Assessment of NK cytotoxicity and interactions with porcine endothelial cells by live-cell imaging in 2D static and 3D microfluidic systems

Comparison of Delfia and microscope-based cytotoxicity assays. (A) Graphical images describe the steps of Delfia assay and (B) the sample preparation for live cell-imaging microscopy. BATDA (Bis(acetoxymethyl)-2,2′:6′,2″-terpyridine-6,6’’-dicarboxylate) is a non-fluorescent compound that permeates cell membranes. TDA (Terbium-based fluorescence) is used as a label in the assay as it chelates produce long-lived fluorescence signals. (C) Endothelial cell monolayer used in the microscope-based assay. Porcine endothelial cells (PAECs) were stained with the endothelial cell marker CD31 (red). The nuclei were stained with Hoechst (cyan) before the live imaging session. Scale bar = 50 μm. © Adapted from Tran et al. Scientific Reports 2024 Figure 1.

Summary

Natural Killer (NK) cells are pivotal in immune responses to viral infections, malignancies, autoimmune diseases, and transplantation. Assessment of NK cell adhesion, migration, and cytotoxicity is fundamental for in vitro studies. the authors of this article, led by GCIR Professor Jörg Seebach, propose a novel live-cell tracking method that addresses these three major aspects of NK cell function using human NK cells and primary porcine aortic endothelial cells (PAECs) in two-dimensional (2D) static assays and an in-house cylindrical 3D microfluidic system. The results showed a significant increase of NK cytotoxicity against pTNF-activated PAECs, with apoptotic cell death observed in the majority of dead cells, while no difference was observed in the conventional Delfia assay. Computed analysis of NK cell trajectories revealed distinct migratory behaviours, including trajectory length, diameter, average speed, and arrest coefficient. In 3D microfluidic experiments, NK cell attachment to pTNF-activated PAECs substantially increased, accompanied by more dead PAECs compared to control conditions. NK cell trajectories showed versatile migration in various directions and interactions with PAECs. This study uniquely demonstrates NK attachment and killing in a 3D system that mimics blood vessel conditions. Their microscope method offers sensitive single-cell level results, addressing diverse aspects of NK functions. It is adaptable for studying other immune and target cells, providing insights into various biological questions.

This work was supported by the Swiss National Science Foundation (SNSF).

Citation: Tran, T., Galdina, V., Urquidi, O. et al. Assessment of NK cytotoxicity and interactions with porcine endothelial cells by live-cell imaging in 2D static and 3D microfluidic systems. Sci Rep 14, 24199 (2024). https://doi.org/10.1038/s41598-024-75217-3

WHY is it important?

Natural killer (NK) cells are critical components of the immune system, responsible for killing infected, cancerous or foreign cells. This study developed a new method to observe NK cell activity and movement using human NK cells and human/porcine endothelial cells. Two different systems were used: a flat 2D model and a more realistic 3D microfluidic model that mimics blood vessels.

The researchers focused on three key functions of NK cells: adhesion, movement and cytotoxicity (ability to kill cells). The 3D model, which more closely mimics the internal environment of blood vessels, provides clearer insights into NK cell behaviour. This technique allowed continuous real-time observation of cell-cell interactions. For example, under fast flow conditions, NK cells could fully stop, adhere to endothelial cells, and kill them. They also moved in various directions, even against the flow, to find more targets—behaviours not visible in conventional 2D systems.

Taken together, this study offers a more lifelike way to investigate NK cell function, which could lead to better therapies in cancer treatment and transplant medicine.

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