Assessment of chimeric antigen receptor T cytotoxicity by droplet microfluidics in vitro

Chimeric antigen receptor T (CAR-T) cells are cytotoxic T cells engineered to specifically kill cancer cells expressing specific target receptor(s). Prior CAR-T efficacy tests include CAR expression analysis by qPCR or ELISA, in vitro measurement of interferon-γ (IFNγ) or interleukin-2 (IL-2), and xenograft models. However, the in vitro measurements did not reflect CAR-T cytotoxicity, whereas xenograft models are low throughput and costly. Here, we presented a robust in vitro droplet microfluidic assay for CAR-T cytotoxicity assessment. This method not only enabled assessment of CAR-T cytotoxic activity under different fluid viscosity conditions, but also facilitated measurement of CAR-T expansion and dissection of mechanism of action via phenotype analysis in vitro. Furthermore, our data suggested that label-free cytotoxicity analysis is feasible by acquiring data before and after treatment. Hence, this study presented a novel in vitro method for assessment of cellular cytotoxicity that could potentially be applied to any cytotoxicity experiment with varying solvent composition. Statement of Significance: This paper presents a robust in vitro droplet microfluidic assay for assessment of cellular cytotoxicity under varying solvent composition. This method rigorously interrogates cell number expansion and reduction with strong statistical power. Phenotype analysis by fluorescence imaging provides mechanistic insight.