Development, validation, and optimization of a versatile electrical stimulator for cardiac tissue engineering

Cardiac tissue engineering (CTE) aims to develop functional substitutes of native myocardium to be exploited as in vitro models for cardiac development and disease research, and ultimately for cardiac repair. For the development of effective cardiac substitutes, alongside the cells and the architectural and biochemical support provided by scaffolds, a paramount role is played by the physical stimuli applied. In vitro, several studies demonstrated that electrical stimulation affects the rate, duration, and number of action potentials of cardiomyocytes, increasing the percentage of spontaneously beating cells, promoting cell–cell coupling and calcium handling, and directing the organization of cardiac cells. In this work, we designed, developed and tested a versatile electrical stimulator, ELETTRA, aimed to provide cardiac-like electrical impulses for CTE applications. Specific requirements guided ELETTRA design: 1) accuracy in mimicking the in vivo pulsatile electric field experienced by human cardiac cells (resting rate = 1.0-1.7 Hz, electric field = 0.1-10.0 V/cm, pulse duration = 1-2 ms); 2) versatility to be used in combination with different bioreactors or experimental setups; 3) ease of use; 4) cost-effectiveness. ELETTRA's core consists of an Arduino Due board running a purpose-built software, interfaced to analog and digital peripherals. A user-friendly interface allows the tuning of stimulation parameters (wave-type = mono/biphasic pulses, frequency = 0.5-10.0 Hz, amplitude = 0.5-12.0 V, pulse duration= 1-10 ms). A sensing resistor allows monitoring of the current flowing between the electrodes. ELETTRA was used in combination with carbon rod electrodes embedded in PDMS culture chambers, manufactured on purpose to deliver the electrical stimulation to two tissue platforms: 1) 2D monolayer of neonatal rat cardiomyocytes cultured on gelatin substrates mimicking the stiffness of the healthy myocardium; 2) 3D fibrin gel constructs, encapsulating neonatal rat cardiomyocytes, cultured on PDMS patterned surfaces designed to replicate the morphology of the native tissue. Preliminary tests confirmed satisfactory stimulation performances, ease of use, and cost-effectiveness (overall cost < € 100). As regards biological tests, electrical stimuli provided by ELETTRA induced the development of conductive and contractile properties in the cultured cardiac cells. In particular, neonatal rat cardiomyocytes cultured on gelatin substrates and electrically stimulated showed improved functional outputs in terms of lower Excitation Threshold (ET) values and higher Maximum Capture Rate (MCR) values (ET = 4 V/cm, MCR = 3 Hz) compared with cells cultured on the same substrate but without electrical stimulation (ET = 6 V/cm, MCR = 2 Hz) and with cells cultured on standard plastic wells without electrical stimulation (ET = 7.8 V/cm, MCR = 2.4 Hz). Minimal and not statistically significant improvement was observed in 3D fibrin constructs, for which further study is needed. In conclusion, ELETTRA delivers a controlled and stable electrical stimulation representing a compelling device for investigating the influence of the electrical stimulation on cardiac development and disease. Moreover, coupled to already existing dynamic culture devices, it could be used to provide combined mechanical and electrical stimulations in a physiologically relevant way, which was demonstrated to be essential for proper differentiation and maturation of cardiomyocytes.