The use of agarose microwells for scalable embryoid body formation and cardiac differentiation of human and murine pluripotent stem cells
The use of agarose microwells for scalable embryoid body formation and cardiac differentiation of human and murine pluripotent stem cells
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DOI：
10.1016/j.biomaterials.2012.12.024
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发表时间：
2013-03-01
期刊：
影响因子：
14
通讯作者：
Gruh, Ina
中科院分区：
文献类型：
--
作者：
Dahlmann, Julia;Kensah, George;Gruh, Ina
在大多数多能干细胞分化方案中，胚状体（EBs）的形成是一个重要步骤。在这里，我们描述了一种快速、直接的软光刻方法，用于从不同模板制备亲水性硅母版，并随后生产图图化琼脂糖- dmem微孔表面，用于可扩展的标准化干细胞聚集和EB形成。无黏附琼脂糖微孔板精确复制了模板的地形，并用于小鼠诱导多能干细胞（iPSCs）和人胚胎干细胞（ESCs）的聚集。通过时间推移分析的直接显微镜评估表明，与常用但耗时的悬挂滴法相比，小鼠iPSCs可以快速形成形状均匀的EBs，在大小分布和收获效率方面具有相似甚至更一致的结果。对于人类ESCs，单细胞接种琼脂糖微孔后获得均匀聚集体，并使用最先进的小分子定向分化方案有效分化为心脏谱系。利用这种基于软光刻的策略，可以以高度可控的方式实现组织工程所需的足够和可重复数量的干细胞衍生心肌细胞。此外，对于任何需要可伸缩和高度标准化聚合的应用程序中的不同单元格类型，它可能都很有用。(C) 2013 Elsevier Ltd.版权所有。
In most pluripotent stem cell differentiation protocols the formation of embryoid bodies (EBs) is an important step. Here we describe a rapid, straightforward soft lithography approach for the preparation of hydrophilic silicon masters from different templates and the subsequent production of patterned agarose-DMEM microwell surfaces for scalable well standardized stem cell aggregation and EB formation. The non-adhesive agarose microwell plates represent an accurate replication of the templates' topography and were used for aggregation of murine induced pluripotent stem cells (iPSCs) and human embryonic stem cells (ESCs). Direct microscopic assessment by time-lapse analysis demonstrated rapid formation of uniformly shaped EBs from murine iPSCs with similar or even more consistent results concerning size distribution and harvesting efficiency compared to the commonly used but time-consuming hanging drop technique. For human ESCs, homogenous aggregates were obtained after single cell inoculation on agarose microwells with efficient differentiation into the cardiac lineage using state-of-the-art protocols for directed differentiation via small molecules. With this soft lithography-based strategy, sufficient and reproducible numbers of stem cell-derived cardiomyocytes necessary for tissue engineering purposes can be realized in a highly controllable manner. Moreover, it might be useful for different cell types in any application that requires scalable and highly standardized aggregation. (C) 2013 Elsevier Ltd. All rights reserved.