Vibrational spectroscopy as a complementary approach to monitor SH-SY5Y cell differentiation

The SH-SY5Y neuroblastoma cell line is a well-established model for investigating neuronal differentiation, typically induced by sequential treatment with retinoic acid and brain-derived neurotrophic factor to promote neurite outgrowth. In the present study, we examined the evolving spectroscopic signatures of SH-SY5Y cells throughout a seven-day differentiation protocol, consisting of five days of retinoic acid followed by two days of brain-derived neurotrophic factor. Using Fourier-transform infrared spectroscopy spectral analyses focused on the 3050-2800 cm −1 , 1800-1500 cm −1 , and 1500-900 cm −1 regions to monitor metabolomic changes across different days in vitro . Distinct spectral profiles emerged between early and late differentiation stages, indicating dynamic biochemical remodelling. Late differentiation stage cells exhibited enhanced signals associated with lipid ester groups and β-sheet protein conformations, whereas early-differentiation-stage cells showed features linked to unsaturated lipid CH stretches, parallel and antiparallel β-sheet arrangements, and phosphate monoester groups from nucleic acids. These findings highlight the sensitivity and utility of Fourier-transform infrared spectroscopy for monitoring and characterizing the biochemical dynamics of SH-SY5Y cell differentiation. • Differentiation of SH-SY5Y cells confirmed by morphology, β-III-tubulin, and FTIR. • Spectroscopic profiles differ across 3050–900 cm -1 regions with cell DIVs. • Lipid, nucleic acid, and protein peak intensities vary with differentiation timepoints. • FTIR-ATR captures metabolomic changes in SH-SY5Y during in vitro differentiation.