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Tschaidse, T.* ; Trefny, M.P.* ; Carlini, E.* ; Andreu-Sanz, D.* ; Michaelides, S.* ; Nguyen, N.T.T.* ; Kobold, S.

Serial killing assay using longitudinal impedance-based tumor cell viability measurement - a useful method to assess T cell performance.

J. Vis. Exp. 2025-December:e69623 (2025)
DOI PMC
Open Access Green as soon as Postprint is submitted to ZB.
Chimeric antigen receptor (CAR) cell therapy has revolutionized the treatment of specific hematologic malignancies. However, a significant portion of patients experience relapse because of antigen loss, antigen downregulation, or T cell exhaustion. These challenges highlight the need for functional assays that can evaluate the killing capacity and persistence of CAR T cells under chronic antigen stimulation. Serial killing assays, which measure the ability of CAR T cells to repeatedly eliminate tumor targets, offer valuable insights into the durability and potency of CAR T cell responses. Here, we present an impedance-based assay using the Real-Time Cell Analysis (RTCA) system to quantify CAR T cell-mediated serial killing in vitro. Tumor cells are repeatedly seeded and allowed to adhere to assay-specific E-plates before the addition of CAR T cells at defined effector-to-target (E:T) ratios. The platform continuously monitors tumor cell viability without labels, capturing dynamic cytotoxicity with high temporal resolution. Core readouts include Cell Index (CI) kinetics, tumor-cell killing rate, and time-to-target clearance. The progressive decline in killing capacity observed upon repeated tumor-target engagements serves as a marker of acquired CAR T cell dysfunction, often termed T cell exhaustion. Together, these metrics allow precise evaluation of CAR T cell function at various E:T ratios and enable direct comparison among different CAR T cell constructs or co-treatments over time. To enhance cost efficiency, we developed a plate-washing procedure that enables the reuse of assay E-plates without compromising assay performance or data integrity. The optimized workflow reduces assay cost while preserving analytical robustness. This approach enables affordable and scalable preclinical assessment of CAR T cell function, facilitating improvements in cell-therapy design.
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Publication type Article: Journal article
Document type Scientific Article
Keywords Real-time; Young-adults; B-cell; Cytotoxicity; Children
ISSN (print) / ISBN 1940-087X
e-ISSN 1940-087X
Journal Journal of Visualized Experiments
Quellenangaben Volume: 2025-December, Issue: 226, Pages: , Article Number: e69623 Supplement: ,
Publisher JoVE
Publishing Place 1 Alewife Center, Ste 200, Cambridge, Ma 02140 Usa
Reviewing status Peer reviewed
Institute(s) Unit for Clinical Pharmacology (KKG-EKLiP)
Grants Fritz-Bender Foundation
Else Kroner-Fresenius-Stiftung (IOLIN)
Hector Foundation
Marie Sklodowska-Curie Training Network for tracking and controlling therapeutic immune cells in cancer - Horizon Programme of The EU
Deutsche Jose Carreras Leukamie Stiftung
Marie Sklodowska-Curie Training Network for Optimizing Adoptive T Cell Therapy of Cancer - Horizon 2020 programme of the European Union
Melanoma Research Alliance
Deutsche Forschungsgemeinschaft (DFG)
Bavarian Cancer Research Center (BZKF)
International doctoral program 'i-Target: immunotargeting of cancer' - Elite Network of Bavaria
European Research Council
Monika Kutzner Foundation
Friedrich-Baur-Stiftung and German Cancer Aid
Bavarian Research Foundation (BAYCELLATOR)
German Cancer Aid
SFB-TRR
EUROSTAR-Programm, European Research Council
Bavarian Ministry for Economic Affairs, Bundesministerium fur Bildung und Forschung
Go-Bio-Initiative
Brigitte and Dr. Konstanze Wegener Foundation
Institutional Strategy LMUexcellent of LMU Munich
Dr. Rurainski-Foundation
Ernst Jung Stiftung
Bruno and Helene Joster Foundation
Wilhelm-Sander-Stiftung
Monika-Kutzner Foundation
Else-Kroner Fresenius Stiftung (IOLIN)