Assessment of 3D bioengineered cardiac tissue model for chemical toxicity research: integration of biomaterials and co-culture cell system

The main aim of this Ph.D. project is to contribute to the development of a 3D bioengineered cardiac tissue replica composed of a 3D-printed poly(urethane) scaffold as support for a co-culture of human induced pluripotent stem cells (hiPSCs)-derived cardiomyocytes (CMs) and Human Coronary Artery Endothelial Cells (HCAECs) embedded in Gelatin-Methacryloyl (GelMA) hydrogel. After integration in a sensorised bioreactor system, the 3D cardiac tissue replica will be used as an in vitro model to test adverse cardiotoxic impacts of pollutants, chemicals, and their mixtures at different concentrations and times of exposition, to investigate the activated chemical pathways and to identify biomarkers of cardiotoxicity. Here, the biological assessment of the 3D bioengineered cardiac tissue replica will be described in association with a preliminary application of the model to study cardiotoxicity. The first part of the work was focused on the differentiation protocol used to obtain CMs from hiPSCs. Afterward, cellular and molecular analyses were conducted to investigate the maturation profile achieved by hiPSC-CMs cultured alone or with endothelial cells (different cell ratio concentrations) in a classical 2D system versus the 3D model (cells embedded in 5% w/v GelMA). Transcriptomic and proteomic analyses highlighted the modulation of different biological, cellular, and molecular processes in the 2D monoculture with respect to the 3D co-culture. Global results highlighted the ability of our 3D bioengineered model to improve CMs maturation due to the mimicking of the human in vivo tissue environment (See graphical abstract, A section). The biological investigation was continued in an improved 3D microenvironment where a poly(urethane) 3D-printed scaffold was implemented as support for the hydrogel to a perfected mimic of the morphological and mechanical properties of the native cardiac extracellular matrix (ECM). By changing the concentration of the GelMA (5% or 10% w/v), the mechanical properties of the hydrogel were affected, providing the opportunity to compare cellular response within materials characterized by different stiffness to simulate the physiological aging of the cardiac tissue. After confirming the absence of differences in terms of viability among cells maintained in the 2 different hydrogel concentrations and the maturation profile shown by CMs cultured in the stiffer matrix (10% w/v of hydrogel), specific omics analyses were carried out. Results reported an impact of hydrogel’s mechanical properties on lipids and amino acids metabolic pathways (See graphical abstract, B section). The final part of the work focused on assessing the 3D bioengineered system as a model for toxicological investigations, starting with a chronic dose-response study of the well-known cardiotoxic compound Doxorubicin (DOX). Analysis of DOX-related impact on cell viability and oxidative stress in 2D versus 3D environment enabled the identification of the 85% effective concentration (EC85), defined as the effective concentration of DOX that produces a biological response in 15% of the population tested. The effect of the selected dose allows for a limited impact on most treated cells, thus ensuring a better investigation of the cellular response after chronic exposure without killing the cells. Starting from the EC85, a small dose-response curve of DOX treatment was performed during 6 months of secondment at the University of Technology of Sydney (UTS, Australia). CMs monoculture in 2D was compared to the 3D bioengineered co-culture of CMs and HCAECs. Acute effects on oxidative stress were investigated, as well as chronic impacts on viability and function, through analysis of beating rates and electrophysiological properties, highlighting different cellular responses as a consequence of the surrounding microenvironment composition in terms of structure organization (2D or 3D) and cellular composition (monoculture or co-culture) (See graphical abstract, C section). This work was supported by the European Union’s Horizon 2020 research and innovation program (grant #101037090 and #814495).