Mechanical Properties at the Nanoscale of Cardiac Organoids Investigated by Scanning Probe Microscopy

Nowadays, the design of advanced <i>in vitro</i> models, allowing effective recapitulation of the complexity of cardiac <i>in vitro</i> pathophysiology, is critical 1) for the definition of the underlying mechanisms, 2) to test the efficacy of novel therapeutic treatments and 3) to move forward in the development of personalized medicine approaches. In this direction, the combination of induced pluripotent stem cell (iPSC) technology with microfabrication approaches, allowed the development of organoids, small dimension 3D structures, recapitulating organ multicellularity, geometrical organization and functionality. Cardiac pathologies often include arrhythmic and fibrotic phenotypes, thus raising the need of <i>in vitro</i> electromechanical measurements, to allow the phenotypization of the <i>in vitro</i> tissue and a functional read-out of the capacity of a treatment to restore the physiological phenotype. At this aim, we evaluated the feasibility to implement scanning probe microscopy and force spectroscopy-based techniques to quantify the pro-fibrotic commitment in our cardiac organoid model (iPSC based, 500 μm diameter spheroids).<br/>In this work, the mechanical properties of cardiac organoids were investigated at the nanoscale by scanning probe microscopy (SPM), and in particular atomic force microscopy (AFM). Force spectroscopy was carried out, after an accurate calibration of the probe mechanical response on a rigid Sapphire sample and a standard two-component polymer sample made of polystyrene (PS) and low-density polyethylene (LDPE). Local single force-distance (FD) curve and the FD curves mapping were carried out onto a surface of an organoid obtained by iPSC line from control or patient affected by known variant causing fibrotic deposition (FIB). Regarding the mechanical properties, obtained from the FD curves mapping down to 1.6x1.6 μm², a clear difference in the elastic modulus distributions on the surface of the organoids was observed, ranging from 25 to 2250 MPa, suggesting a different composition of the myocardium in response to the genetic background. The main experimental analysis focused on the elastic modulus investigation of the surface of the control and FIB cardiac organoids, indicating that AFM measurements can be a useful tool for phenotypization of fibrosis in cardiac organoids.<br/>In addition, Raman spectroscopy was performed to discern the chemical compositions of the organoid. Further morphological analysis was performed by environmental scanning electron microscopy (ESEM), operating at variable pressure (50-200 Pa) allowing to obtain information on the global morphology of the biological samples up to 20k magnifications.<br/>In conclusion, we demonstrated the feasibility to apply SPM techniques to investigate the spatial distribution of the elastic modulus along the surface of the organoid, thus allowing organoids phenotypization, opening the path to exploitation of this method for in vitro modelling scenarios.<br/><b>Acknowledgements</b><br/>Funded by the European Union– Next Generation EU – NRRP M6C2 – Investment 2.1 Enhancement and strengthening of biomedical research in the NHS under the proposal PNRR-MR1-2022-12376524 “Cardiac organoids towards iPSC exploitation for a novel personalized medicine approach to arrhythmogenic cardiomyopathy” – CUP Master B93C22001470008