Training-to-Beat bioreactor for investigating Engineered Cardiac Tissues: design, development & validation

Abstract

In last the decades, advances relevant to the generation of 3D Engineered Cardiac Tissues (ECTs) have been made. In reason of this, ECTs are now considered a great promise for in vitro studies of cardiac development, disease and, eventually, for strategies for the repair of the structure and function of the injured myocardium. Among the several physical stimuli which have been exploited to improve the functionality and maturation of ECTs, a preeminent role has been ascribed to mechanical stimulation. Appropriate mechanical stimulation can be recreated and maintained within bioreactors, which are devices/platforms devoted to mimic the physiological milieu in a monitored/controlled culture environment, where the engineered constructs can be properly stimulated. One main limitation of the bioreactor-based strategy for cardiac tissue engineering applications is that the devices which are currently used are meant to passively apply to ECTs a stimulus predefined by the user, regardless of their level of maturation along the duration of the in vitro culture. In this scenario, and trying to overcome current limitations, a novel bioreactor design has been conceived for the investigation of 3D ECTs with a biomimetic approach. Technically, the here proposed bioreactor is capable (1) to apply native-like or pathologic mechanical stimuli (cyclic strain) by means of a reliable linear actuator operating in a wide range of strains and frequencies, and (2) to monitor in real-time both chemo-physical parameters (e.g. oxygen tension, pH) of the milieu and the mechanical stiffness of ECTs by means of dedicated sensors, eventually adapting the stimulation to the actual stage of maturation of the constructs. As a proof of concept, a first experimental campaign has been carried out with a double aim: (1) to verify the bioreactor feasibility in delivering mechanical cyclical stimulation to 3D fibrin-based, ring shaped Engineered Cardiac Tissues (ECTs); (2) to assess the effect of cyclic strain on tissue maturation, contractility and modification on its mechanical properties. In detail, the bioreactor platform has been preliminarily tested to verify protocols for hold on, sterilization, and control of the delivered mechanical stimuli. Firstly, the suitability of the bioreactor platform in culturing ad-hoc designed constructs, in terms of ease of use and capability in setting the stimulation parameters, has been tested. Then, the observed maturation of ring shaped ECTs subject to sinusoidal cyclic strain within the bioreactor has confirmed the potency of the proposed approach and the instrumental role of mechanical stimulation in ECTs maturation and in the development of an adult-like cardiac phenotype responsive to electrical excitation. Even if further validation steps are required before the implementation of culture strategy fully adaptive in terms of mechanical stimuli applied to the engineered cardiac constructs, the developed bioreactor represents a valuable proof of concept for, in its most advanced operational mode, biomimetic culturing of engineered cardiac constructs