CN114480294A - 一种适合杂交瘤细胞贴壁生长的无血清培养基 - Google Patents
一种适合杂交瘤细胞贴壁生长的无血清培养基 Download PDFInfo
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- CN114480294A CN114480294A CN202111533619.5A CN202111533619A CN114480294A CN 114480294 A CN114480294 A CN 114480294A CN 202111533619 A CN202111533619 A CN 202111533619A CN 114480294 A CN114480294 A CN 114480294A
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- sodium
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Abstract
本发明公开了一种适合杂交瘤细胞贴壁生长的无血清培养基，包括多种氨基酸、维生素、金属盐、辅酶、激素、糖蛋白、基础营养素等多种营养物质。所述无血清培养基中完全不含血清成分，化学成分明确配制简便，原料常见易获得，且成本低廉；该无血清培养基制备方法简单，能够大量稳定生产，极易控制批间差，快速获得高密度细胞及长时间保持细胞活性，完全可取代牛血清培养基用于杂交瘤细胞的贴壁培养，极大精简了杂交瘤细胞培养和单克隆抗体制备的工艺，减少了批间差，降低了成本，也排除了支原体污染的风险。
Description
技术领域
本发明属于生物医药领域，具体涉及一种适合杂交瘤细胞贴壁生长的无血清培养基。
背景技术
目前大多数杂交瘤细胞是在含有胎牛/新生牛/小牛血清的培养基中培养的。但这些培养基存在以下缺陷：(1)培养基中重要成分牛血清一般需国外进口，不仅货期长，且易受贸易环境因素等影响，货源无保障；(2)牛血清批间差较大，每次使用前都须抽样检定，合格后方可购买使用，不仅耗时较长，还增加了检定的工作量，且原料质量无保障；(3)牛血清成分复杂，可能含有的个别成分会抑制细胞的生长，同时也存在支原体污染的风险；(4)牛血清尤其是胎牛血清价格高昂，每100ml培养基需要10～15ml牛血清，成本较高。
因此，筛选出一种专用于杂交瘤细胞培养的无血清培养基，对于降低杂交瘤细胞的贴壁培养和提高培养细胞的质量都具有重要意义。
发明内容
为了解决上述背景技术中的问题，本发明提供了一种适合杂交瘤细胞贴壁生长的无血清培养基，其含有多种氨基酸、维生素、金属盐等营养成分，化学成分明确、使用成本低廉，完全不含动物血清，可获得较高的细胞浓度。
为实现上述发明目的，本发明采用以下技术方案予以实现：
本发明提供了一种适合杂交瘤细胞贴壁生长的无血清培养基，所述无血清培养基包括如下组分：苯丙氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、蛋氨酸、赖氨酸、亮氨酸、组氨酸、胱氨酸、精氨酸、丝氨酸、碳酸氢钠、磷酸二氢钠、磷酸氢二钠、氯化镁、氯化钠、硫酸镁、氯化钾、氯化钙、氢氧化钠、硫酸铜、氯化锰、柠檬酸铁、泛酸钙、叶酸、烟酰胺、维生素B2、硫胺素、生物素、维生素B12、维生素C、维生素A、还原性谷胱甘肽、胸苷、三磷酸腺苷、黄嘌呤、尿嘧啶、硫酸锌、氢化可的松、地塞米松、酵母提取物、纤连蛋白、L-谷氨酰胺。
进一步的，所述无血清培养基包括如下各组分及浓度：苯丙氨酸10-50mg/L、苏氨酸10-50mg/L、色氨酸10-50mg/L、酪氨酸10-50mg/L、缬氨酸10-50mg/L、蛋氨酸10-50mg/L、赖氨酸10-50mg/L、亮氨酸10-50mg/L、组氨酸10-50mg/L、胱氨酸10-50mg/L、精氨酸10-50mg/L、丝氨酸10-50mg/L、碳酸氢钠1000-2000mg/L、磷酸二氢钠100-200mg/L、磷酸氢二钠100-200mg/L、氯化镁10-50mg/L、氯化钠1000-5000mg/L、硫酸镁100-500mg/L、氯化钾100-500mg/L、氯化钙100-200mg/L、氢氧化钠100-1000mg/L、硫酸铜0.1-10mg/L、氯化锰0.1-10mg/L、柠檬酸铁0.1-10mg/L、泛酸钙0.1-10mg/L、叶酸0.1-10mg/L、烟酰胺0.1-10mg/L、维生素B2 0.1-10mg/L、硫胺素0.1-10mg/L、生物素0.1-10mg/L、维生素B120.1-10mg/L、维生素C 0.1-10mg/L、维生素A 0.1-10mg/L、还原性谷胱甘肽0.1-10mg/L、胸苷0.1-10mg/L、三磷酸腺苷0.1-10mg/L、黄嘌呤0.1-10mg/L、尿嘧啶0.1-10mg/L、硫酸锌0.1-10mg/L、氢化可的松0.1-10mg/L、地塞米松0.1-10mg/L、酵母提取物1000-5000mg/L、纤连蛋白0.1-10mg/L、L-谷氨酰胺150-375mg/L。
进一步的，所述无血清培养基的各组分及其浓度为：苯丙氨酸45mg/L、苏氨酸30mg/L、色氨酸35mg/L、酪氨酸45mg/L、缬氨酸24mg/L、蛋氨酸18mg/L、赖氨酸50mg/L、亮氨酸35mg/L、组氨酸40mg/L、胱氨酸20mg/L、精氨酸35mg/L、丝氨酸28mg/L、碳酸氢钠2000mg/L、磷酸二氢钠200mg/L、磷酸氢二钠190mg/L、氯化镁40mg/L、氯化钠5000mg/L、硫酸镁500mg/L、氯化钾500mg/L、氯化钙150mg/L、氢氧化钠1000mg/L、硫酸铜6mg/L、氯化锰5mg/L、柠檬酸铁5mg/L、泛酸钙7mg/L、叶酸10mg/L、烟酰胺6mg/L、维生素B2 8mg/L、硫胺素8mg/L、生物素8mg/L、维生素B12 5mg/L、维生素C10mg/L、维生素A 6mg/L、还原性谷胱甘肽6mg/L、胸苷5mg/L、三磷酸腺苷5mg/L、黄嘌呤8mg/L、尿嘧啶3mg/L、硫酸锌5mg/L、氢化可的松9mg/L、地塞米松10mg/L、酵母提取物5000mg/L、纤连蛋白10mg/L、L-谷氨酰胺375mg/L。
进一步的，所述无血清培养基的各组分及其浓度为：苯丙氨酸15mg/L、苏氨酸12mg/L、色氨酸15mg/L、酪氨酸15mg/L、缬氨酸10mg/L、蛋氨酸10mg/L、赖氨酸15mg/L、亮氨酸20mg/L、组氨酸10mg/L、胱氨酸10mg/L、精氨酸15mg/L、丝氨酸10mg/L、碳酸氢钠1000mg/L、磷酸二氢钠100mg/L、磷酸氢二钠100mg/L、氯化镁15mg/L、氯化钠3000mg/L、硫酸镁200mg/L、氯化钾200mg/L、氯化钙100mg/L、氢氧化钠100mg/L、硫酸铜2mg/L、氯化锰1mg/L、柠檬酸铁1mg/L、泛酸钙1mg/L、叶酸5mg/L、烟酰胺2mg/L、维生素B2 2mg/L、硫胺素1mg/L、生物素1mg/L、维生素B12 2mg/L、维生素C 5mg/L、维生素A1mg/L、还原性谷胱甘肽3mg/L、胸苷2mg/L、三磷酸腺苷2mg/L、黄嘌呤3mg/L、尿嘧啶1mg/L、硫酸锌1mg/L、氢化可的松2mg/L、地塞米松1mg/L、酵母提取物1000mg/L、纤连蛋白50mg/L、L-谷氨酰胺150mg/L。
进一步的，所述无血清培养基的pH值为7.2～7.4。
本发明还提供了所述的无血清培养基的配制方法，具体包括如下步骤：
(1)称量所述无血清培养基的各组分于水中溶解；
(2)溶解后加入抗生素混匀，调节溶液pH值至7.2～7.4；
(3)除菌过滤后密封保存。
进一步的，所述抗生素包括100×青霉素和100×链霉素，其用量为水体积的1％。
本发明还提供了所述的无血清培养基在培养细胞中的应用。
进一步的，所述细胞为杂交瘤细胞。
本发明与现有技术相比，具有以下优点和有益效果：
(1)本发明所述的无血清培养基包括多种氨基酸、维生素、金属盐、辅酶等营养物质，完全不含血清成分，化学成分明确配制简便，原料常见易获得，且成本低廉；
(2)经实验验证，所述的无血清培养基完全可取代牛血清培养基用于杂交瘤细胞的贴壁培养，使杂交瘤细胞无需驯化，直接贴壁培养，
(3)所述无血清培养基可大量稳定生产，极易控制批间差，可快速获得高密度细胞，能长时间保持细胞活性；极大精简了杂交瘤细胞培养和单克隆抗体制备的工艺，减少了批间差，降低了成本，也排除了支原体污染的风险。
附图说明
图1为无血清培养基培养Fer 88-5杂交瘤细胞生长状态；
图2为牛血清培养基培养Fer 88-5杂交瘤细胞生长状态；
图3为两组培养基培养Fer 88-5杂交瘤细胞所产鼠腹水效价。
具体实施方式
为使本发明技术方案和优点更加清晰明了，下面结合实例，对本发明做进一步的详细说明。下述实施例中，如无特殊说明，所使用的实验方法均为常规方法，所用材料、试剂等均可从生物或化学试剂公司购买。
实施例1
一种适合杂交瘤细胞贴壁生长的无血清培养基，包含如下浓度的组分：苯丙氨酸45mg/L、苏氨酸30mg/L、色氨酸35mg/L、酪氨酸45mg/L、缬氨酸24mg/L、蛋氨酸18mg/L、赖氨酸50mg/L、亮氨酸35mg/L、组氨酸40mg/L、胱氨酸20mg/L、精氨酸35mg/L、丝氨酸28mg/L、碳酸氢钠2000mg/L、磷酸二氢钠200mg/L、磷酸氢二钠190mg/L、氯化镁40mg/L、氯化钠5000mg/L、硫酸镁500mg/L、氯化钾500mg/L、氯化钙150mg/L、氢氧化钠1000mg/L、硫酸铜6mg/L、氯化锰5mg/L、柠檬酸铁5mg/L、泛酸钙7mg/L、叶酸10mg/L、烟酰胺6mg/L、维生素B2 8mg/L、硫胺素8mg/L、生物素8mg/L、维生素B12 5mg/L、维生素C 10mg/L、维生素A 6mg/L、还原性谷胱甘肽6mg/L、胸苷5mg/L、三磷酸腺苷5mg/L、黄嘌呤8mg/L、尿嘧啶3mg/L、硫酸锌5mg/L、氢化可的松9mg/L、地塞米松10mg/L、酵母提取物5000mg/L、纤连蛋白10mg/L，L-谷氨酰胺375mg/L。
实施例2
一种适合杂交瘤细胞贴壁生长的无血清培养基，包含如下浓度的组分：苯丙氨酸15mg/L、苏氨酸12mg/L、色氨酸15mg/L、酪氨酸15mg/L、缬氨酸10mg/L、蛋氨酸10mg/L、赖氨酸15mg/L、亮氨酸20mg/L、组氨酸10mg/L、胱氨酸10mg/L、精氨酸15mg/L、丝氨酸10mg/L、碳酸氢钠1000mg/L、磷酸二氢钠100mg/L、磷酸氢二钠100mg/L、氯化镁15mg/L、氯化钠3000mg/L、硫酸镁200mg/L、氯化钾200mg/L、氯化钙100mg/L、氢氧化钠100mg/L、硫酸铜2mg/L、氯化锰1mg/L、柠檬酸铁1mg/L、泛酸钙1mg/L、叶酸5mg/L、烟酰胺2mg/L、维生素B2 2mg/L、硫胺素1mg/L、生物素1mg/L、维生素B12 2mg/L、维生素C 5mg/L、维生素A1mg/L、还原性谷胱甘肽3mg/L、胸苷2mg/L、三磷酸腺苷2mg/L、黄嘌呤3mg/L、尿嘧啶1mg/L、硫酸锌1mg/L、氢化可的松2mg/L、地塞米松1mg/L、酵母提取物1000mg/L、纤连蛋白50mg/L、L-谷氨酰胺150mg/L。
实施例3
(1)配制无血清培养基100ml：按照实施例1的无血清培养基配方依次精确称取各组分，加入100ml纯化水中溶解，搅拌均匀，再加入1ml 100×青霉素(Gibco)、1ml 100×链霉素(Gibco)，混匀，用1M HCl/NaOH调pH值至7.2～7.4，用0.2μm滤膜除菌过滤；
(2)配制牛血清培养基100ml：取RPMI-1640培养基(Sigma)85ml，加入15ml胎牛血清(Gibco)、1ml 100×青霉素(Gibco)、1ml 100×链霉素(Gibco)，混匀；
(3)取无血清培养基和牛血清培养基各25ml，分别加入两个T75细胞培养瓶中(Corning)；复苏2支铁蛋白杂交瘤细胞(Fer 88-5，上海捷门生物)，细胞密度约为1×106cells/ml，分别加入T75培养瓶中培养；5％CO2培养箱37度培养3天，分别观察细胞生长状态。光学显微镜视野下细胞密度分别如图1和图2所示，T75培养瓶37度培养3天后，两组细胞在形态、数量、增值速度、细胞活率、细胞密度等方面无明显差异；各换液1次；
(4)待细胞长满，低速离心收集细胞，1×PBS复溶细胞各24ml，每瓶杂交瘤细胞各打30只Balb/c小鼠(约8周龄，5～6周龄时腹腔注射石蜡油；上海斯莱克实验动物有限责任公司)，按照0.8ml/只(约1×106cells/只)，将Fer 88-5杂交瘤细胞注入小鼠腹腔；
(5)饲养7～10天，待小鼠腹水长满，一次性采集腹水，离心去除杂质沉淀，混匀；
(6)各取1ml小鼠腹水做腹水效价检测，结果如表1和图3所示；
(7)各取30ml小鼠腹水55％硫酸铵沉淀，离心取上清，Protein G亲和层析纯化，收获Fer单克隆抗体并计算得率，结果如表2所示。
腹水效价检测的结果如表1和图3所示，表明无血清培养基组和牛血清培养基组腹水效价均1：>1,000,000，且无血清培养基组效价更高。
表1：两组小鼠腹水效价检测结果(OD450)
| 稀释倍数 | 1,000 | 3,000 | 9,000 | 27,000 | 81,000 | 243,000 | 729,000 | 2,187,000 | NC 1 | NC 2 | NC 3 |
| 无血清培养基组 | 2.425 | 2.413 | 2.288 | 2.269 | 2.016 | 1.567 | 0.852 | 0.328 | 0.062 | 0.069 | 0.066 |
| 牛血清培养基组 | 2.032 | 1.932 | 1.922 | 1.896 | 1.699 | 1.349 | 0.69 | 0.265 | 0.066 | 0.073 | 0.063 |
表2中无血清培养基组抗体得率为3.06mg/ml腹水，稍高于牛血清组2.86mg/ml腹水，表示无血清培养基培养杂交瘤可快速获得高密度细胞，提高小鼠腹水中抗体的得率，从而节省抗体的生产成本。
表2：两组小鼠腹水Protein G亲和纯化结果
综上所述，本发明所述的无血清杂交瘤细胞培养基，可以完全替代含有胎牛血清的培养基，适合杂交瘤细胞的贴壁培养；且该培养基化学成分明确配制简便，成本低廉、可大量稳定生产，极易控制批间差，还有效避免了支原体等的污染。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述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；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (9)
1.一种适合杂交瘤细胞贴壁生长的无血清培养基，其特征在于，所述无血清培养基包括如下组分：苯丙氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、蛋氨酸、赖氨酸、亮氨酸、组氨酸、胱氨酸、精氨酸、丝氨酸、碳酸氢钠、磷酸二氢钠、磷酸氢二钠、氯化镁、氯化钠、硫酸镁、氯化钾、氯化钙、氢氧化钠、硫酸铜、氯化锰、柠檬酸铁、泛酸钙、叶酸、烟酰胺、维生素B2、硫胺素、生物素、维生素B12、维生素C、维生素A、还原性谷胱甘肽、胸苷、三磷酸腺苷、黄嘌呤、尿嘧啶、硫酸锌、氢化可的松、地塞米松、酵母提取物、纤连蛋白、L-谷氨酰胺。
2.根据权利要求1所述的无血清培养基，其特征在于，所述无血清培养基包括如下组分及浓度：苯丙氨酸10-50mg/L、苏氨酸10-50mg/L、色氨酸10-50mg/L、酪氨酸10-50mg/L、缬氨酸10-50mg/L、蛋氨酸10-50mg/L、赖氨酸10-50mg/L、亮氨酸10-50mg/L、组氨酸10-50mg/L、胱氨酸10-50mg/L、精氨酸10-50mg/L、丝氨酸10-50mg/L、碳酸氢钠1000-2000mg/L、磷酸二氢钠100-200mg/L、磷酸氢二钠100-200mg/L、氯化镁10-50mg/L、氯化钠1000-5000mg/L、硫酸镁100-500mg/L、氯化钾100-500mg/L、氯化钙100-200mg/L、氢氧化钠100-1000mg/L、硫酸铜0.1-10mg/L、氯化锰0.1-10mg/L、柠檬酸铁0.1-10mg/L、泛酸钙0.1-10mg/L、叶酸0.1-10mg/L、烟酰胺0.1-10mg/L、维生素B2 0.1-10mg/L、硫胺素0.1-10mg/L、生物素0.1-10mg/L、维生素B12 0.1-10mg/L、维生素C 0.1-10mg/L、维生素A 0.1-10mg/L、还原性谷胱甘肽0.1-10mg/L、胸苷0.1-10mg/L、三磷酸腺苷0.1-10mg/L、黄嘌呤0.1-10mg/L、尿嘧啶0.1-10mg/L、硫酸锌0.1-10mg/L、氢化可的松0.1-10mg/L、地塞米松0.1-10mg/L、酵母提取物1000-5000mg/L、纤连蛋白0.1-10mg/L、L-谷氨酰胺150-375mg/L。
3.根据权利要求2所述的无血清培养基，其特征在于，所述无血清培养基的各组分及其浓度为：苯丙氨酸45mg/L、苏氨酸30mg/L、色氨酸35mg/L、酪氨酸45mg/L、缬氨酸24mg/L、蛋氨酸18mg/L、赖氨酸50mg/L、亮氨酸35mg/L、组氨酸40mg/L、胱氨酸20mg/L、精氨酸35mg/L、丝氨酸28mg/L、碳酸氢钠2000mg/L、磷酸二氢钠200mg/L、磷酸氢二钠190mg/L、氯化镁40mg/L、氯化钠5000mg/L、硫酸镁500mg/L、氯化钾500mg/L、氯化钙150mg/L、氢氧化钠1000mg/L、硫酸铜6mg/L、氯化锰5mg/L、柠檬酸铁5mg/L、泛酸钙7mg/L、叶酸10mg/L、烟酰胺6mg/L、维生素B2 8mg/L、硫胺素8mg/L、生物素8mg/L、维生素B12 5mg/L、维生素C 10mg/L、维生素A6mg/L、还原性谷胱甘肽6mg/L、胸苷5mg/L、三磷酸腺苷5mg/L、黄嘌呤8mg/L、尿嘧啶3mg/L、硫酸锌5mg/L、氢化可的松9mg/L、地塞米松10mg/L、酵母提取物5000mg/L、纤连蛋白10mg/L、L-谷氨酰胺375mg/L。
4.根据权利要求2所述的无血清培养基，其特征在于，所述无血清培养基的各组分及其浓度为：苯丙氨酸15mg/L、苏氨酸12mg/L、色氨酸15mg/L、酪氨酸15mg/L、缬氨酸10mg/L、蛋氨酸10mg/L、赖氨酸15mg/L、亮氨酸20mg/L、组氨酸10mg/L、胱氨酸10mg/L、精氨酸15mg/L、丝氨酸10mg/L、碳酸氢钠1000mg/L、磷酸二氢钠100mg/L、磷酸氢二钠100mg/L、氯化镁15mg/L、氯化钠3000mg/L、硫酸镁200mg/L、氯化钾200mg/L、氯化钙100mg/L、氢氧化钠100mg/L、硫酸铜2mg/L、氯化锰1mg/L、柠檬酸铁1mg/L、泛酸钙1mg/L、叶酸5mg/L、烟酰胺2mg/L、维生素B2 2mg/L、硫胺素1mg/L、生物素1mg/L、维生素B12 2mg/L、维生素C 5mg/L、维生素A 1mg/L、还原性谷胱甘肽3mg/L、胸苷2mg/L、三磷酸腺苷2mg/L、黄嘌呤3mg/L、尿嘧啶1mg/L、硫酸锌1mg/L、氢化可的松2mg/L、地塞米松1mg/L、酵母提取物1000mg/L、纤连蛋白50mg/L、L-谷氨酰胺150mg/L。
5.根据权利要求1所述的无血清培养基，其特征在于，所述无血清培养基的pH值为7.2～7.4。
6.权利要求1-5任一项所述的无血清培养基的配制方法，其特征在于，具体包括如下步骤：
(1)称量无血清培养基的各组分于水中溶解；
(2)溶解后加入抗生素混匀，调节溶液pH值至7.2～7.4；
(3)除菌过滤后密封保存。
7.根据权利要求6所述的无血清培养基的配制方法，其特征在于，所述抗生素包括100×青霉素和100×链霉素，其用量为水体积的1％。
8.权利要求1-5任一项所述的无血清培养基在培养细胞中的应用。
9.根据权利要求8所述的应用，其特征在于，所述细胞为杂交瘤细胞。
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