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

Kuan Un Wong; Jingxuan Shi; Peng Li; Haitao Wang; Yanwei Jia; Chuxia Deng; Lianmei Jiang; Ada Hang-Heng Wong

doi:10.1093/abt/tbac008

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

Antib Ther. 2022 Mar 24;5(2):85-99. doi: 10.1093/abt/tbac008. eCollection 2022 Apr.

Authors

Kuan Un Wong¹, Jingxuan Shi^{2

3

4}, Peng Li^{2

3

5}, Haitao Wang¹, Yanwei Jia^{1

6

7}, Chuxia Deng¹, Lianmei Jiang⁸, Ada Hang-Heng Wong¹

Affiliations

¹ Cancer Centre, Faculty of Health Sciences, University of Macau, Macau SAR, China.
² Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China.
³ Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China.
⁴ University of Chinese Academy of Sciences, Shijingshan District, Beijing 100049, China.
⁵ Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, Guangdong 510005, China.
⁶ State Key Laboratory of Analog and Mixed Signal VLSI, University of Macau, Macau SAR, China.
⁷ Faculty of Science and Technology, University of Macau, Macau SAR, China.
⁸ Department of Molecular Sciences, ARC Excellence Centre for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.

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-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.

Keywords: chimeric antigen receptor T cell therapy; cytotoxicity assessment; droplet microfluidics; immunotherapy; precision cancer therapy.