CN113528429A - 一种适用于hek293细胞的无血清培养基 - Google Patents
一种适用于hek293细胞的无血清培养基 Download PDFInfo
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Abstract
本发明提出了一种适用于HEK293细胞的无血清培养基，包括氨基酸、无机盐、维生素、核苷、添加剂、抗结团剂和稳定剂；每1L培养基含氨基酸275‑1650mg，无机盐5470.2‑37103mg，维生素10.92‑124.4mg，核苷40‑800mg，添加剂1060‑3250mg，抗结团剂100‑1000mg，稳定剂100‑1000mg，余量为水；所述抗结团剂为F68，稳定剂为glutaGRO。本发明培养基无动物源，不含蛋白质或生长因子，支持多种HEK293及其衍生型的细胞，具有抗结团、提高细胞稳定性和延长保质期的作用，并且能在单位时间获得更高的蛋白产量。
Description
技术领域
本发明涉及培养基技术领域，尤其涉及一种适用于HEK293细胞的无血清培养基。
背景技术
从体外生化研究到制药和临床应用等各个领域都需要用到大量高质量的重组蛋白。生产具有复杂翻译后修饰的重组蛋白的技术代表了结构和功能研究的主要挑战。
可用于瞬时转染的哺乳动物细胞系的快速建立和高回收率解决了这一障碍。在最常用的哺乳动物细胞系中，人胚胎肾293(HEK293)细胞因其生长速度快、相对易于操作和高转染效率等优点，而成为研究和规模生产的标准选择。
细胞培养基是决定细胞体外生长代谢最重要、最直接的环境因素。目前大多数合成细胞培养基均需要补充动物血清才能支持细胞的体外生长、增殖，而动物血清的使用带来了动物性病原微生物污染培养物的可能，因此推动了动物细胞无血清培养基的发展。
无血清培养基一般是由基础培养基和替代血清的补充因子组成的。无血清培养技术是细胞培养研究中的一个里程碑。由于无血清培养基全部是由已知成分组成的，不仅为研究和阐明细胞生长、增殖和分化的机制这一生命科学中的根本问题提供了有力的工具，而且为现代生物技术如基因工程、蛋白质工程、细胞工程和单克隆抗体等的应用奠定了基础。
为了使HEK293细胞能够大规模应用于瞬时转染，常将其驯化为悬浮生长；然而在悬浮培养过程中HEK293细胞极易结团，细胞团直接影响细胞活性，导致HEK293细胞无法高密度扩增、连续传代。现有抗结团HEK293细胞无血清培养基中的谷氨酰胺容易降解，并且影响细胞生长，因此急需研发既抗结团，又不影响细胞生长的HEK293细胞无血清培养基。
发明内容
有鉴于此，本发明提出了一种化学成分确定，无动物源，不含蛋白质或生长因子，支持多种HEK293及其衍生型细胞的无血清培养基。
本发明的技术方案是这样实现的：本发明提供了一种适用于HEK293细胞的无血清培养基，包括氨基酸、无机盐、维生素、抗结团剂和稳定剂；每1L培养基含氨基酸275-1650mg，无机盐5500.2-37103mg，维生素10.92-125mg，核苷40-800mg，添加剂1060-3250mg，抗结团剂100-1000mg，稳定剂100-1000mg，余量为水。
在以上技术方案的基础上，优选的，所述抗结团剂为F68，稳定剂为glutaGRO。
在以上技术方案的基础上，优选的，所述氨基酸组分及含量为：甘氨酸10-100mg/L、L-丙氨酸5-50mg/L、L-精氨酸50-200mg/L、L-天冬酰胺10-100mg/L、L-天冬氨酸5-50mg/L、L-盐酸半胱氨酸-H2O 50-200mg/L、L-盐酸胱氨酸10-50mg/L、L-谷氨酸10-100mg/L、L-组氨酸10-50mg/L、L-异亮氨酸10-100mg/L、L-亮氨酸10-100mg/L、L-赖氨酸50-100mg/L、L-蛋氨酸5-50mg/L、L-苯丙氨酸5-50mg/L、L-脯氨酸5-50mg/L、L-丝氨酸5-50mg/L、L-苏氨酸5-50mg/L、L-色氨酸5-50mg/L、L-酪氨酸二钠盐10-100mg/L和L-缬氨酸5-50mg/L。
在以上技术方案的基础上，优选的，所述无机盐组分及含量为：无水氯化钙100-500mg/L、无水硫酸镁50-300mg/L、氯化钾100-500mg/L、碳酸氢钠100-5000mg/L、氯化钠5000-30000mg/L、硫酸镁-7H2O 100-500mg/L、硝酸铁-9H2O 0.1-2mg/L、磷酸二氢钠-H2O50-300mg/L和硫酸锌-7H2O 0.1-1mg/L。
在以上技术方案的基础上，优选的，所述维生素组分及含量为：抗坏血酸10-100mg/L、氯化胆碱0.1-2mg/L、D-泛酸钙0.1-2mg/L、叶酸0.1-2mg/L、烟酰胺0.1-2mg/L、核黄素0.01-0.5mg/L、盐酸硫胺B10.1-2mg/L、维生素B120.1-2mg/L、生物素0.01-0.5mg/L、盐酸吡哆辛0.1-2mg/L、肌醇0.1-5mg/L和硫辛酸0.1-5mg/L。
在以上技术方案的基础上，优选的，所述添加剂组分及含量为：葡萄糖1000-3000mg/L、肝素钠10-50mg/L和丙酮酸钠50-200mg/L。
在以上技术方案的基础上，优选的，所述核苷组分及含量为：腺苷5-100mg/L、胞苷5-100mg/L、鸟苷5-100mg/L、尿苷5-100mg/L、β-脱氧腺苷5-100mg/L、β-脱氧胞苷盐酸盐5-100mg/L、β-脱氧鸟苷5-100mg/L和β-胸苷5-100mg/L。
在以上技术方案的基础上，优选的，所述培养基的pH值为7-7.5。
更进一步优选的，一种适用于HEK293细胞的无血清培养基在多种HEK293细胞及其衍生型细胞中的应用。
本发明的一种适用于HEK293细胞的无血清培养基相对于现有技术具有以下有益效果：
1、本发明的HEK293细胞无血清培养基可明显减少细胞结团现象。
2、本发明的HEK293细胞无血清培养基以glutaGRO替代谷氨酰胺，可提高细胞稳定性并最大限度延长保质期，可以在2-8℃条件下保存6个月。
3、本发明的HEK293细胞无血清培养基可以在单位时间获得更高的蛋白产量。
4、本发明的培养基化学成分确定，无动物源，不含蛋白质或生长因子，支持多种HEK293及其衍生型的细胞，用于快速增殖和高密度培养，支持高效的重组蛋白质表达，表达提高到g/L水平，支持慢病毒和腺病毒的生产，病毒滴度可达1E7TU/ml，易于放大培养规模，适用于平板、摇瓶、TPP管、Wave及其他反应器。
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为本发明的培养基抗结团效果图；
图2为重组蛋白在培养基中的表达量对比图；
图3为293F细胞在培养基中的生长曲线图。
具体实施方式
下面将结合本发明实施方式，对本发明实施方式中的技术方案进行清楚、完整地描述，显然，所描述的实施方式仅仅是本发明一部分实施方式，而不是全部的实施方式。基于本发明中的实施方式，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方式，都属于本发明保护的范围。
实施例一
一种适用于HEK293细胞的无血清培养基，包括氨基酸、无机盐、维生素、抗结团剂和稳定剂。每1L培养基含氨基酸720mg，无机盐29602.6mg，维生素33.93mg，添加剂2615mg，核苷183mg，F68600mg，glutaGRO 200mg，余量为水；具体成分及含量见表1。
将上述各组分依次溶于无菌去离子水中，并用HCl和NaOH调节培养基的pH值为7。
实施例二
一种适用于HEK293细胞的无血清培养基，包括氨基酸、无机盐、维生素、抗结团剂和稳定剂。
每1L培养基含氨基酸1035mg，无机盐13310.9mg，维生素62.5mg，添加剂1060mg，核苷587mg，F68300mg，glutaGRO 400mg，余量为水；具体成分及含量见表1。
将上述各组分依次溶于无菌去离子水中，并用HCl和NaOH调节培养基的pH值为7.5。
实施例三
一种适用于HEK293细胞的无血清培养基，包括氨基酸、无机盐、维生素、抗结团剂和稳定剂。
每1L培养基含氨基酸1235mg，无机盐18751.5mg，维生素77.37mg，添加剂3250mg，核苷40mg，F68800mg，glutaGRO 900mg，余量为水；具体成分及含量见表1。
将上述各组分依次溶于无菌去离子水中，并用HCl和NaOH调节培养基的pH值为7.1。
实施例四
一种适用于HEK293细胞的无血清培养基，包括氨基酸、无机盐、维生素、抗结团剂和稳定剂。
每1L培养基含氨基酸275mg，无机盐5500.2mg，维生素10.92mg，添加剂2150mg，核苷800mg，F68100mg，glutaGRO 100mg，余量为水；具体成分及含量见表1。
将上述各组分依次溶于无菌去离子水中，并用HCl和NaOH调节培养基的pH值为7.2。
实施例五
一种适用于HEK293细胞的无血清培养基，包括氨基酸、无机盐、维生素、抗结团剂和稳定剂。
每1L培养基含氨基酸1650mg，无机盐37103mg，维生素125mg，添加剂1690mg，核苷350mg，F681000mg，glutaGRO 1000mg，余量为水；具体成分及含量见表1。
将上述各组分依次溶于无菌去离子水中，并用HCl和NaOH调节培养基的pH值为7.3。
对比例
以市场上常用的不含稳定剂glutaGRO的EX-CELL 293无血清培养基为对比例。
表1培养基组分含量
备注：上述为配备1L培养基所含的组分及含量，单位mg/L，余量为无菌去离子水。
实验一采用实施例1-5培养基和对比例EX-CELL 293无血清培养基，将已经驯化好的HEK293F细胞在50ml培养皿中连续培养6天，观察细胞生长并进行活细胞计数，结果见图1。
图1-A为EX-CELL 293无血清培养基培养的HEK293T细胞，可见细胞呈结团状；1-B可见HEK293F细胞呈分散、悬浮形式正常生长，没有存在结团现象。图2中可以看出，与对比例相比，本发明实施例培养基接种的HEK293F细胞，在37℃培养6天后，细胞密度达到9.6×106，对比例仅为7.5×106。表明稳定剂glutaGRO不仅不影响细胞生长，还具有促进细胞快速增长的作用。
实验二以人凝血因子Ⅷ、CD20单抗、PD-1单抗3种蛋白在实施例和对比例培养基中培养，观察3种蛋白的表达量，实验步骤如下：
S1，复苏：取同一批次冻存的HEK293细胞在培养基HEK293里复苏。将细胞在37℃培养箱中(120rpm，8％CO2)孵育准备转染并表达目的蛋白。
S2，细胞培养：当细胞活率≥95％且生长处于对数中期时，才开始进行驯化程序；直接将细胞接种到本发明的HEK293细胞无血清培养基和EX-CELL 293无血清培养基中进行培养。
S3，传代培养，每2-3d进行一次传代操作，以保持细胞处于早期对数生长期。细胞接种密度为0.3-0.6×106细胞/mL。当细胞密度达到3-4×106细胞/mL且细胞活率≥95％(2-4d)时，再次传代培养细胞。细胞密度达到约3-4×106个活细胞/mL，活率≥95％时，准备转染质粒。
S4，质粒转染：转染前一天，将细胞按3×106细胞/mL细胞密度接种，并使细胞生长过夜。在第二天(转染当天)，获取包括细胞密度和存活率的数据。进行转染的细胞活率应≥95％。使用新鲜的培养基将细胞稀释至3×106细胞/mL。按照DNA：1.5mg/L、PEI：3mg/L的转染体系准备转染质粒和PEI。用培养基稀释质粒DNA。通过旋转和/或翻转试管混匀。
S5：将含有PEI的试管轻轻翻转4至5次以充分混匀。并用培养基稀释PEI。通过旋转和/或翻转试管来混合。将稀释后的PEI加到稀释后的质粒DNA中。旋转和/或颠倒试管或用移液器轻轻吹打2-3次进行混合。将复合物在室温下孵育约20min。将复合物缓慢添加至细胞摇瓶中，在添加过程中轻轻摇动摇瓶。将摇瓶放回37℃培养箱中按日常条件培养。在转染第二天，在转染后16-22h向摇瓶中添加5％(v/v)293-ProFeed，在添加过程中轻轻晃动摇瓶。将摇瓶放回37℃培养箱。在瞬时表达过程中，维持葡萄糖浓度在4g/L以上。当细胞活率低于60％时收获细胞和表达产物，检测细胞培养液中蛋白浓度。
蛋白表达结果见图3，图3可知，与EX-CELL 293无血清培养基相比，本发明培养基可显著增加蛋白表达量，表达水平高于对比EX-CELL 293无血清培养基。
表2培养基保存条件及时间
| 实施例一 | 实施例二 | 实施例三 | 实施例四 | 实施例五 | 对比例 | |
| 2-8℃ | 183d | 190d | 185d | 193d | 180d | 90d |
表2为本发明培养基保存时长，表中可以看出本发明培养基可以在2-8℃条件下保存180-193d，为对比例的2倍，由此说明glutaGRO不仅可以促进细胞生长，增加重组蛋白表达量，还可以延长保存时长。
以上所述仅为本发明的较佳实施方式而已，并不用以限制本发明，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 (8)
1.一种适用于HEK293细胞的无血清培养基，其特征在于：包括氨基酸、无机盐、维生素、抗结团剂和稳定剂；每1L培养基含氨基酸275-1650mg，无机盐5500.2-37103mg，维生素10.92-125mg，核苷40-800mg，添加剂1060-3250mg，抗结团剂100-1000mg，稳定剂100-1000mg，余量为水；所述抗结团剂为F68，稳定剂为glutaGRO。
2.如权利要求1所述的一种适用于HEK293细胞的无血清培养基，其特征在于：所述氨基酸组分及含量为：甘氨酸10-100mg/L、L-丙氨酸5-50mg/L、L-精氨酸50-200mg/L、L-天冬酰胺10-100mg/L、L-天冬氨酸5-50mg/L、L-盐酸半胱氨酸-H2O 50-200mg/L、L-盐酸胱氨酸10-50mg/L、L-谷氨酸10-100mg/L、L-组氨酸10-50mg/L、L-异亮氨酸10-100mg/L、L-亮氨酸10-100mg/L、L-赖氨酸50-100mg/L、L-蛋氨酸5-50mg/L、L-苯丙氨酸5-50mg/L、L-脯氨酸5-50mg/L、L-丝氨酸5-50mg/L、L-苏氨酸5-50mg/L、L-色氨酸5-50mg/L、L-酪氨酸二钠盐10-100mg/L和L-缬氨酸5-50mg/L。
3.如权利要求1所述的一种适用于HEK293细胞的无血清培养基，其特征在于：所述无机盐组分及含量为：无水氯化钙100-500mg/L、无水硫酸镁50-300mg/L、氯化钾100-500mg/L、碳酸氢钠100-5000mg/L、氯化钠5000-30000mg/L、硫酸镁-7H2O 100-500mg/L、硝酸铁-9H2O0.1-2mg/L、磷酸二氢钠-H2O 50-300mg/L和硫酸锌-7H2O 0.1-1mg/L。
4.如权利要求1所述的一种适用于HEK293细胞的无血清培养基，其特征在于：所述维生素组分及含量为：抗坏血酸10-100mg/L、氯化胆碱0.1-2mg/L、D-泛酸钙0.1-2mg/L、叶酸0.1-2mg/L、烟酰胺0.1-2mg/L、核黄素0.01-0.5mg/L、盐酸硫胺B10.1-2mg/L、维生素B120.1-2mg/L、生物素0.01-0.5mg/L、盐酸吡哆辛0.1-2mg/L、肌醇0.1-5mg/L和硫辛酸0.1-5mg/L。
5.如权利要求1所述的一种适用于HEK293细胞的无血清培养基，其特征在于：所述添加剂组分及含量为：葡萄糖1000-3000mg/L、肝素钠10-50mg/L和丙酮酸钠50-200mg/L。
6.如权利要求1所述的一种适用于HEK293细胞的无血清培养基，其特征在于：所述核苷组分及含量为：腺苷5-100mg/L、胞苷5-100mg/L、鸟苷5-100mg/L、尿苷5-100mg/L、β-脱氧腺苷5-100mg/L、β-脱氧胞苷盐酸盐5-100mg/L、β-脱氧鸟苷5-100mg/L和β-胸苷5-100mg/L。
7.如权利要求1-6任一所述的一种适用于HEK293细胞的无血清培养基，其特征在于，所述培养基的pH值为7-7.5。
8.如权利要求1-6任一所述的一种适用于HEK293细胞的无血清培养基在多种HEK293细胞及其衍生型细胞中的应用。
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