Baca, G. L.; Monticone, R.; Ziman, B.; Rahman, S. M. T.; Parekh, P.; Afrin, S.; Dunn, C.; Telljohan, R.; Yang, D.; Lam, K. W. G.; Killeen, P.; Tsitsipatis, D.; Zagrean, A.-M.; Sung, M.-H.; Greig, N.; Herman, A. B.; Sen, P.; de Cabo, R.; Lakatta, E. G.

Approximately half of the adult sinoatrial node (SAN) consists of non-myocyte populations, indicating that cardiac pacemaking depends on interactions within a multicellular tissue rather than on pacemaker cardiomyocytes alone. Among these, an S100B-associated cell population has been implicated in pacemaker function, yet its identity and physiological roles remain poorly understood. These cells are rare and dispersed throughout the small, structurally complex SAN, making them difficult to observe repeatedly while preserving the native multicellular environment. Here, we established a dissociated multicellular culture of adult mouse SAN tissue on soft collagen-gelatin hydrogels that retains spontaneous electrical activity and permits longitudinal live imaging of S100B-associated cells. Using an S100B-EGFP reporter, we identified at least six reproducible morphological and behavioral phenotypes, including migration, proliferation, phagocytic behavior, and spontaneous self-organization into three-dimensional clusters. Cultures remained spontaneously electrically active for more than 10 days in vitro, with peak activity around day 10. This multicellular culture model bridges the gap between intact SAN preparations and isolated-cell cultures, allowing repeated observation of rare S100B-associated cells within a spontaneously active multicellular environment.