Combining micropatterning and microfluidic gradient generation to study hypoxic extracellular vesicles effects in neuroblastoma

We present an integrated microfluidic platform to study the dose-dependent cellular responses to hypoxia-derived extracellular vesicles (hEVs) in neuroblastoma (NB), a deadly pediatric solid tumor. While the method of generating concentration gradients is well established, our system uniquely combines no-shear microfluidic gradient generation with in situ micropatterning of cell-adhesive and non-adhesive regions on a glass substrate. The device is composed of two polydimethylsiloxane (PDMS) layers: one enabling micropatterning for selective cell positioning, and the other housing the microfluidic network and culture chamber. This configuration supports the culture of SK-N-AS NB cells and human bone marrow-derived mesenchymal stem cells (hMSCs-BM, representative of a common NB metastatic site) under spatially controlled conditions. The platform enables real-time higher-throughput analysis of hEV uptake across a stable gradient within a single experiment. Immunofluorescence (IF) assays reveal that hEV internalization is strongly associated with increased expression of proteins involved in multidrug resistance, stemness, and epithelial-mesenchymal transition (EMT), in both NB cells and hMSCs. Our approach contributes to a better understanding of hEV-mediated signaling in the tumor microenvironment and supports the development of spatially resolved in vitro models for therapeutic evaluation.