Longitudinal Monitoring of Metabolic Gradients in Microreactor Culture Platforms by Raman Spectroscopy

Metabolic heterogeneity within the cell microenvironment is a key driver of cancer progression and resistance to therapy. However, current approaches lack the spatial and temporal resolution required to capture its dynamics in living systems. While recent advances in 3D cell culture models and metabolomic profiling have improved our understanding of the tumor niche, their integration with real-time optical sensing remains underdeveloped. Here, we present an integrated platform combining a 3D-printed microreactor culture chamber with Raman spectroscopy to enable non-invasive, spatially resolved metabolic monitoring of living cell cultures. Our microreactor platform generates controlled oxygen and nutrient cues while simultaneously acquiring label-free Raman spectra, revealing extracellular metabolic fingerprints linked to cell catabolism (e.g., glucose and lactate shifts) and acidification. Analysis across four cell lines uncovered temporal evolution as the dominant source of metabolic variance, while spatial heterogeneity along oxygen gradients is a secondary factor. In particular, diffusion-limited regions exhibited localized acidification and accumulation of stress biomarkers-such as the release of nucleotides-features that cannot be detected using conventional bulk assays. By providing a versatile platform for real-time mapping, this work enables the mechanistic dissection of cell adaptation to microenvironmental stress and supports the prediction of metabolic signatures underlying drug response and treatment outcomes.