ENGINEERING CARDIAC MICROTISSUE IN VITRO: EFFECT OF THE SCAFFOLD AND CULTURE CONDITIONS ON THE FINAL PROPERTIES OF CARDIAC MICRO-MUSCLES

Most living tissues are composed of repeating units on the scale of hundreds of microns, which are ensembles of different cell types with well defined three-dimensional (3-D) micro-architectures and tissue-specific, functional properties. To generate thick and functional engineered tissues, the recreation of these structural features is of great importance in enabling the resulting functions. To address this need recent efforts have been concentrated on bottom-up approaches aimed at generating a larger tissue construct by the assembly of smaller building blocks, which mimics the in vivo tissue structure of repeating functional units. In this PhD thesis a novel bottom-up approach has been applied to produce functional cardiac tissue, starting from the outcomes of recently published works. The overarching goal of this work was to create in vitro functional cardiac ?-tissue by coupling engineered porous ?-scaffold with neonatal rat cells. We hypothesized that such cardiac ?-tissue construct could be used as a functional building unit to obtain a 3D cardiac tissue in vitro. The ?-scaffolds, consisting of gelatine porous micro-beads with a diameters distribution of 75-150 ?m, was colonized by cardiac cell population in dynamic cell seeding condition by means of spinner flask bioreactor. To optimize the micro-tissue functions we varied several culture parameters: spinner culture conditions (duration and type of flow regime) as well as the initial composition of cardiac cell population. We have successfully established that ?-scaffold construct embedded with a specific cardiac cell composition exhibited the important properties of native cardiac tissue, including the assembly of differentiated cardiac cell populations into a 3D syncytium, as well as electrophysiological functionality and responsiveness to external electrical stimulation. Furthermore, it has been tested the possibility to produce a 3D cardiac tissue constructs, of defined size and shape, by exploiting the biological sintering capability of the micro-tissues (?TPs). We conjectured that the cardiac tissue engineered construct developed could be used as a biological model for studying cardiac tissue development and/or disease processes in vitro, and eventually as an implant to repair injured myocardium.