Natural killer (NK) cell cytotoxicity is highly dependent on the ability of NK cells to migrate through the extracellular matrix (ECM) microenvironment. Traditional imaging studies of NK cell migration and cytotoxicity have utilized 2-D surfaces, which do not properly reproduce the structural and mechanical cues that shape the migratory response of NK cells in vivo. In addition, current in vivo imaging does not allow for the accurate long-term single-cell imaging required to dissect the functional heterogeneity of NK cell populations, and importantly, it does not allow studies of human cells. Therefore, it is desirable to implement in vitro migration and killing assays that better mimic in vivo conditions.
We have combined a microwell assay that allows long-term imaging and tracking of small, well-defined populations of NK cells with an interstitial ECM-like matrix to more closely approximate in vivo conditions. The microwells, which are loaded with a gel mixture containing NK and target cells, allows for long-term imaging of NK–target cell interactions within a confined 3-D volume. The microwells were optically sectioned by confocal fluorescence microscopy once every 2 min for 12 h. NK cells were tracked by the Baxter Algorithms to assess motility parameters and interactions with target cells were manually scored for duration and outcome.
We found marked differences in motility between individual cells with a significant fraction of the cells moving slowly and being confined to a small area within the matrix, while other cells moved more freely, probably reflecting local variations in the matrix structure and inherent difference in motility between individual cells. A majority of NK cells also exhibited transient variation in their mobility alternating between periods of migration arrest and random movement. NK cells that alternated between different modes of migration switched on average once every 3 h.
NK cells made fewer and shorter contacts with target cells than in comparable 2-D assays. The difference was particularly pronounced for the process of post-conjugation attachment when NK and target cells separate. The timing of this process is likely influenced by a biomechanical component only present in 3-D environments where the cells are offered multiple anchor points with the matrix that can be used to generate the forces needed to pull apart.
The developed microwell-based assay is suitable for 3-D time-lapse imaging of NK cells migration and cytotoxicity. As it allows for experiments with human cells, it could be used as a complement to in vivo imaging to study the influence of e.g. education and cytokine activation on NK cell heterogeneity in migration and cytotoxicity.