作　　者： ; ; ; ; ;

机构地区： 华东理工大学生物工程学院生物反应器工程国家重点实验室

出　　处： 《生物工程学报》 2005年第4期622-627,共6页

摘　　要： 为了消除造血细胞静态培养中存在的浓度梯度和搅拌悬浮培养时换液引起的波动,为造血细胞体外扩增提供更理想的培养环境和操作方式,利用自主开发的造血细胞重力沉降截留系统结合有溶氧和pH控制的生物反应器进行了脐血造血细胞的灌注培养。两次灌注培养中总细胞分别扩增11·5和18·6倍,扩增倍数最大时,CFU_Mix分别扩增23·2倍和20·4倍、CFU_GM扩增13·9倍和21·5倍、BFU_E扩增8·0倍和6·9倍、CD34+细胞扩增17·1倍和15·4倍。培养到12d时,第一次实验由267×106单个核细胞扩增得到1082×106个总细胞,6·31×106个CFU_GM,6·2×106个CFU_Mix和23×106个CD34+细胞;第二次实验由180×106单个核细胞扩增得到1080×106个总细胞,4·65×106个CFU_GM,11·0×106个CFU_Mix和25·0×106个CD34+细胞,这达到了临床规模,由于控制了较低的溶氧和稳定的培养环境,细胞中干/祖细胞含量显著高于方瓶。但灌注培养到后期细胞密度达到较高后,细胞生长受到抑制,这应该是由细胞密度过高本身所引起。搅拌式反应器中进行灌注培养有利于造血干/祖细胞的进一步扩增,培养得到的细胞中干/祖细胞含量较高,培养规模达到了临床要求,但过高的细胞密度将对造血细胞的生长产生抑制。 To optimize the culture environment and protocol of hematopoietic cells' expansion, avoiding the fluctuation caused by medium changing in stirred culture and concentration gradient in static culture, the hematopoietic cells from cord blood (CB) were cultured in a stirred bioreactor connected with a cell retention system, which is a gravity sedimentation settler designed for hematopoietic cell. Total cells expanded 11.5 and 18.6 fold respectively in the twice perfusion stirred cultures, in which CFU-Mix was expanded 23.2 and 20.4 fold, CFU-GM 13.9 fold and 21.5 fold, BFU-E 8.0 fold and 6.9 fold, CD34~ + cells 17.1 fold and 15.4 fold. After 12-day culture, it was obtained that 1082×10~ 6 total cells, 6.31×10~ 6 CFU-GM, 6.2×10~ 6 CFU-Mix and 23×10~ 6 CD34^+ cells from 267×10~ 6CB mononuclear cells (MNC) in the first culture, and 1080×10~ 6 total cells, 4.65×10~ 6 CFU-GM, 11.0×10~ 6 CFU-Mix, and 25.0×10~ 6 CD34^+ cells from 180×10~ 6 CB MNC. These two cultures met to the clinical scale. Due to the optimized dissolved oxygen(DO) and stable culture environment, the rate of stem/progenitor cells to total cells in the perfusion culture was higher than that in T-flask cell-retention feeding culture. But the cell growth was inhibited in the later phase of perfusion culture, when the cell density is high. The inhibition should be attribute to the high cell density itself. The perfusion culture environment in bioreactor with optimal DO and pH controlling is more favorable for stem/progenitor cells' maintenance and expansion, and the expanded cells' number has reached a clinical scale. But the high cell density in the later phase of perfusion culture caused inhibition to mature hematopoietic cell's growth.

关 键 词： 脐血 造血细胞 生物反应器 灌注培养

领　　域： [生物学]