Drug transport in spheroids on a microfluidic tumor-on-chip platform: An integrated computational and experimental study

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Quantitative prediction of drug transport in three-dimensional tumor spheroids is critical for the design and interpretation of microfluidic spheroid-on-chip assays under perfused conditions. However, this process is governed by coupled advection, diffusion, cellular uptake, and metabolism within structurally heterogeneous spheroid architectures, making accurate modeling challenging. Here, we present a mechanistic transport framework that integrates realistic spheroid geometries reconstructed from histological imaging. The model couples advection–diffusion in the surrounding microchannel with tissue-scale transport through a heterogeneous, densely packed spheroid microenvironment, together with membrane-limited cellular uptake and intracellular metabolic consumption. This formulation resolves spatiotemporal drug concentration distributions under both static and perfused conditions within a unified framework. High-resolution micro particle image velocimetry (micro-PIV) measurements, performed under matched flow conditions, quantify the velocity field and interfacial shear stresses around spheroids in microchannels, providing detailed experimental characterization of the external hydrodynamic environment. The simulated velocity fields were validated against corresponding micro-PIV measurements under matched perfusion conditions, demonstrating good agreement between the computational and experimental flow profiles. In addition, fluorescence-based drug uptake experiments in MG-63 spheroids under static and perfused conditions provided an experimental investigation of perfusion-enhanced transport behavior within the spheroid microenvironment. The combined computational and experimental analysis identifies distinct transport regimes governed by diffusion, perfusion, and cellular metabolism, wherein micro-PIV measurements define the perfusion conditions for the transport analysis, while the computational model reveals how controlled microfluidic perfusion shapes intratumoral concentration gradients and penetration behavior. By directly linking spheroid-scale transport behavior with externally imposed microfluidic flow conditions, the framework provides a mechanistic basis for investigating physiologically relevant operating conditions in spheroid-on-chip systems relevant to microfluidic drug screening applications.