*Purpose: Neutrophil Extracellular Trap (NET) is a vigorous immune reaction of neutrophil. It plays role in xenotransplantation rejection. Functional assay of NET is important; however, it was difficult to mimic in vivo environment. A static 3D co-culture system based on microfluidic chip was designed to study Xeno-immune reaction.

*Methods: Porcine aortic endothelial cell line (MPN3) was seeded inside the microfluidic channel (Fig 1A). Cells were grown under bidirectional perfusion (luminal shear stress: 5 dyne/cm²). Isolated human neutrophil or non-human primate neutrophils were used for the ex-vivo immune reaction. NETs were visualized either with cell permeable/impermeable DNA and/or NET proteins specific antibody by confocal staining and live image recording.

*Results: After 5 days culture, MPN3 cells formed luminal structure. (Fig 1B). Different stages of NET formation were captured in time lapse recording. (Fig. 2A) Citrullinated histone, which is a marker for NET, colocalized with extracellular DNA was shown in the coculture system of human neutrophil with MPN3. (Fig.2B). Human neutrophil adhesion to MPN3 is captured after coculturing human neutrophils (Fig. 3).

*Conclusions: In this microfluidic chip-based co-culture system, we could visualize NET formation both in live recording and confocal staining.

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