CN108103003B - 一种适应pk-15全悬浮生长的无血清培养基及其制备方法和应用于pk-15细胞的全悬浮驯化方法 - Google Patents
一种适应pk-15全悬浮生长的无血清培养基及其制备方法和应用于pk-15细胞的全悬浮驯化方法 Download PDFInfo
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Abstract
本发明涉及一种适应PK‑15全悬浮生长的无血清培养基及其制备方法和应用于pK‑15细胞的全悬浮驯化方法,该培养基包含氨基酸、吐温、亚油酸、维生素E、豆蔻酸、硬脂酸、维生素A、β‑巯基乙醇、氯化钠、葡萄糖、氯化镉、亚油酸、氯化胆碱、肌醇和偏钒酸铵等物质。本发明提供的无血清培养基,能够实现PK‑15细胞全悬浮生长,从而省略微载体成本及消化过程,解决PK‑15细胞微载体悬浮培养过程中难以消化放大的技术问题。
Description
技术领域
本发明属于细胞工程领域,涉及一种无蛋白无水解物成分明确的无血清培养基,尤其涉及一种适用于PK-15细胞全悬浮培养的无血清培养基及其制备方法和应用于pK-15细胞的全悬浮驯化方法。
背景技术
细胞培养是蛋白、多肽类药物、疫苗等生物制品生产中最核心、最基础的技术,细胞培养基为细胞体外培养提供生长所需的营养和适宜的环境,是体外细胞培养的基础和关键因素。一般细胞培养基由无机盐、氨基酸、维生素、碳源、微量元素等成分组成。
PK-15细胞是动物疫苗生产中常用的一种细胞,其主要用来生产猪圆环病毒2型(PCV2)和猪细小病毒(PPV)等。PK-15细胞属于贴壁依赖型细胞,目前技术培养方式有两种:转瓶贴壁培养和微载体悬浮培养,两种方法本质上都属于贴壁培养;培养所用培养基多数为常规培养基(MEM),培养过程需添加8%左右血清,少数厂家使用低血清培养基或者无血清培养基。然而转瓶培养科技含量较低,劳动强度大、占地面积大并且批件差异难以控制,抗原生产过程中外源污染风险较大;而微载体培养虽然极大程度上降低了劳动强度和生产场地,但微载体成本高昂,细胞本质上还是贴壁培养,放大过程需要胰酶消化,由于PK-15细胞属于比较难消化的细胞株,并且反应器中的消化传代放大工艺繁琐、污染风险较大,因此放大过程是PK-15细胞微载体悬浮培养中的一大缺陷;
细胞全悬浮培养是指将贴壁依赖性细胞驯化成非贴壁依耐性细胞,不需要添加微载体及其它贴壁载体,直接将细胞在摇瓶或生物反应其中进行悬浮培养。因此细胞全悬浮培养完全省略了微载体成本及消化过程,可以完全解决微载体悬浮培养过程中的消化放大的技术瓶颈,拥有非常显著优势。然而,PK-15细胞全悬浮培养对细胞培养基要求非常高,需要针定制型的全悬浮培养基,具有专一性。目前,应用于PK-15细胞培养扩增阶段的培养基多为采用5%-10%血清浓度的常规培养基如MEM、DMEM,或者部分采用低血清或无血清培养基,但这些培养基都只是适用于贴壁培养,无法支持PK-15细胞全悬浮培养;这是因为使用普通常规培养基或无血清培养基在培养PK-15细胞过程中会出现细胞严重成团,不生长或生长缓慢等问题。
综上所述,PK-15细胞全悬浮培养的技术难点首先在于需要一个针对性的全悬浮培养基,其次是需要有正确可行的驯化方法。
发明内容
从以上分析可以看出,目前PK-15细胞培养技术使用的是贴壁培养,而使用的培养基也全部是用于贴壁培养,目前还没有适用于PK-15细胞全悬浮培养的细胞培养基,特别是成分明确的无血清培养基。为解决以上问题,本发明了一种针对PK-15全悬浮培养的成分明确的无血清培养基,将目前的生产技术从贴壁培养提高到了科技含量更高、工艺更加先进的全悬浮培养。
本发明提供了一种适应PK-15全悬浮生长的无血清培养基,由如下组分制备而成:
上述适应PK-15全悬浮生长的无血清培养基中配方组分均为商品化的生化试剂。
上述适应PK-15全悬浮生长的无血清培养基的制备方法为:首先将1~10mg吐温80溶解于50毫升注射用水中,然后加入0.15-0.5mg亚油酸、0.2-2mg维生素E、0.2-0.8mg豆蔻酸、0.18-0.5mg硬脂酸、5-50mg维生素A,待其溶解充分,用注射用水补液至500毫升,再向其中加入1-20mgβ-巯基乙醇、2-8g氯化钠、8-15g葡萄糖,搅拌至澄清,用注射用水补液至900毫升,然后加入:
添加完毕后,继续搅拌至完全溶解,再用注射用水补液至1000mL,经过滤、除菌后于4℃避光保存。
本发明将通过上述制备方法得到的培养基对细胞做氨基酸代谢分析发现,L盐酸精氨酸、L天冬氨酸、L天冬酰胺、L异亮氨酸、L亮氨酸和L盐酸赖氨酸为快速消耗型氨基酸,增加这几个氨基酸的用量,对细胞的生长和维持有显著促进作用,因此本发明上述几种物质的添加量偏高;在优选的实施方式中,L盐酸精氨酸为600mg/L、L天冬氨酸为127mg/L、L天冬酰胺为475mg/L、L异亮氨酸为105.6mg/L、L亮氨酸为500mg/L、L盐酸赖氨酸为260mg/L。
应用于对PK-15细胞全悬浮驯化的过程为:以购自ATCC的生长良好的PK-15贴壁细胞为研究基础,将PK-15细胞通过常规方法用胰酶消化后(消化时间至少为8min)以本发明提供的培养基重悬(重悬所用培养基在10~20ml),将重悬后的细胞以1×106-5×106cells/mL的密度接种至125ml摇瓶(摇瓶经过硅化剂预先处理使细胞更难贴壁),再加入本发明提供的培养基30ml,之后将药瓶放于含5%CO2的恒温摇床上培养,培养温度为37℃,摇床转速为100-130rpm;每24h取样用细胞计数仪测定细胞密度及活率,并用倒置显微镜观察细胞状态,且每48h更换本发明提供的培养基,直到PK-15细胞适应悬浮生长,可通过每天观察细胞状态,更换培养基时适当调整摇床转速、培养基体积等培养参数。
本发明对适应全悬浮生长的PK-15细胞做批次式培养,并通过DOE实验设计,从几十种添加物中筛选得到了维生素A、胆固醇、氯化镉、亚油酸等十几种维生素和微量元素类物质,特别是氯化镉、亚油酸、氯化胆碱、肌醇和偏钒酸铵对细胞生长及维持有显著影响作用。
培养基中葡萄糖作为快速消耗型碳源,虽然可以快速为细胞生长提供能量及一些化合物前体,但是其缺点是代谢过程会产生大量乳酸,而乳酸量达到一定浓度后会显著抑制细胞生长;本发明中用细胞更难利用的半乳糖作为缓释型碳源,降低了乳酸的生成量;另一方面培养基中加入的丙酮酸钠,也可减缓葡萄糖的消耗,从而进一步的降低乳酸的生成量。
腺嘌呤、次黄嘌呤和胸苷作为核酸合成的前体,直接加入可被细胞直接利用,省去了细胞从其它物质合成的步骤,对细胞的快速增长有促进作用。
谷氨酰胺作为细胞培养中必须的氨基酸,对细胞生长具有重要作用,但其在水中不稳定,易分解,而分解产生的氨对细胞由毒害作用,本发明用丙谷二肽作为谷氨酰胺的替代物来部分替代谷氨酰胺,增强了培养基的稳定性,减少了氨的生成。
本发明中以磷酸氢二钠和磷酸二氢钾缓冲体系、碳酸氢钠和CO2(来源于摇床所含CO2)缓冲体系、hepes缓冲体系共同作为培养基的缓冲体系,可以更加稳定的维持培养过程中的pH,为细胞提供更好的生长环境。
与现有技术相比,本发明具有以下有益效果:
1、本发明提供的无血清培养基,能够实现PK-15细胞全悬浮生长,从而省略微载体成本及消化过程,解决PK-15细胞微载体悬浮培养过程中难以消化放大的技术问题。
2、本发明提供的无血清培养基,使用的L盐酸精氨酸、L天冬氨酸、L天冬酰胺、L异亮氨酸、L亮氨酸和L盐酸赖氨酸为快速消耗型氨基酸,增加这几个氨基酸的用量,对细胞的生长和维持有显著促进作用。
3、本发明提供的无血清培养基,使用的氯化镉、亚油酸、氯化胆碱、肌醇和偏钒酸铵等维生素和微量元素类物质对细胞生长及维持有显著影响作用。
4、本发明提供的无血清培养基,使用的半乳糖和丙酮酸钠可以降低乳酸的生成量。
5、本发明提供的无血清培养基,使用的丙谷二肽可以增强培养基的稳定性,减少氨的生成。
6、本发明提供的无血清培养基,无蛋白添加,无水解物添加,减少了该培养基的生产成本;且成分明确,产品批间差异小,同时也简化了下游产品的纯化步骤;避免了外源病毒污染,从而降低了安全隐患。
附图说明
图1为本发明实施例PK-15细胞全悬浮细胞生长阶段图,其中(a)为0h密度图,(b)24h密度图,(c)48h密度图。
图2为本发明实施例所提供的PK-15细胞在不同培养基中的细胞生长曲线,其中(a)为微载体培养,(b)为实施例1制备的全悬浮培养基培养。
具体实施方式
下面结合附图和实施例对本发明作进一步详细的说明。
实施例1-3
实施例1-3适应PK-15全悬浮生长的无血清培养基各组分用量参照表1给出的数据,该培养基的具体制备过程为:首先将吐温80溶解于50毫升注射用水中,然后加入不需要依次加入亚油酸、维生素E、豆蔻酸、硬脂酸、维生素A,待其溶解充分,用注射用水补液至500毫升,再向其中加入β-巯基乙醇、氯化钠、葡萄糖,搅拌至澄清,用注射用水补液至900毫升,然后加入剩余组分,添加完毕后,继续搅拌至完全溶解,再用注射用水补液至1000mL,经0.22μm滤器过滤、除菌后于4℃避光保存。
表1实施例1-3适应PK-15全悬浮生长的无血清培养基组分及用量
实施例4
应用于对PK-15细胞全悬浮驯化的过程为:以购自ATCC的生长良好的PK-15贴壁细胞为研究基础,将PK-15细胞通过常规方法用浓度为0.2%的胰酶消化8min左右,之后以实施例2提供的培养基20ml进行重悬,将重悬后的细胞以3×106cells/mL的密度接种至125ml摇瓶(摇瓶经过硅化剂预先处理使细胞更难贴壁),再加入实施例2提供的培养基30ml,之后将药瓶放于含5%CO2的恒温摇床上培养,培养温度为37℃,摇床转速为120rpm;每24h取样用细胞计数仪测定细胞密度及活率,并用倒置显微镜观察细胞状态,且每48h更换培养基,通过每天观察细胞状态,更换培养基时适当调整摇床转速、培养基体积等培养参数,直到PK-15细胞适应悬浮生长,得到PK-15悬浮型细胞。
应用例1
为了考察实施例2提供的培养基在培养全悬浮PK-15时细胞生长阶段的性能,将其与微载体悬浮培养作对比(微载体悬浮培养相对于转瓶培养已经有明显优势,因此本发明直接与微载体悬浮培养做对比)。
本应用例采取的微载体为购自GE的cytodex1载体。
(1)采用微载体悬浮培养PK-15细胞,具体实施方法为:取购自ATCC的生长状态良好的PK-15贴壁细胞,以浓度为0.2%的胰酶消化后加入含3-5g/L cytodex1载体的2L反应器中培养,接种密度约为1×106cells/ml,培养体积为1.8L,培养48h后取样,将微载体上的细胞用胰酶消化后,统计细胞密度及活率,统计结果见表2所示,依据该方法培养三批PK-15细胞。
(2)采用实施例2制备的培养基全悬浮培养PK-15细胞,具体实现方法为:取实施例4制备的PK-15悬浮型细胞,按1×106cells/ml接种于2L反应器,然后加入实施例2中的培养基,培养体积为1.8L,培养48h后取样,统计细胞密度及活率,统计结果见表2所示,依据该方法培养三批PK-15细胞。
采集由实施例2提供的培养基全悬浮培养PK-15细胞生长阶段随培养时间变化的细胞密度图片,如图1所示,从图中也可以看出,随着培养时间延长,PK-15细胞密度是逐渐增大的。
由微载体培养PK-15细胞生长阶段和由实施例2提供的培养基全悬浮培养PK-15细胞生长阶段细胞密度随培养时间的变化曲线,如图2所示,从图中也可以看出,细胞生长阶段全悬浮培养具有显著生长优势。
表2微载体培养PK-15细胞和全悬浮培养PK-15细胞统计结果
2L反应器微载体组 | 2L反应器全悬浮组 | |
第一批 | 5×10<sup>6</sup>cells/ml | 8×10<sup>6</sup>cells/ml |
第二批 | 4.8×10<sup>6</sup>cells/ml | 8.5×10<sup>6</sup>cells/ml |
第三批 | 4.75×10<sup>6</sup>cells/ml | 7.9×10<sup>6</sup>cells/ml |
此外,从表2中统计结果可以看出,采用本发明提供的培养基全悬浮培养的PK-15细胞数量是微载体培养获得细胞数量的1.6倍左右,说明细胞生长阶段全悬浮培养具有显著生长优势,意味着PK-15细胞全悬浮培养具有更快的放大效率和可能更高的产毒量。
应用例2
本应用例将反应容器放大到15L。
本应用例采取的微载体为购自GE的cytodex1载体。
(1)采用微载体悬浮培养PK-15细胞,具体实施方法为:取购自ATCC的生长状态良好的PK-15贴壁细胞,以浓度为0.2%的胰酶消化后加入含3-5g/L cytodex1载体的15L反应器中培养,接种密度约为1×106cells/ml,培养体积为1.8L,培养48h后取样,将微载体上的细胞用胰酶消化后,统计细胞密度及活率,统计结果见表3所示,依据该方法培养三批PK-15细胞。
(2)采用实施例2制备的培养基全悬浮培养PK-15细胞,具体实现方法为:取实施例4制备的PK-15悬浮型细胞,按1×106cells/ml接种于15L反应器,然后加入实施例2中的培养基,培养体积为1.8L,培养48h后取样,统计细胞密度及活率,统计结果见表3所示,依据该方法培养三批PK-15细胞。
表3微载体培养PK-15细胞和全悬浮培养PK-15细胞统计结果
15L反应器微载体组 | 15L反应器全悬浮组 | |
第一批 | 5.2×10<sup>6</sup>cells/ml | 8.3×10<sup>6</sup>cells/ml |
第二批 | 5.3×10<sup>6</sup>cells/ml | 8.1×10<sup>6</sup>cells/ml |
第三批 | 4.9×10<sup>6</sup>cells/ml | 8.3×10<sup>6</sup>cells/ml |
从表3和表2中统计结果对比可以看出,采用本发明提供的培养基全悬浮培养的PK-15细胞所得结果与应用例1中2L反应器结果基本一致,这进一步证明了本发明培养基在应用于PK-15细胞全悬浮培养中生长阶段具有明显优势。
本领域的普通技术人员将会意识到,这里所述的实施例是为了帮助读者理解本发明的原理,应被理解为本发明的保护范围并不局限于这样的特别陈述和实施例。本领域的普通技术人员可以根据本发明公开的这些技术启示做出各种不脱离本发明实质的其它各种具体变形和组合,这些变形和组合仍然在本发明的保护范围内。
Claims (5)
5.利用权利要求1或2所述的适应PK-15全悬浮生长的无血清培养基驯化PK-15细胞全悬浮的方法,其特征在于以PK-15贴壁细胞为基础,将PK-15细胞用胰酶消化后以所述培养基重悬,将重悬后的细胞以1×106cells/mL-5×106cells/mL的密度接种至125ml摇瓶,再加入所述培养基至少30ml,之后将药瓶放于含5%CO2的恒温摇床上培养,培养温度为37℃,摇床转速为100-130rpm;每24h取样用细胞计数仪测定细胞密度及活率,并用倒置显微镜观察细胞状态,且每48h更换所述培养基,直到PK-15细胞适应悬浮生长。
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