脐血细胞在搅拌悬浮生物反应器中的生长特性

Growth characteristics of umbilical cord blood cells in stirred suspension bioreactor

目的:了解脐血细胞在搅拌悬浮生物反应器中的生长特性,为开发搅拌悬浮生物反应器大规模生产造血细胞提供实验依据。方法:实验于2003-06/11在华东理工大学生物反应器工程国家重点实验室动物细胞与组织工程研究室完成。脐血细胞在基本细胞因子组合干细胞因子+白细胞介素3+白细胞介素6的刺激作用下,考察其在静态和动态培养系统中的生长规律,搅拌转速(30,60r/min)对脐血细胞细胞周期分布和凋亡的影响以及不同形状搅拌桨(平翼桨、搅拌棒)对脐血细胞生长的影响。结果:①总细胞的扩增:随着细胞逐步适应体外生存环境,总细胞开始表现出增殖的趋势,且转瓶扩增效果略好于孔板,但差异不显著。②CD34+细胞的扩增:在相同细胞因子组合的培养条件下,动态培养在短期培养(1周左右)过程中表现出了明显的增殖优势,而静态培养更有利于维持造血干/祖细胞状态。③总集落形成细胞的扩增:在短期培养中动态培养系统具有明显的生长优势,随着培养时间的延长,静态培养对总集落形成细胞的维持作用优于动态培养。④不同搅拌转速对脐血细胞细胞周期分布和凋亡的影响:相对于静态培养,CD34+细胞除了G2期含量显著增加外,对其他细胞周期和凋亡无显著影响,随着搅拌转速从30r/min提高到60r/min时,CD34+细胞周期分布和凋亡差异不明显,而细胞周期分布和凋亡比率在静态培养和60r/min的转瓶培养情况下更为相似,表明流体作用力的增强对CD34+细胞的生长行为没有影响。⑤不同搅拌桨形状对造血细胞培养的影响:相同搅拌转速(30r/min)下培养来源相同的脐血细胞,1周后平翼桨转瓶中的单个核细胞数锐减,而搅拌棒转瓶却能保持基本不变,且差异显著(P<0.05)。说明不同搅拌桨形式导致混合方式的差异以及由此产生的不同流体作用力等对造血细胞增殖造成某种程度的影响。结论:动态培养能支持脐血细胞的生长,且造血干/祖细胞的增殖优于静态培养。搅拌转速的增加对脐血细胞细胞周期分布和凋亡无显著影响,而不同的搅拌形式与脐血细胞的增殖有关。

AIM： To understand growth characteristics of umbilical cord blood （UCB） cells in stirred suspension bioreactor, and to give some insights into producing hematopoietic cells in stirred suspension bioreactors. METHODS： The experiment was carried out in Animal Cell and Tissue Engineering Laboratory affiliated to State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology from June to November 2003. Growth behaviors of UCB cells in static and stirred culture systems, effects of agitation rate （30, 60 r per minute） on distribution of cell cycle and apoptosis, and influences of different impellers＇ （flat-blede paddle, stirrer bar） confignrations on proliferation of UCB cells were investigated when UCB cells were stimulated by the cytokine combination of stem cell factor （SCF）＋interhukin （IL）-3＋IL-6. RESULTS： ①Proliferation of total cells： Total cells started to expand when they gradually adapted to ex vivo culture environment. Spinner systems offered proliferation advantage over well-plates and no significant differences were observed.②Expansion of CD34^＋ cells： In short-term culture （about 1 week）, stirred culture showedsignificant growth than that in static control under the same culture condition. The static control was of benefit to maintain hcmatopoietic stem/ancestral cells. ③Expansion of total colony-forming cells （CFC）： In short term culture the stirred culture had significantly growth advantage. With the prolongation of culture time, static culture was better than the stirred culture in maintenance effect of CFC. ④ Effects of different agitation rates on cycle distribution and apoptosis of UCB cells： With the exception of G2 phase, the distribution of other cell cycle and apoptosis of blood cellswere not significantly affected by agitation rates as compared with the static culture. The difference of CD34^＋ cycle distribution and apoptosis was insignificant when the agitation rate increased from 30 r to 60 r per minute. The distribution and apoptosis of cell cycle were similar under static culture and 60 r agitation culture per minute. It was showed that agitation rate had little effects on the growth characteristics of CD34^＋ cells. ⑤Effects of different impellers＇ configurations on proliferation of hematopoietie cells： One week later, the number of mononuclear cells of UCB cells dramatically decreased in spinner flasks with flat-blade paddle under the same agitation rate （30 r per minute） and the same source, while it was kept instant in spinner flasks with stirred bar. The difference was significant （P〈0.05）. The data suggested that different impellers＇ configurations induced resultant shear stress and different fluid effort, which had significant influences on growth of hematopoietic cells. CONCLUSION： Stirred culture can support growth of UCB cells, and lead to greater output of hematopoietic stem and progenitor cells ascompared to static culture. Increased agitation rate has little effects on distribution of cell cycle and apoptoois of UCB cells, while different impellers＇ configurations are related to proliferation of UCB cells.