Raman-based detection of lipid accumulation in 3D steatosis-induced liver cultures

Liver diseases represent a major global health threat due to their high prevalence and potential to progress into severe, irreversible stages. Steatosis, characterised by the excessive accumulation of lipids within the hepatocytes, is one of the most common early manifestations and potentially reversible. Consequently, there is a growing need for physiologically relevant in vitro models and analytical techniques that enable accurate assessment of the disease-associated cellular changes. Three-dimensional (3D) liver spheroid cultures better recapitulate key features of the native liver environment than conventional two-dimensional (2D) cell cultures, yet robust label-free methods for their characterisation remain limited. Here, we evaluate Raman mapping combined with multivariate curve resolution-alternating least squares (MCR-ALS) for the assessment of lipid accumulation in 3D HepG2-LX2 liver spheroids. Raman maps acquired from oleic acid/palmitic acid (OA/PA)-treated and control spheroids were decomposed into chemically meaningful components, enabling reconstruction of spatially resolved lipid-associated concentration profiles. Quantification of these profiles revealed a significant increase in lipid content in the OA/PA-treated spheroids compared to the controls and showed strong agreement with the lipid quantification obtained via BODIPY 493/503 fluorescence imaging, a widely used method for assessing lipid accumulation. In addition, time-resolved experiments demonstrated that the proposed workflow is not only sensitive to binary differences in lipid content, but also to intermediate stages of lipid accumulation. These findings demonstrate that Raman mapping combined with MCR-ALS enables label-free spatially resolved quantification of lipid accumulation in 3D liver spheroid models, providing a promising platform for studying lipid accumulation dynamics and evaluating therapeutic responses in physiologically relevant in vitro models.