CN107988138A - 一种细胞培养用血清替代物 - Google Patents
一种细胞培养用血清替代物 Download PDFInfo
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Abstract
本发明公开了一种细胞培养用血清替代物，包括如下原料：二氯化锰、亚硒酸钠、钼酸铵、柠檬酸铁、酒石酸胆碱、维生素D2、微生素K2、维生素K3、腺嘌呤、盐酸鸟嘌呤、尿嘧啶、亚油酸、胆固醇、胰岛素、氢化可的松、地塞米松、酵母水解物、乳蛋白水解物、酪蛋白水解物、麦芽蛋白胨。本发明的血清替代物，添加到常用的基础培养基里就能培养细胞，可以完全代理血清使用；不含有IgG，大大简化了单抗生产抗体纯化工艺；细胞培养明显延长而细胞不凋零；有利于蛋白和抗体的生产；可成本大幅度降低，可以降低生产成本。
Description
技术领域
本发明涉及一种培养基用血清替代物，具体涉及一种细胞培养用血清替代物。
背景技术
细胞培养经历了几十年的发展，现广泛应用于生物、医疗技术、药品等领域，形成了庞大的产业。一般动物细胞培养常常都要添加牛血清，往往的在用动物细胞培养进行蛋白、抗体以及疫苗生产不采用血清有很多优势：①可以避免牛血清对细胞的抑制和毒性作用，提高细胞密度和蛋白等产品的表达水平；②降低产品的批次间质量差异，提高了实验的重复性和准确性；③无血清培养基成分明确，避免了组分对实验研究的影响，有利于进行细胞代谢的基础研究和特定组分对细胞的作用；④无血清培养基完全采用人工合成的化合物，因此成分确定，可以排除血清中未知成分的干扰和血源性污染，实验结果更为可靠。简化了细胞产品的纯化工作，便于原材料的分析，提高了制品的质量与安全。但无血清培养基的缺点:①细胞在无血清培养基中缺乏牛血清白蛋白的保护作用，容易受到机械和化学因素的损伤，一些重组的组分不稳定，不易保存；②商业化的无血清培养基配方设计因细胞特性的不同而异，因此针对性强，通用性差；③无血清培养基成本相对高，细胞在无血清培养基中需要适应过程，给实验室研究造成了经济负担。
发明内容
本发明所要解决的技术问题是现有技术培养基中含有血清与不含有血清都会对细胞的培养生长造成一定的影响，目的在于提供一种细胞培养用血清替代物，添加到常用的基础培养基里就能培养细胞，可以完全代理血清使用；不含有IgG，大大简化了单抗生产抗体纯化工艺；细胞培养明显延长而细胞不凋零；有利于蛋白和抗体的生产；可成本大幅度降低，可以降低生产成本。
本发明通过下述技术方案实现：
一种细胞培养用血清替代物，包括如下质量浓度的原料：二氯化锰1-100mg/L、亚硒酸钠1-100mg/L、钼酸铵0.001-100mg/L、柠檬酸铁1-100mg/L、酒石酸胆碱1-10mg/L、维生素 D2 0.1-10mg/L、微生素K2 0.1-10mg/L、维生素K3 0.1-10mg/L、腺嘌呤1-10mg/L、盐酸鸟嘌呤1-10mg/L、尿嘧啶1-10mg/L、亚油酸1-10mg/L、胆固醇1-10mg/L、胰岛素0.00001-1mg/L、氢化可的松0.00001-1mg/L、地塞米松0.01-1mg/L、酵母水解物10-1000mg/L、乳蛋白水解物 10-1000mg/L、酪蛋白水解物10-1000mg/L、麦芽蛋白胨10-1000mg/L。
一种细胞培养用血清替代物，包括如下质量浓度的原料：二氯化锰30-70mg/L、亚硒酸钠30-70mg/L、钼酸铵30-70mg/L、柠檬酸铁30-70mg/L、酒石酸胆碱1-6mg/L、维生素D24-10mg/L、微生素K2 0.5-5mg/L、维生素K3 1-7mg/L、腺嘌呤2-6mg/L、盐酸鸟嘌呤4-8mg/L、尿嘧啶3-7mg/L、亚油酸4-6mg//L、胆固醇2-9mg/L、胰岛素0.00001-1mg/L、氢化可的松0.00001-1mg/L、地塞米松0.01-1mg/L、酵母水解物300-700mg/L、乳蛋白水解物300-700mg/L、酪蛋白水解物300-700mg/L、麦芽蛋白胨300-700mg/L。
一种细胞培养用血清替代物，包括如下质量浓度的原料：二氯化锰50mg/L、亚硒酸钠 40mg/L、钼酸铵45mg/L、柠檬酸铁50mg/L、酒石酸胆碱3mg/L、维生素D2 6mg/L、微生素K2 3mg/L、维生素K3 4mg/L、腺嘌呤3mg/L、盐酸鸟嘌呤5mg/L、尿嘧啶6mg/L、亚油酸5mg//L、胆固醇4mg/L、胰岛素0.005mg/L、氢化可的松0.005mg/L、地塞米松0.2mg/L、酵母水解物500mg/L、乳蛋白水解物400mg/L、酪蛋白水解物600mg/L、麦芽蛋白胨500mg/L。
一种细胞培养用血清替代物，包括如下质量浓度的原料：二氯化锰20mg/L、亚硒酸钠 30mg/L、钼酸铵40mg/L、柠檬酸铁20mg/L、酒石酸胆碱2mg/L、维生素D2 0.3mg/L、微生素K2 0.8mg/L、维生素K32mg/L、腺嘌呤8mg/L、盐酸鸟嘌呤2mg/L、尿嘧啶3mg/L、亚油酸2mg/L、胆固醇4mg/L、胰岛素0.2mg/L、氢化可的松0.00001mg/L、地塞米松0.01mg/L、酵母水解物10mg/L、乳蛋白水解物30mg/L、酪蛋白水解物30mg/L、麦芽蛋白胨20mg/L。
一种细胞培养用血清替代物，包括如下质量浓度的原料：二氯化锰100mg/L、亚硒酸钠 90mg/L、钼酸铵80mg/L、柠檬酸铁100mg/L、酒石酸胆碱5mg/L、维生素D2 8mg/L、微生素K2 9mg/L、维生素K3 5mg/L、腺嘌呤4mg/L、盐酸鸟嘌呤8mg/L、尿嘧啶6mg/L、亚油酸 8mg/L、胆固醇7mg/L、胰岛素0.8mg/L、氢化可的松0.001mg/L、地塞米松0.6mg/L、酵母水解物600mg/L、乳蛋白水解物700mg/L、酪蛋白水解物850mg/L、麦芽蛋白胨300mg/L。
本发明与现有技术相比，具有如下的优点和有益效果：
本发明一种细胞培养用血清替代物，添加到常用的基础培养基里就能培养细胞，可以完全代理血清使用；不含有IgG，大大简化了单抗生产抗体纯化工艺；细胞培养明显延长而细胞不凋零；有利于蛋白和抗体的生产；可成本大幅度降低，可以降低生产成本。
附图说明
此处所说明的附图用来提供对本发明实施例的进一步理解，构成本申请的一部分，并不构成对本发明实施例的限定。在附图中：
图1为分别采用添加本发明的培养基与添加血清的培养基培养杂交瘤细胞(PHSA)的生长曲线对比图；
图2为分别采用添加本发明的培养基与添加血清的培养基培养中国仓鼠卵巢细胞(CHO) 的生长曲线对比图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，下面结合实施例，对本发明作进一步的详细说明，本发明的示意性实施方式及其说明仅用于解释本发明，并不作为对本发明的限定。
实施例1
一种细胞培养用血清替代物，包括如下质量浓度的原料：二氯化锰50mg/L、亚硒酸钠 40mg/L、钼酸铵45mg/L、柠檬酸铁50mg/L、酒石酸胆碱3mg/L、维生素D2 6mg/L、微生素K2 3mg/L、维生素K3 4mg/L、腺嘌呤3mg/L、盐酸鸟嘌呤5mg/L、尿嘧啶6mg/L、亚油酸5mg//L、胆固醇4mg/L、胰岛素0.005mg/L、氢化可的松0.005mg/L、地塞米松0.2mg/L、酵母水解物500mg/L、乳蛋白水解物400mg/L、酪蛋白水解物600mg/L、麦芽蛋白胨500mg/L。
实施例2
一种细胞培养用血清替代物，包括如下质量浓度的原料：二氯化锰20mg/L、亚硒酸钠 30mg/L、钼酸铵40mg/L、柠檬酸铁20mg/L、酒石酸胆碱2mg/L、维生素D2 0.3mg/L、微生素K2 0.8mg/L、维生素K32mg/L、腺嘌呤8mg/L、盐酸鸟嘌呤2mg/L、尿嘧啶3mg/L、亚油酸2mg/L、胆固醇4mg/L、胰岛素0.2mg/L、氢化可的松0.00001mg/L、地塞米松0.01mg/L、酵母水解物10mg/L、乳蛋白水解物30mg/L、酪蛋白水解物30mg/L、麦芽蛋白胨20mg/L。
实施例3
一种细胞培养用血清替代物，包括如下质量浓度的原料：二氯化锰100mg/L、亚硒酸钠 90mg/L、钼酸铵80mg/L、柠檬酸铁100mg/L、酒石酸胆碱5mg/L、维生素D2 8mg/L、微生素K2 9mg/L、维生素K3 5mg/L、腺嘌呤4mg/L、盐酸鸟嘌呤8mg/L、尿嘧啶6mg/L、亚油酸 8mg/L、胆固醇7mg/L、胰岛素0.8mg/L、氢化可的松0.001mg/L、地塞米松0.6mg/L、酵母水解物600mg/L、乳蛋白水解物700mg/L、酪蛋白水解物850mg/L、麦芽蛋白胨300mg/L。
实施例4
实验组：采用1640基础培养基，添加本发明实施例1的细胞培养用血清替代物10％；
对照组：采用1640基础培养基，添加10％血清。
用上述实验组与对比组的培养基分别杂交瘤细胞和CHO细胞。
如图1和图2中，小的菱形方块代表实验组，大的正方形块代表对照组。由图1和图2可以看出，杂交瘤细胞和CHO细胞在实验组的培养基中的生长数量与增殖速率明显高于对照组，因此本发明的血清替代物，不仅能够很好地促进细胞的生长增殖，同时能够避免加入血清对细胞造成的影响，提高细胞培养的质量。
以上所述的具体实施方式，对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施方式而已，并不用于限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (5)
1.一种细胞培养用血清替代物，其特征在于，包括如下质量浓度的原料：二氯化锰1-100mg/L、亚硒酸钠1-100mg/L、钼酸铵0.001-100mg/L、柠檬酸铁1-100mg/L、酒石酸胆碱1-10mg/L、维生素D2 0.1-10mg/L、微生素K2 0.1-10mg/L、维生素K3 0.1-10mg/L、腺嘌呤1-10mg/L、盐酸鸟嘌呤1-10mg/L、尿嘧啶1-10mg/L、亚油酸1-10mg/L、胆固醇1-10mg/L、胰岛素0.00001-1mg/L、氢化可的松0.00001-1mg/L、地塞米松0.01-1mg/L、酵母水解物10-1000mg/L、乳蛋白水解物10-1000mg/L、酪蛋白水解物10-1000mg/L、麦芽蛋白胨10-1000mg/L。
2.根据权利要求1所述的一种细胞培养用血清替代物，其特征在于，包括如下质量浓度的原料：二氯化锰30-70mg/L、亚硒酸钠30-70mg/L、钼酸铵30-70mg/L、柠檬酸铁30-70mg/L、酒石酸胆碱1-6mg/L、维生素D2 4-10mg/L、微生素K2 0.5-5mg/L、维生素K3 1-7mg/L、腺嘌呤2-6mg/L、盐酸鸟嘌呤4-8mg/L、尿嘧啶3-7mg/L、亚油酸4-6mg//L、胆固醇2-9mg/L、胰岛素0.00001-1mg/L、氢化可的松0.00001-1mg/L、地塞米松0.01-1mg/L、酵母水解物300-700mg/L、乳蛋白水解物300-700mg/L、酪蛋白水解物300-700mg/L、麦芽蛋白胨300-700mg/L。
3.根据权利要求1所述的一种细胞培养用血清替代物，其特征在于，包括如下质量浓度的原料：二氯化锰50mg/L、亚硒酸钠40mg/L、钼酸铵45mg/L、柠檬酸铁50mg/L、酒石酸胆碱3mg/L、维生素D2 6mg/L、微生素K2 3mg/L、维生素K3 4mg/L、腺嘌呤3mg/L、盐酸鸟嘌呤5mg/L、尿嘧啶6mg/L、亚油酸5mg//L、胆固醇4mg/L、胰岛素0.005mg/L、氢化可的松0.005mg/L、地塞米松0.2mg/L、酵母水解物500mg/L、乳蛋白水解物400mg/L、酪蛋白水解物600mg/L、麦芽蛋白胨500mg/L。
4.根据权利要求1所述的一种细胞培养用血清替代物，其特征在于，包括如下质量浓度的原料：二氯化锰20mg/L、亚硒酸钠30mg/L、钼酸铵40mg/L、柠檬酸铁20mg/L、酒石酸胆碱2mg/L、维生素D2 0.3mg/L、微生素K2 0.8mg/L、维生素K3 2mg/L、腺嘌呤8mg/L、盐酸鸟嘌呤2mg/L、尿嘧啶3mg/L、亚油酸2mg/L、胆固醇4mg/L、胰岛素0.2mg/L、氢化可的松0.00001mg/L、地塞米松0.01mg/L、酵母水解物10mg/L、乳蛋白水解物30mg/L、酪蛋白水解物30mg/L、麦芽蛋白胨20mg/L。
5.根据权利要求1所述的一种细胞培养用血清替代物，其特征在于，包括如下质量浓度的原料：二氯化锰100mg/L、亚硒酸钠90mg/L、钼酸铵80mg/L、柠檬酸铁100mg/L、酒石酸胆碱5mg/L、维生素D2 8mg/L、微生素K2 9mg/L、维生素K3 5mg/L、腺嘌呤4mg/L、盐酸鸟嘌呤8mg/L、尿嘧啶6mg/L、亚油酸8mg/L、胆固醇7mg/L、胰岛素0.8mg/L、氢化可的松0.001mg/L、地塞米松0.6mg/L、酵母水解物600mg/L、乳蛋白水解物700mg/L、酪蛋白水解物850mg/L、麦芽蛋白胨300mg/L。
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