Decoding Cell–Cell Interactions in 2D Blood–Brain Barrier Models Through Molecular Vibrational Spectroscopy and Biology

The blood–brain barrier (BBB) is a coordinated multicellular interface in which intercellular communication shapes responses to injury and metabolic stress. However, the contributions of individual cell types to collective stress adaptation remain poorly understood. Here, we examined molecular and metabolic responses of 2D BBB models comprising endothelial cells, astrocytes, and pericytes exposed to hypoxia or sublethal HgCl₂ toxicity. An integrated platform combining gene and protein expression profiling, label-free vibrational spectroscopic imaging, and machine-learning-based analysis was used to assess stress-induced changes in cell-cycle regulation and metabolism across monoculture and co-culture configurations. Hypoxia broadly suppressed G1/S and G2/M regulators, inducing cell-cycle arrest with limited cell death, consistent with an adaptive response. HgCl₂ caused heterogeneous, model-dependent changes in viability and proliferative capacity. Spectroscopic phenotyping showed that co-cultured cells acquired molecular profiles characteristic of pericytes, indicating a dominant pericyte-driven influence on the cellular community. Fluorescence imaging further revealed intercellular mitochondrial transfer at cell–cell contact sites. These findings identify pericytes as key contributors to stress resistance and BBB cell survival. The developed spectroscopic–molecular platform provides a label-free framework for monitoring cellular communication and stress responses in multicellular BBB models.