Scalable Producing Embryoid Bodies by Rotary Cell Culture System and Constructing Engineered Cardiac Tissue with ES-Derived Cardiomyocytes in Vitro

Abstract

Embryonic stem (ES) cells are of significant interest either as an in vitro model recapitulating early embryonic development or as a renewable source of therapeutically useful cells. ES cells aggregation is important for embryoid bodies (EBs) formation and the subsequent generation of ES cell derivatives. This study was conducted to describe scalable production of EBs by the rotary cell culture system (RCCS, STLV type) and estimate the feasibility of constructing engineered cardiac tissue (ECT). In comparison with suspension culture in a Petri dish, the efficiency of the dynamic process was analyzed with respect to the yield of EB formation and their cardiomyocyte differentiation. Cardiomyocyte differentiation was evaluated by immunohistochemical analysis. After the elementary enrichment by gradient percoll, ES cell-derived cardiomyocytes were applied to construct ECT. Cell gross morphology, spatial distribution, and ultrastructure were evaluated by using histological analysis, confocal laser scanning microscopy, and transmission electron microscopy. Results showed that EB efficiencies in STLV were nearly 1.5-2.0 times higher than that of liquid suspension cultures, and cardiomyocyte differentiation of EBs progressed in a normal course after the dynamic cultivation in STLV. Additionally, the differentiated cultures could be enriched elementarily by gradient percoll. Once cast into the collagen strand, cells grew well and became more matured in Petri dishes. Synchronous contraction of the cell cluster was observed on the surface of the ECT, and cell connection was also established. It was the first report to have beating ES-derived cardiomyocytes on a 3-D collagen scaffold, which might provide a promising model for physiological and pharmacological studies and tissue replacement therapy.

In vitro differentiation of rat embryonic stem cells into functional cardiomyocytes

“…The formation of EBs is the primary way to induce ESC differentiation in vitro [15,37,38], whereas the proper approach for EB formation varies from species to species [39]. In previous studies, researchers have optimized the EB formation method of ESCs from mouse and human [19,[40][41][42], but this method does not work well in rESCs as a majority of the cells die when forming EBs [7,11]. In this study, we have developed a system for the efficient formation of EBs from rESCs by harnessing the application of signal inhibitors and hanging-drop technology.…”

Hydrodynamic modulation of embryonic stem cell differentiation by rotary orbital suspension culture

“…Although EB size can inherently limit the diffusion of oxygen and molecules throughout ESC aggregates, hydrodynamic conditions generally improve nutrient and waste transport. In past studies, increased internal cell viability within EBs cultured in mixed bioreactor systems compared to static culture conditions has been attributed to enhanced access to nutrients and removal of metabolic byproducts from the differentiating ESC spheroids (Cameron et al, 2006;Gerecht-Nir et al, 2004;Schroeder et al, 2005;Wang et al, 2006). Transport properties are an important, yet often overlooked consideration for EB differentiation studies due to the fact that even small molecules do not readily diffuse from the surrounding media homogeneously within the 3-D cell spheroids (Carpenedo et al, 2009;Sachlos and Auguste, 2008).…”

Developmental biology and tissue engineering

“…The use of cellular spheroids as building blocks of tissues proved successful in recent cardiac TE experiments, which employed cardiomyocyte spheroids derived from embryonic stem cells (Wang et al, 2006).…”