CN116656607B - 一种t细胞无血清培养基及其应用 - Google Patents
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Abstract
本发明涉及一种T细胞无血清培养基及其应用,所述培养基包含终浓度为100~1000μg/mL的烷基葡糖苷(APG)和体积终浓度为0.05~0.2%的吐温‑80。从人外周血中分离出PBMC,在进行T细胞激活培养的第0天在培养体系中加入APG和吐温‑80进行培养,可以有效提高从PBMC中诱导激活的T细胞的慢病毒转导效率,同时不影响T细胞扩增能力和活性状态,为临床治疗过程中获得更多CAR‑T细胞提供了有力手段。
Description
技术领域
本发明涉及细胞培养基领域,具体为一种T细胞无血清培养基及其应用。
背景技术
与传统肿瘤治疗方法,放疗、化疗和手术治疗方法相比,嵌合抗原受体T (CAR-T)细胞疗法被认为是治疗复发或难治性肿瘤的有效解决方案,尤其是血液系统恶性肿瘤,已经取得令人瞩目的治疗效果。CAR-T细胞疗法主要过程是从病人体内分离出T细胞,在体外进行基因改造,使其获得识别并攻击具有特定靶点肿瘤的功能,然后在体外大量扩增,再回输到病人体内。经过改造的CAR-T细胞接触到特定靶点的肿瘤细胞后迅速活化,扩增,通过分泌颗粒酶、穿孔素和一型干扰素等方式杀伤肿瘤细胞,达到帮助病人清除肿瘤的治疗效果。对T细胞进行基因改造的过程中,主要是通过慢病毒将编码目的基因的序列整合到其基因组上,使其表达识别特异肿瘤靶点的T细胞受体。与一般体细胞相比,T淋巴细胞存在难以进行慢病毒转导的问题。因此,如何提高T细胞慢病毒转导效率,是改进CAR-T细胞疗法的重要途径。
发明内容
针对现有技术的不足,本发明提供一种T细胞无血清培养基及其应用,以弥补现有技术的不足。
为了解决上述技术问题,本发明提供如下技术方案:
一种T细胞无血清培养基,包括基础培养基和添加复合物,
所述的添加复合物包括:终浓度为100~1000μg/mL的APG和体积终浓度为0.05~0.2%的吐温-80。
优选的,所述的添加复合物包括:终浓度为500μg/mL的APG和体积终浓度为0.12%的吐温-80。
优选的,所述的基础培养基为康宁KBM581无血清培养基、OpTmizer T细胞无血清培养基或依科赛T细胞无血清培养基的任意一种。
上述任一所述的一种T细胞无血清培养基的应用,用于T细胞的培养。
优选的,用于T细胞的慢病毒转导培养。
本发明的有益效果:本发明提供的T细胞无血清培养基通过在基础培养基中添加终浓度为100~1000μg/mL的APG和体积终浓度为0.05~0.2%的吐温-80,可以有效提高从PBMC中诱导激活的T细胞的慢病毒转导效率,同时不影响T细胞扩增能力和活性状态。
本发明提供了一种增强T细胞体外培养过程中转导效率的组合物,为临床治疗过程中获得更多CAR-T细胞提供了有力手段。
本发明的作用机理:T细胞体外转导过程中,除了病毒和转导试剂的特性外,培养基成分也是影响转导效率的重要因素。烷基葡糖苷(Alkyl polyglucoside;APG),分子量320,由葡萄糖的半缩醛羟基和脂肪醇羟基在催化作用下脱水而成,是一种非离子型两亲性分子,因此很可能和细胞膜相互作用促进生物大分子(比如核酸、病毒)的递送,经实验验证,加入培养基中后可促进慢病毒转导入T细胞。此外,APG与其它表面活性剂复配有明显增效作用。除了常用的阴离子表面活性剂LAS,AES外,其他的如吐温-80、甜菜碱、氧化胺等进行复配,复配后的产品不但可以降低表面活性剂的刺激性,还可以提高表面活性。吐温-80,又名聚山梨醇-80,是一种非离子型表面活性剂,有增加细胞膜通透性的作用,可辅助APG发挥作用。本发明在培养基中添加APG和吐温-80,可协同作用提高慢病毒转导效率。
附图说明
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。
在附图中:
图1是本发明实施例1中阴性对照流式细胞检测图;
图2是本发明实施例1中0μg/mL APG+0%吐温-80组合条件下流式细胞检测图;
图3是本发明实施例1中100μg/mL APG+0.05%吐温-80组合条件下流式细胞检测图;
图4是本发明实施例1中500μg/mL APG+0.12%吐温-80组合条件下流式细胞检测图;
图5是本发明实施例1中1000μg/mL APG+0.2%吐温-80组合条件下流式细胞检测图;
图6是本发明实施例2中阴性对照流式细胞检测图;
图7是本发明实施例2中0μg/mL APG+0%吐温-80组合条件下流式细胞检测图;
图8是本发明实施例2中100μg/mL APG+0.05%吐温-80组合条件下流式细胞检测图;
图9是本发明实施例2中500μg/mL APG+0.12%吐温-80组合条件下流式细胞检测图;
图10是本发明实施例2中1000μg/mL APG+0.2%吐温-80组合条件下流式细胞检测图;
图11是本发明实施例3中阴性对照流式细胞检测图;
图12是本发明实施例3中0μg/mL APG+0%吐温-80组合条件下流式细胞检测图;
图13是本发明实施例3中100μg/mL APG+0.05%吐温-80组合条件下流式细胞检测图;
图14是本发明实施例3中500μg/mL APG+0.12%吐温-80组合条件下流式细胞检测图;
图15是本发明实施例3中1000μg/mL APG+0.2%吐温-80组合条件下流式细胞检测图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,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,都属于本发明保护的范围。
一、实验试剂:
康宁KBM581无血清培养基(CORNING康宁,货号88-581-CM);
OpTmizer T细胞无血清培养基(Thermofisher 赛默飞,货号A10221);
依科赛T细胞无血清培养基(依科赛,货号TE000-N032);
抗人CD3抗体(Biolegend百进生物,货号317236);
抗人CD28抗体(Biolegend百进生物,货号302934);
rhIL-2(艾策生物,货号AC52388);
烷基葡糖苷(Alkyl polyglucoside;APG)(尤尼威尔,货号14674);
吐温-80(Sigma Aldrich西格玛,货号P4780-100ML);
慢病毒(吉凯基因 慢病毒制备载体编号GV365);
病毒感染试剂(吉凯基因,货号REVG005)。
二、实验方法:
(1)T细胞培养和慢病毒转导:
使用密度离心法从人全血中新鲜分离PBMC用于T细胞的激活扩增。第-1天(接种细胞前一天),用PBS将1μg/mL抗人CD3抗体和0.5μg/mL抗人CD28抗体包被于12孔板中。第0天,将分离好的PBMC细胞悬液转移至准备好的包被了抗人CD3抗体和抗人CD28抗体的12孔板中,使用T细胞培养基调整细胞密度为0.8×106个/mL,添加300IU/mL rhIL-2,添加APG(终浓度100~1000μg/mL)和吐温-80(体积终浓度0.05~0.2%),放入37℃,5%的二氧化碳培养箱内进行细胞激活。第1天,按照MOI=3(一个细胞对应3个病毒数)加入慢病毒和病毒感染试剂;第3天,将细胞转入六孔板,补加培养基2~3mL;此后每两至三天,取样计数,以0.7×106个/mL细胞密度进行传代,直到第10天,进行感染效率检测。
(2)T细胞的细胞活力及增殖效率检测:
在培养的第5,7,10天取样进行计数,获得细胞活率和细胞增殖的数据。
(3)T细胞转导效率检测:
在细胞培养第10天时,进行细胞转导效率检测。取约1.0×106个细胞,400 g离心5分钟,去上清后用1mL PBS洗一遍,0.5mL PBS重悬,流式细胞仪进行检测分析。阴性对照为未进行转导的1.0×106个细胞,同样操作后进行流式检测分析。
三、实施例和实验结果:
下面结合具体实施例对本发明进行具体说明。
实施例1:
完全按照上述实验方法对T细胞进行培养与鉴定,第0天开始向康宁KBM581无血清培养基中加入300IU/mL rhIL-2,不同浓度组合的APG和吐温-80,继续培养至D10。
细胞活率实验结果为:D10时,0μg/mL APG+0%吐温-80组合条件下细胞活率为78.3%,100μg/mL APG+0.05%吐温-80组合条件下细胞活率为76.1%,500μg/mL APG+0.12%吐温-80组合条件下细胞活率为80.4%,1000μg/mL APG+0.2%吐温-80组合条件下细胞活率为81.2%,因此,添加本发明中的组合物不影响T细胞体外转导过程中的细胞活率。
细胞扩增实验结果为:D10时,0μg/mL APG+0%吐温-80组合条件下细胞扩增倍数为120,100μg/mL APG+0.05%吐温-80组合条件下细胞活扩增倍数为121,500μg/mL APG+0.12%吐温-80组合条件下细胞扩增倍数为125,1000μg/mL APG +0.2%吐温-80组合条件下细胞扩增倍数为118,因此,添加本发明中的组合物不影响T细胞体外转导过程中的细胞扩增。
实验结果如图1~图5:D10时,0μg/mL APG+0%吐温-80组合条件下转导效率为40.9%,100μg/mL APG+0.05%吐温-80组合条件下转导效率为54.4%,500μg/mL APG+0.12%吐温-80组合条件下转导效率为64.6%,1000μg/mL APG+0.2%吐温-80组合条件下转导效率为51.0%,因此,添加了本发明中的组合物可以提高T细胞转导效率,其中500μg/mL APG+0.12%吐温-80组合转导效率提高效果最佳。
实施例2:
完全按照上述实验方法对T细胞进行培养与鉴定,第0天开始向OpTmizer T细胞无血清培养基中加入300IU/mL rhIL-2,不同浓度组合的APG和吐温-80,继续培养至D10。
细胞活率实验结果为:D10时,0μg/mL APG+0%吐温-80组合条件下细胞活率为85.2%,100μg/mL APG+0.05%吐温-80组合条件下细胞活率为81.6%,500μg/mL APG+0.12%吐温-80组合条件下细胞活率为86.3%,1000μg/mL APG+0.2%吐温-80组合条件下细胞活率为86.1%,因此,添加本发明中的组合物不影响T细胞体外转导过程中的细胞活率。
细胞扩增实验结果为:D10时,0μg/mL APG+0%吐温-80组合条件下细胞扩增倍数为142,100μg/mL APG+0.05%吐温-80组合条件下细胞活扩增倍数为140,500μg/mL APG+0.12%吐温-80组合条件下细胞扩增倍数为145,1000μg/mL APG +0.2%吐温-80组合条件下细胞扩增倍数为139,因此,添加本发明中的组合物不影响T细胞体外转导过程中的细胞扩增。
实验结果如如图6~图10:D10时,0μg/mL APG+0%吐温-80组合条件下转导效率为55.4%,100μg/mL APG+0.05%吐温-80组合条件下转导效率为61.5%,500μg/mL APG+0.12%吐温-80组合条件下转导效率为69.9%,1000μg/mL APG+0.2%吐温-80组合条件下转导效率为69.5%,因此,添加了本发明中的组合物可以提高T细胞转导效率,其中500μg/mL APG+0.12%吐温-80组合转导效率提高效果最佳。
实施例3:
完全按照上述实验方法对T细胞进行培养与鉴定,第0天开始向依科赛T细胞无血清培养基中加入300IU/mL rhIL-2,不同浓度组合的APG和吐温-80,继续培养至D10。
细胞活率实验结果为:D10时,0μg/mL APG+0%吐温-80组合条件下细胞活率为92.3%,100μg/mL APG+0.05%吐温-80组合条件下细胞活率为93.6%,500μg/mL APG+0.12%吐温-80组合条件下细胞活率为96.1%,1000μg/mL APG+0.2%吐温-80组合条件下细胞活率为92.6%,因此,添加本发明中的组合物不影响T细胞体外转导过程中的细胞活率。
细胞扩增实验结果为:D10时,0μg/mL APG+0%吐温-80组合条件下细胞扩增倍数为183,100μg/mL APG+0.05%吐温-80组合条件下细胞活扩增倍数为170,500μg/mL APG+0.12%吐温-80组合条件下细胞扩增倍数为182,1000μg/mL APG+0.2%吐温-80组合条件下细胞扩增倍数为185,因此,添加本发明中的组合物不影响T细胞体外转导过程中的细胞扩增。
实验结果如图11~图15:D10时,0μg/mL APG+0%吐温-80组合条件下转导效率为52.2%,100μg/mL APG+0.05%吐温-80组合条件下转导效率为66.8%,500μg/mL APG+0.12%吐温-80组合条件下转导效率为73.8%,1000μg/mL APG+0.2%吐温-80组合条件下转导效率为71.5%,因此,添加了本发明中的组合物可以提高T细胞转导效率,其中500μg/mL APG+0.12%吐温-80组合转导效率提高效果最佳。
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程方法物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程方法物品或者设备所固有的要素。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各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。凡在本发明的精神和原则之内,所作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 (3)
1.一种T细胞无血清培养基,其特征在于,由基础培养基和添加复合物组成,
所述的添加复合物为:终浓度为100~1000μg/mL的APG和体积终浓度为0.05~0.2%的吐温-80;
所述的基础培养基为康宁KBM581无血清培养基、OpTmizer T细胞无血清培养基或依科赛T细胞无血清培养基的任意一种。
2.根据权利要求1所述的一种T细胞无血清培养基,其特征在于,所述的添加复合物为:终浓度为500μg/mL的APG和体积终浓度为0.12%的吐温-80。
3.根据权利要求1或2任一所述的一种T细胞无血清培养基的应用,其特征在于,用于T细胞的慢病毒转导培养。
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