Versatile electrical stimulator for cardiac tissue engineering—Investigation of charge-balanced monophasic and biphasic electrical stimulations

The application of biomimetic physical stimuli replicating the in vivo dynamic microenvironment is crucial for the in vitro development of functional cardiac tissues. In particular, pulsed electrical stimulation (ES) has been shown to improve the functional properties of in vitro cultured cardiomyocytes. However, commercially available electrical stimulators are expensive and cumbersome devices while customized solutions often allow limited parameter tunability, constraining the investigation of different ES protocols. The goal of this study was to develop a versatile compact electrical stimulator (ELETTRA) for biomimetic cardiac tissue engineering approaches, designed for delivering controlled parallelizable ES at a competitive cost. ELETTRA is based on an open-source micro-controller running custom software and is combinable with different cell/tissue culture set-ups, allowing simultaneously testing different ES patterns on multiple samples. In particular, customized culture chambers were appositely designed and manufactured for investigating the influence of monophasic and biphasic pulsed ES on cardiac cell monolayers. Finite element analysis was performed for characterizing the spatial distributions of the electrical field and the current density within the culture chamber. Performance tests confirmed the accuracy, compliance, and reliability of the ES parameters delivered by ELETTRA. Biological tests were performed on neonatal rat cardiac cells, electrically stimulated for 4 days, by comparing, for the first time, the monophasic waveform (electric field = 5 V/cm) to biphasic waveforms by matching either the absolute value of the electric field variation (biphasic ES at ±2.5 V/cm) or the total delivered charge (biphasic ES at ±5 V/cm). Findings suggested that monophasic ES at 5 V/cm and, particularly, charge-balanced biphasic ES at ±5 V/cm were effective in enhancing electrical functionality of stimulated cardiac cells and in promoting synchronous contraction.


Lumped-parameter model assumptions
The three parameters of the Randles Cell (Re, Rp, Cp) were evaluated considering the materials and geometry of the chamber described in section 2.2.
To determine Re it must be taken into account the conductivity of the solution σ and the geometry of the electrolyte where the current flows in. For an electrode area A exposed to the electrolyte carrying a uniform current, being d the spacing between the electrodes, the solution resistance is calculated as follows (Tandon et al., 2008) where σ is the conductivity of the solution and A is the area of the electrode exposed to the solution.
The value of A was calculated assuming that the cylindrical carbon rod electrodes in the chamber expose 2/3 of their lateral surface to the electrolyte: Considering the length of the portion exposed by the electrode to the electrolyte l = 20 mm, the electrode radius r = 1.5 mm a the interelectrodic distance d = 1 cm, the area resulted A = 1.26 cm 2 .
Considering the value for the conductivity of the culture media reported in literature (

Movie analysis
From the recorded movies of the electrically paced samples, cardiomyocyte contractility was assessed by measuring the peak amplitude (PA) of the contractions, defined as the maximum displacement of each CM during a contraction, and the contraction time delay (CTD), defined as the maximum time delay between the contractions of different CMs following a single pacing pulse.
For this purpose, the movies, acquired using a 10X objective lens at 30 fps with the live-imaging microscope incubator (ZEISS X91, Olympus, Japan), were analyzed with TrackMate, a Fiji software (NIH, USA) tracking plugin, and processed with a custom Matlab code. Briefly, the movies in Fiji were divided in frames, each frame corresponded to 0.033 s of movies. In TrackMate, the estimated object diameter was set at 15 µm and the quality threshold at 50, using the LoG detector. The program tracked the movement of points through the different frames and generated their trajectory using the Simple LAP tracker. The trajectories were eliminated if they had gaps of tracking, or their displacements were greater than 10 µm between two consecutive frames, thus false trajectories were excluded. Ten trajectories per sample were randomly selected manually, to ensure that the trajectory corresponded exactly to the movement of the cells. For each trajectory, 4 cell contractions were recorded. The X and Y coordinates of the trajectories were exported to an Excel file and the signals were processed with a custom Matlab code. For each trajectory, the displacement magnitude was obtained considering as reference the X and Y coordinates of the points of an instant in which the cells were relaxed.
To calculate the PA, for each trajectory the local maxima of the displacement magnitude were extracted and then averaged, obtaining 10 PA values for each movie (Supplementary Figure S4).
To calculate the CTD, for each paced contraction, the time intervals between the trajectories' peaks were calculated and the maximum time interval value was considered as the CTD, obtaining 4 CTD values for each movie (Supplementary Figure S4). Values were grouped for each experimental condition and expressed as mean ± SD.
Supplementary Table S1. Electrical conductivity and relative permittivity values of the modelled subdomains.