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目的:探讨可用于临床治疗功能成熟的DC体外扩增的优化培养方案。方法:胎牛血清培养基联合细胞因子rhGM-CSF(100ng/mL)和rhIL-4(50 ng/mL)扩增人外周血分离的单个核细胞,细胞培养分别按5×106/mL、6×106/mL和7×106/mL的密度,加入6孔培养板。第6d加入rhTNF-a(100 ng/mL)联合培养,分别于第6 d,第9 d和12 d收获细胞。从形态学、细胞表面标志方面进行鉴定。结果:显微镜观察,经过9 d诱导后,培养细胞具有典型树突细胞外形。流式细胞仪分析,6×106/mL密度的细胞培养组培养到第9天最宜。结论:细胞具有典型的DC的形态特征,细胞表型及功能实验证实其DC的特性,说明建立的血清培养基联合细胞因子rhGM-CSF、rhIL-4和rhTNF-a体外诱导DC的方法是切实可行的。
Objective: To explore the optimal culture program for the in vitro expansion of DC with clinical function. Methods: Human peripheral blood mononuclear cells (PBMCs) were isolated from fetal bovine serum with rhGM-CSF (100ng / mL) and rhIL-4 (50ng / mL) × 106 / mL and a density of 7 × 106 / mL, added to a 6-well culture plate. On the 6th day, rhTNF-a (100 ng / mL) was added to co-culture, and the cells were harvested on the 6th day, the 9th day and the 12th day respectively. From morphological, cell surface markers identified. Results: Microscopically, cultured cells showed typical dendritic cell shape after 9 days induction. Flow cytometry analysis, 6 × 106 / mL density cell culture group cultured to the best on the 9th day. CONCLUSION: The cells have typical morphological features of DC. The cell characteristics of DCs were confirmed by cell phenotype and function test. The results showed that the established serum culture medium combined with cytokines rhGM-CSF, rhIL-4 and rhTNF-a induces DC in vitro feasible.