Unperturbed dye-based imaging of spontaneous synchronized calcium activity in iPSC-derived neuronal cultures

Synchronous calcium (Ca²⁺) bursting is a hallmark of neuronal network maturation. While microelectrode array (MEA) recordings are routinely used to generate population-averaged measurements on this functional network activity, live cell Ca²⁺-imaging offers single-cell resolved, contextual data. Unfortunately, most electrophysiologically active cells are hypersensitive to medium exchange, which is standard practice in most sensor dye-based Ca²⁺-imaging protocols. Here, we found that the use of conditioned imaging medium preserves spontaneous network activity of iPSC-derived glutamatergic and motor neuron cultures. The effect was consistent across different cell lines and seeding densities and allowed for the faithful detection of disease-specific phenotypes, as shown using a KCNQ2-related epilepsy model. Our findings thus provide a simple, robust strategy to measure spontaneous network activity in Ca²⁺-imaging experiments, broadening the utility of this technique for functional phenotyping, disease modeling, and drug screening with cellular resolution.