Background Since the FDA approval of an NK cell-based cancer immunotherapy, the heterogeneity of primary NK cell populations has been of increasing interest. How can we reveal the properties of single cells in analyses of large cell populations? Large-number functional single cell experiments provide the key for that question, since they allow the user to monitor different properties of killing events during an NK killing assay – revealing individual cell properties. In recent years, advances have been made in tools for high-throughput analysis. However, these technologies mostly collect data that are not associated with individual cells. Moreover, the data obtained are rarely directly associated with the properties and functions of individual cells. Therefore, there is an urgent need for technologies that enable the study of large cell populations at the single cell level under in vivo-like conditions. In addition, technologies that enable the isolation of single living cells for downstream processes increase the value many times over.

Methods ARRALYZE is a digital cell biology platform that meets all of the above requirements by using highly miniaturized glass well arrays for functional single cell screening. Our micrometer-sized glass wells accommodate hundreds to thousands of wells on the footprint of a microscope slide. In a controlled manner, cells are placed in these wells, continuously imaged with brightfield and fluorescence microscopy for high-throughput analysis and time-lapse video, and then isolated for further study while they are still viable. The platform enables controlled co-cultivation of different cell types, making it ideal for immune cell killing assays and other functional studies.

Results In our paper, the performance of the platform is demonstrated using K562 target cells and primary NK cells as an example system. Both cell types are stained with two different colors and co-cultured in the presence of a death dye. The color change from the target cell by the death dye shows a killing event. By monitoring the contents of each well, we can study the kinetics of the killing events to gain insight into immune cell motility and behavior, as well as their effectiveness in identifying and eliminating target cells.

Conclusions Our contribution will be of great benefit to participants in the fields of immunotherapy and related fields.