CN113583955A - 一种因子法扩增t细胞的培养基及培养方法 - Google Patents
一种因子法扩增t细胞的培养基及培养方法 Download PDFInfo
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Abstract
本发明公开了一种因子法扩增T细胞的培养基及培养方法,包括包被培养液1;包被培养液2;第一阶段培养液;第二阶段培养液;第三阶段培养基及第四阶段培养基等共五个阶段,利用所述方法可以提高扩增效率,得到的T细胞纯度良好,且得到的T细胞亚型种类多。
Description
技术领域
本发明涉及细胞制备技术领域,具体涉及一种因子法扩增T细胞的培养基及培养方法。
背景技术
T细胞即胸腺依赖性淋巴细胞,是淋巴细胞的主要组分,它具有多种生物学功能,如直接杀伤靶细胞、辅助或抑制B细胞产生抗体,对特异性抗原和促有丝分裂原的应答反应以及产生细胞因子等。T 细胞产生的免疫应答是细胞免疫,细胞免疫的效应形式主要有两种:与靶细胞特异性结合,破坏靶细胞膜,直接杀伤靶细胞;另一种是释放淋巴因子,最终使免疫效应扩大和增强。
目前大家所说的免疫细胞治疗通常是指T细胞治疗法,通过体外培养、增殖、激活,回输体内即可诱导自体免疫应答。有杀伤肿瘤细胞作用的T细胞经激活后在体内大多数变为记忆细胞储存在淋巴组织内,为彻底清除肿瘤细胞和防治转移复发提供了长期保护。
目前大多数T细胞的生产是扩增的T细胞在最后阶段被纯化,通常是通过磁分选来消除其他免疫细胞,如NK、B或DC细胞。安全高效的生产方法是获得大量功能性T细胞的关键,而T细胞与生长失活的滋养细胞(如PBMC 或K562细胞系)共培养是目前最常用的一种策略,对T细胞的活化和扩增有显著影响。尽管细胞系如K562共培养帮助T细胞扩增取得了显著的效果,但这些用肿瘤细胞系进行扩增的方法仍然不确定是否存在安全隐患,本发明提供一种用因子法扩增T细胞的培养基及培养方法。
发明内容
发明目的:为避免T细胞扩增使用生长失活的滋养细胞,本发明提供一种用因子法扩增T细胞的方案,包括包被培养液1;包被培养液2;第一阶段培养液;第二阶段培养液;第三阶段培养基及第四阶段培养基等共五个阶段,用本方案的得到的细胞数量多且T细胞亚型更多。
本发明的有益效果如下:
以本发明提供的因子法扩增T细胞的培养基及培养方法,扩增效率高、纯度良好,且得到的T细胞亚型种类多,更有利于T细胞发挥作用。
附图说明
图1是流式检测实施例及其对比组的CD3+CD56-阳性率的结果图;
图2是流式检测阴性组的CD3+CD56-阳性率的结果图;
图3 实施例T细胞种类检测阳性结果图;
图4 实施例T细胞种类检测阴性组结果图。
具体实施方式
为了使本技术领域的人员更好的理解本发明方案,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整的描述。
一种因子法扩增T细胞的培养基及培养方法,采用五个阶段对T细胞分别进行逐步的刺激、丰富及扩增,所述外周血也可以包括脐带血。
实施例
本实施例提供T细胞的培养基及培养方法。
本实施例采用的包被培养液1;包被培养液2;第一阶段培养液;第二阶段培养液;第三阶段培养基及第四阶段培养基,包被液以10ml为例,培养液以50ml为例:
包被培养液1中CD3、CD28的终浓度比为1∶1,具体配方为:10mL K581基础培养基中加入终浓度为5ug/mL的CD3、终浓度为5ug/mL的CD28。
包被培养液2中10mL K581基础培养基中加入DLL1的终浓度为5ug/mL。
所述第一阶段培养液中IL-2、IL-12、IFN-γ的终浓度比为10∶1∶1;具体配方为:45mL K581基础培养基中加入5 mL自体灭火血浆并加入终浓度为1200U/mL的IL-2、终浓度为50ug/mL的IL-12、终浓度为50ug/mL的IFN-γ。
所述第二阶段培养液中IL-2、IL-4、IL-27、TGF-B的终浓度比为10∶1∶1∶0.5;具体配方为:45mL K581基础培养基中加入5 mL自体灭火血浆并加入终浓度为1000U/mL的 IL-2、终浓度为40ug/mL的IL-4、终浓度为40ug/mL的IL-27、终浓度为20ug/mL的TGF-B。
所述第三阶段培养液中IL-2、IL-1b、IL-6、IL-23的终浓度比为10∶1∶1∶1;具体配方为:45mL K581基础培养基中加入2.5mL自体灭火血浆并加入终浓度为800U/mL的IL-2、终浓度为16ug/mL的 IL-1b、终浓度为16ug/mL的IL-6、终浓度为16ug/mL的 IL-23。
所述第四阶段培养液中45mL K581基础培养基中加入2.5mL自体灭火血浆并加入终浓度为500U/mL的 IL-2。
本实施例采用T细胞的扩增方法包括:
Day0-day1使用包被培养液1包被T25 T细胞扩增培养瓶置于4℃冰箱;
Day1接种外周血单核细胞,使用第一阶段培养液,单个核细胞接种量为1×106/mL,接种10mL,期间不换液;
Day2使用包被培养液2包被新的T25 T细胞扩增培养瓶置于4℃冰箱;
Day3将接种细胞转移至新的T25 T细胞扩增培养瓶并使用第二阶段培养液,期间不换液;
Day4将第二阶段培养液更换为第三阶段培养基;
Day5将第三阶段培养液更换为第四阶段培养基,隔1天对细胞进行计数并补加第四阶段培养基;
为了做比较,本实施例还设置了对比组,对比组一与本实施例的区别仅在于不使用包被培养液2,对比组二与本实施例的区别仅在于第一阶段培养液的因子浓度减半:45mLK581基础培养基中加入5 mL自体灭火血浆并加入终浓度为600U/mL的IL-2、终浓度为25ug/mL的IL-12、终浓度为25ug/mL的IFN-γ。
实施例的结果测试
CD3+CD56-阳性率的检测:取实施例及其对比组最终所得细胞,收集细胞到50mL离心管中1000r/min离心5min,弃上清后用1mL含2%(v/v)血清的PBS重悬,加入抗体CD3-FITC、CD56-APC,4℃孵育30min后用含2%血清的PBS洗一遍。然后以5×106 /ml的密度重悬细胞,使用Agilent NovoCyte 流式仪细胞仪检测并分析其分化效率。在检测过程中,以未用流式抗体染色的细胞作为阴性对照。
在第14天对T细胞的种类进行检测,检测指标为CD4+、CD8+及其他细胞三大类。CD4+、CD8+阳性率的检测:取实施例及其对比组最终所得细胞,收集细胞到50mL离心管中1000r/min离心5min,弃上清后用1mL含2%(v/v)血清的PBS重悬,加入抗体CD8-PE、CD4-APC,4℃孵育30min后用含2%血清的PBS洗一遍。然后以5×106 /ml的密度重悬细胞,使用Agilent NovoCyte 流式仪细胞仪检测并分析其分化效率。在检测过程中,以未用流式抗体染色的细胞作为阴性对照。
结果实施例1及其对比组测试结果如图1所示,流式细胞分析发现实施例1的CD3+CD56-阳性率上升到74.26% (其对比组1为43.82%,对比组2为58.87%);得到的T细胞种类丰富,其中实施例1中CD4+细胞含62.36%、CD8+细胞含15.61%、其他种类T细胞共22.03%。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
显然,所描述的实施例仅仅本发明的一部分实施例,而不是全部的实施例。基于本发明中的实施例,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例,都应当属于本发明保护的范围。
Claims (9)
1.一种因子法扩增T细胞的培养基,其特征在于,包括包被培养液1;包被培养液2;第一阶段培养液;第二阶段培养液;第三阶段培养基及第四阶段培养基;所述包被培养液1包括K581基础培养基、CD3、CD28;包被培养液2包括K581基础培养基、DLL1;所述第一阶段培养液包括K581基础培养基、IL-2、IL-12、IFN-γ、自体灭活血浆;所述第二阶段培养液包括K581基础培养基、IL-2、IL-4、IL-27、TGF-B、自体灭活血浆;所述第三阶段培养液包括K581基础培养基、IL-2、IL-1b、IL-6、IL-23、自体灭活血浆;所述第四阶段培养液包括K581基础培养基、IL-2、自体灭活血浆。
2.根据权利要求1所述的一种因子法扩增T细胞的培养基,其特征在于,所述包括包被培养液1中CD3、CD28的终浓度为1-10ug/mL;所述包括包被培养液2中DLL1的终浓度为1-10ug/mL;所述的第一阶段培养液中IL-2的终浓度为1000-2000U/mL、IL-12的终浓度为5-100ng/mL、IFN-γ的终浓度为50-250ng/mL、自体灭活血浆的体积分数为10%;所述的第二阶段培养液中IL-2的终浓度为1000-2000U/mL、IL-4的终浓度为5-100ng/mL、IL-27的终浓度为2-100ng/mL、TGF-B的终浓度为2-50ng/mL、自体灭活血浆的体积分数为10%;所述的第二阶段培养液中IL-2的终浓度为1000-2000U/mL、IL-4的终浓度为5-100ng/mL、IL-27的终浓度为2-100ng/mL、TGF-B的终浓度为2-100ng/mL、自体灭活血浆的体积分数为10%;所述的第三阶段培养液中IL-2的终浓度为1000-2000U/mL、IL-6的终浓度为5-50ng/mL、IL-23的终浓度为2-100ng/mL、TGF-B的终浓度为2-50ng/mL、自体灭活血浆的体积分数为5%;所述的第四阶段培养液中IL-2的终浓度为1000-2000U/mL、自体灭活血浆的体积分数为5%。
3.根据权利要求1所述的一种因子法扩增T细胞的培养基,其特征在于,所述包被培养液1中CD3、CD28的终浓度比为1∶1;所述第一阶段培养液中IL-2、IL-12、IFN-γ的终浓度比为10∶1∶1;所述第二阶段培养液中IL-2、IL-4、IL-27、TGF-B的终浓度比为10∶1∶1∶0.5;所述第三阶段培养液中IL-2、IL-1b、IL-6、IL-23的终浓度比为10∶1∶1∶1。
4.根据权利要求1所述的一种因子法扩增T细胞的培养基,其特征在于,所述包被培养液1中CD3的终浓度为5ug/mL最优、CD28的终浓度为5ug/mL最优;所述包被培养液2中DLL1的终浓度为5ug/mL最优;所述第一阶段培养液中IL-2的终浓度为1200U/mL最优、IL-12的终浓度为50ug/mL最优、IFN-γ终浓度为50ug/mL最优;所述第二阶段培养液中IL-2的终浓度为1000U/mL最优、IL-4的终浓度为40ug/mL最优、IL-27的终浓度为40ug/mL最优、TGF-B的终浓度为20ug/mL最优;第三阶段培养液中IL-2的终浓度为800U/mL最优、IL-1b的终浓度为16ug/mL最优、IL-6的终浓度为16ug/mL最优、IL-23的终浓度为16ug/mL最优;第四阶段培养液中IL-2的终浓度为500U/mL最优。
5.一种因子法扩增T细胞的方法,其特征在于,包括:如下步骤:
S1:包被阶段:包被培养液1包被需要使用的T细胞扩增培养瓶;
S2:刺激阶段:第一阶段培养液刺激T细胞;
S3:二次包被阶段:包被培养液2包被新的T细胞扩增培养瓶;
S4:丰富阶段:第二阶段培养液、第三阶段培养液丰富T细胞亚型;
S5:扩增阶段:第四阶段培养液扩增T细胞。
6.根据权利要求5所述的一种因子法扩增T细胞的方法,其特征在于,提前一天使用包被液包被T细胞扩增瓶在4℃包被1天。
7.根据权利要求5所述的一种因子法扩增T细胞的方法,其特征在于,所述包被阶段的包被培养液1包被的的第0天至第1天,不适用任何培养基;所述刺激阶段使用的第一阶段培养液激活T细胞,从第1天至第3天,期间不换液,第3天细胞转移至包被培养液2包被的新板中;所述二次包被阶段使用包被培养液2包被T细胞培养瓶为T细胞扩增提供类似体内胸腺环境,培养细胞的第2天至第3天,不适用任何培养基;所述丰富阶段使用第二阶段培养液、第三阶段培养液丰富T细胞亚型,从第3天至第4天使用第二阶段培养液,从第4天至第5天使用第三阶段培养液;所述扩增阶段使用第四阶段培养液扩增T细胞,从第5起,隔1天对细胞进行计数并补加培养基。
8.根据权利要求5所述的一种因子法扩增T细胞的方法,其特征在于,单个核细胞接种量为1×106-2×106/ml,起始接种10ml。
9.根据权利要求5所述的一种因子法扩增T细胞的方法,其特征在于,所述外周血也包括脐带血。
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