Label-Free Hyperspectral Raman Imaging and Analysis of a 3D Spheroid Model for Liver Fibrosis Study

An aberrant wound-healing response to chronic liver injury results in excessive accumulation of extracellular matrix proteins, including collagen, leading to liver fibrosis, a process in which hepatocytes and hepatic stellate cells (HSCs) play key roles. In this study, we present HepG2 and LX-2 cocultured spheroids as a three-dimensional in vitro model of liver fibrosis and highlight the potential of hyperspectral Raman imaging as a label-free analytical approach for assessing fibrosis-associated responses. HepG2 cells and LX-2 cells served as hepatocytes and HSCs, respectively, within the coculture spheroids. Hyperspectral Raman imaging allowed for the acquisition of fingerprint spectra from individual cells, revealing distinct cytochrome-related band patterns that facilitated cell-type segmentation without labeling. To induce fibrosis, spheroids were treated with fibrosis activation factors: free fatty acids (FFAs) and transforming growth factor-β (TGF-β). FFA administration induced steatotic features in the coculture spheroids, as demonstrated by a direct correlation between the increasing lipid band intensity in the Raman spectra and FFA concentrations. Concurrently, a reduced cytochrome-related band intensity was observed in these spheroids. Furthermore, LX-2 spheroids but not HepG2 spheroids exhibited signs of fibrosis after TGF-β activation. Raman analysis revealed that the amide I and III bands reflected extracellular matrix, especially collagen accumulation, applied as fibrosis markers. Notably, in the 2:1 coculture spheroids, we observed enhanced fibrosis-related bands in the LX-2-enriched regions. The consistency between Raman imaging results and immunofluorescent images underscores the potential of hyperspectral Raman imaging as a sensitive and noninvasive method for assessing liver fibrosis.