CN116515737B - 一种hek293细胞和cho细胞通用培养基及应用 - Google Patents
一种hek293细胞和cho细胞通用培养基及应用 Download PDFInfo
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- CN116515737B CN116515737B CN202310773274.3A CN202310773274A CN116515737B CN 116515737 B CN116515737 B CN 116515737B CN 202310773274 A CN202310773274 A CN 202310773274A CN 116515737 B CN116515737 B CN 116515737B
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Abstract
本发明提出了一种HEK293细胞和CHO细胞通用培养基及应用，该培养基无血清、无蛋白、化学成分明确，可以稳定贮存，且细胞克隆率、汇合度高。本发明的克隆培养基是针对单细胞克隆应用开发，可显著提高单克隆增殖率，保持良好的细胞状态，并快速筛选出优质克隆；优势在于不含蛋白类不稳定成分，化学成分限定，利于批次间的稳定性、重复性、高效性；市场上普遍存在的克隆培养基针对的是CHO细胞，本发明克隆培养基，既可应用于CHO细胞，还可应用于HEK293细胞，从而增加细胞株上应用的通用性。
Description
技术领域
本发明涉及细胞培养基领域，具体为一种HEK293细胞和CHO细胞通用培养基及应用。
背景技术
哺乳动物细胞需要稳定持续生长，并且能够通过外源基因导入以实现目的基因的表达，需要通过层层筛选、培养优化获得。HEK293细胞（人胚胎肾细胞）和CHO细胞（中华仓鼠卵巢细胞）是基因工程技术中常用的哺乳动物细胞，广泛应用于重组蛋白的生产，其具有诸多优点：①具有较高的耐受剪切力和渗透压能力，表达水平较高；②对表达产物进行准确的转录后糖基化修饰；③可在生物反应器中实现大规模高密度无血清悬浮培养；④胞外分泌功能利于下游产物的分离纯化；⑤HEK293细胞具有更高的生长密度和更快的生长速度优势，易培养，易转染。
在细胞培养过程中，群体细胞经过多次培养，细胞间存在异质性，目的基因的表达出现差异性，为了将高产优质的细胞从细胞群体中筛选出来，通常会进行群体细胞的单克隆筛选，将高产优质的单细胞从细胞群体中筛选出来，进行扩增，建立细胞库。
相比较群体细胞而言，单细胞培育具有极大的挑战，因此培育单细胞的克隆培养基直接影响单细胞的扩增及后续生物制品的产量、质量和安全。
CN111040986A（公开日2020.04.21），一种悬浮培养细胞单克隆筛选培养方法。本发明提供了一种促贴壁培养基和一种悬浮驯育培养基，分别可用于恢复悬浮细胞贴壁特性和辅助细胞悬浮培养驯化，其培养基需额外添加血清来促进。
CN112391335A（公开日2021.02.23），涉及一种单克隆细胞培养基、应用及培养单克隆细胞的方法。通过基础培养基和条件培养基的配合使用，使得本发明提供的单克隆细胞培养基有丰富的营养物质。其内容主要提供一种单克隆细胞培养基含有胎牛血清白蛋白或转铁蛋白等。
CN113122495A（公开日2021.07.16），一种用于中国仓鼠卵巢细胞克隆形成的培养基。本发明提供的培养基不含血清、不含动物来源的组分，但含有植物水解物成分。
目前市场上普遍应用的克隆培养基国外EX-CELL®CHO克隆培养基（C6366，西格玛sigma），国内QuaMono™ Plus CHO 克隆培养基（A11602，康晟生物Quacell），均含有植物水解物成分。
应良好制造规范GMP（Good Manufacturing Practice）的要求，无血清培养基的发展主要聚焦在两个方向：无动物来源，化学成分明确。化学成分明确培养基是目前被公认为最安全和理想的培养基。
发明内容
针对现有技术的不足，本发明提供一种HEK293细胞和CHO细胞通用培养基及应用，该培养基无血清、无蛋白、化学成分明确，可以稳定储存，且细胞克隆率、汇合度高，以弥补现有技术的不足。
为了解决上述技术问题，本发明提供如下技术方案：
一种HEK293细胞和CHO细胞通用培养基，包括氨基酸、维生素、微量元素、无机盐、核苷类和其他成分，
所述氨基酸组分终浓度分别为：L-盐酸精氨酸100~729.5mg/L、L-天冬酰胺91.79~495 mg/L、L-丙氨酸10~436.5mg/L、L-谷氨酸20~200mg/L、L-甘氨酸9.92~200mg/L、L-盐酸组氨酸36.67~500mg/L、L-异亮氨酸50~1000mg/L、L-亮氨酸50~1000mg/L、L-盐酸赖氨酸59.5~410mg/L、L-甲硫氨酸10~800mg/L、L-苯丙氨酸24.93~600mg/L、L-脯氨酸50~300mg/L、L-丝氨酸87.4~500mg/L、L-苏氨酸30~300mg/L、L-色氨酸10.5~500mg/L、L-酪氨酸钠盐50~500mg/L、L-缬氨酸20~200mg/L、L-盐酸半胱氨酸10~500mg/L、L-丙氨酰-L-谷氨酰胺5.2~54.1mg/L；
所述维生素组分终浓度分别为：氯化胆碱10~100mg/L、叶酸3.6~50mg/L、肌醇10~100mg/L、烟酰胺1~15.8mg/L、泛酸钙0.5~30mg/L、核黄素0.1~30mg/L、生物素0.01~20mg/L、维生素B12 0.1~60mg/L、对氨基苯甲酸0.1~50mg/L、盐酸腐胺2~30mg/L、硫辛酸0.01~20mg/L、L-抗坏血酸10~466.8mg/L；
所述微量元素组分终浓度分别为：一水合硫酸锰0.000001~0.0001mg/L、八水合二氯氧化锆0.00000236~0.0001509mg/L、六水合三氯化铬0.000003~0.00026mg/L、六水合氯化钴0.00008~0.00065mg/L、氯化亚锡0.0000001~0.001mg/L、四水钼酸铵0.00001~0.0003mg/L、氯化镉0.0000002~0.00002mg/L、氯化铜二水合物0.0001~0.01mg/L、氯化铝0.00001~0.001mg/L、硝酸银0.0000000061~0.0000007mg/L、硫酸镍0.0000175~0.001mg/L、醋酸钡0.00000001~0.0000001mg/L、亚硒酸钠0.001~0.01652mg/L；
所述无机盐组分终浓度分别为：氯化钠4000~8000mg/L、氯化钾10~250mg/L、硫酸镁10~102.8mg/L、氯化锌0.1~30mg/L、磷酸二氢钠100~1000mg/L、氯化钙100~500mg/L、碳酸氢钠1550~8000mg/L、柠檬酸铁0.5~100mg/L；
所述核苷类组分终浓度分别为：腺苷0.1~10mg/L、胞苷0.1~10mg/L、鸟苷0.1~10mg/L、胸苷0.1~10mg/L、尿苷0.1~10mg/L、2'-脱氧鸟苷水合物0.1~10mg/L、次黄嘌呤0.1~10mg/L；
所述其他成分组分终浓度分别为：亚油酸钠0.1~50mg/L、葡萄糖2000~10000mg/L、丙酮酸钠5~500mg/L、盐酸乙醇胺0.5~150mg/L。
优选的，所述氨基酸组分终浓度分别为：L-盐酸精氨酸100mg/L、L-天冬酰胺150mg/L、L-丙氨酸10mg/L、L-谷氨酸20mg/L、L-甘氨酸24.5mg/L、L-盐酸组氨酸53mg/L、L-异亮氨酸750mg/L、L-亮氨酸360.5mg/L、L-盐酸赖氨酸155.6mg/L、L-甲硫氨酸20mg/L、L-苯丙氨酸36.5mg/L、L-脯氨酸52.3mg/L、L-丝氨酸87.4mg/L、L-苏氨酸75mg/L、L-色氨酸10.5mg/L、L-酪氨酸钠盐82mg/L、L-缬氨酸30mg/L、L-盐酸半胱氨酸55mg/L、L-丙氨酰-L-谷氨酰胺10mg/L；
所述维生素组分终浓度分别为：氯化胆碱14.8mg/L、叶酸5mg/L、肌醇25mg/L、烟酰胺1mg/L、泛酸钙3mg/L、核黄素0.5mg/L、生物素0.1mg/L、维生素B12 42mg/L、对氨基苯甲酸0.1mg/L、盐酸腐胺2mg/L、硫辛酸0.1mg/L、L-抗坏血酸200mg/L；
所述微量元素组分终浓度分别为：一水合硫酸锰0.000023mg/L、八水合二氯氧化锆0.000095mg/L、六水合三氯化铬0.000045mg/L、六水合氯化钴0.00065mg/L、氯化亚锡0.00000609mg/L、四水钼酸铵0.0001mg/L、氯化镉0.0000018mg/L、氯化铜二水合物0.00102mg/L、氯化铝0.00059mg/L、硝酸银0.0000001mg/L、硫酸镍0.00059mg/L、醋酸钡0.00000014mg/L、亚硒酸钠0.01mg/L；
所述无机盐组分终浓度分别为：氯化钠5490mg/L、氯化钾169mg/L、硫酸镁50mg/L、氯化锌0.1mg/L、磷酸二氢钠540mg/L、氯化钙100mg/L、碳酸氢钠2000mg/L、柠檬酸铁0.5mg/L；
所述核苷类组分终浓度分别为：腺苷0.86mg/L、胞苷0.86mg/L、鸟苷0.86mg/L、胸苷0.86mg/L、尿苷0.86mg/L、2'-脱氧鸟苷水合物0.86mg/L、次黄嘌呤5.5mg/L；
所述其他成分组分终浓度分别为：亚油酸钠0.1mg/L、葡萄糖2000mg/L、丙酮酸钠200mg/L、盐酸乙醇胺15mg/L。
优选的，所述的HEK293细胞为HEK293F细胞。
优选的，所述的CHO细胞为CHO-K1细胞。
上述的任意一种HEK293细胞和CHO细胞通用培养基的应用，用于HEK293细胞或CHO细胞的培养。
优选的，用于HEK293细胞或CHO细胞的单克隆培养。
与现有技术相比，本发明的有益效果如下：
本发明提出了一种HEK293细胞和CHO细胞通用培养基及应用，该培养基无血清、无蛋白、化学成分明确，可以稳定贮存，且细胞克隆率、汇合度高。本发明的克隆培养基是针对单细胞克隆应用开发，可显著提高单克隆增殖率，保持良好的细胞状态，并快速筛选出优质克隆；优势在于不含蛋白类不稳定成分，化学成分限定，利于批次间的稳定性、重复性、高效性；市场上普遍存在的克隆培养基针对的是CHO细胞，本发明克隆培养基，既可应用于CHO细胞，还可应用于HEK293细胞，从而增加细胞株上应用的通用性。
本发明的作用机理：本发明的核苷类物质，能够影响细胞周期，虽然CHO等细胞，能够自身合成核苷酸，但添加到培养基中，比从头合成产生核苷酸所需的能量更少，对于单个细胞的生长，有促进作用，搭配其他各种微量元素，及多种氨基酸，能够代替水解物中的某些成分，从而实现本发明培养基的独特性能。
附图说明
附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与本发明的实施例一起用于解释本发明，并不构成对本发明的限制。在附图中：
图1为CHO-K1细胞在Cellvento® 4CHO-C克隆培养基上D14天克隆生长图；
图2为CHO-K1细胞在本发明实施例1通用培养基上D14天克隆生长图；
图3为CHO-K1细胞在QuaMono™ Plus CHO克隆培养基上D14天克隆生长图；
图4为HEK293F细胞在Cellvento® 4CHO-C克隆培养基上D14天克隆生长图；
图5为HEK293F细胞在本发明实施例1通用培养基上D14天克隆生长图；
图6为HEK293F细胞在QuaMono™ Plus CHO克隆培养基上D14天克隆生长图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
一、实验试剂：
以下实施例中所述的HEK293和CHO细胞通用型克隆培养基组成（包括氨基酸、维生素、无机盐、微量元素、核苷类和其他分子化合物各成分）均采购自西格玛（Sigma）；商业化培养基QuaMono™ Plus CHO 克隆培养基（康晟生物Quacell、A11602）；商业化培养基Cellvento® 4CHO-C克隆培养基（默克Merck、14390C）；细胞株HEK293F、CHO-K1的原始购买来源为ATCC（美国模式培养物集存库）。
二、实验方法：
1、实验组培养基的配置方法如下：
（1）将1L用量实施例1~9任一所述终浓度的物质成分，混匀；
（2）将步骤（1）的通用培养基混合物溶解于最终配制体积80%的注射用水中，搅拌约1小时，物料溶解后，加入一定量的盐酸或者氢氧化钠调pH至7.0~7.2以促进培养基混合物的溶解，继续搅拌约0.5小时，然后用注射用水定容，用0.22μm无菌膜过滤，4℃保存备用。
2、对照组培养基：一种是QuaMono™ Plus CHO 克隆培养基，含有植物水解物成分；另一种是国外新产品Cellvento®4CHO-C克隆培养基，是一种化学成分限定、无动物成分的培养基，用于替代含有植物水解物成分的EX-CELL®CHO（C6366，西格玛sigma）克隆培养基。
3、本发明的通用培养基单克隆操作方法，包括如下步骤：
（1）按照标准的细胞复苏及传代培养步骤，将复苏后的细胞培养至活率大于90%以上；
（2）采用梯度稀释法，细胞稀释每次10倍稀释，连续稀释至103活细胞数/mL备用，最终稀释至0.5~1细胞数/200µL；
（3）用多通道排枪吸取稀释液200µL/孔加入96孔板中，96孔板推荐使用无菌透明平底材质；放置于静置培养箱中培养，调整好参数，尽量减少晃动；铺板数可视情况而定，本发明所涉及的实验铺板数为两块；
（4）在第7天（D07）补加30µL/孔通用培养基，可补充蒸发量和促进克隆快速增殖，第14天（D14）采用CloneSelect Imager细胞生长分析系统（简称CSI）仪器扫板，进行克隆数据统计。
4、对照组培养基使用方法：
（1）QuaMono™ Plus CHO克隆培养基使用按其说明书操作；
（2）Cellvento®4CHO-C克隆培养基使用上述本发明的通用培养基单克隆操作方法。
实施例1：各种物质成分在通用培养基的终浓度见下表1。
表1
实施例2：各种物质成分在通用培养基的终浓度见下表2。
表2
实施例3：各种物质成分在通用培养基的终浓度见下表3。
表3
实施例4：各种物质成分在通用培养基的终浓度见下表4。
表4
实施例5：各种物质成分在通用培养基的终浓度见下表5。
表5
实施例6：各种物质成分在通用培养基的终浓度见下表6。
表6
实施例7：各种物质成分在通用培养基的终浓度见下表7。
表7
实施例8：各种物质成分在通用培养基的终浓度见下表8。
表8
实施例9：各种物质成分在通用培养基的终浓度见下表9。
表9
如图1~图6所示，其中图2、图5位本发明实施1的通用培养基培养细胞在D14的镜下图，相较于对照组的图1/3/4/6，本发明通用培养基培养的CHO-K1细胞和HEK293F细胞在96孔板呈现的镜下细胞多，另外图2的CHO-K1细胞的生长情况汇合度高。
根据下表10结果可知：
本发明通用培养基在克隆培养CHO-K1细胞上，相对于商业培养基Cellvento®4CHO-C克隆培养基在克隆率上均高出，最高高出约21%，具有克隆优势，对于商业培养基QuaMono™ Plus CHO 克隆培养基克隆率结果没有明显差异，但胜在本发明克隆培养基不含有水解物，成分稳定。克隆细胞生长汇合度方面接近和高于两个商业化培养基。
HKE 293F细胞上，由于其生长特性，不做汇合度比较，克隆率上本发明克隆培养基相对于商业培养基接近和高出，其中最优实施例1的克隆率高Cellvento® 4CHO-C克隆培养基约20%，高QuaMono™ Plus CHO 克隆培养基约30%。突出本发明克隆培养基具有有明显优势。
表10
本发明提供一种HEK293和CHO细胞通用型且无血清、无蛋白、化学成分明确的通用培养基，解决市场上普遍存在的含水解物成分的克隆培养基，且满足HEK293和CHO细胞通用型克隆培养。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程方法物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程方法物品或者设备所固有的要素。
最后应说明的是：以上所述仅为本发明的优选实施例而已，并不用于限制本发明，尽管参照前述实施例对本发明进行了详细的说明，对于本领域的技术人员来说，其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内，所作的任何修改等同替换改进等，均应包含在本发明的保护范围之内。
Claims (5)
1.一种HEK293细胞和CHO细胞通用单克隆培养基，由氨基酸、维生素、微量元素、无机盐、核苷类和其他成分组成，其特征在于，
所述氨基酸组分终浓度分别为：L-盐酸精氨酸100mg/L、L-天冬酰胺150mg/L、L-丙氨酸10mg/L、L-谷氨酸20mg/L、L-甘氨酸24.5mg/L、L-盐酸组氨酸53mg/L、L-异亮氨酸750mg/L、L-亮氨酸360.5mg/L、L-盐酸赖氨酸155.6mg/L、L-甲硫氨酸20mg/L、L-苯丙氨酸36.5mg/L、L-脯氨酸52.3mg/L、L-丝氨酸87.4mg/L、L-苏氨酸75mg/L、L-色氨酸10.5mg/L、L-酪氨酸钠盐82mg/L、L-缬氨酸30mg/L、L-盐酸半胱氨酸55mg/L、L-丙氨酰-L-谷氨酰胺10mg/L；
所述维生素组分终浓度分别为：氯化胆碱14.8mg/L、叶酸5mg/L、肌醇25mg/L、烟酰胺1mg/L、泛酸钙3mg/L、核黄素0.5mg/L、生物素0.1mg/L、维生素B12 42mg/L、对氨基苯甲酸0.1mg/L、盐酸腐胺2mg/L、硫辛酸0.1mg/L、L-抗坏血酸200mg/L；
所述微量元素组分终浓度分别为：一水合硫酸锰0.000023mg/L、八水合二氯氧化锆0.000095mg/L、六水合三氯化铬0.000045mg/L、六水合氯化钴0.00065mg/L、氯化亚锡0.00000609mg/L、四水钼酸铵0.0001mg/L、氯化镉0.0000018mg/L、氯化铜二水合物0.00102mg/L、氯化铝0.00059mg/L、硝酸银0.0000001mg/L、硫酸镍0.00059mg/L、醋酸钡0.00000014mg/L、亚硒酸钠0.01mg/L；
所述无机盐组分终浓度分别为：氯化钠5490mg/L、氯化钾169mg/L、硫酸镁50mg/L、氯化锌0.1mg/L、磷酸二氢钠540mg/L、氯化钙100mg/L、碳酸氢钠2000mg/L、柠檬酸铁0.5mg/L；
所述核苷类组分终浓度分别为：腺苷0.86mg/L、胞苷0.86mg/L、鸟苷0.86mg/L、胸苷0.86mg/L、尿苷0.86mg/L、2'-脱氧鸟苷水合物0.86mg/L、次黄嘌呤5.5mg/L；
所述其他成分组分终浓度分别为：亚油酸钠0.1mg/L、葡萄糖2000mg/L、丙酮酸钠200mg/L、盐酸乙醇胺15mg/L。
2.根据权利要求1所述的一种HEK293细胞和CHO细胞通用单克隆培养基，其特征在于，所述的HEK293细胞为HEK293F细胞。
3.根据权利要求1所述的一种HEK293细胞和CHO细胞通用单克隆培养基，其特征在于，所述的CHO细胞为CHO-K1细胞。
4.根据权利要求1所述的一种HEK293细胞和CHO细胞通用单克隆培养基的应用，其特征在于，用于HEK293细胞或CHO细胞的培养。
5.根据权利要求4所述的一种HEK293细胞和CHO细胞通用单克隆培养基的应用，其特征在于，用于HEK293细胞或CHO细胞的单克隆培养。
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