Objective: To explore an optimal method for granulocyte cell production from umbilical cord blood mononuclear cells. Methods: Erythrocytes were precipitated by hydroxyethyl starch. Mononuclear cells were isolated through Ficoll density gradient centrifugation. Different media, additives and cultivation model were chosen for granulocyte induction. Cell morphology was observed by microscopy, and cell phenotype was detected by flow cytometry. The CD18 expression of granulocytes was tested by immunofluorescence assay, and phagocytosis test was executed as well. Results: Compared to fetal bovine serum (FBS) treatment group, cell viability, counts and differentiation rate of granulocytes induced by X-VIVO(TM) 15 combined with TPO, SCF, G-CSF but without FBS were superior. And X-VIVO(TM)15 medium was better than SCGM medium at effectiveness and cost. Using two-stage mode of hematopoietic stem cell expansion followed by granulocyte induction with X-VIVO(TM)15 combining TPO, SCF and G-CSF, cell proliferation was nearly 132 times at day 21. Flow cytometry showed that the differentiation was lagged in 2-stage mode than in direct induction mode, CD15 expression was (69.60± 1.06) % vs (97.73±0.39) %; Wright-Giemsa staining demonstrated mature granulocytes; immunofluorescence showed the expression of lysosomal proteins CD18. A strong phagocytic function of mature granulocytes was demonstrated by phagotrophic efficiency of (51.43±0.05) %. And granulocyte had chemotaxis ability under the role of chemotactic factor IL-8. Conclusion: Optimized culture media and cultivation mode are achieved for functional granulocytes induction in vitro.
目的： 探索脐血来源单个核细胞体外诱导分化为粒系细胞的方法。 方法： 采用羟乙基淀粉沉降红细胞，淋巴细胞分离液分离单个核细胞。选择不同的培养基、添加剂以及培养模式诱导粒系细胞分化，显微镜观察细胞形态，流式细胞术检测细胞表型，免疫荧光测定粒系细胞CD18表达，并检测细胞吞噬功能。 结果： 采用X-VIVO(TM)15中添加细胞因子TPO、SCF、G-CSF诱导粒系细胞，细胞存活率、细胞数、粒系细胞分化效率均优于添加胎牛血清组。与SCGM培养基诱导粒系细胞相比，X-VIVO(TM)15培养基效果更佳，且成本低。采用造血干细胞扩增和在基础培养基X-VIVO(TM)15中添加细胞因子TPO、SCF、G-CSF诱导粒系细胞的两阶段扩增、诱导模式，21 d细胞扩增倍数近132倍；流式细胞术检测表明，粒系细胞分化效率滞后于直接诱导模式，粒系标志CD15表达分别为（69.60±1.06）%和（97.73±0.39）%；瑞氏-吉姆萨染色可见成熟的分叶核粒细胞；免疫荧光方法检测显示溶酶体蛋白CD18的表达；成熟的粒细胞具有较强吞噬墨汁的功能，吞噬效率为（51.43±0.05）%。且在细胞趋化因子IL-8作用下，粒细胞具有趋化作用。 结论： 优化了诱导粒系细胞培养体系和培养模式，获得了具有一定功能的粒系细胞。.
Keywords: Cord blood; Granulocyte cells; Mononuclear cells.