CN112626013A - 一种无血清培养基及其应用 - Google Patents
一种无血清培养基及其应用 Download PDFInfo
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- CN112626013A CN112626013A CN202011536036.3A CN202011536036A CN112626013A CN 112626013 A CN112626013 A CN 112626013A CN 202011536036 A CN202011536036 A CN 202011536036A CN 112626013 A CN112626013 A CN 112626013A
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- hydrolysate
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- sericin
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Abstract
本发明公开了一种无血清培养基及其应用，属于分子生物学领域。本发明是为了减小利用血清培养细胞导致的潜在的风险。无血清培养基的配方为，所述培养基以500ml计，由以下成分组成：基础培养基DMEM、二氯化锰、亚硒酸钠、钼酸铵、柠檬酸铁、酒石酸胆碱、维生素D2L、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、丝胶酶水解物和丝胶碱水解物。无血清培养基提高细胞培养结果的一致性，有利于体外培养细胞的分化，可提高目的蛋白的表达量并使产品易于分离纯化，组分稳定，可大量生产，可以促进细胞增殖。
Description
技术领域
本发明属于分子生物学领域，具体涉及一种无血清培养基及其应用。
背景技术
细胞培养基是人工模拟细胞在体内的生长环境，是提供细胞营养和促进细胞生长增殖的物质基础。培养基主要包括天然细胞培养基、合成细胞培养基和无血清细胞培养基等。细胞培养基是细胞培养的基础，它为动物细胞的健康快速成长提供营养物质。天然细胞培养基是人们早期采用的细胞培养基，直接取自于动物组织提取液或体液，如血浆凝块、血清、淋巴液等，营养价值高，但成分复杂，差异大、不稳定，来源也受到限制；目前最常用的是合成细胞培养基，其是用化学成分明确的试剂配制的培养基，组分稳定，主要包括糖类、必需氨基酸、维生素、无机盐类等。由于天然培养基的一些营养成分不能被合成细胞培养基完全代替，因此一般需在合成细胞培养基中添加一定比例的小牛血清。
血清的主要作用大致可以概括为以下几点：提供氨基酸、维生素、无机物、脂类物质、核酸衍生物等基本营养物质；提供激素和各种生长因子；提供携带重要低分子量物质的结合蛋白，如转铁蛋白携带铁，白蛋白携带维生素、脂肪、以及激素等；提供促接触和伸展因子使细胞贴壁免受机械损伤，对培养中的细胞起到某些保护作用。血清是非常复杂的混合物，成分多样，为细胞培养带来很多便利，但同时也带来了很多问题。
血清的成份可能有几百种之多，目前对其准确的成份、含量及其作用机制仍不清楚，尤其是对其中一些多肽类生长因子、激素和脂类等尚未充分认识，给研究工作带来许多困难；血清都是批量生产，各批量之间差异很大，要保证每批血清的相似性比较困难，从而使实验的标准化和连续性受到限制；不能排除血清中含有易变物质；对大多数细胞，在体内状态，血清不是它们接触的生理学液体，只是在损伤愈合以及血液凝固过程中才接触血清，因此使用血清有可能改变某种细胞在体内的正常状态，血清可能促进某些细胞如成纤维细胞的生长同时抑制另一类细胞如表皮细胞的生长；血清含一些对细胞产生毒性的物质，如多胺氧化酶，能与来自高度繁殖细胞的多胺反应(如精胺、亚精胺)形成有细胞毒性作用的聚精胺；取材中可能带入支原体、病毒等，对细胞产生潜在安全隐患；血清的使用使得实验和生产的标准化困难，其中的蛋白质使得某些转基因蛋白生物药品生产中分离纯化工作很难完成；大规模生产中，血清来源困难，价格昂贵，是构成生产成本的主要部分之一；为了使与传染源相关的风险减少到最低，存在多个国家间限制进出口生物材料的国际法规。
发明内容
本发明的目的是为了提供一种无血清的细胞培养基，为了解决如何减小利用血清培养细胞导致的潜在的风险，本发明提供了一种无血清的培养基，所述培养基以500mL计，由以下成分组成：500mL基础培养基DMEM、二氯化锰50mg/L、亚硒酸钠30mg/L、钼酸铵0.001mg/L、柠檬酸铁1mg/L、酒石酸胆碱8mg/L、维生素D210mg/L、微生素K20.4/L、维生素K31mg/L、腺嘌呤5mg/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、丝胶酶水解物10-100μg/L和丝胶碱水解物1-10μg/L。
进一步地限定，所述培养基以500mL计，由以下成分组成：500mL基础培养基DMEM、二氯化锰50mg/L、亚硒酸钠30mg/L、钼酸铵0.001mg/L、柠檬酸铁1mg/L、酒石酸胆碱8mg/L、维生素D210mg/L、微生素K20.4/L、维生素K31mg/L、腺嘌呤5mg/L、盐酸鸟嘌呤1mg/L、尿嘧啶1mg/L、亚油酸1mg/L、胆固醇1mg/L、胰岛素0.00001mg/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10mg/L、酪蛋白水解物10mg/L、麦芽蛋白胨10mg、丝胶酶水解物10μg/L和丝胶碱水解物1μg/L。
进一步地限定，所述培养基以500mL计，由以下成分组成：500mL基础培养基DMEM、二氯化锰50mg/L、亚硒酸钠30mg/L、钼酸铵0.001mg/L、柠檬酸铁1mg/L、酒石酸胆碱8mg/L、维生素D210mg/L、微生素K20.4/L、维生素K31mg/L、腺嘌呤5mg/L、盐酸鸟嘌呤10mg/L、尿嘧啶10mg/L、亚油酸10mg/L、胆固醇10mg/L、胰岛素1mg/L、氢化可的松1mg/L、地塞米松1mg/L、酵母水解物1000mg/L、乳蛋白水解物1000mg/L、酪蛋白水解物1000mg/L、麦芽蛋白胨1000mg、丝胶酶水解物100μg/L和丝胶碱水解物10μg/L。
本发明还提供了一种上述的无血清培养基在培养上皮细胞中的应用。
本发明还提供了一种上述的无血清培养基在培养鸡胚成纤维细胞中的应用。
本发明还提供了一种上述的无血清培养基在培养肝癌细胞中的应用。
本发明还提供了一种上述的无血清培养基在培养原代细胞中的应用。
有益效果：1)避免血清不同批次间的质量差异，提高细胞培养实验结果的一致性。
2)避免血清对细胞的毒性作用和血清源性污染。
3)避免血清组分对实验研究的影响。
4)有利于体外培养细胞的分化。
5)可提高目的蛋白的表达量并使产品易于分离纯化。
6)组分稳定，可大量生产。
7)不含有丝分裂原抑制剂，可以促进细胞增殖。
附图说明
图1为用本发明的无血清培养基培养293T细胞(上皮细胞)的结果图；
图2为用本发明的无血清培养基培养DF1(鸡胚成纤维细胞)的结果图；
图3为用本发明的无血清培养基培养SNU387(肝癌细胞)的结果图；
图4为用本发明的无血清培养基培养骨髓间充质细胞(原代细胞)的结果图。
具体实施例
实施例1.
无血清培养基的配方如下：是在基本人工合成培养基DMEM的基础上，添加了不同浓度的替代血清的补充因子，包括胰岛素、亚硒酸钠、转铁蛋白、细胞贴壁因子等，同时还按比例加入一定量的微量元素、维生素和脂类等低分子量物质。具体配方：所述培养基以500ml计，由以下成分组成：500mL基础培养基DMEM、二氯化锰50mg/L、亚硒酸钠30mg/L、钼酸铵0.001mg/L、柠檬酸铁1mg/L、酒石酸胆碱8mg/L、维生素D210mg/L、微生素K20.4/L、维生素K31mg/L、腺嘌呤5mg/L、盐酸鸟嘌呤1mg/L、尿嘧啶1mg/L、亚油酸1mg/L、胆固醇1mg/L、胰岛素0.00001mg/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10mg/L、酪蛋白水解物10mg/L、麦芽蛋白胨10mg、丝胶酶水解物10μg/L和丝胶碱水解物1μg/L。
实施例2.
无血清培养基的配方如下：是在基本人工合成培养基DMEM的基础上，添加了不同浓度的替代血清的补充因子，包括胰岛素、亚硒酸钠、转铁蛋白、细胞贴壁因子等，同时还按比例加入一定量的微量元素、维生素和脂类等低分子量物质。具体配方：所述培养基以500ml计，由以下成分组成：500mL基础培养基DMEM、二氯化锰50mg/L、亚硒酸钠30mg/L、钼酸铵0.001mg/L、柠檬酸铁1mg/L、酒石酸胆碱8mg/L、维生素D210mg/L、微生素K20.4/L、维生素K31mg/L、腺嘌呤5mg/L、盐酸鸟嘌呤10mg/L、尿嘧啶10mg/L、亚油酸10mg/L、胆固醇10mg/L、胰岛素1mg/L、氢化可的松1mg/L、地塞米松1mg/L、酵母水解物1000mg/L、乳蛋白水解物1000mg/L、酪蛋白水解物1000mg/L、麦芽蛋白胨1000mg、丝胶酶水解物100μg/L和丝胶碱水解物10μg/L。
培养细胞的结果：
利用本发明的无血清培养基，培养293T细胞(上皮细胞)、DF1(鸡胚成纤维细胞)、SNU387(肝癌细胞)和骨髓间充质细胞(原代细胞)。
细胞培养条件：10^6各细胞，于37摄氏度，5％二氧化碳，95％空气，无血清培养基，1％双抗条件下培养传代5次，结果如图1-4所示，第五次传代后可见细胞贴壁98％以上，少见漂浮细胞，形态正常未发生改变。
Claims (7)
1.一种无血清的培养基，其特征在于，所述培养基以500mL计，由以下成分组成：500mL基础培养基DMEM、二氯化锰50mg/L、亚硒酸钠30mg/L、钼酸铵0.001mg/L、柠檬酸铁1mg/L、酒石酸胆碱8mg/L、维生素D210mg/L、微生素K20.4/L、维生素K31mg/L、腺嘌呤5mg/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、丝胶酶水解物10-100μg/L和丝胶碱水解物1-10μg/L。
2.根据权利要求1所述的培养基，其特征在于，所述培养基以500mL计，由以下成分组成：500mL基础培养基DMEM、二氯化锰50mg/L、亚硒酸钠30mg/L、钼酸铵0.001mg/L、柠檬酸铁1mg/L、酒石酸胆碱8mg/L、维生素D210mg/L、微生素K20.4/L、维生素K31mg/L、腺嘌呤5mg/L、盐酸鸟嘌呤1mg/L、尿嘧啶1mg/L、亚油酸1mg/L、胆固醇1mg/L、胰岛素0.00001mg/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10mg/L、酪蛋白水解物10mg/L、麦芽蛋白胨10mg、丝胶酶水解物10μg/L和丝胶碱水解物1μg/L。
3.根据权利要求1所述的培养基，其特征在于，所述培养基以500mL计，由以下成分组成：500mL基础培养基DMEM、二氯化锰50mg/L、亚硒酸钠30mg/L、钼酸铵0.001mg/L、柠檬酸铁1mg/L、酒石酸胆碱8mg/L、维生素D210mg/L、微生素K20.4/L、维生素K31mg/L、腺嘌呤5mg/L、盐酸鸟嘌呤10mg/L、尿嘧啶10mg/L、亚油酸10mg/L、胆固醇10mg/L、胰岛素1mg/L、氢化可的松1mg/L、地塞米松1mg/L、酵母水解物1000mg/L、乳蛋白水解物1000mg/L、酪蛋白水解物1000mg/L、麦芽蛋白胨1000mg、丝胶酶水解物100μg/L和丝胶碱水解物10μg/L。
4.权利要求1-3任意一项所述的培养基在培养上皮细胞中的应用。
5.权利要求1-3任意一项所述的培养基在培养鸡胚成纤维细胞中的应用。
6.权利要求1-3任意一项所述的培养基在培养肝癌细胞中的应用。
7.权利要求1-3任意一项所述的培养基在培养原代细胞中的应用。
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