A photopatternable multiplex GelMA platform for studying small EV-brain endothelial interactions and direction-resolved glioblastoma spheroid outgrowth

Gelatin methacryloyl (GelMA) photopatterning enables spatial tumour-endothelial cultures, but formulation selection and imaging readouts must account for endothelial responses and heterogeneous tumour outgrowth. Four GelMA/Irgacure 2959 formulations (3 or 5% w/v polymer; 0.5 or 1% w/v photoinitiator) were screened using freeze-dried scanning electron microscopy, hydrated rheology, metabolic activity and expression of five endothelial-associated genes. The 3% GelMA/0.5% photoinitiator formulation (Gel3PI0.5) yielded the highest mean biological readouts in brain microvascular endothelial cells (BMECs). An ordered array of U87-containing microconstructs was photopatterned using this formulation and assembled onto a separately prepared BMEC-laden compartment. Endothelial marker retention and area-normalised impedance characterised the resulting static construct. A small extracellular-vesicle (EV) fraction isolated directly from human amniotic fluid was evaluated by bulk quantitative polymerase chain reaction and sector-based imaging. Two complementary descriptors quantified the 90th percentile distance of U87-positive pixels from the aggregate centre (P90) and their area occupancy within a 500 µm radius (C₅₀₀). At Day 8, 50 µg mL^-1 EV-associated protein increased P90 relative to untreated BMEC-free cultures without a resolved C₅₀₀ difference; in BMEC-containing cultures, 75 versus 25 µg mL^-1 increased C₅₀₀ without a resolved P90 difference. Thus, radial extent and local occupancy captured different condition-associated outgrowth features. Within the 5% polymer pair, lower initiator loading improved mean metabolic activity without a corresponding decrease in representative storage modulus, supporting biological criteria for material selection. The unmatched doses across endothelial contexts support within-arm and predefined-condition comparisons; they do not isolate an endothelial-by-EV interaction or establish functional blood-brain barrier behaviour.