CN113005082B - 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T细胞无血清培养基由基础培养基和添加成分混合配制而成，所述添加成分由重组人胰岛素、重组人转铁蛋白、重组人血清白蛋白、胸腺嘧啶、牛磺酸和亚叶酸组成，所述基础培养基是IMDM、DMEM、DMEM/F12、aMEM、RPMI1640、M199中的一种或几种的混合。本发明的培养基不仅化学成分明确，无血清，无人源和动物源成分，而且加入的成分种类更少，简化了配方，性能优异，T细胞培养10至12天可扩增400倍至1000倍，终末细胞活率可达85％‑95％。
Description
技术领域
本发明涉及细胞培养技术领域，具体地说，涉及一种T细胞无血清培养基及其应用。
背景技术
T细胞是淋巴细胞的主要组分之一，它具有多种生物学功能，如直接杀伤靶细胞，辅助其它淋巴细胞发挥功能，对特异性抗原或促有丝分裂原的应答反应以及产生细胞因子，是身体抵御疾病感染、防止肿瘤形成的主要免疫细胞之一。在体外富集、活化、扩增这类免疫细胞，可用于回输治疗多种疾病，包括恶性肿瘤、感染、自身免疫病等。在这个过程中，使用安全可靠、化学成分明确的培养基对T细胞进行扩增，避免可能的人源、动物源病原体的引入风险，同时避免T细胞扩增体系汇总不明成分可能引起的机体免疫反应，对后续T细胞治疗产品的临床应用非常关键。
传统的T细胞培养基包含一定比例的血清。这种培养基的问题在于：1)血清批次间差异导致T细胞扩增效果不稳定；2)含异种动物(一般为牛)来源的血清成分，存在引入异种动物病原体感染的风险，增加了临床使用的风险；3)血清成分复杂，其中含有多种蛋白、生长因子等，其中有些成分会导致T细胞的过度激活和耗竭，不能达到最佳的T细胞扩增效果；4)培养体系中异种动物源成分的可能残留，增加了细胞治疗产品质量控制和临床申报的困难。
现有的商业化T细胞无血清培养基存在以下问题：1)配方成分复杂，除基础培养基外，需添加至少十几种其它成分；2)大部分需添加人血清来源的人源白蛋白，增加了成分的复杂程度、原料来源的不稳定性及生产制造的难度，不能做到真正的化学成分明确，临床使用具有很大的不确定性。
专利文献CN108642006A，公开日2018.10.12，公开了一种T细胞无血清培养基及其使用方法，所述T细胞无血清培养基包括用于细胞生长培养的基础培养基，以及其他添加成分，所述其他添加成分包括乙醇胺、硫酸酮、硝酸铁、硫酸锌、亚硒酸钠、丙酮酸钠、胰岛素、转铁蛋白、谷氨酰胺、血清白蛋白、硫代甘油和L-维生素C。取得的有益效果在于：在开发的整个培养基体系中没有引入非人源性蛋白，可对来源安全可靠的血液样本中的T细胞进行选择性地高效扩增。
上述专利文献公开的T细胞培养基，血清白蛋白使用的是酵母表达生产的重组人血清白蛋白，转铁蛋白使用的是酵母表达生产的重组人转铁蛋白，人胰岛素是使用酵母为载体生产出来的药用级的胰岛素，因而避免了培养基混入人源成分或其他动物来源成分。但仍然存在添加成分种类较多的缺陷，扩增效率稍显不足，细胞质量有待提高。因此有必要提供成分更简单，扩增效率高，培养的细胞质量更高的无血清及无人源、无动物源成分的T细胞培养基。
发明内容
本发明的目的是针对现有技术中的不足，提供一种成分更简单，扩增效率高，培养的细胞质量更高的无血清及无人源、无动物源成分的T细胞培养基。
本发明的再一的目的是，提供所述T细胞无血清培养基的制备方法。
本发明的另一的目的是，提供所述T细胞无血清培养基的用途。
为实现上述第一个目的，本发明采取的技术方案是：
一种T细胞无血清培养基，所述T细胞无血清培养基由基础培养基和添加成分混合配制而成，所述添加成分由重组人胰岛素、重组人转铁蛋白、重组人血清白蛋白、胸腺嘧啶、牛磺酸和亚叶酸组成。
作为本发明的一种优选实施方案，每升所述T细胞无血清培养基中：所述重组人胰岛素为2.5-50mg，所述重组人转铁蛋白为5-100mg，所述重组人血清白蛋白为0.2-5g，所述胸腺嘧啶为0.2-20mg，牛磺酸为0.1-10mg，所述亚叶酸为0.5-50mg。
作为本发明的另一种优选实施方案，所述基础培养基是IMDM、DMEM、DMEM/F12、aMEM、RPMI1640、M199中的一种或几种的混合。
作为其中的一个优选例，所述基础培养基是IMDM、DMEM/F12和RPMI1640三种培养基的混合。
更优选地，所述IMDM、DMEM/F12和RPMI1640三种培养基的体积比为1:1:1。
作为其中的另一个优选例，所述基础培养基是IMDM和RPMI1640两种培养基的混合。
更优选地，所述IMDM和RPMI1640两种培养基的体积比为1:1。
为实现上述第二个目的，本发明采取的技术方案是：
如上任一所述的T细胞无血清培养基的制备方法。
为实现上述第三个目的，本发明采取的技术方案是：
如上任一所述的T细胞无血清培养基在培养T细胞中的应用。
本发明优点在于：
1、本发明的T细胞培养基化学成分明确，无血清，无人源和动物源成分，添加的人胰岛素、人转铁蛋白、人血清白蛋白均为重组表达产品，和传统的含血清及成分不明确的T细胞培养基相比，具有更高的科研和临床应用价值。
2、本发明的T细胞培养基通过配方的优化，去除传统配方中互相冲突的成分，简化了配方，添加成分种类更少，在科研和可能的临床应用中具有更高的稳定性和确定性。
3、本发明的T细胞培养基性能优异，T细胞培养10至12天可扩增400倍至1000倍，终末细胞活率可达85％-95％。
附图说明
图1：对应实施例1，为本发明所述T细胞无血清培养基中激活扩增的PBMC中T细胞增殖曲线。其中A为商品化的T细胞无血清培养基，B为本发明所述T细胞无血清培养基。
图2：对应实施例1，为本发明所述T细胞无血清培养基中激活扩增的PBMC中T细胞增殖过程中的细胞活率数据。其中A为商品化的T细胞无血清培养基，B为本发明所述T细胞无血清培养基。
图3：对应实施例2，为本发明所述T细胞无血清培养基中激活扩增的PBMC中T细胞增殖曲线。其中A为商品化的T细胞无血清培养基，B为本发明所述T细胞无血清培养基。
图4：对应实施例2，为本发明所述T细胞无血清培养基中激活扩增的PBMC中T细胞增殖过程中的细胞活率数据。其中A为商品化的T细胞无血清培养基，B为本发明所述T细胞无血清培养基。
具体实施方式
下面结合附图对本发明提供的具体实施方式作详细说明。
以下实施例中材料来源为：
IMDM(Gibco，货号12440053)、DMEM/F12(Gibco，货号11320082)、RPMI1640(Gibco，货号31870082)、所述商业化T细胞无血清培养基为X-Vivo-15(Lonza，货号BE02-060F)。
重组人胰岛素(Sigma，货号91077c)、重组人转铁蛋白(Sigma，货号T3705)、重组人血清白蛋白(Sigma，货号SAE0072)、胸腺嘧啶(Sigma，货号T0895)、牛磺酸(Sigma，货号T8691)、亚叶酸(Sigma，货号47612)、CD3抗体(Biolegend，货号317325)、CD28抗体(Biolegend，货号302933)、IL2(Peprotech，货号200-02)。
实施例中其他所用的方法和试剂，除非另有说明，否则为本领域常规的方法和试剂。
实施例1人外周血单核细胞(PBMC)中T细胞的激活扩增(一)
本实施例中，使用IMDM、DMEM/F12和RPMI1640三种培养基按体积比1:1:1混合配制成基础培养基，然后向其中添加终浓度为2.5g/L的重组人胰岛素，5mg/L的重组人转铁蛋白，0.2g/L的重组人血清白蛋白，0.2mg/L的胸腺嘧啶、0.2mg/L的牛磺酸、1mg/L的亚叶酸。由上述方法配制的T细胞无血清培养基用于后续T细胞激活扩增实验。
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。将分离好的PBMC细胞悬液转移至准备好的6孔板培养皿中，接种密度1x106/ml，添加终浓度10μg/mL的CD3抗体，终浓度5μg/mL的CD28抗体，及终浓度100IU/mL的IL2，并放入37℃，5％二氧化碳培养箱内继续培养。分别于培养的第0天、第5天、第6天、第7天、第8天、第9天、第10天观察细胞增殖情况并对细胞进行计数，获得细胞增殖曲线和细胞活率的数据。细胞增殖曲线如图1，细胞活率数据如图2。数据显示培养第10天时，T细胞扩增402倍，活率达到87％。
实施例2：人外周血单核细胞(PBMC)中T细胞的激活扩增(二)
本实施例中，使用IMDM和RPMI1640两种培养基按体积比1:1混合配制成基础培养基，然后向其中添加终浓度为5g/L的重组人胰岛素，20mg/L的重组人转铁蛋白，1g/L的重组人血清白蛋白，1mg/L的胸腺嘧啶、1mg/L的牛磺酸、10mg/L的亚叶酸。由上述方法配制的T细胞无血清培养基用于后续T细胞激活扩增实验。
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。将分离好的PBMC细胞悬液转移至准备好的6孔板培养皿中，接种密度1x106/ml，添加终浓度10μg/mL的CD3抗体，终浓度5μg/mL的CD28抗体，及终浓度100IU/mL的IL2，并放入37℃，5％二氧化碳培养箱内继续培养。分别于培养的第0天、第5天、第6天、第7天、第8天、第9天、第10天、第11天、第12天观察细胞增殖情况并对细胞进行计数，获得细胞增殖曲线和细胞活率的数据。细胞增殖曲线如图3，细胞活率数据如图4。数据显示培养第12天时，T细胞扩增825倍，活率达到92％。
实施例3本发明T细胞无血清培养基(一)
使用IMDM、DMEM/F12和RPMI1640三种培养基按体积比1:1:1混合配制成基础培养基，然后向其中添加终浓度为2.5g/L的重组人胰岛素，5mg/L的重组人转铁蛋白，0.2g/L的重组人血清白蛋白，0.2mg/L的胸腺嘧啶、0.2mg/L的牛磺酸、1mg/L的亚叶酸。
实施例4本发明T细胞无血清培养基(二)
使用IMDM和RPMI1640两种培养基按体积比1:1混合配制成基础培养基，然后向其中添加终浓度为5g/L的重组人胰岛素，20mg/L的重组人转铁蛋白，1g/L的重组人血清白蛋白，1mg/L的胸腺嘧啶、1mg/L的牛磺酸、10mg/L的亚叶酸。
实施例5本发明T细胞无血清培养基(三)
使用IMDM、DMEM/F12和RPMI1640三种培养基按体积比1:1:1混合配制成基础培养基，然后向其中添加终浓度为50g/L的重组人胰岛素，100mg/L的重组人转铁蛋白，4.5g/L的重组人血清白蛋白，20mg/L的胸腺嘧啶、10mg/L的牛磺酸、50mg/L的亚叶酸。
实施例6本发明T细胞无血清培养基(四)
使用DMEM、DMEM/F12和RPMI1640三种培养基按体积比1:1:1混合配制成基础培养基，然后向其中添加终浓度为50g/L的重组人胰岛素，100mg/L的重组人转铁蛋白，4.5g/L的重组人血清白蛋白，20mg/L的胸腺嘧啶、10mg/L的牛磺酸、50mg/L的亚叶酸。
实施例7本发明T细胞无血清培养基(五)
使用IMDM和RPMI1640两种培养基按体积比1:1混合配制成基础培养基，然后向其中添加终浓度为20g/L的重组人胰岛素，50mg/L的重组人转铁蛋白，5g/L的重组人血清白蛋白，10mg/L的胸腺嘧啶、0.1mg/L的牛磺酸、0.5mg/L的亚叶酸。
实施例8本发明T细胞无血清培养基(六)
使用IMDM、aMEM和M199三种培养基按体积比1:1:1混合配制成基础培养基，然后向其中添加终浓度为50g/L的重组人胰岛素，100mg/L的重组人转铁蛋白，4.5g/L的重组人血清白蛋白，20mg/L的胸腺嘧啶、5mg/L的牛磺酸、40mg/L的亚叶酸。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员，在不脱离本发明方法的前提下，还可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。
Claims (6)
1.一种T细胞无血清培养基，其特征在于，所述T细胞无血清培养基由基础培养基和添加成分混合配制而成，所述添加成分由重组人胰岛素、重组人转铁蛋白、重组人血清白蛋白、胸腺嘧啶、牛磺酸和亚叶酸组成，每升所述T细胞无血清培养基中：所述重组人胰岛素为2.5-50mg，所述重组人转铁蛋白为5-100mg，所述重组人血清白蛋白为0.2-5g，所述胸腺嘧啶为0.2-20mg，所述牛磺酸为0.1-10mg，所述亚叶酸为0.5-50mg。
2.根据权利要求1所述的T细胞无血清培养基，其特征在于，所述基础培养基是IMDM、DMEM、DMEM/F12、aMEM、RPMI1640、M199中的一种或几种的混合。
3.根据权利要求2所述的T细胞无血清培养基，其特征在于，所述基础培养基是IMDM、DMEM/F12和RPMI1640三种培养基的混合。
4.根据权利要求3所述的T细胞无血清培养基，其特征在于，所述IMDM、DMEM/F12和RPMI1640三种培养基的体积比为1:1:1。
5.根据权利要求2所述的T细胞无血清培养基，其特征在于，所述基础培养基是IMDM和RPMI1640两种培养基的混合。
6.根据权利要求5所述的T细胞无血清培养基，其特征在于，所述IMDM和RPMI1640两种培养基的体积比为1:1。
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