Kryukov Olga, Ruvinov Emil, Cohen Smadar
1 Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev , Beer Sheva, Israel .
Tissue Eng Part C Methods. 2014 Nov;20(11):886-94. doi: 10.1089/ten.TEC.2013.0528. Epub 2014 Mar 31.
The therapeutic application of autologous cardiac-derived progenitor cells (CPCs) requires a large cell quantity generated under defined conditions. Herein, we investigated the applicability of a three-dimensional (3D) perfusion cultivation system to facilitate the expansion of CPCs harvested from human heart biopsies and characterized by a relatively high percentage of c-kit(+) cells. The cells were seeded in macroporous alginate scaffolds and after cultivation for 7 days under static conditions, some of the constructs were transferred into a perfusion bioreactor, which was operated for an additional 14 days. A robust and highly reproducible human CPC (hCPC) expansion of more than seven-fold was achieved under the 3D perfusion culture conditions, while under static conditions, the expansion of CPCs was limited only to the first 7 days, after which it leveled-off. On day 21 of perfusion cultivation, the expanded cells exhibited a higher expression level of the progenitor marker c-kit, suggesting that the c-kit-positive CPCs are the main cell population undergoing proliferation. The profile of the spontaneous differentiation in the perfused construct was different from that in the static cultivated constructs; genes typical for cardiac and endothelial cell lineages were more widely expressed in the perfused constructs. By contrast, the differentiation to osteogenic (Von Kossa staining and alkaline phosphatase activity) and adipogenic (Oil Red staining) lineages was reduced in the perfused constructs compared with static cultivated constructs. Collectively, our results indicate that 3D perfusion cultivation mode is an appropriate system for robust expansion of human CPCs while maintaining their progenitor state and differentiation potential into the cardiovascular cell lineages.
自体心脏来源祖细胞（CPCs）的治疗应用需要在特定条件下产生大量细胞。在此，我们研究了三维（3D）灌注培养系统用于促进从人心脏活检组织中获取的、具有相对较高比例c-kit(+)细胞的CPCs扩增的适用性。将细胞接种于大孔藻酸盐支架中，在静态条件下培养7天后，部分构建体转移至灌注生物反应器中，再运行14天。在3D灌注培养条件下，人CPC（hCPC）实现了超过7倍的强劲且高度可重复的扩增，而在静态条件下，CPCs的扩增仅局限于前7天，之后便趋于平稳。在灌注培养第21天时，扩增后的细胞显示出更高水平的祖细胞标志物c-kit表达，表明c-kit阳性CPCs是主要的增殖细胞群体。灌注构建体中的自发分化情况与静态培养构建体不同；心脏和内皮细胞谱系的典型基因在灌注构建体中表达更为广泛。相比之下，与静态培养构建体相比，灌注构建体中向成骨（Von Kossa染色和碱性磷酸酶活性）和脂肪生成（油红染色）谱系的分化减少。总体而言，我们的结果表明，3D灌注培养模式是一种适用于强劲扩增人CPCs的系统，同时能维持其祖细胞状态以及向心血管细胞谱系的分化潜能。
