CN114480286A - 无血清悬浮型lmh细胞系及其制备方法和应用 - Google Patents
无血清悬浮型lmh细胞系及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供一种无血清悬浮型LMH细胞系及其制备方法和应用。本发明采用添加氨基酸、维生素、无机盐、微量元素以及其他成分的DMEM/F12培养基的无血清培养基配方制备无血清悬浮型LMH细胞系,该无血清培养基配方能够用于贴壁培养型LMH细胞传代培养,经过逐代传代培养使其能适应采用该无血清培养基配方进行悬浮培养的方式,有助于LMH细胞的规模化生产,有利于病毒的复制和表达,促进病毒疫苗的规模化生产。
Description
技术领域
本发明属于生物技术领域,具体涉及一种无血清悬浮型LMH细胞系及其制备方法和应用。
背景技术
鸡肝癌细胞(LMH细胞)已成为生产禽类腺病毒(Fowl Adenovirus,FADV)、鸡传染性喉气管炎病毒(Infectious laryngotracheitis virus,ILTV)等常用的细胞系之一。贴壁型LMH细胞在培养过程中,贴壁困难,易聚团生长,通常需使用含10%(v/v)及以上的胎牛血清的培养基进行培养,且由于转瓶、细胞工厂等贴壁细胞培养容器的限制,生产成本一直居高不下,致使用该细胞系生产的产品无法规模化放大。另外,采用含血清成分的培养基对贴壁型LMH细胞培养的过程中,血清化学成分不确定,且批次间质量不稳定,以致于用此种细胞生产的生物制品,质量难以控制。
相对于采用含血清培养基进行贴壁培养而言,采用无血清培养基进行悬浮培养的工艺,其培养基成分明确,可大规模培养,占地面积小,单位体积能生产更多的生物制品,从而提高产能,降低成本。随着高密度细胞培养容器的推广及生物制品质量安全标准的提高,可悬浮的细胞具有了更广泛的应用前景。然而,目前适用于无血清悬浮培养工艺的LMH细胞及无血清培养基鲜有报道。
发明内容
基于上述技术问题,本发明的目的之一是提供一种无血清悬浮培养型LMH细胞系的制备方法,采用该制备方法,能获得生长状态稳定、分散性好的悬浮培养型LMH细胞,适用于LMH细胞以及病毒的规模化培养。
本发明的目的可以通过以下技术方案实现:
一种无血清悬浮培养型LMH细胞系的制备方法,所述制备方法包括采用无血清培养基对贴壁培养型LMH细胞进行传代培养制备无血清悬浮培养型LMH细胞系的步骤;
所述无血清培养基包含氨基酸、维生素、盐、微量元素、葡萄糖、β-巯基乙醇、重组人胰岛素和Pluronic F-68,以及DMEM/F12培养基。
在其中一些实施例中,所述氨基酸选自L-丙氨酸、L-精氨酸盐酸盐、L-谷氨酸、L-谷氨酰胺、甘氨酸、L-组氨酸盐酸盐水合物、L-异亮氨酸、L-亮氨酸、L-丝氨酸、L-苏氨酸、天冬酰胺一水合物、天冬氨酸、L-半胱氨酸一水合物、L-赖氨酸盐酸盐、甲硫氨酸、L-苯丙氨酸、L-脯氨酸、L-色氨酸和L-缬氨酸中的一种或者多种。
在其中一些实施例中,所述维生素选自泛酸钙、氯化胆碱、生物素、对氨基苯甲酸、抗坏血酸和生育酚中的一种或者多种。
在其中一些实施例中,所述盐选自氯化钠、无水硫酸镁、氯化钾、碳酸氢钠、磷酸二氢钠、磷酸氢二钠和丙酮酸钠中的一种或者多种;
在其中一些实施例中,所述微量元素选自亚硒酸钠、四水合钼酸铵和七水硫酸锌中的一种或者多种。
在其中一些实施例中,所述无血清培养基包含DMEM/F12培养基以及如下浓度的组分:L-丙氨酸133mg/L-1680mg/L、L-精氨酸盐酸盐60mg/L-1525mg/L、L-谷氨酸25mg/L-1375mg/L、L-谷氨酰胺150mg/L-1300mg/L、甘氨酸5mg/L-1300mg/L、L-组氨酸盐酸盐水合物60mg/L-1525mg/L、L-异亮氨酸20mg/L-1466mg/L、L-亮氨酸30mg/L-1400mg/L、L-丝氨酸110mg/L-1428mg/L、L-苏氨酸60mg/L-1417mg/L、天冬酰胺一水合物80mg/L-1390mg/L、天冬氨酸50mg/L-1359mg/L、L-半胱氨酸一水合物150mg/L-1352mg/L、L-赖氨酸盐酸盐90mg/L-1333mg/L、甲硫氨酸50mg/L-1346mg/L、L-苯丙氨酸20mg/L-1296mg/L、L-脯氨酸27mg/L-1261mg/L、L-色氨酸40mg/L-1500mg/L、L-缬氨酸58mg/L-1394mg/L、泛酸钙1.0mg/L-50mg/L、氯化胆碱0.5mg/L-5mg/L、生物素0.05mg/L-0.9mg/L、对氨基苯甲酸0.02mg/L-0.2mg/L、抗坏血酸0.01mg/L-0.5mg/L、生育酚0.05mg/L-0.95mg/L、氯化钠2200mg/L-7500mg/L、无水硫酸镁300mg/L-1800mg/L、氯化钾100mg/L-790mg/L、碳酸氢钠1600mg/L-4200mg/L、磷酸二氢钠129mg/L-1300mg/L、磷酸氢二钠68mg/L-744mg/L、亚硒酸钠1.7mg/L-6mg/L、四水合钼酸铵0.5mg/L-2mg/L、七水硫酸锌0.6mg/L-4.1mg/L、葡萄糖2000mg/L-8000mg/L、β-巯基乙醇0.4mg/L-25mg/L、丙酮酸钠200mg/L-800mg/L、重组人胰岛素2mg/L-10mg/L,和PluronicF-68 500mg/L-2400mg/L。
在其中一些实施例中,所述无血清培养基包含DMEM/F12培养基以及如下浓度的组分:L-丙氨酸400mg/L-500mg/L、L-精氨酸盐酸盐120mg/L-200mg/L、L-谷氨酸100mg/L-200mg/L、L-谷氨酰胺500mg/L-650mg/L、甘氨酸80mg/L-150mg/L、L-组氨酸盐酸盐水合物85mg/L-150mg/L、L-异亮氨酸100mg/L-200mg/L、L-亮氨酸100mg/L-200mg/L、L-丝氨酸110mg/L-200mg/L、L-苏氨酸100mg/L-200mg/L、天冬酰胺一水合物100mg/L-200mg/L、天冬氨酸100mg/L-200mg/L、L-半胱氨酸一水合物250mg/L-400mg/L、L-赖氨酸盐酸盐90mg/L-200mg/L、甲硫氨酸50mg/L-150mg/L、L-苯丙氨酸60mg/L-170mg/L、L-脯氨酸50mg/L-120mg/L、L-色氨酸40mg/L-100mg/L、L-缬氨酸58mg/L-100mg/L、泛酸钙10mg/L-35mg/L、氯化胆碱1mg/L-3mg/L、生物素0.5mg/L-2.9mg/L、对氨基苯甲酸0.1mg/L-0.2mg/L、抗坏血酸0.1mg/L-0.3mg/L、生育酚0.1mg/L-0.5mg/L、氯化钠4000mg/L-5500mg/L、无水硫酸镁650mg/L-850mg/L、氯化钾100mg/L-220mg/L、碳酸氢钠2500mg/L-3200mg/L、磷酸二氢钠900mg/L-1100mg/L、磷酸氢二钠300mg/L-400mg/L、亚硒酸钠2mg/L-4mg/L、四水合钼酸铵0.5mg/L-2mg/L、七水硫酸锌0.6mg/L-4.1mg/L、葡萄糖3000mg/L-5000mg/L、β-巯基乙醇1mg/L-5mg/L、丙酮酸钠200mg/L-400mg/L、重组人胰岛素2mg/L-10mg/L,和Pluronic F-68 800mg/L-1500mg/L。
在其中一些实施例中,传代培养的代数对应所得传代培养物中的细胞密度与当代次接种细胞密度之比≥3且所述传代培养物中细胞活率>90%的代次。
在其中一些实施例中,传代培养的过程中,每次传代培养的细胞接种密度为0.5×106个/mL-2.5×106个/mL。
在其中一些实施例中,传代培养的过程中,每次传代培养的条件包括:旋转速度为60rpm-200rpm,旋转轨道直径为10mm-50mm,CO2体积含量5%-10%,温度为35℃-39℃,湿度>60%,时长为2天-8天。
一种无血清悬浮培养型LMH细胞系,其通过所述的制备方法获得。
一种家禽病毒疫苗的制备方法,所述制备方法包括:制备家禽病毒的步骤和将所述家禽病毒制备成疫苗的步骤,
制备所述家禽病毒的步骤包括:采用所述的无血清悬浮培养型LMH细胞系为宿主细胞,接种家禽病毒,培养。
在其中一些实施例中,培养所采用的培养基包含所述的无血清培养基。
在其中一些实施例中,培养的温度为30℃-37℃。
在其中一些实施例中,接毒所采用的接毒量为100-10-5MOI。
在其中一些实施例中,在所述宿主细胞的密度为2.0×106个/mL-20.0×106个/mL的条件下接毒所述家禽病毒。
在其中一些实施例中,所述家禽病毒包含H9亚型禽流感病毒、禽腺病毒、水禽星状病毒、禽减蛋综合征病毒、禽呼肠孤病毒、鸡传染性喉气管炎病毒、鸡传染性法氏囊病毒、鸡传染性支气管炎病毒和禽副粘病毒。
与传统技术相比,本发明具有以下有益效果:
本发明采用添加氨基酸、维生素、无机盐、微量元素以及其他成分的DMEM/F12培养基作为无血清培养基配方对贴壁培养型LMH细胞进行传代培养,,经过逐代传代培养使其能适应采用该无血清培养基配方进行悬浮培养的方式,有助于LMH细胞的规模化生产,有利于病毒的复制和表达,促进病毒疫苗的规模化生产。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明实施例1制备的无血清悬浮培养型LMH细胞形态图;
图2为本发明实施例1无血清悬浮培养型LMH细胞按照1.5×106cells/mL接种,生长至最高细胞密度时的活细胞数及活率变化情况。
具体实施方式
为了便于理解本发明,下面将对本发明进行更详细的描述。但是,应当理解,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施方式或实施例。相反地,提供这些实施方式或实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施方式或实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”的可选范围包括两个或两个以上相关所列项目中任一个,也包括相关所列项目的任意的和所有的组合,所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。
本发明中,“第一方面”、“第二方面”、“第三方面”等仅用于描述目的,不能理解为指示或暗示相对重要性或数量,也不能理解为隐含指明所指示的技术特征的重要性或数量。
本发明中,以开放式描述的技术特征中,包括所列举特征组成的封闭式技术方案,也包括包含所列举特征的开放式技术方案。
本发明中,涉及到数值区间,如无特别说明,则包括数值区间的两个端点。
本发明中涉及的百分比含量,如无特别说明,对于固液混合和固相-固相混合均指质量百分比,对于液相-液相混合指体积百分比。
本发明中涉及的百分比浓度,如无特别说明,均指终浓度。所述终浓度,指添加成分在添加该成分后的体系中的占比。
本发明中的温度参数,如无特别限定,既允许为恒温处理,也允许在一定温度区间内进行处理。所述的恒温处理允许温度在仪器控制的精度范围内进行波动。
下述实验方法如无特别说明,均为常规方法。所使用细胞株具有鉴定为美国典型培养物保藏中心(ATCC)编号为CRL-2117的细胞系的特征。其他所用实验材料如无特别说明,均可从商业公司获取。
其中,“接种密度”通常是指,当细胞生长到高密度,培养基营养成分不能满足细胞的生长时,再把高密度的细胞按照比例稀释成低密度的过程,一般接种密度在0.1×106cells/mL-2.0×106cells/mL。
本发明所述的“最高细胞密度”是指培养基能够满足细胞生长的最高生长密度。“生长培养基”是指用于细胞从低密度生长到高密度的无血清细胞培养基。“生产培养基”是指可以满足病毒有效复制的病毒生产液。
本发明的“生长培养基”和“生产培养基”可以是本领域已知的、任何一款或者几款可以用于LMH悬浮细胞生长和生产的培养基。
在本发明的一些实施方案中,所述“生长培养基”与“生产培养基”可以是同一种培养基,也可以是不同的培养基。
其中,所述“无血清培养”是指在培养过程中不添加牛血清,只靠培养基提供的营养成分满足细胞生长的培养方式。
传统的用于动物细胞生长的方法,其培养采用的无血清培养基,例如CN1962857A公开的无血清培养基,其主要是针对哺乳动物细胞,并且配方复杂,也不适用例如鸡肝癌细胞之类的非哺乳动物细胞。为此,本发明提供一种适用于鸡肝癌细胞且无血清培养基配方简单的方法。
第一方面,本发明提供一种无血清悬浮培养型LMH细胞系的制备方法,所述制备方法包括采用所述的无血清培养基对贴壁培养型LMH细胞进行传代培养制备无血清悬浮培养型LMH细胞系的步骤;
所述无血清培养基包含氨基酸、维生素、盐、微量元素、葡萄糖、β-巯基乙醇、重组人胰岛素和Pluronic F-68,以及DMEM/F12培养基。
在其中一个示例中,所述氨基酸选自L-丙氨酸、L-精氨酸盐酸盐、L-谷氨酸、L-谷氨酰胺、甘氨酸、L-组氨酸盐酸盐水合物、L-异亮氨酸、L-亮氨酸、L-丝氨酸、L-苏氨酸、天冬酰胺一水合物、天冬氨酸、L-半胱氨酸一水合物、L-赖氨酸盐酸盐、甲硫氨酸、L-苯丙氨酸、L-脯氨酸、L-色氨酸和L-缬氨酸中的一种或者多种。
在其中一个示例中,所述维生素选自泛酸钙、氯化胆碱、生物素、对氨基苯甲酸、抗坏血酸和生育酚中的一种或者多种。
在其中一个示例中,所述盐选自氯化钠、无水硫酸镁、氯化钾、碳酸氢钠、磷酸二氢钠、磷酸氢二钠和丙酮酸钠中的一种或者多种。
在其中一个示例中,所述微量元素选自亚硒酸钠、四水合钼酸铵和七水硫酸锌中的一种或者多种。
本发明涉及上述无血清培养基在LMH细胞培养中的用途。本发明提供的培养基能够用于传代培养贴壁培养型LMH细胞为无血清悬浮培养型LMH细胞,当然可以理解为,其既可以用于培养无血清悬浮培养型LMH细胞,也可以用于培养贴壁培养型LMH细胞。培养既可以包括将其作为生长培养基进行培养,也可以将其作为生产培养基进行培养。
在其中一个示例中,所述无血清培养基包含DMEM/F12培养基以及如下浓度的组分:L-丙氨酸133mg/L-1680mg/L、L-精氨酸盐酸盐60mg/L-1525mg/L、L-谷氨酸25mg/L-1375mg/L、L-谷氨酰胺150mg/L-1300mg/L、甘氨酸5mg/L-1300mg/L、L-组氨酸盐酸盐水合物60mg/L-1525mg/L、L-异亮氨酸20mg/L-1466mg/L、L-亮氨酸30mg/L-1400mg/L、L-丝氨酸110mg/L-1428mg/L、L-苏氨酸60mg/L-1417mg/L、天冬酰胺一水合物80mg/L-1390mg/L、天冬氨酸50mg/L-1359mg/L、L-半胱氨酸一水合物150mg/L-1352mg/L、L-赖氨酸盐酸盐90mg/L-1333mg/L、甲硫氨酸50mg/L-1346mg/L、L-苯丙氨酸20mg/L-1296mg/L、L-脯氨酸27mg/L-1261mg/L、L-色氨酸40mg/L-1500mg/L、L-缬氨酸58mg/L-1394mg/L、泛酸钙1.0mg/L-50mg/L、氯化胆碱0.5mg/L-5mg/L、生物素0.05mg/L-0.9mg/L、对氨基苯甲酸0.02mg/L-0.2mg/L、抗坏血酸0.01mg/L-0.5mg/L、生育酚0.05mg/L-0.95mg/L、氯化钠2200mg/L-7500mg/L、无水硫酸镁300mg/L-1800mg/L、氯化钾100mg/L-790mg/L、碳酸氢钠1600mg/L-4200mg/L、磷酸二氢钠129mg/L-1300mg/L、磷酸氢二钠68mg/L-744mg/L、亚硒酸钠1.7mg/L-6mg/L、四水合钼酸铵0.5mg/L-2mg/L、七水硫酸锌0.6mg/L-4.1mg/L、葡萄糖2000mg/L-8000mg/L、β-巯基乙醇0.4mg/L-25mg/L、丙酮酸钠200mg/L-800mg/L、重组人胰岛素2mg/L-10mg/L,和PluronicF-68 500mg/L-2400mg/L。
在其中一个示例中,所述无血清培养基包含DMEM/F12培养基以及如下浓度的组分:L-丙氨酸400mg/L-500mg/L、L-精氨酸盐酸盐120mg/L-200mg/L、L-谷氨酸100mg/L-200mg/L、L-谷氨酰胺500mg/L-650mg/L、甘氨酸80mg/L-150mg/L、L-组氨酸盐酸盐水合物85mg/L-150mg/L、L-异亮氨酸100mg/L-200mg/L、L-亮氨酸100mg/L-200mg/L、L-丝氨酸110mg/L-200mg/L、L-苏氨酸100mg/L-200mg/L、天冬酰胺一水合物100mg/L-200mg/L、天冬氨酸100mg/L-200mg/L、L-半胱氨酸一水合物250mg/L-400mg/L、L-赖氨酸盐酸盐90mg/L-200mg/L、甲硫氨酸50mg/L-150mg/L、L-苯丙氨酸60mg/L-170mg/L、L-脯氨酸50mg/L-120mg/L、L-色氨酸40mg/L-100mg/L、L-缬氨酸58mg/L-100mg/L、泛酸钙10mg/L-35mg/L、氯化胆碱1mg/L-3mg/L、生物素0.5mg/L-2.9mg/L、对氨基苯甲酸0.1mg/L-0.2mg/L、抗坏血酸0.1mg/L-0.3mg/L、生育酚0.1mg/L-0.5mg/L、氯化钠4000mg/L-5500mg/L、无水硫酸镁650mg/L-850mg/L、氯化钾100mg/L-220mg/L、碳酸氢钠2500mg/L-3200mg/L、磷酸二氢钠900mg/L-1100mg/L、磷酸氢二钠300mg/L-400mg/L、亚硒酸钠2mg/L-4mg/L、四水合钼酸铵0.5mg/L-2mg/L、七水硫酸锌0.6mg/L-4.1mg/L、葡萄糖3000mg/L-5000mg/L、β-巯基乙醇1mg/L-5mg/L、丙酮酸钠200mg/L-400mg/L、重组人胰岛素2mg/L-10mg/L,和Pluronic F-68 800mg/L-1500mg/L。
本发明所述的贴壁培养型LMH细胞例如为ATCC CRL-2117。
本发明所述的贴壁培养型LMH细胞,其获取来源不限,可以通过含血清培养基培养得到,也可以是采用其他类型的培养基培养得到。
在其中一个示例中,传代培养的代数对应所得传代培养物中的细胞密度与当代次接种细胞密度之比≥3且所述传代培养物中细胞活率>90%的代次。例如:在F3代能够满足所得传代培养物中的细胞密度与该代次接种细胞密度之比≥3且所述传代培养物中细胞活率>90%,则传代培养所需的代数为3,即传代培养3次可获得无血清悬浮培养型LMH细胞系。
在其中一个示例中,传代培养的过程中,每次传代培养的细胞接种密度为0.5×106个/mL-2.5×106个/mL。例如0.5×106个/mL、1×106个/mL、1.5×106个/mL、2×106个/mL、2.5×106个/mL。优选地,细胞接种密度为1.0×106个/mL-2.0×106个/mL。
在其中一个示例中,传代培养的过程中,每次传代培养的条件包括:旋转速度为60rpm-200rpm,旋转轨道直径为10mm-50mm,CO2体积含量5%-10%,湿度>60%,温度为35℃-39℃,时长为2天-8天。每次传代培养的条件中:旋转速度例如60rpm、80rpm、100rpm、120rpm、140rpm、160rpm、180rpm、200rpm,优选为100rpm-140rpm,更优选为120rpm;旋转轨道直径例如为10mm、20mm、30mm、40mm、50mm,优选地20mm-30mm,更优选为25mm;CO2体积含量例如5%、5.5%、6%、6.5%、7%、7.5%、8%、8.5%、9%、9.5%、10%,优选为5%-6%,更优选为5%;湿度例如为65%、70%、75%、80%、85%,优选为75%-85%,更优选为80%;温度例如35℃、36℃、37℃、38℃、39℃,优选为36.5℃-37.5℃,更优选为37℃;时长例如为2天、3天、4天、5天、6天、7天、8天,优选为3天-4天,更优选为3天。
第二方面,本发明提供一种无血清悬浮培养型LMH细胞系,其通过所述的制备方法获得。
本发明的提供的无血清悬浮培养型LMH细胞系,其密度为3.0×106个/mL-50.0×106个/mL(优选为3.0×106个/mL-15.0×106个/mL,最优选为10.0×106个/mL)的悬浮液可使用程序降温盒于-80℃冰箱中保存24h,然后转移冻存管至液氮罐中保存。
第三方面,本发明提供一种家禽病毒疫苗的制备方法,所述制备方法包括:制备家禽病毒的步骤和将所述家禽病毒制备成疫苗的步骤,
制备所述家禽病毒的步骤包括:采用所述的无血清悬浮培养型LMH细胞系为宿主细胞,接种家禽病毒,培养。
在其中一个示例中,培养所采用的培养基包含所述的无血清培养基。在该培养基中,细胞生长3天-10天可达到最高细胞密度。
在其中一个示例中,培养的温度为30℃-37℃,例如30℃、31℃、32℃、33℃、34℃、35℃、36℃、37℃。
在其中一个示例中,接毒所采用的接毒量为100-10-5MOI。接毒量例如为100MOI、10-1MOI、10-2MOI、10-3MOI、10-4MOI、10-5MOI。优选为10-1-10-3MOI,更优选为10-2MOI。
在其中一个示例中,在所述宿主细胞的密度为2.0×106个/mL-20.0×106个/mL的条件下接毒所述家禽病毒。所述宿主细胞的密度例如为2×106个/mL、4×106个/mL、6×106个/mL、8×106个/mL、10×106个/mL、12×106个/mL、14×106个/mL、16×106个/mL、18×106个/mL、20×106个/mL,优选为4.0×106个/mL-8.0×106个/mL,更优选为6.0×106个/mL。可以理解的是,该宿主细胞的密度可以是直接培养宿主细胞达到密度,也可以是直接培养宿主细胞经稀释后达到的密度。稀释可以采用如上所述的无血清培养基。
本发明所述的家禽病毒对应LMH细胞上敏感的病毒,包括但不限于:H9亚型禽流感病毒、禽腺病毒、水禽星状病毒、禽减蛋综合征病毒、禽呼肠孤病毒、鸡传染性喉气管炎病毒、鸡传染性法氏囊病毒、鸡传染性支气管炎病毒和禽副粘病毒。
本发明制备家禽病毒的步骤中,培养的装置可以包括但不限于摇瓶和反应器,例如采用工作体积1L及以上的反应器,包含搅拌式生物反应器和激流式生物反应器,其可自动控制培养温度、pH、溶氧、搅拌速度等参数,容积为2L-3000L;培养温度为30-39℃、pH6.5-7.4、溶氧20%-80%、搅拌速度20-200r/min,生物反应器在使用前需调试。
可以理解的是,制备所述家禽病毒的步骤中,可以稀释/补加生产培养基。稀释/补加生产培养基是指,LMH细胞按照一定的接种密度接种细胞后,通过2-8天的生长,细胞密度可以达到2.0×106个/mL-20.0×106个/mL之间,补加原有工作体积1-5倍的生产培养基或者采用其他稀释方式,使细胞密度降低到1.0×106个/mL-10.0×106个/mL之间,通过1-7天的病毒生产,收获病毒液。当然,也可以接毒后再补加原有工作体积1-5倍的生产培养基。
可以理解的是,将所述家禽病毒制备成疫苗的步骤例如包括:对培养所得产物中所述家禽病毒进行纯化。
实施例1
一、实验材料
1、细胞株:来源于ATCC的贴壁培养型LMH细胞株。
2、培养基:DMEM/F12培养基,在DMEM/F12培养基中添加了氨基酸、维生素、无机盐、微量元素等组分制备无血清培养基(见表1)。
二、实验方法
1、将冻存的贴壁培养型LMH细胞复苏,来源:ATCC,编号为CRL-2117。
2、将复苏的贴壁培养型LMH细胞置于T75cm2flask(培养瓶),加入15mL含10%(v/v)胎牛血清的DMEM/F12完全培养基,置于37℃,5%CO2,湿度≥80%的培养箱中培养4天,使细胞达到80%-100%汇合状态。
3、弃除培养基,并加入1mL 0.25%Trypsin(胰蛋白酶)-EDTA,使贴壁培养型LMH细胞消化脱离培养瓶容器表面,然后加入15mL含10%胎牛血清的DMEM/F12完全培养基终止胰酶消化。
4、将上述消化后的贴壁培养型LMH细胞用移液管吹散,用细胞计数仪计算单位体积的细胞密度和细胞活率,按照1:3(v:v)的接种密度接种于T225cm2flask,加入45mL含10%胎牛血清的DMEM/F12完全培养基,置于37℃,5%CO2的培养箱中培养4天,使细胞达到80%-100%汇合状态。
5、每4天进行传代,重复步骤3和4扩增贴壁培养型LMH细胞,选择足够量状态良好的贴壁培养型LMH细胞,弃除培养基并加0.25%Trypsin-EDTA使LMH细胞消化脱离培养瓶表面,然后加入25mL含10%胎牛血清的DMEM/F12完全培养基终止胰酶消化,1000rpm离心5min,去上清收集LMH细胞。
6、使用无血清培养基(见表1)重悬贴壁培养型LMH细胞至125mL悬浮培养摇瓶中,吹散细胞团使得细胞呈分散状态。
7、调整细胞接种密度在1.5×106cells/mL,培养体积40mL。
8、放于37℃、5%CO2、湿度≥80%、轨道直径为25mm、转速为120rpm的摇床中培养。
9、细胞培养3天,密度在2.5×106cells/mL,活率在85%以上,进行传代培养。
10、摇瓶培养的LMH细胞悬液离心,去上清收集LMH细胞,重复步骤6、7、8。
11、每3天按照1.5×106cells/mL传代一次,观察细胞结团以及分散情况,直至细胞密度≥4.5×106cells/mL,,活率在90%以上,分散性良好且无较大团块,细胞最高密度接近20.0×106cells/mL,获得无血清悬浮培养型LMH细胞系。
12、将上述步骤制备的无血清悬浮培养型LMH细胞系,按照1.5×106cells/mL浓度接种至无血清培养基中,记录生长至最高细胞密度时的活细胞数及细胞活率变化情况。
无血清悬浮培养型LMH细胞系在无血清培养基中的细胞形态见图1(培养第1天的细胞),无血清悬浮培养型LMH细胞系的生长变化情况见图2。
实施例2
本实施例是实施例1的变化例,变化之处主要是采用的无血清培养基配方不同,具体见表1。其余参照同实施例1。
实施例3
本实施例是实施例1的变化例,变化之处主要是采用的无血清培养基配方不同,具体见表1。其余参照同实施例1。
实施例4
本实施例是实施例1的变化例,变化之处主要是采用的无血清培养基配方不同,具体见表1。其余参照同实施例1。
实施例5
本实施例是实施例1的变化例,变化之处主要是采用的无血清培养基配方不同,具体见表1。其余参照同实施例1。
表1、无血清培养基(单位:mg/L)
表2、实施例1至5所得结果汇总
实施例6、禽腺病毒在无血清悬浮培养型LMH细胞中的测试
一、实验材料
1、细胞株:实施例1至实施例5中的无血清悬浮培养型LMH细胞。
2、培养基:实施例1至实施例5中所述的无血清培养基(见表1)。
二、实验方法
以采用实施例1提供的无血清悬浮培养型LMH细胞和无血清培养基为例,对实验方法进行说明,实验方法包括以下步骤:
1、培养基的配制:实施例1提供的无血清培养基,除菌过滤后4℃避光保存。
2、细胞的复苏:从液氮罐中取出冻存的无血清悬浮培养型LMH细胞,37℃融化后加入到30mL无血清培养基中,1000rpm离心,丢弃上清液,重悬无血清悬浮培养型LMH细胞于新鲜1×的无血清培养基中,置于37℃,5%CO2培养箱进行培养。
3、传代:新复苏的血清悬浮培养型LMH细胞实验前需要传代3次,每隔3天传代一次,接种密度1.5×106cells/mL,连续传代3次,然后进行接毒。
4、接毒:无血清悬浮培养型LMH细胞传代至第3代第3天时细胞密度达到6.0×106cells/mL,使用无血清培养基作为生产培养基,将细胞密度稀释至为3.0×106cells/mL,接种禽腺病毒,接毒量MOI为10-1,35℃条件下培养至收获。
5、测毒:按照半数组织(细胞)培养感染剂量测试病毒的TCID50。
以实施例2至实施例5提供的无血清培养基和无血清悬浮培养型LMH细胞进行的测试参照上述步骤1至步骤5。测试结果见表3。
实施例7、禽腺病毒在无血清悬浮型LMH细胞的测试
一、实验材料
1、细胞株:实施例1至实施例5中的无血清悬浮培养型LMH细胞。
2、生长培养基:实施例1至实施例5中提供的无血清培养基(见表1),除菌过滤后4℃避光保存。
3、生产培养基:DMEM/F12培养基。
二、实验方法
以采用实施例1提供的无血清悬浮培养型LMH细胞和无血清培养基为例,对实验方法进行说明,实验方法包括以下步骤:
1、培养基的配制:实施例1提供的无血清培养基,除菌过滤后4℃避光保存。
2、细胞的复苏:从液氮罐中取出冻存的无血清悬浮培养型LMH细胞,37℃融化后加入到30mL培养基中,1000rpm离心,丢弃上清液,重悬LMH悬浮细胞于悬浮生长培养基中,置于37℃,5%CO2培养箱进行培养。
3、传代:新复苏的细胞实验前需要传代3次,每隔3天传代一次,接种密度是1.5×106cells/mL,连续传代3次,然后进行接毒。
4、接毒:细胞传代至第3代第3天时细胞密度达到6.0×106cells/mL,使用DMEM/F12培养基作为生产培养基,将细胞密度稀释至为3.0×106cells/mL,接种禽腺病毒,接毒量MOI为10-1,35℃条件下培养至收获。
5、测毒:按照半数组织(细胞)培养感染剂量测试病毒的TCID50。
以实施例2至实施例5提供的无血清培养基和无血清悬浮培养型LMH细胞进行的测试参照上述步骤1至步骤5。测试结果见表3。
表3、实施例1至5对应的测试结果汇总
实施例8
本实施例是实施例1的对比例,相对于实施例1的主要变化在于无血清培养基的配方不同,具体地,本对比例的无血清培养基中采用盐酸硫氨代替对氨基苯甲酸。
参照实施例1,采用实施例8提供的无血清培养基制备无血清悬浮培养型LMH细胞系。
采用实施例1提供的无血清培养基培养实施例1驯化得到的悬浮型细胞系,采用实施例8提供的无血清培养基培养实施例8驯化得到的悬浮型细胞系,培养5代(每代3天),统计细胞培养情况。
根据表4可知,相对于实施例1,尽管实施例8的无血清培养基也驯化得到的悬浮型细胞系,但是该悬浮型细胞系随着传代代次的增加,悬浮培养中会出现结团。
表4
以上所述实施例仅表达了本发明的几种实施方式,便于具体和详细地理解本发明的技术方案,但并不能因此而理解为对发明专利保护范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
应当理解,本领域技术人员在本发明提供的技术方案的基础上,通过合乎逻辑的分析、推理或者有限的试验得到的技术方案,均在本发明所述附权利要求的保护范围内。因此,本发明专利的保护范围应以所附权利要求的内容为准,说明书及附图可以用于解释权利要求的内容。
Claims (10)
1.一种无血清悬浮培养型LMH细胞系的制备方法,其特征在于,所述制备方法包括采用无血清培养基对贴壁培养型LMH细胞进行传代培养制备无血清悬浮培养型LMH细胞系的步骤;
所述无血清培养基包含氨基酸、维生素、盐、微量元素、葡萄糖、β-巯基乙醇、重组人胰岛素和Pluronic F-68,以及DMEM/F12培养基。
2.根据权利要求1所述的无血清悬浮培养型LMH细胞系的制备方法,其特征在于,所述氨基酸选自L-丙氨酸、L-精氨酸盐酸盐、L-谷氨酸、L-谷氨酰胺、甘氨酸、L-组氨酸盐酸盐水合物、L-异亮氨酸、L-亮氨酸、L-丝氨酸、L-苏氨酸、天冬酰胺一水合物、天冬氨酸、L-半胱氨酸一水合物、L-赖氨酸盐酸盐、甲硫氨酸、L-苯丙氨酸、L-脯氨酸、L-色氨酸和L-缬氨酸中的一种或者多种;
或/和,所述维生素选自泛酸钙、氯化胆碱、生物素、对氨基苯甲酸、抗坏血酸和生育酚中的一种或者多种;
或/和,所述盐选自氯化钠、无水硫酸镁、氯化钾、碳酸氢钠、磷酸二氢钠、磷酸氢二钠和丙酮酸钠中的一种或者多种;
或/和,所述微量元素选自亚硒酸钠、四水合钼酸铵和七水硫酸锌中的一种或者多种。
3.根据权利要求2所述的无血清悬浮培养型LMH细胞系的制备方法,其特征在于,所述无血清培养基包含DMEM/F12培养基以及如下浓度的组分:L-丙氨酸133mg/L-1680mg/L、L-精氨酸盐酸盐60mg/L-1525mg/L、L-谷氨酸25mg/L-1375mg/L、L-谷氨酰胺150mg/L-1300mg/L、甘氨酸5mg/L-1300mg/L、L-组氨酸盐酸盐水合物60mg/L-1525mg/L、L-异亮氨酸20mg/L-1466mg/L、L-亮氨酸30mg/L-1400mg/L、L-丝氨酸110mg/L-1428mg/L、L-苏氨酸60mg/L-1417mg/L、天冬酰胺一水合物80mg/L-1390mg/L、天冬氨酸50mg/L-1359mg/L、L-半胱氨酸一水合物150mg/L-1352mg/L、L-赖氨酸盐酸盐90mg/L-1333mg/L、甲硫氨酸50mg/L-1346mg/L、L-苯丙氨酸20mg/L-1296mg/L、L-脯氨酸27mg/L-1261mg/L、L-色氨酸40mg/L-1500mg/L、L-缬氨酸58mg/L-1394mg/L、泛酸钙1.0mg/L-50mg/L、氯化胆碱0.5mg/L-5mg/L、生物素0.05mg/L-0.9mg/L、对氨基苯甲酸0.02mg/L-0.2mg/L、抗坏血酸0.01mg/L-0.5mg/L、生育酚0.05mg/L-0.95mg/L、氯化钠2200mg/L-7500mg/L、无水硫酸镁300mg/L-1800mg/L、氯化钾100mg/L-790mg/L、碳酸氢钠1600mg/L-4200mg/L、磷酸二氢钠129mg/L-1300mg/L、磷酸氢二钠68mg/L-744mg/L、亚硒酸钠1.7mg/L-6mg/L、四水合钼酸铵0.5mg/L-2mg/L、七水硫酸锌0.6mg/L-4.1mg/L、葡萄糖2000mg/L-8000mg/L、β-巯基乙醇0.4mg/L-25mg/L、丙酮酸钠200mg/L-800mg/L、重组人胰岛素2mg/L-10mg/L,和Pluronic F-68 500mg/L-2400mg/L;
可选地,所述无血清培养基包含DMEM/F12培养基以及如下浓度的组分:L-丙氨酸400mg/L-500mg/L、L-精氨酸盐酸盐120mg/L-200mg/L、L-谷氨酸100mg/L-200mg/L、L-谷氨酰胺500mg/L-650mg/L、甘氨酸80mg/L-150mg/L、L-组氨酸盐酸盐水合物85mg/L-150mg/L、L-异亮氨酸100mg/L-200mg/L、L-亮氨酸100mg/L-200mg/L、L-丝氨酸110mg/L-200mg/L、L-苏氨酸100mg/L-200mg/L、天冬酰胺一水合物100mg/L-200mg/L、天冬氨酸100mg/L-200mg/L、L-半胱氨酸一水合物250mg/L-400mg/L、L-赖氨酸盐酸盐90mg/L-200mg/L、甲硫氨酸50mg/L-150mg/L、L-苯丙氨酸60mg/L-170mg/L、L-脯氨酸50mg/L-120mg/L、L-色氨酸40mg/L-100mg/L、L-缬氨酸58mg/L-100mg/L、泛酸钙10mg/L-35mg/L、氯化胆碱1mg/L-3mg/L、生物素0.5mg/L-2.9mg/L、对氨基苯甲酸0.1mg/L-0.2mg/L、抗坏血酸0.1mg/L-0.3mg/L、生育酚0.1mg/L-0.5mg/L、氯化钠4000mg/L-5500mg/L、无水硫酸镁650mg/L-850mg/L、氯化钾100mg/L-220mg/L、碳酸氢钠2500mg/L-3200mg/L、磷酸二氢钠900mg/L-1100mg/L、磷酸氢二钠300mg/L-400mg/L、亚硒酸钠2mg/L-4mg/L、四水合钼酸铵0.5mg/L-2mg/L、七水硫酸锌0.6mg/L-4.1mg/L、葡萄糖3000mg/L-5000mg/L、β-巯基乙醇1mg/L-5mg/L、丙酮酸钠200mg/L-400mg/L、重组人胰岛素2mg/L-10mg/L,和Pluronic F-68 800mg/L-1500mg/L。
4.根据权利要求1至3任一项所述的无血清悬浮培养型LMH细胞系的制备方法,其特征在于,传代培养的代数对应所得传代培养物中的细胞密度与当代次接种细胞密度之比≥3且所述传代培养物中细胞活率>90%的代次。
5.根据权利要求1至3任一项所述的无血清悬浮培养型LMH细胞系的制备方法,其特征在于,传代培养的过程中,每次传代培养的细胞接种密度为0.5×106个/mL-2.5×106个/mL,或/和,每次传代培养的条件包括:旋转速度为60rpm-200rpm,旋转轨道直径为10mm-50mm,CO2体积含量5%-10%,温度为35℃-39℃,湿度>60%,时长为2天-8天。
6.一种无血清悬浮培养型LMH细胞系,其特征在于,其通过权利要求1至5任一项所述的制备方法获得。
7.一种家禽病毒疫苗的制备方法,其特征在于,所述制备方法包括:制备家禽病毒的步骤和将所述家禽病毒制备成疫苗的步骤,
制备所述家禽病毒的步骤包括:采用权利要求6所述的无血清悬浮培养型LMH细胞系为宿主细胞,接种家禽病毒,培养。
8.根据权利要求7所述的家禽病毒疫苗的制备方法,其特征在于,培养所采用的培养基包含权利要求1至3任一项定义的所述的无血清培养基。
9.根据权利要求7或者8所述的家禽病毒疫苗的制备方法,其特征在于,培养的温度为30℃-37℃,或/和,接毒所采用的接毒量为100-10-5MOI,或/和,在所述宿主细胞的密度为2.0×106个/mL-20.0×106个/mL的条件下接毒所述家禽病毒。
10.根据权利要求7或者8所述的家禽病毒疫苗的制备方法,其特征在于,所述家禽病毒包含H9亚型禽流感病毒、禽腺病毒、水禽星状病毒、禽减蛋综合征病毒、禽呼肠孤病毒、鸡传染性喉气管炎病毒、鸡传染性法氏囊病毒、鸡传染性支气管炎病毒和禽副粘病毒。
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