CN115612658A - 一种st细胞悬浮驯化的培养基及其应用 - Google Patents
一种st细胞悬浮驯化的培养基及其应用 Download PDFInfo
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Abstract
本发明提出了一种ST细胞悬浮驯化的培养基及其应用,利用本发明的培养基可以使ST细胞的悬浮驯化过程更简单,细胞增殖快,驯化周期短,不易发生结团,细胞活率高,尤其可以适用于疫苗领域,提高生产效率。
Description
技术领域
本发明涉及生物技术领域。具体地,本发明涉及一种ST细胞悬浮驯化的培养基及其应用。
背景技术
ST细胞为猪睾丸细胞(swine testis),可进行连续传代培养,培养过程中增殖较为缓慢,而且细胞消化传代中容易成团。ST细胞可以用于扩增病毒,实现疫苗生产。然而,ST细胞在疫苗生产过程中存在培养放大不便、培养周期长等问题,制约着ST细胞的推广应用。
因此,目前ST细胞的悬浮驯化培养方法仍有待研究。
发明内容
本发明旨在至少在一定程度上解决上述技术问题之一或至少提供一种有用的商业选择。为此,本发明的一个目的在于提出一种ST细胞自悬浮培养基,使细胞的悬浮驯化过程更简单,悬浮细胞在疫苗生产过程中,与贴壁细胞进行对比,生产效率大大提高。
在本发明的一个方面,本发明提出了一种用于悬浮培养ST细胞的培养基。根据本发明的实施例,所述培养基包括:DMEM培养基;添加剂,所述添加剂包括:终浓度为110mg/L的L-盐酸精氨酸;终浓度为500mg/L的L-门冬酰胺;终浓度为300mg/L的L-天门冬氨酸;终浓度为150mg/L的L-半胱氨酸盐酸盐一水;终浓度为30mg/L的L-胱氨酸二盐酸盐;终浓度为600mg/L的L-谷氨酸;终浓度为585mg/L的L-谷氨酰胺;终浓度为210mg/L的L-盐酸组氨酸一水;终浓度为170mg/L的甘氨酸;终浓度为200mg/L的L-异亮氨酸;终浓度为400mg/L的L-亮氨酸;终浓度为355mg/L的L-盐酸赖氨酸;终浓度为170mg/L的L-甲硫氨酸;终浓度为250mg/L的L-苯丙氨酸;终浓度为150mg/L的L-脯氨酸;终浓度为100mg/L的L-丝氨酸;终浓度为134mg/L的L-苏氨酸;终浓度为80mg/L的L-色氨酸;终浓度为450mg/L的L-酪氨酸二钠盐无水;终浓度为300mg/L的L-缬氨酸;终浓度为1.9mg/L的D-生物素;终浓度为70mg/L的氯化胆碱;终浓度为16mg/L的叶酸;终浓度为53mg/L的肌醇;终浓度为21mg/L的烟酰胺;终浓度为0.3mg/L的维生素B2;终浓度为5mg/L的维生素B1;终浓度为11mg/L的维生素B12;终浓度为15.5mg/L的四盐酸精胺;终浓度为0.9mg/L的还原型谷胱甘肽;终浓度为0.66mg/L的腺嘌呤硫酸盐;终浓度为0.66mg/L的鸟苷;终浓度为16mg/L的二水氯化钙;终浓度为4.8mg/L的七水硫酸亚铁;终浓度为537mg/L的无水磷酸氢二钠;终浓度为298mg/L的一水磷酸二氢钠;终浓度为1.8mg/L的七水硫酸锌;终浓度为3500mg/L的D-无水葡萄糖;终浓度为3000mg/L的大豆水解物;终浓度为3000mg/L的酵母水解物;终浓度为100mg/L的丙酮酸钠;终浓度为1000mg/L的F68;终浓度为0.5mg/L的九水硝酸铁;终浓度为12mg/L的维生素 C 磷酸酯镁;终浓度为28mg/L的乙醇胺盐酸盐;终浓度为0.00034mg/L的硝酸银;终浓度为0.00098936mg/L的偏钒酸铵;终浓度为0.00000726mg/L的偏钒酸钠;终浓度为0.00366mg/L的氯化镉;终浓度为0.00046mg/L的六水氯化铬;终浓度为0.00106mg/L的二氧化锗;终浓度为0.00024mg/L的溴化钾;终浓度为0.00034mg/L的碘化钾;终浓度为0.00034mg/L的四水氯化锰;终浓度为0.00026mg/L的硫酸镍;终浓度为0.00242mg/L的氯化铷;终浓度为0.0002mg/L的氯化亚锡;终浓度为0.168mg/L的氯化铝;终浓度为0.00644mg/L的氧氯化锆;终浓度为0.0051mg/L的乙酸钡;终浓度为0.00476mg/L的六水氯化钴;终浓度为0.005mg/L的五水硫酸铜;终浓度为169.6002mg/L的六水氯化镁;终浓度为0.0165mg/L的氯化锂;终浓度为0.006mg/L的钼酸铵;终浓度为0.626mg/L的九水偏硅酸钠;终浓度为0.0346mg/L的亚硒酸钠;终浓度为0.0084mg/L的氟化钠;终浓度为0.66mg/L的1,4-丁二胺二盐酸盐;终浓度为0.42mg/L的硫辛酸;终浓度为0.00132mg/L的维生素E;终浓度为0.072mg/L的氢化可的松;终浓度为0.037488mg/L的亚油酸;终浓度为2mg/L的对氨基苯甲酸;终浓度为1.16mg/L的重组人胰岛素;终浓度为52.6mg/L的柠檬酸铁;终浓度为0.004394703mg/L的乙醇胺;终浓度为1.318410875mg/L的胰岛素;终浓度为0.488021295mg/L的胆固醇;终浓度为0.155279503mg/L的维生素E醋酸酯;终浓度为0.022182786mg/L的肉豆蔻酸;终浓度为0.022182786mg/L的油酸;终浓度为0.022182786mg/L的棕榈酸;终浓度为0.022182786mg/L的硬脂酸;终浓度为4.880212955mg/L的吐温80。
本发明的发明人结合之前在细胞培养领域积累的丰富经验,进行了大量的筛选和优化工作,意外地获得了一种悬浮培养ST细胞的培养基,利用该培养基可以实现ST细胞自悬浮培养,细胞增殖快,不易发生结团,细胞活率高,尤其可以适用于疫苗领域,提高生产效率。
在本发明的另一方面,本发明提出了前面所述用于悬浮培养ST细胞的培养基在悬浮培养ST细胞和扩增病毒中的应用。利用前面所述培养基可以实现ST细胞悬浮培养,细胞增殖快,不易发生结团,细胞活率高,也可以适用于病毒扩增,应用前景广泛。
在本发明的又一方面,本发明提出了一种悬浮培养ST细胞的方法。根据本发明的实施例,所述方法包括:将ST细胞接种于前面所述用于悬浮培养ST细胞的培养基中;对所述ST细胞进行悬浮培养。由此,可以实现ST细胞悬浮培养,细胞增殖快,驯化周期短,不易发生结团,细胞活率高,生产效率高。
在本发明的又一方面,本发明提出了一种扩增病毒的方法。根据本发明的实施例,所述方法包括:按照前面所述悬浮培养ST细胞的方法,对ST细胞进行悬浮培养,得到培养液;和在所述培养液中接种病毒,以便对所述病毒进行扩增。由此,采用根据本发明实施例的方法培养所得ST细胞培养液有助于实现病毒增殖,提高病毒效价,从而更好地应用于疫苗病毒的扩增生产。
根据本发明的实施例,所述病毒选自猪瘟病毒、猪伪狂犬病毒,猪细小病毒、猪传染性胃肠炎病毒。
在本发明的又一方面,本发明提出了一种制备疫苗的方法。根据本发明的实施例,所述方法包括:按照前面所述扩增病毒的方法扩增病毒。由此,利用根据本发明实施例的方法可以获得高病毒效价且高产量的疫苗,并且制备方法操作简便,产能高,适于规模化生产应用。
根据本发明的实施例,所述方法进一步包括:将所述扩增病毒所得到的病毒液进行减毒或者灭活处理,以便获得所述疫苗。由此,可以避免病毒进入机体产生不良反应。
在本发明的又一方面,本发明提出了一种疫苗。根据本发明的实施例,所述疫苗是通过前面所述制备疫苗的方法获得的。由此,根据本发明实施例的疫苗安全、有效,适于推广应用。
术语“疫苗”是指含有有效诱导受试者的抗特定病原体或疾病的治疗程度的免疫性的活性组分的试剂或组合物,在此是治疗性地针对感染病毒所引发的疾病。疫苗可以包括药学上可接受的载体、稀释剂和/或佐剂。
在本发明的又一方面,本发明提出了前面所述疫苗在制备药物中的用途。根据本发明的实施例,所述药物用于治疗或者预防病毒相关疾病。
本发明的疫苗可以通过标准途径给药,所述途径包括但不限于胃肠外(例如,静脉内、椎管内的、皮下或肌肉内)、口服、粘膜(例如鼻内)或局部途径。
本发明的附加方面和优点将在下面的描述中部分给出,部分将从下面的描述中变得明显,或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解,其中:
图1显示了根据本发明一个实施例的ST细胞用ST-SFM1培养基悬浮培养的细胞密度、活率与时间关系曲线;
图2显示了根据本发明一个实施例的ST细胞用ST-SFM2培养基悬浮培养的细胞密度、活率与时间关系曲线;
图3显示了根据本发明一个实施例的ST细胞用DMEM+10%NBS培养基悬浮培养的细胞密度、活率与时间关系曲线;
图4显示了根据本发明一个实施例的ST细胞分别采用ST-SFM1培养基、ST-SFM2培养基和DMEM+10%NBS培养基的电镜图;
图5显示了根据本发明一个实施例的ST悬浮细胞接种猪瘟病毒效价检测分析图。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品,其中,重组胰岛素采用通化东宝生产的重组胰岛素。
实施例1
1、配置培养基
(1)在DMEM培养基基础上,分别补充如下成分,制成ST-SFM1和ST-SFM2培养基,其中ST-SFM1为本发明配方,ST-SFM2为对照无血清培养基。
其中,DMEM培养基的组成如下:
(2)配置DMEM培养基,并添加10%NBS作为血清对照培养基。
2、悬浮驯化方法:
将ST贴壁细胞消化计数后,以1.0×106的细胞量接种于250ml摇瓶,分别使用DMEM+10%NBS、ST-SFM1培养基和ST-SFM2培养基进行培养,在37℃、5%CO2培养箱内进行悬浮培养,转速控制在130-150rpm范围内,培养72h后,采用Countstar Biotech自动细胞计数仪检测细胞活率、细胞密度,并按照1.0×106cells/ml的密度传代,进行连续培养。
检测结果显示在图1~图3中,其中,图1显示了ST细胞用ST-SFM1培养基悬浮培养的细胞密度、活率与时间关系曲线,图2显示了ST细胞用ST-SFM2培养基悬浮培养的细胞密度、活率与时间关系曲线,图3显示了ST细胞用DMEM+10%NBS培养基悬浮培养的细胞密度、活率与时间关系曲线。通过图1~图3 的结果,可以看出ST-SFM1培养基能快速实现ST细胞的悬浮驯化,驯化周期在20天左右,之后的72h细胞密度达9.0×106cells/ml以上,与常规逐步减少血清的驯化方法相比,操作更简便;使用DMEM+10%NBS培养基和ST-SFM2培养基驯化效果不佳,细胞活率低、增殖倍数低。如图4所示,使用DMEM+10%NBS培养基和ST-SFM2培养基容易出现细胞结团,而ST-SFM1培养基更利于ST细胞悬浮生长,不易出现细胞结团、细胞死亡等问题,细胞分散性好、活率高,可用于后续的工业化大规模生产。
3、ST-SFM培养基生产应用
悬浮驯化的ST细胞稳定传代后,用ST-SFM1培养基按照1.5×106cells/ml的密度接种至5L反应器,按1%比例同步接种猪瘟病毒(CSFV),进行连续培养,每3天进行细胞沉降一次,收获70%的上清抗原液,采用免疫荧光法对病毒半数组织感染量进行测定。结果如图5所示,使用ST-SFM1培养基悬浮培养的ST细胞,在无血清条件下三收的FAID50均大于5.0,符合猪瘟疫苗质量标准,表明该ST-SFM1培养基在猪瘟疫苗生产中具有良好的应用前景。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (7)
1.一种用于悬浮培养ST细胞的培养基,其特征在于,包括:
DMEM培养基;
添加剂,所述添加剂包括:
终浓度为110mg/L的L-盐酸精氨酸;
终浓度为500mg/L的L-门冬酰胺;
终浓度为300mg/L的L-天门冬氨酸;
终浓度为150mg/L的L-半胱氨酸盐酸盐一水;
终浓度为30mg/L的L-胱氨酸二盐酸盐;
终浓度为600mg/L的L-谷氨酸;
终浓度为585mg/L的L-谷氨酰胺;
终浓度为210mg/L的L-盐酸组氨酸一水;
终浓度为170mg/L的甘氨酸;
终浓度为200mg/L的L-异亮氨酸;
终浓度为400mg/L的L-亮氨酸;
终浓度为355mg/L的L-盐酸赖氨酸;
终浓度为170mg/L的L-甲硫氨酸;
终浓度为250mg/L的L-苯丙氨酸;
终浓度为150mg/L的L-脯氨酸;
终浓度为100mg/L的L-丝氨酸;
终浓度为134mg/L的L-苏氨酸;
终浓度为80mg/L的L-色氨酸;
终浓度为450mg/L的L-酪氨酸二钠盐无水;
终浓度为300mg/L的L-缬氨酸;
终浓度为1.9mg/L的D-生物素;
终浓度为70mg/L的氯化胆碱;
终浓度为16mg/L的叶酸;
终浓度为53mg/L的肌醇;
终浓度为21mg/L的烟酰胺;
终浓度为0.3mg/L的维生素B2;
终浓度为5mg/L的维生素B1;
终浓度为11mg/L的维生素B12;
终浓度为15.5mg/L的四盐酸精胺;
终浓度为0.9mg/L的还原型谷胱甘肽;
终浓度为0.66mg/L的腺嘌呤硫酸盐;
终浓度为0.66mg/L的鸟苷;
终浓度为16mg/L的二水氯化钙;
终浓度为4.8mg/L的七水硫酸亚铁;
终浓度为537mg/L的无水磷酸氢二钠;
终浓度为298mg/L的一水磷酸二氢钠;
终浓度为1.8mg/L的七水硫酸锌;
终浓度为3500mg/L的D-无水葡萄糖;
终浓度为3000mg/L的大豆水解物;
终浓度为3000mg/L的酵母水解物;
终浓度为100mg/L的丙酮酸钠;
终浓度为1000mg/L的F68;
终浓度为0.5mg/L的九水硝酸铁;
终浓度为12mg/L的维生素 C 磷酸酯镁;
终浓度为28mg/L的乙醇胺盐酸盐;
终浓度为0.00034mg/L的硝酸银;
终浓度为0.00098936mg/L的偏钒酸铵;
终浓度为0.00000726mg/L的偏钒酸钠;
终浓度为0.00366mg/L的氯化镉;
终浓度为0.00046mg/L的六水氯化铬;
终浓度为0.00106mg/L的二氧化锗;
终浓度为0.00024mg/L的溴化钾;
终浓度为0.00034mg/L的碘化钾;
终浓度为0.00034mg/L的四水氯化锰;
终浓度为0.00026mg/L的硫酸镍;
终浓度为0.00242mg/L的氯化铷;
终浓度为0.0002mg/L的氯化亚锡;
终浓度为0.168mg/L的氯化铝;
终浓度为0.00644mg/L的氧氯化锆;
终浓度为0.0051mg/L的乙酸钡;
终浓度为0.00476mg/L的六水氯化钴;
终浓度为0.005mg/L的五水硫酸铜;
终浓度为169.6002mg/L的六水氯化镁;
终浓度为0.0165mg/L的氯化锂;
终浓度为0.006mg/L的钼酸铵;
终浓度为0.626mg/L的九水偏硅酸钠;
终浓度为0.0346mg/L的亚硒酸钠;
终浓度为0.0084mg/L的氟化钠;
终浓度为0.66mg/L的1,4-丁二胺二盐酸盐;
终浓度为0.42mg/L的硫辛酸;
终浓度为0.00132mg/L的维生素E;
终浓度为0.072mg/L的氢化可的松;
终浓度为0.037488mg/L的亚油酸;
终浓度为2mg/L的对氨基苯甲酸;
终浓度为1.16mg/L的重组人胰岛素;
终浓度为52.6mg/L的柠檬酸铁;
终浓度为0.004394703mg/L的乙醇胺;
终浓度为1.318410875mg/L的胰岛素;
终浓度为0.488021295mg/L的胆固醇;
终浓度为0.155279503mg/L的维生素E醋酸酯;
终浓度为0.022182786mg/L的肉豆蔻酸;
终浓度为0.022182786mg/L的油酸;
终浓度为0.022182786mg/L的棕榈酸;
终浓度为0.022182786mg/L的硬脂酸;
终浓度为4.880212955mg/L的吐温80。
2.权利要求1所述用于悬浮培养ST细胞的培养基在悬浮培养ST细胞和扩增病毒中的应用。
3.一种悬浮培养ST细胞的方法,其特征在于,包括:
将ST细胞接种于权利要求1所述用于悬浮培养ST细胞的培养基中;
对所述ST细胞进行悬浮培养。
4.一种扩增病毒的方法,其特征在于,包括:
按照权利要求3所述的方法,对ST细胞进行悬浮培养,得到培养液;和
在所述培养液中接种病毒,以便对所述病毒进行扩增。
5.根据权利要求4所述的方法,其特征在于,所述病毒选自猪瘟病毒、猪伪狂犬病毒,猪细小病毒、猪传染性胃肠炎病毒。
6.一种制备疫苗的方法,其特征在于,包括:按照权利要求4或5所述的方法扩增病毒。
7.根据权利要求6所述的方法,其特征在于,进一步包括:
将所述扩增病毒所得到的病毒液进行减毒或者灭活处理,以便获得所述疫苗。
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