CN108424875A - 一种人源细胞培养基及其制备方法 - Google Patents
一种人源细胞培养基及其制备方法 Download PDFInfo
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- CN108424875A CN108424875A CN201810360784.7A CN201810360784A CN108424875A CN 108424875 A CN108424875 A CN 108424875A CN 201810360784 A CN201810360784 A CN 201810360784A CN 108424875 A CN108424875 A CN 108424875A
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Abstract
本发明公开了一种人源细胞培养基,包括氨基酸、维生素、无机盐、次黄嘌呤、胸苷、葡萄糖、丙酮酸钠、亚油酸、硫辛酸、腐胺、4‑羟乙基哌嗪乙磺酸。本发明通过对培养基成分的改进,根据人源细胞系的生长、代谢特点,增加了盐酸吡哆醇、硫酸铜、硫酸亚铁、硫酸锌、氯化镁、磷酸氢二钠、次黄嘌呤钠、亚油酸、硫辛酸、腐胺、胸苷等组分,更贴近于人体内细胞生长的微环境,能够促进人源细胞生物酶的合成。优化了培养基各组分的浓度,降低了新组份带来的不稳定性,更好的保持了培养细胞的生物特性,可减少或者免去细胞培养中血清的使用,经过测试该培养基能适合贴壁培养及悬浮培养,并适用于大多数人源细胞的培养。
Description
技术领域
本发明涉及用于细胞培养的组合物,特别涉及一种人源细胞培养基及其制备方法。
背景技术
自建立组织和细胞培养技术以来,利用人源细胞开展现代生物制药,特别是利用人源细胞进行组织工程研究或生物治疗,不断蓬勃发展。在这些技术中,研制适宜的培养基是其中最核心、最基础的关键技术。
到目前为止,体外扩增人源细胞最常使用添加胎牛血清的基础培养基。但是,利用胎牛血清培养细胞也带来了不少问题。首先,胎牛血清蛋白属异种蛋白,用胎牛血清培养的细胞用于治疗后,可能导致被治疗者产生感染,具有潜在危险。其次,胎牛血清可能携带传播性病原体。此外,在牛的饲养过程中持续使用抗生素,也存在感染的风险。另外,胎牛血清的缺点还包括成分的不确定性、批次间的不稳定性,存储时间短,供应时间存在周期性等。
由于血清培养细胞存在的诸多问题,促使产生了继天然培养基、合成培养基之后的第三类培养基——无血清培养基。细胞工程中无血清培养技术的应用,在很大程度上可以减少甚至避免含血清培养所带来的不利。
无血清培养基具有以下明显的优缺点:
无血清培养基的优点:
(1)可避免血清批次间的不稳定性,提高细胞培养和实验结果的可重复性。
(2)避免血清培养的细胞用于治疗后引起人体的免疫反应及感染问题。
(3)避免血清受到污染引入有害的病毒。
(4)化学成分相对比较明确,可避免血清中包含的不确定组分对实验研究的影响。
(5)易于生产,不存在胎牛血清生产的周期性。
无血清培养基的缺点:
(1)细胞在无血清培养基中易受某些机械因素和化学因素的影响,培养基的保存和应用不如传统的合成培养基方便。
(2)成本较高。
(3)针对性强,一种无血清培养基可能仅适合某一类细胞的培养。
发明内容
本发明要解决的技术问题是提供一种适于多种类型人源细胞培养的培养基及其制备方法。
为了解决上述技术问题,本发明的技术方案为:
一种人源细胞培养基,包括氨基酸、维生素、无机盐、次黄嘌呤、胸苷、葡萄糖、丙酮酸钠、亚油酸、硫辛酸、腐胺、4-羟乙基哌嗪乙磺酸。
优选的,所述氨基酸包括甘氨酸、L-缬氨酸、L-异亮氨酸、L-亮氨酸、L-苯丙氨酸、L-甲硫氨酸、L-色氨酸、L-苏氨酸、L-丝氨酸、L-丙氨酸、L-脯氨酸、L-胱氨酸、L-酪氨酸二钠盐、L-赖氨酸盐酸盐、L-精氨酸盐酸盐、L-组氨酸盐酸盐、L-谷氨酰胺、L-谷氨酸、L-天门冬酰胺或L-天门冬氨酸中的一种或多种;
所述维生素包括叶酸、硫胺素、核黄素、维生素B12、生物素、泛酸钙、盐酸吡哆醛、盐酸吡哆醇、肌醇、氯化胆硷或烟酰胺中的一种或多种;
所述无机盐包括氯化钠、碳酸氢钠、二水合磷酸二氢钠、磷酸氢二钠、五水合亚硒酸钠、氯化钾、硝酸钾、二水合氯化钙、七水合硫酸亚铁、七水合硫酸锌、五水合硫酸铜、氯化镁或二水合硫酸镁中的一种或多种;
优选的,所述甘氨酸浓度为0.27~0.41mmol/L;所述L-缬氨酸浓度为0.53~0.80mmol/L;所述L-异亮氨酸浓度为0.52~0.78mmol/L;所述L-亮氨酸浓度为0.53~0.79mmol/L;所述L-苯丙氨酸浓度为0.26~0.39mmol/L;所述L-甲硫氨酸浓度为0.13~0.20mmol/L;所述L-色氨酸浓度为0.05~0.08mmol/L;所述L-苏氨酸浓度为0.53~0.79mmol/L;所述L-丝氨酸浓度为0.27~0.41mmol/L;所述L-丙氨酸浓度为0.20~0.29mmol/L;所述L-脯氨酸浓度为0.27~0.41mmol/L;所述L-胱氨酸浓度为0.22~0.33mmol/L;所述L-酪氨酸二钠盐浓度为0.30~0.45mmol/L;所述L-赖氨酸盐酸盐浓度为0.54~0.81mmol/L;所述L-精氨酸盐酸盐浓度为0.41~0.62mmol/L;所述L-组氨酸盐酸盐浓度为0.14~0.22mmol/L;所述L-谷氨酰胺浓度为2.72~4.08mmol/L;所述L-谷氨酸浓度为0.34~0.51mmol/L;所述L-天门冬酰胺浓度为0.14~0.21mmol/L;所述L-天门冬氨酸浓度为0.16~0.24mmol/L;所述叶酸浓度为6.3E-03~9.4E-03mmol/L;所述硫胺素浓度为7.7E-03~1.2E-02mmol/L;所述核黄素浓度为7.0E-04~1.0E-03mmol/L;所述维生素B12浓度为1.7E-04~2.6E-04mmol/L;所述生物素浓度为3.9E-05~5.8E-05mmol/L;所述泛酸钙浓度为5.5E-03~8.3E-03mmol/L;所述盐酸吡哆醛浓度为1.3E-02~1.9E-02mmol/L;所述盐酸吡哆醇浓度为4.7E-05~7.0E-05mmol/L;所述肌醇浓度为4.2E-02~6.2E-02mmol/L;所述氯化胆硷浓度为3.4E-02~5.1E-02mmol/L;所述烟酰胺浓度为2.1E-02~3.2E-02mmol/L;所述氯化钠浓度为71~106mmol/L;所述碳酸氢钠浓度为25~38mmol/L;所述二水合磷酸二氢钠浓度为0.58~0.87mmol/L;所述磷酸氢二钠浓度为0.16~0.24mmol/L;所述五水合亚硒酸钠浓度为4.2E-05~6.3E-05mmol/L;所述氯化钾浓度为3.3~4.9mmol/L;所述硝酸钾浓度为4.8E-04~7.2E-04mmol/L;所述二水合氯化钙浓度为1.0~1.5mmol/L;所述七水合硫酸亚铁浓度为4.8E-04~7.2E-04mmol/L;所述七水合硫酸锌浓度为4.8E-04~7.2E-04mmol/L;所述五水合硫酸铜浓度为1.6E-06~2.4E-06mmol/L;所述氯化镁浓度为0.10~0.14mmol/L;所述二水合硫酸镁浓度为0.73~1.09mmol/L;所述胸苷浓度为4.6E-04~6.9E-04mmol/L;所述次黄嘌呤浓度为4.8E-03~7.2E-03mmol/L;所述葡萄糖浓度为18~26mmol/L;所述丙酮酸钠浓度为0.8~1.2mmol/L;所述亚油酸浓度为4.8E-05~7.2E-05mmol/L;所述硫辛酸浓度为1.6E-04~2.4E-04mmol/L;所述腐胺浓度为1.6E-04~2.4E-04mmol/L;所述4-羟乙基哌嗪乙磺酸浓度为16~24mmol/L;。
优选的,所述培养基PH值为6.8~7.4。
优选的,所述培养基渗透压为280-330mOsm/kg。
一种制备上述人源细胞培养基的方法,包括如下步骤:
(1)称取如权利要求3所述的培养基各组分;
(2)加入纯水搅拌混合均匀后定容;
(3)调节溶液pH值至7.2;
(4)将溶液过滤除菌,冷藏保存。
使用上述人源细胞培养基培养细胞的方法,包括如下步骤:
(1)在所述的人源细胞培养基中,加入体积百分数0%-20%的胎牛血清;
(2)将细胞加入生理盐水离心清理后,接种到培养瓶中,加入步骤(1)的培养基;
(3)在37℃,5%二氧化碳条件下培养到悬浮细胞达到1~2*106/ml时传代。
本发明有益效果:
(1)通过对培养基成分的改进,根据人源细胞系的生长、代谢特点,增加了盐酸吡哆醇、硫酸铜、硫酸亚铁、硫酸锌、氯化镁、磷酸氢二钠、次黄嘌呤钠、亚油酸、硫辛酸、腐胺、胸苷等组分,更贴近于人体内细胞生长的微环境,能够促进人源细胞生物酶的合成。
(2)优化了培养基各组分的浓度,降低了新组份带来的不稳定性,更好的保持了培养细胞的生物特性,可减少或者免去细胞培养中血清的使用,经过测试该培养基能适合贴壁培养及悬浮培养,并适用于大多数人源细胞的培养。
附图说明
图1-A为使用本发明的人源细胞培养基培养脐带间充质干细胞至第6代时的细胞形态图;
图1-B为使用对照组伊思柯夫改良培养液(IMDM)培养脐带间充质干细胞至第6代时的细胞形态图;
图2-A为使用本发明的人源细胞培养基培养脐带间充质干细胞至第6代时的细胞对表面标记分子CD90的表达情况;
图2-B为使用对照组伊思柯夫改良培养液(IMDM)培养脐带间充质干细胞至第6代时的细胞对表面标记分子CD90的表达情况;
图2-C为使用本发明的人源细胞培养基培养脐带间充质干细胞至第6代时的细胞对表面标记分子CD73的表达情况;
图2-D为使用对照组伊思柯夫改良培养液(IMDM)培养脐带间充质干细胞至第6代时的细胞对表面标记分子CD73的表达情况;
图2-E为使用本发明的人源细胞培养基培养脐带间充质干细胞至第6代时的细胞对表面标记分子HLA-DR的表达情况;
图2-F为使用对照组伊思柯夫改良培养液(IMDM)培养脐带间充质干细胞至第6代时的细胞对表面标记分子HLA-DR的表达情况;
图3-A为使用本发明的人源细胞培养基培养脐静脉内皮细胞至第5代时的细胞对表面标记分子CD31的表达情况;
图3-B为使用对照组伊思柯夫改良培养液(IMDM)培养脐静脉内皮细胞至第5代时的细胞对表面标记分子CD31的表达情况;
图3-C为使用本发明的人源细胞培养基培养脐静脉内皮细胞至第5代时的细胞对表面标记分子血管内皮钙粘蛋白(VE-Cadherin)的表达情况;
图3-D为使用对照组伊思柯夫改良培养液(IMDM)培养脐静脉内皮细胞至第5代时的细胞对表面标记分子血管内皮钙粘蛋白(VE-Cadherin)的表达情况;
图4-A为使用本发明的人源细胞培养基培养脐静脉内皮细胞至第8代时的细胞形态图;
图4-B为使用对照组伊思柯夫改良培养液(IMDM)培养脐静脉内皮细胞至第8代时的细胞形态图;
图5-A为使用本发明的人源细胞培养基培养免疫细胞培养21天时的细胞对表面标记分子CD3+的表达情况;
图5-B为使用对照组RPMI-1640培养液培养脐带间充质干细胞培养21天时的细胞对表面标记分子CD3+的表达情况;
图5-C为使用本发明的人源细胞培养基培养免疫细胞培养21天时的细胞对表面标记分子CD56+的表达情况;
图5-D为使用对照组RPMI-1640培养液培养脐带间充质干细胞培养21天时的细胞对表面标记分子CD56+的表达情况;
图5-E为使用本发明的人源细胞培养基培养免疫细胞培养21天时的细胞对表面标记分子CD3+CD56+的表达情况;
图5-F为使用对照组RPMI-1640培养液培养免疫细胞培养21天时的细胞对表面标记分子CD3+CD56+的表达情况。
具体实施方式
下面结合附图对发明的具体实施方式作进一步说明。在此需要说明的是,对于这些实施方式的说明用于帮助理解本发明,但并不构成对本发明的限定。此外,下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
实施例1
一种用于培养人源细胞的培养基,由20种氨基酸、11种维生素、13种无机盐、2种碱基和核苷、葡萄糖等2种能量代谢物质、亚油酸等2种脂类及前体物质及腐胺等其他3种成分组成。
其中,氨基酸成分的配方为:甘氨酸0.27~0.41mmol/L;L-缬氨酸0.53~0.80mmol/L;L-异亮氨酸0.52~0.78mmol/L;L-亮氨酸0.53~0.79mmol/L;L-苯丙氨酸0.26~0.39mmol/L;L-甲硫氨酸0.13~0.20mmol/L;L-色氨酸0.05~0.08mmol/L;L-苏氨酸0.53~0.79mmol/L;L-丝氨酸0.27~0.41mmol/L;L-丙氨酸0.20~0.29mmol/L;L-脯氨酸0.27~0.41mmol/L;L-胱氨酸0.22~0.33mmol/L;L-酪氨酸二钠盐0.30~0.45mmol/L;L-赖氨酸盐酸盐0.54~0.81mmol/L;L-精氨酸盐酸盐0.41~0.62mmol/L;L-组氨酸盐酸盐0.14~0.22mmol/L;L-谷氨酰胺2.72~4.08mmol/L;L-谷氨酸0.34~0.51mmol/L;L-天门冬酰胺0.14~0.21mmol/L;L-天门冬氨酸0.16~0.24mmol/L。
其中,维生素成分的配方为:叶酸6.3E-03~9.4E-03mmol/L;硫胺素7.7E-03~1.2E-02mmol/L;核黄素7.0E-04~1.0E-03mmol/L;维生素B121.7E-04~2.6E-04mmol/L;生物素3.9E-05~5.8E-05mmol/L;泛酸钙5.5E-03~8.3E-03mmol/L;盐酸吡哆醛1.3E-02~1.9E-02mmol/L;盐酸吡哆醇4.7E-05~7.0E-05mmol/L;肌醇4.2E-02~6.2E-02mmol/L;氯化胆硷3.4E-02~5.1E-02mmol/L;烟酰胺2.1E-02~3.2E-02mmol/L。
其中,无机盐成分的配方为:氯化钠(NaCl)71~106mmol/L;碳酸氢钠(NaHCO3)25~38mmol/L;二水合磷酸二氢钠(NaH2PO4·2H2O)0.58~0.87mmol/L;磷酸氢二钠(Na2HPO4)0.16~0.24mmol/L;五水合亚硒酸钠(Na2SeO3·5H20)4.2E-05~6.3E-05mmol/L;氯化钾(KCl)3.3~4.9mmol/L;硝酸钾(KNO3)4.8E-04~7.2E-04mmol/L;二水合氯化钙(CaCl2·2H2O)1.0~1.5mmol/L;七水合硫酸亚铁(FeSO4·7H2O)4.8E-04~7.2E-04mmol/L;七水合硫酸锌(ZnSO4·7H2O)4.8E-04~7.2E-04mmol/L;五水合硫酸铜(CuSO4·5H2O)1.6E-06~2.4E-06mmol/L;氯化镁(MgCl2)0.10~0.14mmol/L;二水合硫酸镁(MgSO4·2H2O)0.73~1.09mmol/L。
其中,碱基的配方为:胸苷4.6E-04~6.9E-04mmol/L。
其中,核苷的配方为:次黄嘌呤4.8E-03~7.2E-03mmol/L。
其中,能量代谢类成分的配方为:葡萄糖18~26mmol/L;丙酮酸钠0.8~1.2mmol/L。
其中,脂类的配方为:亚油酸4.8E-05~7.2E-05mmol/L。
其中,前体类成分的配方为:硫辛酸1.6E-04~2.4E-04mmol/L。
其中,培养基中其他成分的配方为:腐胺1.6E-04~2.4E-04mmol/L;4-羟乙基哌嗪乙磺酸(HEPES)16~24mmol/L。
其中,上述培养基的PH值为6.8~7.4。
其中,上述培养基的渗透压为280-330mOsm/kg。
实施例2
制备1000mL人源细胞培养基:
(1)称取甘氨酸0.34mmol、L-缬氨酸0.66mmol、L-异亮氨酸0.65mmol、L-亮氨酸0.66mmol、L-苯丙氨酸0.32mmol、L-甲硫氨酸0.16mmol、L-色氨酸0.06mmol、L-苏氨酸0.66mmol、L-丝氨酸0.34mmol、L-丙氨酸0.25mmol、L-脯氨酸0.34mmol、L-胱氨酸0.28mmol、L-酪氨酸二钠盐0.38mmol、L-赖氨酸盐酸盐0.68mmol、L-精氨酸盐酸盐0.52mmol、L-组氨酸盐酸盐0.18mmol、L-谷氨酰胺3.4mmol、L-谷氨酸0.43mmol、L-天门冬酰胺0.17mmol、L-天门冬氨酸0.2mmol、叶酸7.8E-03mmol、硫胺素9.7E-03mmol、核黄素8.7E-04mmol、维生素B122.1E-04mmol、生物素4.8E-05mmol、泛酸钙6.9E-03mmol、盐酸吡哆醛1.6E-02mmol、盐酸吡哆醇5.8E-05mmol、肌醇5.2E-02mmol、氯化胆硷4.3E-02mmol、烟酰胺2.6E-02mmol、氯化钠(NaCl)88mmol、碳酸氢钠(NaHCO3)32mmol、二水合磷酸二氢钠(NaH2PO4·2H2O)0.72mmol、磷酸氢二钠(Na2HPO4)0.2mmol、五水合亚硒酸钠(Na2SeO3·5H20)5.2E-05mmol、氯化钾(KCl)4.1mmol、硝酸钾(KNO3)6E-04mmol、二水合氯化钙(CaCl2·2H2O)1.25mmol、七水合硫酸亚铁(FeSO4·7H2O)6E-04mmol、七水合硫酸锌(ZnSO4·7H2O)6E-04mmol、五水合硫酸铜(CuSO4·5H2O)2E-06mmol、氯化镁(MgCl2)0.12mmol、二水合硫酸镁(MgSO4·2H2O)0.9mmol、胸苷5.8E-04mmol、次黄嘌呤6E-03mmol、葡萄糖22mmol、丙酮酸钠1mmol、亚油酸6E-05mmol、硫辛酸2E-04mmol、腐胺2E-04mmol、4-羟乙基哌嗪乙磺酸(HEPES)20mmol;
(2)将称取的上述成分加入800mL纯水搅拌混匀后,定容至1000mL。
(3)可以使用但不限于用1mol/L的NaOH或1mol/L的盐酸,将上述溶液pH值调节至7.2
(4)可以使用但不限于用0.22μm滤膜,将上述溶液过滤除菌,冷藏保存,优选4℃保存。
实施例3
使用由实施例2制备的人源细胞培养基培养脐带间充质干细胞的方法:
(1)在通过实施例2制备的人源细胞培养基中添加体积分数7%的胎牛血清(FBS);
为了对比试验效果,采用对照组培养基:用伊思柯夫改良培养液(IMDM)作为基础培养基,添加体积分数7%胎牛血清(FBS);
(2)取第3代脐带间充质干细胞,加入生理盐水离心清洗后,接分别种至两个培养瓶中,分别添加实验组、对照组培养基;
(3)在37℃,5%二氧化碳(CO2)条件下培养培养至90%融合时传代。
如图1所示,为传代至第6代时的细胞形态图。其中A为采用本发明的人源细胞培养基,B为采用伊思柯夫改良培养液(IMDM)作为培养基。可以看到采用本发明的人源细胞培养基细胞生长速度较快,传代至第6代时的细胞密度明显大于采用伊思柯夫改良培养液(IMDM)作为基础培养基的对照组。
对培养至第6代的细胞进行流式鉴定,具体操作步骤如下:
取第6代细胞,达到80%~90%融合时用胰蛋白酶/乙二胺四乙酸(EDTA)消化液消化,制成1×106/ml细胞悬液。分别加入鼠抗人单克隆抗体CD90-PE、CD73-FITC、HLA-DR-PE各10μL,充分混匀,室温下反应30分钟,每管加入1.5mL磷酸缓冲盐溶液(PBS),1200r/min离心5分钟,弃上清,每管加入100μL磷酸缓冲盐溶液(PBS),流式细胞仪检测。
如图2所示,图2-A为使用本发明的人源细胞培养基培养脐带间充质干细胞至第6代时的细胞对表面标记分子CD90的表达情况;图2-B为使用对照组伊思柯夫改良培养液(IMDM)培养脐带间充质干细胞至第6代时的细胞对表面标记分子CD90的表达情况;图2-C为使用本发明的人源细胞培养基培养脐带间充质干细胞至第6代时的细胞对表面标记分子CD73的表达情况;图2-D为使用对照组伊思柯夫改良培养液(IMDM)培养脐带间充质干细胞至第6代时的细胞对表面标记分子CD73的表达情况;图2-E为使用本发明的人源细胞培养基培养脐带间充质干细胞至第6代时的细胞对表面标记分子HLA-DR的表达情况;图2-F为使用对照组伊思柯夫改良培养液(IMDM)培养脐带间充质干细胞至第6代时的细胞对表面标记分子HLA-DR的表达情况。
脐带间充质干细胞表面分子标志检测结果如下表:
实验结果表明,经多次传代后,实验组培养基较对照组更能保持脐带间充质干细胞的增殖及性状。
实施例4
使用由实施例2制备的人源细胞培养基脐静脉内皮细胞的方法:
(1)在通过实施例2制备的人源细胞培养基中添加内皮细胞生长添加物(ECGS)、50μg/ml肝素钠和体积分数7%的胎牛血清(FBS);
为了对比试验效果,采用对照组培养基:用伊思柯夫改良培养液(IMDM)作为基础培养基,添加内皮细胞生长添加物(ECGS)、50μg/ml肝素钠和体积分数7%的胎牛血清(FBS);
(2)取第3代脐静脉内皮细胞,加入生理盐水离心清洗后,接分别种至两个培养瓶中,分别添加实验组、对照组培养基;
(3)在37℃,5%二氧化碳(CO2)条件下培养培养至80%-90%融合时传代。
细胞培养至第5代,进行测试,具体测试步骤如下:
(1)提前准备将显微镜盖玻片(Microscope Cover Glass)放入24孔板中,铺上0.25%明胶,放至2小时后,晾干待用。
(2)将脐静脉内皮细胞(Huvec细胞)按一定比例种与准备的24孔板中,待细胞基本长满,取出24孔板,弃去培养液,用杜氏磷酸盐缓冲液(DPBS)洗涤两次,5分钟/次。
(3)用4%多聚甲醛(PFA)室温固定15分钟,杜氏磷酸盐缓冲液(DPBS)清洗细胞涂片3次,5分钟/次。
(4)0.5%Triton-X100(供应商:ACROS ORGANICS,CAS:9002-93-1)室温破膜5分钟,杜氏磷酸盐缓冲液(DPBS)清洗细胞涂片3次,5分钟/次。
(5)1%牛血清白蛋白(BSA)于37℃封闭90min。
(6)分别孵育一抗CD31工作液(用1%牛血清白蛋白(BSA)按1:100稀释)和血管内皮钙粘蛋白(VE-Cadherin)工作液(用1%牛血清白蛋白(BSA)按1:100稀释),4℃过夜。
(7)次日细胞涂片复温30分钟,杜氏磷酸盐缓冲液(DPBS)清洗细胞涂片3次,10分钟/次,分别加入不同荧光二抗(用1%牛血清白蛋白(BSA)按1:1000稀释)37℃避光孵育60分钟。杜氏磷酸盐缓冲液(DPBS)清洗细胞涂片3次,10分钟/次。
(8)加入4',6-二脒基-2-苯基吲哚(DAPI),室温避光孵育5分钟。杜氏磷酸盐缓冲液(DPBS)清洗细胞涂片3次,10分钟/次。
(9)50%甘油封片,显微镜观察拍照。
如图3所示,图3-A为使用本发明的人源细胞培养基培养脐静脉内皮细胞至第5代时的细胞对表面标记分子CD31的表达情况;图3-B为使用对照组伊思柯夫改良培养液(IMDM)培养脐静脉内皮细胞至第5代时的细胞对表面标记分子CD31的表达情况;图3-C为使用本发明的人源细胞培养基培养脐静脉内皮细胞至第5代时的细胞对表面标记分子血管内皮钙粘蛋白(VE-Cadherin)的表达情况;图3-D为使用对照组伊思柯夫改良培养液(IMDM)培养脐静脉内皮细胞至第5代时的细胞对表面标记分子血管内皮钙粘蛋白(VE-Cadherin)的表达情况。
如图4所示,继续培养脐静脉内皮细胞传代至第8代时,细胞形态如下:图4-A为使用本发明的人源细胞培养基培养脐静脉内皮细胞至第8代时的细胞形态图;图4-B为使用对照组伊思柯夫改良培养液(IMDM)培养脐静脉内皮细胞至第8代时的细胞形态图。由图可知,使用本发明的人源细胞培养基培养脐静脉内皮细胞至第8代时的细胞密度明显高于使用对照组伊思柯夫改良培养液(IMDM)的培养情况。
实施例5
使用由实施例2制备的人源细胞培养基免疫细胞的方法:
(1)在通过实施例2制备的人源细胞培养基中添加5%的胎牛血清(FBS);
为了对比试验效果,采用对照组培养基:用RPMI-1640培养液作为基础培养基,添加5%的胎牛血清(FBS);
(2)无菌条件下取足月剖腹产或足月正常产乙肝病毒检测阴性孕妇的脐带血50~80mL,放于含CPDA1复合抗凝剂的采血袋中,4℃保存;
(3)将脐带血用生理盐水进行1:1稀释;
(4)将稀释好的脐带血沿管壁缓慢加入含Ficoll Hypaque人淋巴细胞分离液上层(两者比例为1:1),其相对密度为1.077g/L。梯度离心,500g/min,30min;
(5)取中间云雾状的单个核细胞层,用生理盐水稀释混匀后分至两个50mL离心管;分别用实验组及对照组培养基稀释后离心洗涤2次,每次300g/min,5min;
(6)弃上清后分别用培养基重新悬浮细胞调整密度成0.8~1.5×106/mL;
(7)在细胞悬液中添加重组人γ干扰素(IFN-γ)终浓度为1000U/ml,移入培养瓶中,置于37℃,5%二氧化碳(CO2)饱和湿度培养箱中培养24小时;
(8)添加人CD3单克隆抗体,人重组白介素1和人重组白介素2,终浓度分别为100ng/mL,100U/mL和500U/mL;
(9)继续培养48~72小时后显微镜下细胞计数,调节细胞密度为1×105/ml;
(10)每隔72小时按上述相同条件扩大培养一次,培养至第21天时收集细胞待用;
进行流式鉴定,具体操作步骤如下:
分别将免疫细胞制成1×106/ml细胞悬液,分别加入鼠抗人单克隆抗体CD3-FITC、CD56-PE各10μL,充分混匀,室温下反应30min,每管加入1.5mL磷酸缓冲盐溶液(PBS),1200r/min离心5min,弃上清,每管加入100μL磷酸缓冲盐溶液(PBS),流式细胞仪检测。
如图5所示,图5-A为使用本发明的人源细胞培养基培养免疫细胞培养21天时的细胞对表面标记分子CD3+的表达情况;图5-B为使用对照组RPMI-1640培养液培养脐带间充质干细胞培养21天时的细胞对表面标记分子CD3+的表达情况;图5-C为使用本发明的人源细胞培养基培养免疫细胞培养21天时的细胞对表面标记分子CD56+的表达情况;图5-D为使用对照组RPMI-1640培养液培养脐带间充质干细胞培养21天时的细胞对表面标记分子CD56+的表达情况;图5-E为使用本发明的人源细胞培养基培养免疫细胞培养21天时的细胞对表面标记分子CD3+CD56+的表达情况;图5-F为使用对照组RPMI-1640培养液培养免疫细胞培养21天时的细胞对表面标记分子CD3+CD56+的表达情况。
细胞表面分子标志CD3+、CD56+、CD3+CD56+检测结果如下表所示:
表面分子标记 | 实验组阳性率 | 对照组阳性率 |
CD3+ | 80.68% | 91.11% |
CD56+ | 57.64% | 21.88% |
CD3+CD56+ | 38.11% | 14.01% |
实验结果表明,经多次传代后,实验组培养基较对照组更能保持免疫细胞的增殖及性状。
以上结合附图对本发明的实施方式作了详细说明,但本发明不限于所描述的实施方式。对于本领域的技术人员而言,在不脱离本发明原理和精神的情况下,对这些实施方式进行多种变化、修改、替换和变型,仍落入本发明的保护范围内。
Claims (7)
1.一种人源细胞培养基,其特征在于:包括氨基酸、维生素、无机盐、次黄嘌呤、胸苷、葡萄糖、丙酮酸钠、亚油酸、硫辛酸、腐胺、4-羟乙基哌嗪乙磺酸。
2.根据权利要求1所述的人源细胞培养基,其特征在于:
所述氨基酸包括甘氨酸、L-缬氨酸、L-异亮氨酸、L-亮氨酸、L-苯丙氨酸、L-甲硫氨酸、L-色氨酸、L-苏氨酸、L-丝氨酸、L-丙氨酸、L-脯氨酸、L-胱氨酸、L-酪氨酸二钠盐、L-赖氨酸盐酸盐、L-精氨酸盐酸盐、L-组氨酸盐酸盐、L-谷氨酰胺、L-谷氨酸、L-天门冬酰胺或L-天门冬氨酸中的一种或多种;
所述维生素包括叶酸、硫胺素、核黄素、维生素B12、生物素、泛酸钙、盐酸吡哆醛、盐酸吡哆醇、肌醇、氯化胆硷或烟酰胺中的一种或多种;
所述无机盐包括氯化钠、碳酸氢钠、二水合磷酸二氢钠、磷酸氢二钠、五水合亚硒酸钠、氯化钾、硝酸钾、二水合氯化钙、七水合硫酸亚铁、七水合硫酸锌、五水合硫酸铜、氯化镁或二水合硫酸镁中的一种或多种。
3.根据权利要求2所述的人源细胞培养基,其特征在于:所述甘氨酸浓度为0.27~0.41mmol/L;所述L-缬氨酸浓度为0.53~0.80mmol/L;所述L-异亮氨酸浓度为0.52~0.78mmol/L;所述L-亮氨酸浓度为0.53~0.79mmol/L;所述L-苯丙氨酸浓度为0.26~0.39mmol/L;所述L-甲硫氨酸浓度为0.13~0.20mmol/L;所述L-色氨酸浓度为0.05~0.08mmol/L;所述L-苏氨酸浓度为0.53~0.79mmol/L;所述L-丝氨酸浓度为0.27~0.41mmol/L;所述L-丙氨酸浓度为0.20~0.29mmol/L;所述L-脯氨酸浓度为0.27~0.41mmol/L;所述L-胱氨酸浓度为0.22~0.33mmol/L;所述L-酪氨酸二钠盐浓度为0.30~0.45mmol/L;所述L-赖氨酸盐酸盐浓度为0.54~0.81mmol/L;所述L-精氨酸盐酸盐浓度为0.41~0.62mmol/L;所述L-组氨酸盐酸盐浓度为0.14~0.22mmol/L;所述L-谷氨酰胺浓度为2.72~4.08mmol/L;所述L-谷氨酸浓度为0.34~0.51mmol/L;所述L-天门冬酰胺浓度为0.14~0.21mmol/L;所述L-天门冬氨酸浓度为0.16~0.24mmol/L;所述叶酸浓度为6.3E-03~9.4E-03mmol/L;所述硫胺素浓度为7.7E-03~1.2E-02mmol/L;所述核黄素浓度为7.0E-04~1.0E-03mmol/L;所述维生素B12浓度为1.7E-04~2.6E-04mmol/L;所述生物素浓度为3.9E-05~5.8E-05mmol/L;所述泛酸钙浓度为5.5E-03~8.3E-03mmol/L;所述盐酸吡哆醛浓度为1.3E-02~1.9E-02mmol/L;所述盐酸吡哆醇浓度为4.7E-05~7.0E-05mmol/L;所述肌醇浓度为4.2E-02~6.2E-02mmol/L;所述氯化胆硷浓度为3.4E-02~5.1E-02mmol/L;所述烟酰胺浓度为2.1E-02~3.2E-02mmol/L;所述氯化钠浓度为71~106mmol/L;所述碳酸氢钠浓度为25~38mmol/L;所述二水合磷酸二氢钠浓度为0.58~0.87mmol/L;所述铃酸氢二钠浓度为0.16~0.24mmol/L;所述五水合亚硒酸钠浓度为4.2E-05~6.3E-05mmol/L;所述氯化钾浓度为3.3~4.9mmol/L;所述硝酸钾浓度为4.8E-04~7.2E-04mmol/L;所述二水合氯化钙浓度为1.0~1.5mmol/L;所述七水合硫酸亚铁浓度为4.8E-04~7.2E-04mmol/L;所述七水合硫酸锌浓度为4.8E-04~7.2E-04mmol/L;所述五水合硫酸铜浓度为1.6E-06~2.4E-06mmol/L;所述氯化镁浓度为0.10~0.14mmol/L;所述二水合硫酸镁浓度为0.73~1.09mmol/L;所述胸苷浓度为4.6E-04~6.9E-04mmol/L;所述次黄嘌呤浓度为4.8E-03~7.2E-03mmol/L;所述葡萄糖浓度为18~26mmol/L;所述丙酮酸钠浓度为0.8~1.2mmol/L;所述亚油酸浓度为4.8E-05~7.2E-05mmol/L;所述硫辛酸浓度为1.6E-04~2.4E-04mmol/L;所述腐胺浓度为1.6E-04~2.4E-04mmol/L;所述4-羟乙基哌嗪乙磺酸浓度为16~24mmol/L。
4.根据权利要求1~3中任一权利要求所述的人源细胞培养基,其特征在于:所述培养基PH值为6.8~7.4。
5.根据权利要求1~4中任一权利要求所述的人源细胞培养基,其特征在于:所述培养基渗透压为280-330mOsm/kg。
6.一种制备如权利要求1~3所述的人源细胞培养基的方法,其特征在于:包括如下步骤:
(1)称取如权利要求3所述的培养基各组分;
(2)加入纯水搅拌混合均匀后定容;
(3)调节溶液pH值至7.2;
(4)将溶液过滤除菌,冷藏保存。
7.使用如权利要求6所述的人源细胞培养基培养细胞的方法,其特征在于:包括如下步骤:
(1)在如权利要求1~3所述的人源细胞培养基中再加入体积百分数0%-20%的胎牛血清;
(2)将细胞加入生理盐水离心清洗后,接种到培养瓶中,加入步骤(1)的培养基;
(3)在37℃,5%二氧化碳条件下培养到悬浮细胞达到1~2*106/ml时传代。
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CN115786254A (zh) * | 2022-11-30 | 2023-03-14 | 海南苏生生物科技有限公司 | 一种促进体外培养细胞生成外泌体的培养基及其诱导方法 |
CN115786254B (zh) * | 2022-11-30 | 2023-09-29 | 海南苏生生物科技有限公司 | 一种促进体外培养细胞生成外泌体的培养基及其诱导方法 |
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