Serial Killing Assay Using Longitudinal Impedance-Based Tumor Cell Viability Measurement - A Useful Method to Assess T Cell Performance

Summary

This protocol presents an impedance-based real-time assay to quantify CAR T cell serial killing capacity and persistence under chronic antigen stimulation, incorporating a plate-washing method for cost-effective reuse of assay plates.

Abstract

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.

Introduction

Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer immunotherapy with remarkable clinical success across multiple hematologic malignancies such as chronic lymphoid leukemia, acute lymphoblastic leukemia, large B-cell lymphoma and multiple myeloma^1,2,3,4,5. Since the first regulatory approvals, the number of CAR T cell products and treated patients has expanded steadily⁶. Depending on the specific product and clinical indication, typical infusion doses range from....

Protocol

NOTE: This protocol has been optimized for the E-Plate 96 and requires the xCELLigence RTCA system. Adherent tumor cells are seeded overnight in advance to reach the logarithmic growth phase at the time of CAR T cell addition. Depending on the tumor cell line and seeding density, this interval may be shortened to 6 - 9 h, allowing same-day effector addition. If the latter workflow is performed, the nominal day-by-day timeline described in the protocol is altered, and users should adapt the schedule accordingly. Co-cultures are maintained for 48 h before CAR T cells are transferred onto freshly plated tumor cells, requiring two plate positions on the xCELLigence instru....

Results

Successful execution of the protocol yields reproducible impedance-based readouts that reflect tumor cell viability and CAR T cell cytotoxicity over time. Cell growth and cytotoxicity were assessed using the commercial RTCA system. Data were expressed as normalized cell index (NCI), calculated by dividing the CI at each time point by the CI at a selected normalization time. The normalization time is set immediately before the addition of effector cells. Time to clearance is defined as the interval until NCI reaches zero .......

Discussion

This protocol describes a method for evaluating CAR T cell-mediated cytotoxicity over multiple rounds of antigen exposure using the xCELLigence RTCA platform. By enabling longitudinal measurement of tumor cell viability via impedance, this approach captures repetitive cytotoxic responses - referred to as serial killing - in a label-free and continuous format. The protocol is designed to monitor how T cell effector function changes over time in response to successive tumor cell re-challenges, providing insight into parame.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BxPC3	ATCC	CRL-1687
E-Plate 96	Agilent	300600910
RTCA software Pro (Basic)	Agilent	N/A	Version 2.6.1
xCELLigence RTCA MP - (Multiple Plates)	Agilent	N/A