1 Department of Chemical and Biological Engineering, State University of New York at Buffalo , Buffalo, New York.
Tissue Eng Part A. 2014 Feb;20(3-4):588-99. doi: 10.1089/ten.TEA.2013.0219. Epub 2013 Nov 28.
A prerequisite for the realization of human pluripotent stem cell (hPSC) therapies is the development of bioprocesses for generating clinically relevant quantities of undifferentiated hPSCs and their derivatives under xeno-free conditions. Microcarrier stirred-suspension bioreactors are an appealing modality for the scalable expansion and directed differentiation of hPSCs. Comparative analyses of commercially available microcarriers clearly show the need for developing synthetic substrates supporting the adhesion and growth of hPSCs in three-dimensional cultures under agitation-induced shear. Moreover, the low seeding efficiencies during microcarrier loading with hPSC clusters poses a significant process bottleneck. To that end, a novel protocol was developed increasing hPSC seeding efficiency from 30% to over 80% and substantially shortening the duration of microcarrier loading. Importantly, this method was combined with the engineering of polystyrene microcarriers by surface conjugation of a vitronectin-derived peptide, which was previously shown to support the growth of human embryonic stem cells. Cells proliferated on peptide-conjugated beads in static culture but widespread detachment was observed after exposure to stirring. This prompted additional treatment of the microcarriers with a synthetic polymer commonly used to enhance cell adhesion. hPSCs were successfully cultivated on these microcarriers in stirred suspension vessels for multiple consecutive passages with attachment efficiencies close to 40%. Cultured cells exhibited on average a 24-fold increase in concentration per 6-day passage, over 85% viability, and maintained a normal karyotype and the expression of pluripotency markers such as Nanog, Oct4, and SSEA4. When subjected to spontaneous differentiation in embryoid body cultures or directed differentiation to the three embryonic germ layers, the cells adopted respective fates displaying relevant markers. Lastly, engineered microcarriers were successfully utilized for the expansion and differentiation of hPSCs to mesoderm progeny in stirred suspension vessels. Hence, we demonstrate a strategy for the facile engineering of xeno-free microcarriers for stirred-suspension cultivation of hPSCs. Our findings support the use of microcarrier bioreactors for the scalable, xeno-free propagation and differentiation of human stem cells intended for therapies.
实现人类多能干细胞（hPSC）治疗的前提是开发生物工艺，以便在无动物源条件下生成临床相关数量的未分化 hPSC 及其衍生物。微载体搅拌悬浮生物反应器是可扩展地扩增和定向分化 hPSC 的一种有吸引力的方式。对市售微载体的比较分析清楚地表明，需要开发合成基质，以在搅拌诱导的剪切下支持三维培养中的 hPSC 粘附和生长。此外，hPSC 集落加载到微载体时的低接种效率是一个显著的工艺瓶颈。为此，开发了一种新方案，将 hPSC 的接种效率从 30%提高到 80%以上，并大大缩短了微载体加载的时间。重要的是，该方法与通过表面缀合先前显示支持人胚胎干细胞生长的 vitronectin 衍生肽来工程化聚苯乙烯微载体相结合。细胞在静态培养中在肽缀合珠上增殖，但在暴露于搅拌后观察到广泛的脱落。这促使对微载体进行额外的处理，使用一种常用于增强细胞粘附的合成聚合物。hPSC 可成功地在搅拌悬浮容器中的这些微载体上进行连续多代培养，附着效率接近 40%。培养细胞的浓度平均每 6 天传代增加 24 倍，存活率超过 85%，并保持正常核型和多能性标志物的表达，如 Nanog、Oct4 和 SSEA4。当在胚状体培养中进行自发分化或定向分化为三个胚胎胚层时，细胞采用各自的命运并显示出相关标志物。最后，工程化的微载体成功地用于 hPSC 的扩增和分化，以产生中胚层祖细胞，在搅拌悬浮容器中。因此，我们展示了一种用于搅拌悬浮培养 hPSC 的无动物源微载体的简便工程策略。我们的研究结果支持使用微载体生物反应器进行可扩展的、无动物源的人类干细胞增殖和分化，以用于治疗。
