Shear-Induced CROSS (Cellular RedOx Spreading Shield) Assembly Sustains Neurotrophic Extracellular Vesicle Production...

Stem cell-derived neuron-glia models provide a robust platform for studying brain physiology, developing therapeutics, and exploring biocomputation. Extracellular vesicles (EVs) containing neurotrophic factors, also derived from stem cells, have the capacity to facilitate the formation of neural networks within these systems. However, their bioactivity is often compromised by the propagation of oxidative stress between cells during their manufacture, limiting reproducibility. Here, the Cellular Redox Spreading Shield (CROSS) is introduced as a droplet microfluidic-assembled antioxidant crystal-loaded microgel that sustains antioxidant activity for up to 6-7 days in stem cell cultures. In mesenchymal stromal cell (MSC) cultures, CROSS mitigates oxidation propagation and preserves potent neurotrophic EV production. These EVs, specifically enriched with neurotrophic microRNAs, enhance neural stem cell differentiation into neuron-glia networks, characterized by increased synaptic density and functional connectivity, as determined by calcium transient imaging combined with graph theory. In contrast, EVs from untreated, oxidatively stressed MSCs impair neural stem cell differentiation and network formation. This work highlights the importance of CROSS in stabilizing cellular production of neurotrophic EVs, with broad implications for neural tissue regeneration and biohybrid technologies.