A validated in vitro approach for low-frequency alternating-current stimulation of stem cells: From electrode characterization to biological responses

Electrical stimulation remains a promising but insufficiently standardized approach in regenerative medicine, as many in vitro studies lack physically defined stimulation systems and quantitative characterization of cellular electric-field exposure. Here, we present a measurement-based framework integrating real-time electrical recordings, impedance-informed simulations, and electrode characterization under experimental conditions. Human adipose-derived stem cells were stimulated for three days at 20 Hz using sinusoidal alternating current at applied voltages of 1–3 Vrms. Simulations revealed spatially heterogeneous electric-field distributions, with exposure predominantly below 5 Vm⁻¹ at 1 Vrms and shifting toward 5–15 Vm⁻¹ at higher amplitudes. Moderate stimulation (1–2 Vrms) was not associated with detectable reductions in metabolic activity and was accompanied by maintained functional behaviour and stable reference gene expression, whereas 3 Vrms coincided with electrochemical interface alterations and reduced gene stability, suggesting the onset of stress-associated cellular responses. These findings identify a system-specific, biologically compatible and functionally active stimulation window and demonstrate that cellular responses are governed by the effective electrical dose rather than nominal voltage alone. The presented framework enables reproducible, impedance-controlled and quantitatively defined in vitro alternating-current stimulation.