Multiplexed cell-based assays for evaluating the structure and function of excitable cells

The flexibility and accessibility of induced pluripotent stem cell technology has allowed complex human biology to be reproduced in vitro at high throughput scales. Indeed, rapid advances in stem cell technology have led to widespread adoption for the development of in vitro models of neuron and cardiomyocyte electrophysiology to be used in screening applications in drug discovery and safety. Furthermore, advanced cell preparations, such as organoids, are under investigation with aims toward establishing mature human phenotypes in vitro. For the development and validation of relevant in vitro neuronal and cardiac models, it is critical to evaluate the structure and function of neuronal synapses and networks, as well as cardiomyocyte viability, electrophysiology, and contractility. The objective of this work is to develop and validate a multiplexed structure-function assay as an efficient approach for evaluating neuronal and cardiomyocyte models in vitro. A planar grid of microelectrodes embedded in the substrate of each well interfaces with cultured cellular networks to continuously monitor both electrophysiological function and structural viability. The electrodes detect the raw electrical activity from the cells to identify changes in function, while structural effects, such as morphological changes and cell viability, are detected as changes in impedance at the cell-electrode interface. Here, we characterized and validated this multiplexed assay using known control compounds that differentially affected cell structure and function. For iPSC-derived neuronal models, all compounds tested (DMSO, glutamate, tributyltin, ionomycin, and Triton X-100) altered functional spiking activity. Glutamate, ionomycin, and tributyltin all produced a dose- and time-dependent decrease in viability, as measured via impedance, with Triton X-100 serving as the positive control for complete loss of membrane integrity. For cardiomyocytes, all compounds (E-4031, nifedipine, isoproterenol, doxorubicin, and blebbistatin) affected aspects of electrical or contractile function, but only doxorubicin decreased cell viability. These results support the continued development and use of human iPSC-derived neuronal and cardiomyocyte assays for high throughput drug discovery and safety assessment.