Chi Zhan-You, Jiang Hua, Cai Hai-Bo, Tan Wen-Song, Dai Gan-Ce
The State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
Sheng Wu Gong Cheng Xue Bao. 2005 Jul;21(4):622-7.
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 x 10(6) total cells, 6.31 x 10(6) CFU-GM, 6.2 x 10(6) CFU-Mix and 23 x 10(6) CD34+ cells from 267 x 10(6) CB mononuclear cells (MNC) in the first culture, and 1080 x 10(6) total cells, 4.65 x 10(6) CFU-GM, 11.0 x 10(6) CFU-Mix, and 25.0 x 10(6) CD34+ cells from 180 x 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.
为优化造血细胞扩增的培养环境和方案，避免搅拌培养中换液引起的波动以及静态培养中的浓度梯度，将脐血（CB）中的造血细胞在与细胞保留系统相连的搅拌生物反应器中培养，该细胞保留系统是专为造血细胞设计的重力沉降沉降器。在两次灌注搅拌培养中，总细胞分别扩增了11.5倍和18.6倍，其中CFU-Mix分别扩增了23.2倍和20.4倍，CFU-GM扩增了13.9倍和21.5倍，BFU-E扩增了8.0倍和6.9倍，CD34+细胞扩增了17.1倍和15.4倍。培养12天后，第一次培养中从267×10⁶个CB单个核细胞（MNC）中获得了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，第二次培养中从180×10⁶个CB MNC中获得了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。这两次培养达到了临床规模。由于溶解氧（DO）得到优化且培养环境稳定，灌注培养中干/祖细胞与总细胞的比例高于T型瓶细胞保留补料培养。但在灌注培养后期，当细胞密度较高时，细胞生长受到抑制。这种抑制应归因于高细胞密度本身。具有最佳DO和pH控制的生物反应器中的灌注培养环境更有利于干/祖细胞的维持和扩增，且扩增细胞数量已达到临床规模。但灌注培养后期的高细胞密度对成熟造血细胞的生长产生了抑制。
