Search

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Despite recent major advances including reprogramming and directed cardiac differentiation of human cells, therapeutic application of in vitro engineered myocardial tissue is still not feasible due to the inability to construct functional large vascularized contractile tissue patches based on clinically applicable and fully defined matrix components. Typical matrices with preformed porous 3D structure cannot be applied due to the obvious lack of migratory capacity of cardiomyocytes (CM). We have therefore developed a fully defined in situ hydrogelation system based on alginate (Alg) and hyaluronic acid (HyA), in which their aldehyde and hydrazide-derivatives enable covalent hydrazone cross-linking of polysaccharides in the presence of viable myocytes. By varying degrees of derivatization, concentrations and composition of blends in a modular system, mechanophysical properties of the resulting hydrogels are easily adjustable. The hydrogel allowed for the generation of contractile bioartificial cardiac tissue from CM-enriched neonatal rat heart cells, which resembles native myocardium. A combination of HyA and highly purified human collagen I led to significantly increased active contraction force compared to collagen, only. Therefore, our in situ cross-linking hydrogels represent a valuable toolbox for the fine-tuning of engineered cardiac tissue's mechanical properties and improved functionality, facilitating clinical translation toward therapeutic heart muscle reconstruction., (Copyright © 2012 Elsevier Ltd. All rights reserved.)