A simplified workflow for end-point validation of real-time electrical cell-substrate impedance sensing of retinoic acid stimulated neurogenesis in human SH-SY5Y cells in vitro

Abstract Objective Retinoic acid (RA) is known to transition proliferating SH-SY5Y neuroblastoma cells towards functional neurons. However, the activity of RA is restricted due to its photosensitivity. The aim of the study was to establish a real-time, long-term (9-day) protocol for the screening and identification of novel small molecule compounds that can induce or enhance SH-SY5Y differentiation and subsequently aid in the development of synthetic neurotrophic factors. Results A differentiation baseline for SH-SY5Y cells was established. Cells were seeded and exposed to repeated spikes of RA using the xCELLigence real-time cell analyser single plate (RTCA-SP) for real-time monitoring and identification of differentiation activity over a 9-day period in order to be more representative of differentiation timelines. Conclusion RA-induced growth inhibition was identified by the xCELLigence analysis and confirmed by end-point metabolic and phenotypic analysis. Change in cellular morphology and neurite outgrowth length was identified by end-point fluorescence detection followed by computational analysis. It was possible to confirm SH-SY5Y phenotypic differentiation using the combination of technologies employed.