Decoding Cell–Cell Interactions in Neurovascular Unit Co-Cultures 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 neurovascular unit (NVU) co-culture 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 mono- 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 revealed that the triple co-culture exhibited spectral features resembling those of pericyte monocultures, although the contributions of cell–cell interactions and differences in culture conditions could not be distinguished. Fluorescence imaging further revealed mitochondria-associated signals at cell–cell contact sites, suggesting a possible association of mitochondrial material with regions of close cellular interaction. The presence of pericytes was associated with a more stress-resistant cellular phenotype in the investigated co-culture configurations. The developed spectroscopic–molecular platform provides a label-free framework for characterizing cellular communication and stress responses in multicellular NVU co-culture models.