Design of Artificial Myocardial Microtissues

Abstract

Cultivation technologies promoting organization of mammalian cells in three dimensions are essential for gene-function analyses as well as drug testing and represent the first step toward the design of tissue replacements and bioartificial organs. Embedded in a three-dimensional environment, cells are expected to develop tissue-like higher order intercellular structures (cell-cell contacts, extracellular matrix) that orchestrate cellular functions including proliferation, differentiation, apoptosis, and angiogenesis with unmatched quality. We have refined the hanging drop cultivation technology to pioneer beating heart microtissues derived from pure primary rat and mouse cardiomyocyte cultures as well as mixed populations reflecting the cell type composition of rodent hearts. Phenotypic characterization combined with detailed analysis of muscle-specific cell traits, extracellular matrix components, as well as endogenous vascular endothelial growth factor (VEGF) expression profiles of heart microtissues revealed (1). a linear cell number-microtissue size correlation, (2). intermicrotissue superstructures, (3). retention of key cardiomyocyte-specific cell qualities, (4). a sophisticated extracellular matrix, and (5). a high degree of self-organization exemplified by the tendency of muscle structures to assemble at the periphery of these myocardial spheroids. Furthermore (6). myocardial spheroids support endogenous VEGF expression in a size-dependent manner that will likely promote vascularization of heart microtissues produced from defined cell mixtures as well as support connection to the host vascular system after implantation. As cardiomyocytes are known to be refractory to current transfection technologies we have designed lentivirus-based transduction strategies to lead the way for genetic engineering of myocardial microtissues in a clinical setting.

3D‐model of adult cardiac stem cells promotes cardiac differentiation and resistance to oxidative stress

“…Moreover, studies suggest that grafts could induce arrythmias [Smits et al, 2003;Menasche et al, 2003;Perin et al, 2003] and exacerbate conduction abnormalities in the failing heart [Chen et al, 2006]. Other undesirable consequences of transplantation include immunogenic responses [Kofidis et al, 2005], abnormal tissue architecture [Whittaker et al, 2003], neoplastic predisposition, and accelerated cell death [Muller-Ehmsen et al, 2002].3D cultures of neonatal and adult cardiac myocytes in artificial scaffolds have been engineered to develop functional micro-tissues [Akins et al, 1999;Baar et al, 2004;Kelm et al, 2004] for investigations of the vasculature and perfusion in cultured myocardium [Carrier et al, 2002;Watzka et al, 2004] and for repair of damaged myocardium [Hoerstrup et al, 2000;Yang et al, 2001]. Although the 3D-models containing differentiated cardiomyocytes provide a direct source of contractile tissue to repair damaged myocardium, the development of these systems has been far from adequate.…”

Coculture of Spheroids/2D Cell Layers Using a Miniaturized Patterned Platform as a Versatile Method to Produce Scaffold‐Free Tissue Engineering Building Blocks

“…As expected, in the 3D Direct condition, a high number of Saos-2 can be observed in the spheroid. (2) and Dil/Dio (3) spheroid staining, at days 1 and 3 of culture. c) Cell viability assessment by Live/Dead staining re-adhered spheroids, after 24 hours incubation.…”

“…Also, they play an important role in cancer research, drug discovery, and tissue engineering. [1] Spheroids are progressively finding application in tissue engineering strategies as building block units to be used in a multiplicity of approaches targeting the regeneration of different organs and tissues, [2][3][4][5] including bioprinting and biofabrication microfluidic devices, [19] production of smart drug delivery systems, [20] biocompatible polymeric microparticles with nearly 100% drug encapsulation yield, [21] bioactive spherical structures with shape memory capability, [22] and high-throughput generation of shapecontrolled microgels. [23] Other promising application of these platforms involves the easy in situ analyzes of cellular response.…”

Formation of three‐dimensional fetal myocardial tissue cultures from rat for long‐term cultivation

“…Scaffold-free cell sheet stacking after cultivation of monolayer patches on a thermosensitive culturing membrane showed promising mechanical properties (Shimizu et al, 2003). Recently, the scaffold-free generation of beating three-dimensional "myocardial microtissues" was reported by Kelm and coworkers (Kelm et al, 2004). A cultivation technique as hanging drops resulted in aggregation of dissociated rat heart cells to three-dimensional spherical clusters up to 200 m in diameter.…”

“…Numerous different tissue-like culture models are established by using various culture techniques, biological and nonbiological scaffolds, culture media, and cell sources (Zimmermann et al, 2000;Shimizu et al, 2003;Shin et al, 2003;Kelm et al, 2004;Kofidis et al, 2004). The aim of our study was to develop an easy-to-produce threedimensional scaffold-free culture model with homogenously distributed cardiomyocytes that could serve as a robust test system for basic research in vitro and in vivo.…”