不同培养基对流式细胞仪分选外周血T细胞的影响

Influence of Various Medium to Avidity and Proliferation of T Cells Sorted by FACS

目的:通过探讨用于流式分选的T细胞体外扩增的无血清培养基,提高过继细胞的增殖能力和活性。方法:采用人外周血淋巴细胞分离管制备外周血单个核细胞,再用流式细胞分选仪从6例健康志愿者的外周血单个核细胞中分选CD3;T细胞到4种常用的培养基中:X-VIVO 15、KBM 581、TexMACS GMP和10%FBS/1640,观察并记录培养细胞的状态和体外增殖能力。于第3天,第6天和第8天,通过胎盼蓝染色后进行活细胞计数。于第8天用凋亡试剂盒检测扩增细胞的凋亡情况,并用流式细胞分析仪检测细胞的免疫表型。结果:X-VIVO 15、TexMACS GMP和10%FBS/1640作为流式细胞分选的接收液仅少量细胞碎片,而分选在KBM 581的细胞大量死亡,显著高于X-VIVO 15组(P<0.05)。X-VIVO 15中扩增的细胞数量最多,增殖检测结果显示活细胞在X-VIVO 15中快速增殖且细胞凋亡率显著低于KBM 581和TexMACS GMP(P<0.05)。4种培养基扩增的细胞主要呈现效应记忆型。其中,X-VIVO 15中效应记忆型T细胞比例显著高于TexMACS GMP(P<0.05)。TexMACS中效应细胞比例显著高于10%FBS/1640(P<0.05)。结论:X-VIVO 15无血清培养基扩增流式分选的T细胞具有高增殖能力、细胞活性和记忆表型,适用于经流式分选后细胞的体外扩增。

Objective:To improve the proliferation and activity of adoptive T cell by screening serum-free media for in vitro expansion of flow sorting T cells.Methods:Peripheral blood mononuclear cells from six healthy volunteers were prepared by gradient centrifugation using peripheral blood lymphocyte separation tube.The CD3^(+)T cells from the peripheral blood mononuclear cells were then sorted by flow cytometry into four commonly Lymphocyte culture medium,including X-VIVO 15,KBM 581,TexMACS GMP serum-free media and RPMI 1640 with ten percent of fetal bovine serum complete medium.The status of the cultured cells and the proliferation ability of the cells in vitro was observed and recorded.On the 3rd,6th and 8th days,viable cell counts were tested by trypan blue staining.On the 8th day,apoptosis was detected by the Annexin V:FITC Apoptosis Detection Kit.The immune phenotype of the expanded cells in vitro was detected by the flow cytometer.Results:A small amount of cell debris was observed when X-VIVO 15,TexMACS GMP and RPMI 1640 with ten percent of fetal bovine serum medium were used as the receiving medium for flow cytometry,while a large number of dead cells were observed in samples sorted in KBM 581(P<0.05).The number of cells expanded in X-VIVO 15 medium is more than that of the other three media,and proliferation testing shows that live cells proliferate rapidly in X-VIVO 15 medium.Rate of apoptosis is significantly lower than that of KBM 581 and TexMACS GMP(P<0.05).The expanded cells mainly show effect memory type in the four different cultural environments.The rate of effect memory T cells in X-VIVO 15 medium was higher than that in TexMACS GMP(P<0.05).The rate of terminal effector T cells in TexMACS GMP medium was higher than that in 10%FBS/1640.Conclusions:Flow-sorted T cells show higher proliferation capacity,viability and memory phenotype in X-VIVO 15 media,suggesting that X-VIVO 15 media is suitable for in vitro expansion of cells after flow-sorting.