Assessment of chimeric antigen receptor T (CAR-T) cytotoxicity by droplet microfluidics in vitro

Abstract

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-gamma; (IFNgamma) 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 cell-kill-cell experiment with varying solvent composition.

Engineered Microphysiological Systems for Testing Effectiveness of Cell-Based Cancer Immunotherapies

“…For example, both lymphocytes and tumor spheroids can be combined inside hanging drops, such as a study which used this technique to assess the cytotoxicity of CAR T cells recognizing the human epidermal growth factor receptor 2 (HER2) in breast cancer [ 35 , 39 ]. Oil and water immersion microfluidic techniques can also be used to combine cancer cells with lymphocytes within droplets in order to image cytotoxicity or perform small-volume cytokine sampling [ 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 ].…”