Protocol step #	Critical Steps
2.4	Place the carbon rods 1 - 2 cm apart or at the smallest possible distance to avoid high-energy stimulation and cell death.
2.6	Test the connections between the platinum wire and the carbon once the stimulation chamber is made for the first time. Link the stimulation chamber to the electrical stimulator and then set a desired voltage. Place a volt lead onto each of the two carbon rods. The output voltage from the stimulation chamber should be the same voltage read on the voltmeter. Any incorrect readings indicate the possible coating of the platinum wire with PDMS or another defective connection in the system.
3.1	Single cells were used to seed the biowire. For dissociation, the incubation time with collagenase is critical and should be determined by the age of the cells and the differentiation protocol (EBs versus monolayers). In this protocol, an incubation time of 2 h is recommended for day 21 EBs and 30 min for monolayers. For cells of other ages, the collagenase digestion time might need optimization by monitoring cell viability and contractile function post-digestion.
3.3	Trypsin can damage the cells; thus, limit the incubation duration to no longer than 5 min.
3.5	Avoid introducing contaminants by holding only the end of the syringe to attach the syringe to the needle tip. The number of plunges is determined by the absence of large clumps of cells. Minimize needle disruption to avoid cell death. Avoid bubble formation. In case of visible clumps after needle disruption, prolong the incubation time in collagenase in subsequent isolations instead of increasing the incubation time in trypsin or the number of plunges.
3.6	Remove the supernatant completely prior to the addition of gel. Residual medium may dilute the ratio of cell:gel and affect gel polymerization.
3.7	Maintain the concentrations of the collagen gel components when scaling up or down. Keep in mind that stock concentrations may vary according to the batch or preparation of the reagents.
3.11	To ensure that PDMS microwells stay at the bottom of the Petri dish, make sure that the Petri dish is dry and use a pair of sterile forceps to push the PDMS microwells down.
4.2	Ensure that all biowires are in place. Cover the biowires completely with medium in the electrical stimulation chamber. Arrange all biowires perpendicular to the carbon rods.
4.3	Avoid any damage to the platinum wire by covering the lid gently. Use tape to secure the lid properly on the plate in case the lid does not close fully. When closing the incubator doors, place the thinner portion of the wires from the electrical stimulator at the door closing points to ensure proper sealing of the incubator.
4.4	Disconnect the electrical stimulator for the medium changes. Use a P1000 to remove only the top portion of the old medium and then add fresh medium. Ensure that the biowires are always submerged under medium.