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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 möglich sobald Postprint bei der ZB eingereicht worden ist.
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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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Schlagwörter Real-time; Young-adults; B-cell; Cytotoxicity; Children
ISSN (print) / ISBN 1940-087X
e-ISSN 1940-087X
Zeitschrift Journal of Visualized Experiments
Quellenangaben Band: 2025-December, Heft: 226, Seiten: , Artikelnummer: e69623 Supplement: ,
Verlag JoVE
Verlagsort 1 Alewife Center, Ste 200, Cambridge, Ma 02140 Usa
Begutachtungsstatus Peer reviewed
Institut(e) Unit for Clinical Pharmacology (KKG-EKLiP)
Förderungen 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)