## 2 Materials and Methods
### Whole-Heart Decellularization through Coronary Perfusion  
The rat hearts (\(n=20\)) were decellularized using a modified [PERSON] experimental device, previously presented in our studies. The experimental device respects the principle described by [PERSON]. The decellularization solution is aspirated from the decellularization chamber by the peristaltic pump and reintroduced into the heart via the cannula. The perfusion pressure of the heart is the pressure on the discharge branch of the peristaltic pump and it is monitored by the pressure transducer. The operation of the peristaltic pump is controlled by the automation system. The output of this system reaches a power amplifier and via a solid-state relay it controls the peristaltic pump function [26; 27].  
Half of the hearts (\(n=10\)) were decellularized in the absence of the electric field (control group) and the other half (\(n=10\)) in the presence of an alternating rectangular electric field. The hearts were perfused with 1% sodium dodecyl sulfate (SDS; Thermo Scientific, Waltham, MA, USA) in deionized water for 16 h at about 80 mmHg. A peristaltic pump was used for the perfusion of the heart, recirculating the decellularization solution during the entire course of the experiment. One advantage of this system is a relatively decreased volume of decellularization solution used, which is approximately 150 mL. The heart was totally covered with solution within the glass container, constituting the electrochemical cell. Two stainless steel plate-type electrodes (the surface of each electrode was approximately 14 cm\({}^{2}\)) were placed on the container to generate the alternative electric field required for decellularization. The two electrodes were reachable from the exterior of the electrochemical cell. For generation of alternative electric field, a generator of functions was used, programmed to produce a rectangular electric signal, with a constant frequency (20 hHz) and amplitude (100 mA), corresponding to 7.14 mA/cm\({}^{2}\) current density. Due to the electrical field behavior of the SDS molecule, it was assumed that the detergent would more efficiently penetrate the cellular membranes, allowing for a faster decellularization. During all the decellularization experiment procedures, 10-uL samples were taken from the decellularization solution every 30 min for spectrophotometric measurement of the DNA and total protein concentrations. The hearts were weighed before the decellularization process, with no significant differences between the two groups. For the decellularization process, the aortic cannula was inserted into the ascending aorta and secured with a surgical suture to allow for retrograde coronary perfusion with a flow of 8 mL/min (Figure 2a-g).  
Figure 1: Heart explantation. (**a**,**b**) Removal of the heart from the chest. (**c**) Preparation of the heart to be washed with isotonic saline solution containing anticoagulant. (**d**) Attachment of a needle into the ascending aorta to allow for retrograde coronary perfusion.  
It is to be noted that after its use, the detergent should be removed from the remaining extracellular matrix (ECM), washing it with phosphate buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, USA) and deionized water. The hearts were perfused and stored with 10,000 IU/mL penicillin-streptomycin(Pen/Strep 10,000 IU/mL; Sigma-Aldrich, St. Louis, MO, USA) in PBS before recellularization.