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000308512 1001_ $$aTschaidse, Tengis$$b0
000308512 245__ $$aSerial Killing Assay Using Longitudinal Impedance-Based Tumor Cell Viability Measurement - A Useful Method to Assess T Cell Performance.
000308512 260__ $$aCambridge, MA$$bJoVE$$c2025
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000308512 520__ $$aChimeric antigen receptor (CAR) cell therapy has revolutionized the treatment of specific hematologic malignancies. However, a significant portion of patients experience relapse because of antigen loss, antigen downregulation, or T cell exhaustion. These challenges highlight the need for functional assays that can evaluate the killing capacity and persistence of CAR T cells under chronic antigen stimulation. Serial killing assays, which measure the ability of CAR T cells to repeatedly eliminate tumor targets, offer valuable insights into the durability and potency of CAR T cell responses. Here, we present an impedance-based assay using the Real-Time Cell Analysis (RTCA) system to quantify CAR T cell-mediated serial killing in vitro. Tumor cells are repeatedly seeded and allowed to adhere to assay-specific E-plates before the addition of CAR T cells at defined effector-to-target (E:T) ratios. The platform continuously monitors tumor cell viability without labels, capturing dynamic cytotoxicity with high temporal resolution. Core readouts include Cell Index (CI) kinetics, tumor-cell killing rate, and time-to-target clearance. The progressive decline in killing capacity observed upon repeated tumor-target engagements serves as a marker of acquired CAR T cell dysfunction, often termed T cell exhaustion. Together, these metrics allow precise evaluation of CAR T cell function at various E:T ratios and enable direct comparison among different CAR T cell constructs or co-treatments over time. To enhance cost efficiency, we developed a plate-washing procedure that enables the reuse of assay E-plates without compromising assay performance or data integrity. The optimized workflow reduces assay cost while preserving analytical robustness. This approach enables affordable and scalable preclinical assessment of CAR T cell function, facilitating improvements in cell-therapy design.
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000308512 650_7 $$2NLM Chemicals$$aReceptors, Chimeric Antigen
000308512 650_2 $$2MeSH$$aHumans
000308512 650_2 $$2MeSH$$aT-Lymphocytes: immunology
000308512 650_2 $$2MeSH$$aT-Lymphocytes: cytology
000308512 650_2 $$2MeSH$$aCell Survival: immunology
000308512 650_2 $$2MeSH$$aReceptors, Chimeric Antigen: immunology
000308512 650_2 $$2MeSH$$aCell Line, Tumor
000308512 650_2 $$2MeSH$$aElectric Impedance
000308512 650_2 $$2MeSH$$aImmunotherapy, Adoptive: methods
000308512 7001_ $$aTrefny, Marcel P$$b1
000308512 7001_ $$aCarlini, Emanuele$$b2
000308512 7001_ $$aAndreu-Sanz, David$$b3
000308512 7001_ $$aMichaelides, Stefanos$$b4
000308512 7001_ $$0P:(DE-He78)abb10265fc5b7b424eee557e979d490f$$aNguyen, Mai Thi Ngoc$$b5
000308512 7001_ $$0P:(DE-HGF)0$$aKobold, Sebastian$$b6
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