Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity

Abstract

Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon–glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.

A quantitative model for the regulation of innate immune activation

“…Our results on immune cell heterogeneity are consistent with experimental studies. Investigations of the patterns of target cell killing have observed widely varying responses for individual NK cells: some kill many targets efficiently, while others are inactive [38][39][40][41][42][43] . This finding holds not only for diverse primary cells [38][39][40] , but also for NK cell lines [41][42][43] , where cells in the population would be predicted to have homogeneous expression and density of receptors.…”

Mechanical Regulation of the Cytotoxic Activity of Natural Killer Cells

“…the number of killed target cells (41). It was found that there is a small but extremely active sub-population NK cells, which was called "serial killer" NK cells, due to their ability to eliminate a number of target cells each (42). This in vitro detection of the "serial-killer" population mirrors a prevalent model by which only a minority of NK cells is responsible for tumor elimination.…”

Analysis of Leukocyte Behaviors on Microfluidic Chips

“…One of the advantages of microchamber‐based chips is the convenience in cell loading which usually relies on merely gravity. For example, the mixture of activated primary natural killer (NK) cells and chronic myeloid leukemia cells was introduced into a chip and cells were allowed to settle into the microchambers ( Figure A) . After culturing for several days, the intrinsic cytotoxicity heterogeneity of NK cells was detected on the chip by high‐throughput tracking.…”

“…A) The silicon–glass microchamber chip for NK cytotoxicity study: i) illustration of the microchamber chip; ii) detailed view of the silicon–glass microchamber chip; iii) time lapse images of NK cells killing different target cells. Reproduced under the terms of the CC‐BY 4.0 license . Copyright 2016, the Authors.…”

“…Specifically, microfluidic chips are more than suitable for leukocyte behavior analysis due to their merits in leukocyte microenvironment recapitulation and precise control of immunological chemoattractants . So far, various microfluidic chip–based leukocyte behavior studies have been performed, including leukocyte enumeration, separation, identification, migration, and cytotoxicity screening . These microfluidic chips allowed us to better understand how leukocytes function in the body, to diagnose various diseases without bulk equipments, and to develop more effective treatment for multiple diseases.…”