg/L、维生素B65.5mg/L、维生素B70.45mg/L,将添加组分加入至基础培养基DMEM/F12中。
培养基3:由基础培养基DMEM/F12+添加组分组成,所述添加组分由以下各成分组成:乙二胺四乙酸二钠10mg/L、二水合乙醇胺盐酸盐30mg/L、乙二胺四乙酸铁纳25mg/L、rhEGF 0.02mg/L、重组胰岛素10mg/L、重组纤连蛋白5mg/L、亚油酸钠0.5mg/L、肉豆蔻酸0.5mg/L、硬脂酸0.8mg/L、肌醇80mg/L、维生素B610mg/L、维生素B70.8mg/L,将添加组分加入至基础培养基DMEM/F12中。
商品化培养基MEM(购自上海信帆生物科技有限公司)添加10%(V/V)新生牛血清作为对照组。
2、细胞培养和检测
2.1培养
将Vero细胞株分别接种到培养基1、培养基2和培养基3中培养,培养方法和参数如下:用上述配置好的培养基在100mm培养皿中培养Vero细胞株,细胞接种密度为1.0×104/cm2,培养条件:37℃,5%CO2,培养72小时后,将细胞消化收集并计数后,以上述相同方法继续传代培养两次。
同时,以商品化培养基MEM(购自Gibco公司)添加10%(V/V)胎牛血清按照上述方法培养Vero细胞,作为对照。
2.2检测
当每代次细胞生长至72h左右,终止实验,并将所有实验孔细胞用胰酶消化收集并计数。
3、结果
细胞连续传代三次的技术结果如图1所示,与市售基础一样既MEM(购自Gibco公司)相比,使用本发明的培养基,能够明显增强细胞的扩增能力。
细胞连续传代三次后理论收获的活细胞总数结果如图2所示,与市售基础一样既MEM(购自Gibco公司)相比,使用本发明的培养基,连续传代三次培养,可收获的活细胞总数约为对照组的约4倍。
4、结论
以上结果说明使用本发明所提供的培养基培养细胞,能够在不添加血清的情况下,显著提高细胞的扩增效率。
本发明开发了一种适用于Vero细胞的无血清培养基,细胞在培养基中生长良好、能够明显增强细胞的扩增能力、连续传代三次培养可收获的活细胞总数多,有利于提高生物制品生产的稳定性,降低了后期纯化工艺复杂程度,降低了生产成本。本发明通过在基础培养基中添加乙二胺四乙酸二钠、二水合乙醇胺盐酸盐、乙二胺四乙酸铁纳、rhEGF、重组胰岛素、重组纤连蛋白、亚油酸钠、肉豆蔻酸、硬脂酸、肌醇、维生素B6、维生素B7多种营养因子替代血清功能,并确定了其各成分的之间的浓度,成分明确、稳定,排除了血清培养的不稳定性,同时各因子之间协同作用,能够高效的扩增Vero细胞,使得细胞汇合度更优、收获的活细胞总数更多、显著提高细胞的扩增效率。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员,在不脱离本发明原理的前提下,还可以做出若干改进和补充,这些改进和补充也应视为本发明的保护范围。
Claims (7)
1.一种Vero细胞无血清培养基,其特征在于,包括基础培养基和添加组分,所述基础培养基是:DMEM/F12,所述添加组分由以下成分组成:乙二胺四乙酸二钠、二水合乙醇胺盐酸盐、乙二胺四乙酸铁纳、rhEGF、重组胰岛素、重组纤连蛋白、亚油酸钠、肉豆蔻酸、硬脂酸、肌醇、维生素B6、维生素B7,所述各成分的终浓度分别为:乙二胺四乙酸二钠1~10mg/L、二水合乙醇胺盐酸盐5~30mg/L、乙二胺四乙酸铁纳2~25mg/L、rhEGF 0.005~0.02mg/L、重组胰岛素1~10mg/L、重组纤连蛋白0.1~5mg/L、亚油酸钠0.05~0.5mg/L、肉豆蔻酸0.05~0.5mg/L、硬脂酸0.05~0.8mg/L、肌醇6~80mg/L、维生素B6 1~10mg/L、维生素B7 0.1~0.8mg/L。
2.根据权利要求1所述的Vero细胞无血清培养基,其特征在于,所述各成分的终浓度分别为:乙二胺四乙酸二钠3~8mg/L、二水合乙醇胺盐酸盐15~20mg/L、乙二胺四乙酸铁纳12~15mg/L、rhEGF 0.01~0.015mg/L、重组胰岛素3~8mg/L、重组纤连蛋白2.1~3mg/L、亚油酸钠0.15~0.4mg/L、肉豆蔻酸0.15~0.4mg/L、硬脂酸0.35~0.5mg/L、肌醇36~50mg/L、维生素B6 3~8mg/L、维生素B7 0.4~0.5mg/L。
3.根据权利要求1所述的Vero细胞无血清培养基,其特征在于,所述各成分的终浓度分别为:乙二胺四乙酸二钠5.5mg/L、二水合乙醇胺盐酸盐17.5mg/L、乙二胺四乙酸铁纳13.5mg/L、rhEGF 0.0125mg/L、重组胰岛素5.5mg/L、重组纤连蛋白2.55mg/L、亚油酸钠0.05~0.5mg/L、肉豆蔻酸0.275mg/L、硬脂酸0.425mg/L、肌醇43mg/L、维生素B6 5.5mg/L、维生素B7 0.45mg/L。
4.权利要求1-3任一所述的Vero细胞无血清培养基在培养Vero细胞中的应用。
5.权利要求1-3任一所述的Vero细胞无血清培养基在制备疫苗中的应用。
6.根据权利要求5所述的应用,其特征在于,所述疫苗包括乙型脑炎疫苗、脊髓灰质炎疫苗、狂犬病疫苗。
7.权利要求1-3任一所述的Vero细胞无血清培养基培养乙脑病毒或脊髓灰质炎病毒或狂犬病毒的方法,其特征在于,包括如下步骤:
(1)Vero细胞的复苏;
(2)Vero细胞的传代扩增;
(3)在Vero细胞上接种乙脑病毒毒株或脊髓灰质炎病毒毒株或狂犬病毒毒株;
(4)收获感染细胞培养上清液,病毒液进行澄清过滤,即得到所述的乙脑病毒液或脊髓灰质炎病毒液或狂犬病毒液;
其中步骤(2)中的传代扩增和步骤(3)中病毒的增殖过程均采用权利要求1-4任一所述的无血清培养基。
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