摘要目的 观察泊洛沙姆188(P188)对体外三维(3D)培养诱导脐血单个核细胞向巨核细胞分化的影响.方法 将分离的脐血单个核细胞分别接种于细胞瓶和细胞培养袋中,后者采用WIGGENS摇床模拟生物反应器进行3D培养.在巨核细胞诱导培养基中加入P188体外培养14d,观察细胞形态、计数细胞数并计算细胞存活率,采用流式细胞术观察巨核细胞表面标志表达情况.结果 与采用传统的细胞培养瓶二维(2D)培养诱导巨核细胞相比,2D+P188培养组巨核系CD41+、CD41+/CD61+、CD61+细胞数明显增加(P值均＜0.01);在3D培养中加入P188,细胞体积变大,核形状不规则,胞质含紫红色颗粒,细胞分化更接近成熟.2D培养、3D培养及3D+P188培养组组间巨核细胞表面标志CD41、CD41/CD61、CD61表达水平差异有统计学意义(P值均＜0.01).LSD-t检验两两比较显示,与2D培养相比,3D培养诱导巨核细胞存活率及细胞数均降低(P值分别为0.018、0.027),3D+P188培养组细胞数、细胞存活率与2D和3D培养组比较差异均无统计学意义(P值均＞0.05).而3D培养组巨核细胞CD41/CD61表达水平为(36.30±1.27)％,高于2D培养组的(23.95±1.34)％(P=0.002),3D+P188培养组CD41/CD61表达水平更高[(59.45±1.20)％].结论 3D培养有利于巨核系祖细胞诱导分化,但细胞存活率低,加入P188,细胞生存状态好,且诱导效率更高.

abstractsObjective To observe the effect of poloxamer 188 (P188) on megakaryocyte cultivation and induction from cord blood mononuclear cells in order to obtain more megakaryocyte progenitor cells (MPC).Methods The cord blood mononuclear cells were isolated and inoculated in cell culture bag or cell culture flask respectively.The WIGGENS shaker and cell culture bags were used to mimick WAVE Bioreactor for three-dimensional (3D) cell culture,and the P188 was added to induction medium,The cells were detected for morphology,surface marker,viability,and number on day 14.Results In the two-dimensional (2D) culture,CD41 +,CD41 +/CD61 +,CD61 + megakaryocytic numbers increased significantly after adding P188 (all P＜ 0.01).And in the 3D culture of adding P188,the cell volume became larger and the nuclear shape was irregular,the cytoplasm appeared magenta granules,and the megakaryocyte cells became more mature.By 3D culture,the expression of CD41/CD61 was (36.30± 1.27)％ vs (23.95± 1.34)％,hence the differentiation for MPC was significantly higher than that in the 2D group (P ＜ 0.01).Furthermore,adding P188 in 3D culture resulted in highest differentiation efficiency for MPC [(59.45± 1.20)％].There were no significantly differences in terms of cell viability and cell number among 3D culture containing P188,2D and 3D culture groups (all P ＞ 0.05).Conclusion 3D culture was beneficial for the differentiation of MPC,but the cell viability was lower than 2D group;However,the satisfied cell growth and better induction efficiency were obtained by adding of P188,which might provide a new method of megakaryocytes production for clinical application.