Electric field stimulation integrated into perfusion bioreactor for cardiac tissue engineering.

We describe herein the features of a novel cultivation system, combining electrical stimulation with medium perfusion for producing thick, functional cardiac patches. A custom-made electrical stimulator was integrated via inserting two carbon rod electrodes into a perfusion bioreactor, housing multiple neonatal Sprague-Dawley rat cardiac cell constructs between two 96% open-pore-area fixing nets. The stimulator produced adjustable stimulation waveform (i.e., duty cycle, number of stimulating channels, maximum stimulation amplitude, etc.), specially designed for cardiac cell stimulation. The cell constructs were subjected to a homogenous fluid flow regime and electrical stimulation under conditions optimal for cell excitation. The stimulation threshold in the bioreactor was set by first determining its value in a Petri dish under a microscope, and then matching the current density in the two cultivation systems by constructing electric field models. The models were built by Comsol Multiphysics software using the exact three-dimensional geometry of the two cultivation systems. These models illustrate, for the first time, the local electric conditions required for cardiomyocyte field excitation and they confirmed the uniformity of the electrical field around the cell constructs. Bioreactor cultivation for only 4 days under perfusion and continuous electrical stimulus (74.4 mA/cm², 2 ms, bipolar, 1 Hz) promoted cell elongation and striation in the cell constructs and enhanced the expression level of Connexin-43, the gap junction protein responsible for cell-cell coupling. These results thus confirm the validity of the electrical field model in predicting the optimal electrical stimulation in a rather complex cultivation system, a perfusion bioreactor.