Tunable self-assembling cellular microarray for single-neutrophil vital and suicidal extracellular traps.

Neutrophils, the innate immune system's first line of defense, function in pathogen removal through diverse cellular responses. One critical response is neutrophil extracellular trap (NET) formation, which, despite its importance in inflammation, remains poorly understood and lacks high-resolution tools to effectively study its multiple forms: vital and suicidal. To begin addressing this gap, we introduce a tunable micropatterned platform that enables single-cell analysis of NETosis with spatial precision. Using photopatterned bacterial extracellular vesicles (bEVs) on a functionalized surface, we achieved controlled alignment of human peripheral blood neutrophils and demonstrated that engineered patterns control key NETosis outcomes. Meanwhile, phorbol 12-myristate 13-acetate (PMA) stimulation induced uniform suicidal NETosis. This approach supports additional applications, enabling tunable neutrophil patterning and live-cell imaging for intracellular investigation. Additionally, there is flexibility for different extracellular vesicle and nanoparticle patterning and pre-stimulation studies. Here, we present a novel system to quantify and manipulate NETosis pathways at the single-cell level, opening new avenues to explore immune heterogeneity and develop targeted therapies.