Tissue culture is a powerful tool to model and study human disease. Human induced pluripotent stem cells (hiPSCs) can be differentiated into cardiomyocytes and matured for functional assays interrogating contractile health as a function of pharmacologic or genetic perturbation. Long-term culture requires sufficient sterile technique, and many protocols use penicillin/streptomycin (P/S) to protect against bacterial contamination. However, P/S decreases contractile output. To quantify this effect, we cultured hiPSC (WTC11) derived cardiomyocytes (hiPSC-CMs) with or without P/S and assayed force production and calcium signaling. Cells were cultured without P/S until day 21 post-differentiation, with day 16 to 19 lactate purification. Cells were then replated either into tissue culture dishes, onto compliant patterns, or into Engineered Heart Tissues (EHTs), with or without 1X P/S for 14 days. 2-D culture was assayed with Cal-Brite590. The force and kinetics of activation and relaxation were measured at pCa 5.7 and 4 in myofibrils purified from the cells on compliant patterns. EHTs were cast in fibrin, composed of 500K hiPSC-CMs and 50K HS27A stromal cells per tissue. EHTs cultured without P/S generated higher peak twitch force compared to those cultured in P/S (1.88 ± 0.3 vs. 0.64 ± 0.1 mN/mm 2 ), a 65.9% decrease. Time to peak force was slower with P/S (246 ± 6.1 vs. 232 ± 9.3 ms) and relaxation was not significantly different. Myofibrils cultured in P/S generated significantly lower force (pCa 5.7, 96% decrease and pCa 4, 94% decrease). Interestingly, 2-D culture suggests faster calcium rise with P/S (142.4 vs. 177.7ms), higher peak (48.8%), and slower time to 90% relaxation (410.3 vs. 398.8ms). In our other experiments, force loss due to P/S decreased our signal-noise ratio, which masked the effects of a small molecule myosin modulator on kinetics and force. These findings suggest caution when using penicillin/streptomycin.
Asencio et al. (2026) studied this question.