Gerecht-Nir Sharon, Cohen Smadar, Itskovitz-Eldor Joseph
Biotechnology Interdisciplinary Unit, Technion - Israel Institute of Technology, Haifa, Israel.
Biotechnol Bioeng. 2004 Jun 5;86(5):493-502. doi: 10.1002/bit.20045.
The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy and tissue engineering depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe for the first time the formation of differentiating human embryoid bodies (hEBs) in rotating bioreactors to try and control their agglomeration. The efficacy of the dynamic process compared to static cultivation in Petri dishes was analyzed with respect to the yield of hEB formation and differentiation. Quantitative analyses of hEBs, DNA and protein contents, and viable cell concentration, as measures for culture cellularity and scale-up, revealed 3-fold enhancement in generation of hEBs compared to the static culture. Other metabolic indices such as glucose consumption, lactic acid production, and pH pointed to efficient cell expansion and differentiation in the dynamic cultures. The type of rotating vessel had a significant impact on the process of hEB formation and agglomeration. In the slow turning lateral vessel (STLV), hEBs were smaller in size and no large necrotic centers were seen, even after 1-month cultivation. In the high aspect rotating vessel (HARV), hEB agglomeration was massive. The appearance of representative tissues derived from the three germ layers as well as primitive neuronal tube organization, blood vessel formation, and specific-endocrine secretion indicated that the initial developmental events are not altered in the dynamically formed hEBs. Collectively, our study defines the culture conditions in which control over the aggregation of differentiating hESCs is obtained, thus enabling scaleable cell production for clinical and industrial applications.
人类胚胎干细胞(hESCs)有望为细胞治疗和组织工程提供无限的细胞供应,这取决于是否有可控的生物过程来实现其扩增和分化。我们首次描述了在旋转生物反应器中形成分化的人类胚状体(hEBs),以试图控制其聚集。针对hEB形成和分化的产量,分析了与在培养皿中静态培养相比,动态过程的效果。对hEBs、DNA和蛋白质含量以及活细胞浓度进行定量分析,作为培养细胞密度和放大培养的指标,结果显示与静态培养相比,hEB的生成量提高了3倍。其他代谢指标,如葡萄糖消耗、乳酸产生和pH值,表明动态培养中细胞的有效扩增和分化。旋转容器的类型对hEB的形成和聚集过程有显著影响。在慢速旋转侧容器(STLV)中,hEBs尺寸较小,即使培养1个月后也未观察到大型坏死中心。在高长宽比旋转容器(HARV)中,hEB聚集大量。源自三个胚层的代表性组织的出现以及原始神经管组织、血管形成和特定内分泌分泌表明,动态形成的hEBs中初始发育事件未改变。总体而言,我们的研究确定了能够控制分化的hESCs聚集的培养条件,从而实现用于临床和工业应用的可扩展细胞生产。