CN113755427A - 一种优化后的bhk21细胞无血清悬浮培养基 - Google Patents
一种优化后的bhk21细胞无血清悬浮培养基 Download PDFInfo
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- CN113755427A CN113755427A CN202111191092.2A CN202111191092A CN113755427A CN 113755427 A CN113755427 A CN 113755427A CN 202111191092 A CN202111191092 A CN 202111191092A CN 113755427 A CN113755427 A CN 113755427A
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Abstract
本发明公开了一种优化后的BHK21细胞无血清悬浮培养基,包括下列原料组成,氨基酸组;维生素组;无机盐与微量元素组;脂类、糖类与缓冲剂组;本发明中优化后的BHK21细胞无血清悬浮培养基培养BHK21悬浮细胞不仅能获得更高的细胞密度,并且在同一阶段相同密度条件下,细胞拥有更高的细胞活率、更小的细胞直径与更低的乳酸积累值,即拥有更好的细胞状态。而细胞状态直接决定了后续病毒扩增的效果。
Description
技术领域
本发明涉及细胞培养生物技术领域,尤其涉及一种优化后的BHK21细胞无血清悬浮培养基。
背景技术
口蹄疫是偶蹄动物携带的一种急性、热性和可快速远距离传播的传染病,感染对象是猪、牛、羊等主要畜种及其他家养和野生的偶蹄动物,易感动物多达70多种。传播快、感染性强,给畜牧业造成巨大的经济损失,严重影响国际贸易。素有“政治经济病”之称。由于其防控口蹄疫的措施主要是扑杀发病动物和应用质量可靠的灭活疫苗免疫易感动物。因此高质量安全的灭活疫苗对于口蹄疫防控至关重要。口蹄疫疫苗的制备通常采用BHK-21细胞作为基质。
BHK21细胞又称幼仓鼠肾成纤维细胞,该细胞系是从1日龄的金仓鼠肾脏建立的亲本,经过连续培养,通过单细胞分离技术而获得。该细胞被广泛应用于口蹄疫、狂犬病毒、乙脑病毒等病毒疫苗的生产基质。然而在口蹄疫疫苗的制作过程中容易残留培养基质细胞时所用的血清、水解乳蛋白,基质细胞蛋白等多种非目的蛋白成分。这些异源蛋白不仅导致注苗动物经常出现不同程度的不良反应,轻者减食、停食或发生流产,重者发生死亡,而且能够裂解病毒粒子,降低疫苗效力。
本实验室早期开发了一种BHK21细胞化学定义的CD培养基,该培养基支持细胞快速高密培养,但培养后期细胞超过1E7后出现乳酸高度积累及培养基pH急剧下降的现象,导致细胞悬崖式死亡。因此,需要一种用来解决上述问题的优化后的BHK21培养基。
发明内容
本发明的提供一种优化后的BHK21细胞无血清悬浮培养基。
本发明的方案是:
一种优化后的BHK21细胞无血清悬浮培养基,包括下列原料组成,氨基酸组;维生素组;无机盐与微量元素组;脂类、糖类与缓冲剂组。
作为优选的技术方案,各物质含量如下,
氨基酸组
维生素组
无机盐与微量元素组
脂类、水解物与添加物组
作为优选的技术方案,所述核苷酸包括次黄嘌呤和胸苷,能促进细胞的核苷酸合成,保证细胞的生长;次黄嘌呤和胸苷成分太高,会抑制细胞生长;
本发明还公开一种制备优化后的BHK21细胞无血清悬浮培养基的方法,包括下列步骤:
按配比称量培养基中各组成分;将称量好的氨基酸组;维生素组;无机盐与微量元素组;脂类、糖类与缓冲剂组溶解,溶解后用0.22μm的滤膜正压过滤除菌,获得优化后的BHK21细胞无血清悬浮培养基。
本发明还公开了一种所述的优化后的BHK21细胞无血清悬浮培养基在兽类疫苗细胞悬浮培养中的应用。
由于采用了上述技术方案一种优化后的BHK21细胞无血清悬浮培养基,包括下列步骤:按配比称量培养基中各组成分;将称量好的氨基酸组;维生素组;无机盐与微量元素组;脂类、糖类与缓冲剂组溶解,溶解后用0.22μm的滤膜正压过滤除菌,获得优化后的BHK21细胞无血清悬浮培养基。
本发明的有益效果在于:
1.稳定性强,培养效果增强,能够高的提高活细胞密度与活率,并且降低了其乳酸的积累,并且仍能使细胞生长超过1E7并维持相当一段时间的高细胞活率;
2.本发明的培养基培养BHK21细胞不仅能获得更高的细胞密度,并且在同一阶段相同密度条件下,细胞拥有更高的细胞活率、更小的细胞直径与更低的乳酸积累值,即拥有更好的细胞状态。而细胞状态直接决定了后续病毒扩增的效果。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,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是实施例1的3次不同批次复苏的BHK21细胞在优化前后的培养基中的生长情况图;
图2是F40代前同一批次的BHK21细胞在不同批次配制的优化前后培养基中的生长曲线图;
图3是F40代后同一批次的BHK21细胞在优化前后的培养基中的生长曲线图。
具体实施方式
为了弥补以上不足,本发明提供了一种优化后的BHK21细胞无血清悬浮培养基以解决上述背景技术中的问题。
一种优化后的BHK21细胞无血清悬浮培养基,包括下列原料组成,氨基酸组;维生素组;无机盐与微量元素组;脂类、糖类与缓冲剂组。
各物质含量如下,
氨基酸组
维生素组
无机盐与微量元素组
脂类、水解物与添加物组
所述核苷酸包括次黄嘌呤和胸苷,能促进细胞的核苷酸合成,保证细胞的生长;次黄嘌呤和胸苷成分太高,会抑制细胞生长;
本发明还公开一种制备优化后的BHK21细胞无血清悬浮培养基的方法,包括下列步骤:
按配比称量培养基中各组成分;将称量好的氨基酸组;维生素组;无机盐与微量元素组;脂类、糖类与缓冲剂组溶解,溶解后用0.22μm的滤膜正压过滤除菌,获得优化后的BHK21细胞无血清悬浮培养基。
本发明还公开了一种所述的优化后的BHK21细胞无血清悬浮培养基在兽类疫苗细胞悬浮培养中的应用。
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体实施例,进一步阐述本发明。
实施例1:
一种优化后的BHK21细胞无血清培养基各物料浓度如下:
氨基酸组
维生素组
成份 | 含量范围mg/L | 成份 | 含量范围mg/L |
α-硫辛酸 | 1.8 | 烟酰胺 | 4.5 |
氰钴胺素 | 1.1 | 硫胺盐酸盐 | 3.5 |
生物素 | 2.8 | 核黄素 | 0.36 |
叶酸 | 3.9 | 对氨基苯甲酸 | 1.6 |
维生素C | 23 | 吡哆醇 | 3.8 |
泛酸钙 | 2.8 | 氯化胆碱 | 86 |
肌醇 | 88 |
无机盐与微量元素组
脂类、水解物与添加物组
成份 | 含量范围mg/L | 成份 | 含量范围mg/L |
亚油酸 | 0.15 | P188 | 2000 |
丙酮酸钠 | 180 | HEPES | 1600 |
葡萄糖 | 6200 | 豌豆水解物 | 2000 |
柠檬酸铁铵 | 55 | 核苷酸 | 18 |
上述物料准确称量溶解后经0.22μm的无菌过滤膜过滤,使用3批新复苏的F3-F10的BHK21细胞,以3E5的接种密度,30ml的体系培养于上海多宁的125ml摇瓶中,再放入上海多宁的DNSI0101摇床中培养,摇床设定参数为:8%CO2、36.5℃和110rmp,整个培养过程无需补糖补料,只在接种后的第三天开始每天取样记录活细胞密度、雷度血气pH和Cedex生化参数即可。
如图1所示,3批BHK21细胞在优化后的本实施例的培养基中的最高活细胞密度能达到1.4E7-1.6E7,均高于优化前的1.2E7-1.4E7。
实施例2:
一种优化后的BHK21细胞无血清培养基各物料浓度如下:
氨基酸组
维生素组
无机盐与微量元素组
成份 | 含量范围mg/L | 成份 | 含量范围mg/L |
氯化钾 | 680 | 硫酸亚铁 | 0.5 |
碳酸氢钠 | 2200 | 硅酸钠 | 0.48 |
氯化钙 | 60 | 氯化锰 | 0.01 |
磷酸氢二钠 | 600 | 偏钒酸钠 | 0.002 |
氯化亚锡 | 0.05 | 钼酸钠 | 0.005 |
硫酸锌 | 2.6 | 亚硒酸钠 | 0.005 |
硫酸镁 | 76 | 硫酸铜 | 0.1 |
氯化铝 | 0.01 | 氯化钴 | 0.05 |
氯化锂 | 0.02 | 二氧化锗 | 0.03 |
脂类、水解物与添加物组
成份 | 含量范围mg/L | 成份 | 含量范围mg/L |
亚油酸 | 0.15 | P188 | 2000 |
丙酮酸钠 | 180 | HEPES | 1600 |
葡萄糖 | 6200 | 豌豆水解物 | 2000 |
柠檬酸铁铵 | 55 | 核苷酸 | 18 |
上述物料准确称量溶解后经0.22μm的无菌过滤膜过滤,使用新复苏的F3-F10的BHK21细胞,以3E5的接种密度,30ml的体系培养于上海多宁的125ml摇瓶中,再放入上海多宁DNSI0101的摇床中培养,摇床设定参数为:8%CO2、36.5℃和110rmp,整个培养过程无需补糖补料,只在接种后的第三天开始每天取样记录活细胞密度、雷度血气pH和Cedex生化参数即可。
实施例3:
一种优化后的BHK21细胞无血清培养基各物料浓度如下:
氨基酸组
维生素组
成份 | 含量范围mg/L | 成份 | 含量范围mg/L |
α-硫辛酸 | 1.8 | 烟酰胺 | 4.5 |
氰钴胺素 | 1.1 | 硫胺盐酸盐 | 3.5 |
生物素 | 2.8 | 核黄素 | 0.36 |
叶酸 | 3.9 | 对氨基苯甲酸 | 1.6 |
维生素C | 23.5 | 吡哆醇 | 3.8 |
泛酸钙 | 2.8 | 氯化胆碱 | 86 |
肌醇 | 90 |
无机盐与微量元素组
成份 | 含量范围mg/L | 成份 | 含量范围mg/L |
氯化钾 | 680 | 硫酸亚铁 | 0.5 |
碳酸氢钠 | 2200 | 硅酸钠 | 0.48 |
氯化钙 | 60 | 氯化锰 | 0.01 |
磷酸氢二钠 | 600 | 偏钒酸钠 | 0.002 |
氯化亚锡 | 0.05 | 钼酸钠 | 0.005 |
硫酸锌 | 2.6 | 亚硒酸钠 | 0.005 |
硫酸镁 | 76 | 硫酸铜 | 0.1 |
氯化铝 | 0.01 | 氯化钴 | 0.05 |
氯化锂 | 0.02 | 二氧化锗 | 0.03 |
脂类、水解物与添加物组
上述物料准确称量溶解后经0.22μm的无菌过滤膜过滤,使用新复苏的F3-F10的BHK21细胞,以3E5的接种密度,30ml的体系培养于上海多宁的125ml摇瓶中,再放入上海多宁的DNSI0101摇床中培养,摇床设定参数为:8%CO2、36.5℃和110rmp,整个培养过程无需补糖补料,只在接种后的第三天开始每天取样记录活细胞密度、雷度血气pH和Cedex生化参数即可。
如图1所示,不同批次复苏的BHK21细胞在实施例1后的培养基中的生长曲线结果显示,不同批次复苏的BHK21种子均能在优化后的培养基中稳定的生长,生长曲线基本一致。
如图2所示,同批次复苏的BHK21细胞在实施例1、实施例2、实施例3不同批次配制的优化后的培养基中的生长曲线结果显示,在该培养基中培养的细胞均能达到最高活细胞密度1.4E7-1.6E7,具有很好的批次稳定性。
本发明通过实验培养后的BHK21细胞在复苏后传代第40代的时候细胞出现老化,图3展示的是老化后的细胞在优化前后的培养基中生长情况,可以看出优化后的培养基虽然达不到正常的最高活细胞密度,但是细胞最高活细胞密度依然可以达到1E7以上并维持相当一段时间的高活力,直接反映了优化后的培养基具有更好的细胞培养效果。
如表1所示,3批复苏的种子3E5接种在125ml摇瓶中培养5天时细胞密度达到最大值,对比优化前后的培养基,该细胞在优化后的实施例1培养基中拥有更高的活细胞密度、更高的活率、更小的细胞直径、更优的环境pH、更低的乳酸积累。
D5 | 活细胞密度 | 细胞活率 | 平均细胞直径 | pH | 乳酸 | |
1号种子 | 优化前 | 123 | 97.32 | 14.66 | 6.6 | 2.6 |
实施例1 | 153 | 98.2 | 14.24 | 6.75 | 2.25 | |
2号种子 | 优化前 | 123 | 97.63 | 14.8 | 6.6 | 2.53 |
实施例1 | 151 | 98.32 | 14.4 | 6.71 | 2.33 | |
3号种子 | 优化前 | 136 | 97 | 14.6 | 6.57 | 3.34 |
实施例1 | 156 | 99.31 | 14.32 | 6.65 | 2.61 |
以上显示和描述了本发明的基本原理、主要特征及本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (5)
1.一种优化后的BHK21细胞无血清悬浮培养基,其特征在于,包括下列原料组成:氨基酸组;维生素组;无机盐与微量元素组;脂类、糖类与缓冲剂组。
3.如权利要求1或2中所述的优化后的BHK21细胞无血清悬浮培养基,其特征在于:所述核苷酸包括次黄嘌呤和胸苷。
4.一种制备如权利要求1至2中所述的优化后的BHK21细胞无血清悬浮培养基的方法,其特征在于,包括下列步骤:
按配比称量培养基中各组成分;将称量好的氨基酸组;维生素组;无机盐与微量元素组;脂类、糖类与缓冲剂组溶解,溶解后用0.22μm的滤膜正压过滤除菌,获得BHK21细胞无血清悬浮培养基。
5.一种如权利要求1至3所述的优化后的BHK21细胞无血清悬浮培养基在兽类疫苗细胞悬浮培养中的应用。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115433705A (zh) * | 2022-10-27 | 2022-12-06 | 天信和(苏州)生物科技有限公司 | 用于培养bhk21细胞的化学限定培养基、添加剂及其应用 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102268403A (zh) * | 2011-08-01 | 2011-12-07 | 上海米迪生物技术有限公司 | 适于幼仓鼠肾细胞大规模单细胞悬浮培养的无血清培养基 |
CN103555658A (zh) * | 2013-11-07 | 2014-02-05 | 令世鑫 | 一种bhk-21细胞全悬浮培养的无血清培养基 |
CN105018416A (zh) * | 2014-07-15 | 2015-11-04 | 内蒙古金源康生物工程有限公司 | 一种悬浮培养bhk-21细胞的无血清无动物源培养基及其配制方法 |
CN107435037A (zh) * | 2016-05-27 | 2017-12-05 | 上海倍谙基生物科技有限公司 | 一种用于bhk细胞的无血清培养基 |
CN110592000A (zh) * | 2019-08-13 | 2019-12-20 | 苏州易迈吉生物医药科技有限公司 | 一种支持bhk细胞高密度悬浮培养的无血清培养基 |
-
2021
- 2021-10-13 CN CN202111191092.2A patent/CN113755427A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102268403A (zh) * | 2011-08-01 | 2011-12-07 | 上海米迪生物技术有限公司 | 适于幼仓鼠肾细胞大规模单细胞悬浮培养的无血清培养基 |
CN103555658A (zh) * | 2013-11-07 | 2014-02-05 | 令世鑫 | 一种bhk-21细胞全悬浮培养的无血清培养基 |
CN105018416A (zh) * | 2014-07-15 | 2015-11-04 | 内蒙古金源康生物工程有限公司 | 一种悬浮培养bhk-21细胞的无血清无动物源培养基及其配制方法 |
CN107435037A (zh) * | 2016-05-27 | 2017-12-05 | 上海倍谙基生物科技有限公司 | 一种用于bhk细胞的无血清培养基 |
CN110592000A (zh) * | 2019-08-13 | 2019-12-20 | 苏州易迈吉生物医药科技有限公司 | 一种支持bhk细胞高密度悬浮培养的无血清培养基 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115433705A (zh) * | 2022-10-27 | 2022-12-06 | 天信和(苏州)生物科技有限公司 | 用于培养bhk21细胞的化学限定培养基、添加剂及其应用 |
CN115433705B (zh) * | 2022-10-27 | 2023-03-03 | 天信和(苏州)生物科技有限公司 | 用于培养bhk21细胞的化学限定培养基、添加剂及其应用 |
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