CN112226416A - 一种杂交瘤细胞培养用培养基添加剂 - Google Patents
一种杂交瘤细胞培养用培养基添加剂 Download PDFInfo
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- CN112226416A CN112226416A CN202011157822.2A CN202011157822A CN112226416A CN 112226416 A CN112226416 A CN 112226416A CN 202011157822 A CN202011157822 A CN 202011157822A CN 112226416 A CN112226416 A CN 112226416A
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- culture medium
- hybridoma
- salt
- hybridoma cell
- insulin
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Abstract
本发明提出了一种杂交瘤细胞培养用培养基添加剂，包括以下组分，转铁蛋白、胰岛素、丙酮酸钠、纤粘连蛋白、谷氨酰胺、油酸、亚油酸、谷胱甘肽、钙盐、镁盐、锌盐、和硒盐，其中胰岛素为重组人源或鼠源胰岛素；本发明在细胞增殖处于低密度时能够有效的维持杂交瘤细胞生长活性，避免其由于密度过低死亡，从而通过提高细胞低密度时的存活率以提高细胞融合效率，获得更高杂交瘤细胞克隆成功率，同时在杂交瘤细胞正常培养生长过程中，提升其生长速度，减少杂交瘤细胞生长时间以缩短实验周期。
Description
技术领域
本发明涉及杂交瘤细胞技术领域，尤其涉及一种杂交瘤细胞培养用培养基添加剂。
背景技术
目前，通过小鼠脾细胞与骨髓瘤细胞融合获得杂交瘤细胞，再进一步筛选得到纯化的单克隆细胞，培养细胞以生产抗体，生产出的抗体即为单克隆抗体。单抗技术一直是人类免疫相关实验进步的基础，通过培养杂交瘤细胞，可以获得大量特异性、灵敏度等特性都相当均一的抗体。单抗相比于多克隆抗体具有产品批次稳定性好、产品特性稳定、后期投入低、周期短、可以快速获得大量抗体、适合大规模生产等特性，因此一直以来是抗体制备技术中不可或缺的一部分。
单抗技术特性决定了杂交瘤细胞活性以及脾细胞与骨髓瘤细胞的融合是能否高效获得抗体的关键所在，自单抗技术开发以来，有诸多技术致力于提高杂交瘤细胞活性，如合适的细胞比例、添加合适的饲养层、优化的融合剂配方等方法，均对提高杂交瘤细胞活性出了重要贡献。但实际操作过程中，依然会出现融合效率低下、细胞活性较差、生长速度缓慢、无法高密度培养、培养过程尤其是高密度培养时中容易大批量死亡等现象。
发明内容
有鉴于此，本发明提出了一种能够有效的维持细胞活性，提高细胞融合效率的杂交瘤细胞培养用培养基添加剂。
本发明的技术方案是这样实现的：本发明提供了一种杂交瘤细胞培养用培养基添加剂，包括以下组分及其配置浓度为，转铁蛋白10-30mg/L，胰岛素20-50mg/L，谷氨酰胺60-70g/L，油酸20-30mg/L，亚油酸5-15mg/L，谷胱甘肽100-200mg/L，钙盐100-150mg/L，镁盐5-15mg/L，锌盐20-30mg/L和硒盐5-15mg/L。
在以上技术方案的基础上，优选的，还包括以下组分及其配置浓度为，丙酮酸钠10-20g/L。
在以上技术方案的基础上，优选的，还包括以下组分及其配置浓度为，纤粘连蛋白100-1000mg/L。
在以上技术方案的基础上，优选的，胰岛素为重组人源或鼠源胰岛素。
在以上技术方案的基础上，优选的，谷氨酰胺为L-谷氨酰胺或其衍生物。
在以上技术方案的基础上，优选的，谷胱甘肽为还原性或氧化型谷胱甘肽。
在以上技术方案的基础上，优选的，钙盐为氯化钙或碳酸氢钙，镁盐为氯化镁或硫酸镁，锌盐为氯化锌或者硫酸锌，硒盐为亚硒酸钾或亚硒酸钠或二者混合液。
在以上技术方案的基础上，优选的，其应用于血清培养基或无血清培养基。
在以上技术方案的基础上，优选的，添加剂溶液配置完成后按1:100比例加入培养基中即可。
本发明的一种杂交瘤细胞培养用培养基添加剂相对于现有技术具有以下有益效果：
(1)本发明在细胞增殖处于低密度时能够有效的维持杂交瘤细胞生长活性，避免其由于密度过低死亡，从而通过提高细胞低密度时的存活率以提高细胞融合效率，获得更高杂交瘤细胞克隆成功率，同时在杂交瘤细胞正常培养生长过程中，提升其生长速度，减少杂交瘤细胞生长时间以缩短实验周期。
(2)本发明在细胞增殖至高密度时仍能有效的维持杂交瘤细胞生长活性，避免其由于培养基营养不足或未及时换液而较早凋亡，从而延长培养基使用时间节省培养基同时保持良好的细胞活性，获得更高密度的细胞培养效果。
(3)本发明应用于含血清培养基内时，能够取得和采用无血清培养基时一样的良好效果，扩大了本发明的适用范围，提高了杂交瘤细胞的培养效率。
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1：
本发明的一种杂交瘤细胞培养用培养基添加剂，包括以下组分及其配置浓度为，转铁蛋白10mg/L，胰岛素20mg/L，丙酮酸钠10g/L，纤粘连蛋白100mg/L，谷氨酰胺60g/L，二氨基苯甲酸10g/L，油酸20mg/L，亚油酸5mg/L，乙醇胺10mg/L，腐胺10mg/L，谷胱甘肽100mg/L，氨基乙醇100ul/L，钙盐100mg/L，镁盐5mg/L，锌盐20mg/L和硒盐5mg/L。
其中，胰岛素为重组人源或鼠源胰岛素；谷氨酰胺为L-谷氨酰胺或其衍生物；二氨基苯甲酸为邻氨基苯甲酸、间氨基苯甲酸或对氨基苯甲酸；腐胺为丁二胺或丁二胺盐酸盐；谷胱甘肽为还原性或氧化型谷胱甘肽；钙盐为氯化钙或碳酸氢钙，镁盐为氯化镁或硫酸镁，锌盐为氯化锌或者硫酸锌，硒盐为亚硒酸钾或亚硒酸钠或二者混合液。
第二方面，其用途为应用于血清培养基或无血清培养基。
第三方面，其使用方法为添加剂溶液配置完成后按1:100比例加入培养基中即可。
实施例2：
本发明的一种杂交瘤细胞培养用培养基添加剂，包括以下组分及其配置浓度为，转铁蛋白20mg/L，胰岛素35mg/L，丙酮酸钠15g/L，纤粘连蛋白500mg/L，谷氨酰胺65g/L，二氨基苯甲酸15g/L，油酸25mg/L，亚油酸10mg/L，乙醇胺15mg/L，腐胺15mg/L，谷胱甘肽150mg/L，氨基乙醇150ul/L，钙盐125mg/L，镁盐10mg/L，锌盐25mg/L和硒盐10mg/L。实施例1中的其他条件保持不变。
实施例3：
本发明的一种杂交瘤细胞培养用培养基添加剂，包括以下组分及其配置浓度为，转铁蛋白30mg/L，胰岛素50mg/L，丙酮酸钠20g/L，纤粘连蛋白1000mg/L，谷氨酰胺70g/L，二氨基苯甲酸20g/L，油酸30mg/L，亚油15mg/L，乙醇胺20mg/L，腐胺20mg/L，谷胱甘肽200mg/L，氨基乙醇200ul/L，钙盐150mg/L，镁盐15mg/L，锌盐30mg/L和硒盐15mg/L。实施例1中的其他条件保持不变。
本发明中的胰岛素能够促进葡萄糖进入细胞，调节细胞对葡萄糖、氨基酸和脂类的摄取、利用和储存，同时抑制糖原、蛋白质和脂肪的分解。需要注意的是，胰岛素对细胞的促生长作用主要是通过其与细胞膜上胰岛素样生长因子(Insulin-like growth factors，IGFs)受体相互作用而介导的，而对细胞的代谢调节作用主要是通过其与细胞膜上的胰岛素受体(Insulin receptor，IR)相互作用而介导的。此外，胰岛素也可通过IGFs与IR的介导，发挥抗凋亡作用。
本发明中的转铁蛋白能够通过与转铁蛋白受体(Transferrin Receptor，TfR)相互作用，从而与三价铁离子可逆性结合，调节铁离子转运及代谢，维持细胞内的铁平衡，其主要的意义在于维持细胞的生长和增殖，即类似于生长因子的功能。同时，转铁蛋白也是一种重要的胞外抗氧化剂，可以避免细胞外环境中自由基的产生，防止细胞受到伤害。
胰岛素-转铁蛋白体系能够大幅提高细胞融合生长效率和新陈代谢速率，相较于单一组分的使用效果，二者配合使用时使用时效果更佳。其中，胰岛素采用重组人源或鼠源胰岛素，有助于针对性的作用于小鼠脾细胞与骨髓瘤细胞，避免细胞融合产生杂质抗原，从而提高了单抗的纯度，同时也避免了血清组分对杂交瘤细胞活性产生的不良影响。
另外，丙酮酸钠是细胞代谢所需能量物质，丙酮酸在细胞三羧酸循环中处于重要地位，添加适量有助于提高细胞代谢效率从而提高细胞活性；由于谷氨酰胺在培养基保存过程中会逐步降解，添加适量的丙酮酸钠有助于培养基营养环境稳定。纤粘连蛋白可以促进细胞贴壁，使细胞不易漂浮同时维持较好的生长状态。钙、镁、锌、硒均为细胞分裂增殖所需微量元素，添加适量微量元素可以提高细胞增殖效率从而提高细胞克隆成功率。谷胱甘肽添加可以有效的维持细胞培养基中的氧化还原电势，避免活性氧自由基等影响细胞状态的物质对细胞生长造成影响。其他物质均为细胞构成的成分，如油酸及亚油酸可以构成细胞膜。
综上所述，本发明的配方一方面促进细胞分裂增殖，另一方面维持了正常的细胞培养环境，还为细胞提供了足够多能量及所需组分，全方位为细胞快速增殖和分泌创造最优环境，具有优异的促进细胞增殖和分泌能力的作用，同时也能保证细胞高密度生长时营养充足。
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 (9)
1.一种杂交瘤细胞培养用培养基添加剂，其特征在于：包括以下组分及其配置浓度为，转铁蛋白10-30mg/L，胰岛素20-50mg/L，谷氨酰胺60-70g/L，油酸20-30mg/L，亚油酸5-15mg/L，谷胱甘肽100-200mg/L，钙盐100-150mg/L，镁盐5-15mg/L，锌盐20-30mg/L和硒盐5-15mg/L。
2.根据权利要求1所述的一种杂交瘤细胞培养用培养基添加剂，其特征在于：还包括以下组分及其配置浓度为，丙酮酸钠10-20g/L。
3.根据权利要求1所述的一种杂交瘤细胞培养用培养基添加剂，其特征在于：所述胰岛素为重组人源或鼠源胰岛素。
4.根据权利要求1所述的一种杂交瘤细胞培养用培养基添加剂，其特征在于：所述谷氨酰胺为L-谷氨酰胺或其衍生物。
5.根据权利要求1所述的一种杂交瘤细胞培养用培养基添加剂，其特征在于：还包括以下组分及其配置浓度为，纤粘连蛋白100-1000mg/L。
6.根据权利要求1所述的一种杂交瘤细胞培养用培养基添加剂，其特征在于：所述谷胱甘肽为还原性或氧化型谷胱甘肽。
7.根据权利要求1所述的一种杂交瘤细胞培养用培养基添加剂，其特征在于：所述钙盐为氯化钙或碳酸氢钙，所述镁盐为氯化镁或硫酸镁，所述锌盐为氯化锌或者硫酸锌，所述硒盐为亚硒酸钾或亚硒酸钠或二者混合液。
8.根据权利要求1所述的一种杂交瘤细胞培养用培养基添加剂，其特征在于：其应用于血清培养基或无血清培养基。
9.根据权利要求1所述的一种杂交瘤细胞培养用培养基添加剂，其特征在于：所述添加剂溶液配置完成后按1:100比例加入培养基中即可。
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