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 approximately 2 × 10⁶to 5 × 10⁸CAR⁺ T cells per patient⁷. High infusion doses are necessary to achieve effective tumor clearance and long-term disease control, but are associated with clinical and practical challenges. Large cell numbers may increase the risk of serious side effects like cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), both of which can be dose-dependent⁸. In addition, manufacturing sufficient numbers of functional CAR T cells is challenging from heavily pretreated or lymphodepleted patients, and the process can be logistically demanding and costly for treatment centers⁹.

Despite these challenges, CD19-targeted CAR T cell therapies achieve 70%-90% complete remission rates in patients with relapsed or refractory B-cell malignancies, demonstrating a transformative clinical impact^10,11,12. However, CAR T cell therapy faces relevant challenges that limit its long-term efficacy. Key obstacles include antigen loss or downregulation, insufficient CAR T cell persistence and functional exhaustion⁹. CAR T cell exhaustion - characterized by decreased proliferative capacity, impaired anti-tumor activity, and poor persistence - represents a vital cause of nonresponse and relapse¹³. Exhausted CAR T cells come with reduced effector function, persistent expression of inhibitory receptors, and defective cytokine production due to chronic antigen exposure and immunosuppressive tumor microenvironments¹⁴. Clinical studies have demonstrated that loss of functional CAR T cell persistence is associated with an increased risk of relapse^11,15,16,17. Because prolonged in vivo activity appears to be a major predictor of therapeutic success, understanding the durability and sustained killing capacity of CAR T cell products is an important goal in both preclinical and translational research. Of particular interest is the ability of CAR T cells to repeatedly recognize and lyse tumor cells over extended periods - so called 'serial killing' - as this property provides a functional measure of persistence and resistance to exhaustion¹⁸.

This article provides a detailed protocol for performing serial killing assays using the xCELLigence Real-Time Cell Analysis (RTCA) impedance platform, including an optional washing procedure that enables re-use of assay plates, further referred to as E-plates, thus improving cost-efficiency without compromising assay performance. The rationale for developing such a protocol arises from limitations in conventional cytotoxicity assays, which typically offer only static, endpoint readouts. Common approaches, such as chromium-51 release, lactate dehydrogenase (LDH) assays, or flow cytometry-based viability assays require labels, involve labor-intensive workflows, and are limited to defined time points^18,19. More importantly, they cannot easily capture dynamic killing kinetics over prolonged stimulation or multiple rounds of antigen exposure, a situation directly linked to CAR T cell persistence and functional exhaustion¹⁸.

The xCELLigence RTCA platform addresses these gaps by providing label-free, real-time, kinetic monitoring of target cell viability via electrical impedance²⁰. Adherent tumor target cells are seeded on gold microelectrode-coated E-plates, where their attachment and proliferation increase the measured impedance, reported as the Cell Index (CI)²¹. Upon CAR T-mediated killing, target cells are killed and detach, leading to a rapid drop in impedance²⁰. This real-time readout allows continuous assessment of cytotoxicity over hours or days without the need for any additional labels or reagents²². CAR T cells, being non-adherent, do not contribute to the impedance signal, allowing for a clean separation of effector and target readouts²³. Because data are recorded continuously, researchers can conduct multi-round stimulation protocols where CAR T cells are repeatedly transferred and exposed to fresh tumor target cells, allowing direct measurement of how killing capacity evolves over time²⁴. This mimics the repeated antigen encounters CAR T cells experience in vivo and provides a functional readout of their cytotoxic capacities.

This impedance-based serial killing assay is ideally suited for researchers evaluating CAR T cell function in preclinical settings, including construct optimization, functional comparisons, and potency testing. This protocol requires the xCELLigence RTCA system and the use of E-Plate 96. While broadly applicable to adherent tumor models, it is not directly suited for suspension cultures without immobilization or tethering strategies. Because impedance measurements alone reflect only target-cell adherence and viability, complementary analyses such as flow cytometry or cytokine assays are recommended to fully characterize CAR T cell activation, proliferation, and exhaustion dynamics. The protocol includes designated steps that allow harvesting of cells and supernatants for these additional readouts, enabling a more comprehensive assessment of CAR T cell functionality.

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 instrument and alternating tumor cell plating and effector addition every other day. CAR T cells should be generated according to institutional SOPs or established protocols. Briefly, peripheral blood T cells are isolated under approved ethical standards, transduced under appropriate biosafety conditions, and assessed for transduction efficiency and viability by flow cytometry or equivalent QC assays. xCELLigence assays should not be performed when CAR transduction efficiency is below 20%, as lower efficiencies may lead to increased background and unspecific killing by untransduced cells, which can distort tumor growth curves. All cell handling should be performed under sterile conditions in a laminar flow hood. Media can be used to the laboratory's standard or as recommended by the supplier. Unless specified otherwise, cells are incubated at 37 °C in a humidified atmosphere containing 5% CO₂. While optimized for CAR T cells, this method can also be applied to other suspension-based effector cells and adherent targets.

1. Preparation and blanking (Day 1)

1. Plate preparation
   1. Add 50 µL of tumor medium to the bottom of each well of the E-plate 96.
      ​NOTE: Be careful not to scratch the bottom of the well as this might cause damage to the plate.
   2. Place the E-plate in the incubator, positioning it on the same machine slot to be used for measurements.
   3. Incubate the E-Plate for 30 min at 37 °C to bring it to the appropriate assay temperature and pH.
2. Instrument setup
   1. Meanwhile, launch the RTCA Software Pro (Version 2.6.1) and select a file path to save the experiment.
   2. Enter the experiment name in the "RTCA Operator" field and save.
   3. Select the wells to be measured.
   4. Assign cell names under the Cell tab and treatment types under the Treatment tab.
3. Measurement schedule
   1. Set up the schedule with the following steps:
      Step Name = Blank ; Sweeps = 1 ; Interval = 1 ; Duration = 0:00
      Step Name = Tumor Cells ; Sweeps = 250-300 ; Interval = 15 min; Duration = minimum 24 h
      Step Name = Treatment ; Sweeps = up to 2500 ; Interval = 5 min; Duration = minimum 72 h
   2. Press Apply and ensure that Auto is not selected for any step.
4. Blanking step
   1. After the 30 min incubation of the E-Plate at 37 °C, press the PLAY/RUN to initiate the blank step.
   2. Wait until the software marks the step DONE (approx. 1-2 min).
   3. Unlock the cradle and remove the E-plate only when ready to proceed with cell seeding.

2. Round 1: Tumor cell seeding (Day 1)

1. Seeding tumor cells
   1. Prepare a suspension of tumor cells in 50 µL of appropriate medium per well.
   2. Remove the E-plate from the incubator and seed the cells in triplicates.
2. Starting measurement
   1. Immediately return the E-plate to the machine and lock the cradle.
   2. Wait for 5 -10 min before the first measurement to equilibrate the E-plate temperature and pH, then click the PLAY/RUN button to start the measurement (Step 2 of schedule = Tumor Cells).
   3. Incubate the E-plate overnight to allow cell adherence. Monitor the non-normalized Cell Index (CI). When the CI enters the log-phase growth stage, proceed to the next step. This phase depends on the cell line used and can be influenced by the number of seeded cells. BxPC3 cells (ATCC CRL-1687) were seeded at 20,000 cells per well, with log-phase growth typically reached after 6-18 h.

3. Round 1 : CAR T cell treatment (Day 2)

1. Preparation and seeding of T cells
   1. Prepare the required number of CAR T cells in 100 µL/well in human T cell medium without expansion cytokines (e.g. IL-2, IL-7, IL-15 etc.).
      NOTE: CAR transduction efficiency is measured once at the beginning of the experiment. The desired effector-to-target (E:T) ratio is set according to the number of CAR⁺ T cells relative to the initially plated tumor cells. For example, for a 1:1 E:T ratio with 50 % CAR⁺ T cells and 20,000 BxPC3 tumor cells, 40,000 total T cells should be plated. The E:T ratio therefore refers to the initial condition at the start of the serial killing assay; unless additional measurements are performed, changes in E:T during later rounds remain unknown. Changes in T cell proliferation or survival over time, and hence in the E:T ratio, are considered part of the serial killing readout. If T cells are split between rounds, the E:T is recalculated proportionally (e.g., a 1:1 split after round 3 reduces an initial 2:1 E:T to 1:1 for round 4). If E:T dynamics between rounds are of interest, optional additional wells can be plated for intermediate measurements of CAR % or effector function. Removing anti-CD3/CD28 T cell stimulation beads by magnetic separation can help to reduce unspecific killing. It is also recommended to use T cells in the logarithmic growth phase (typically between 5- and 20-days post-stimulation) and to expand cells with cytokines at least two days prior to the assay, rather than the day before.
   2. Adjust CAR transduction efficiency across experimental groups to the lowest common percentage by mixing with untransduced T cells, based on transduction efficiency analysis.
      NOTE: If all groups have equal transduction efficiency, no adjustment is needed. If CAR⁺ percentages differ, the number of T cells to plate is calculated so that each group starts with the same effective number of CAR⁺ cells and CAR^- cells. This is done by multiplying the desired CAR⁺ cell number (based on the E:T ratio and tumor seeding) by 100 and dividing by the measured CAR⁺ percentage for each group, then adding untransduced T cells if needed to match total T cell numbers. For example, if one group has 50% CAR⁺ T cells, 40,000 T cells are plated (20,000 CAR⁺ + 20,000 CAR^-), while a group with 60% CAR⁺ T cells would plate 33,333 T cells to provide 20,000 CAR⁺ cells, adding 6,667 untransduced T cells to match the total T cell number of the first group (total 40,000).
   3. Optionally, if the cytokine or compound treatment is to be investigated, add 20 µL of a solution consisting of human T cell medium and the desired cytokine or compound at 11x the intended final concentration. This volume is added on top of the standard 200 µL already present in each well (50 µL for blanking, 50 µL for tumor cell seeding, and up to 100 µL for T cell addition). According to the manufacturer, the E-Plate 96 supports a maximum volume of 243 µL ± 5 µL per well, making a total volume of 220 µL acceptable for such optional treatments.
2. T cell seeding and measurement
   1. Remove the E-plate by unlocking the cradle only when ready to add T cells.
   2. Seed prepared T cells directly onto the tumor cells.
   3. Lock the cradle inside the measurement device and abort Step 2 in the RTCA software.
      NOTE: If step 2 is not aborted, the system will continue to measure the previous step and stop only when that step is completed, which results in loss of data points.
   4. Wait for Step 2 to be marked DONE.
   5. Click the PLAY/RUN button to start step 3 (Treatment) and confirm the prompt by clicking OK.
      ​NOTE: Allowing a 15-30 min equilibrium period after handling (e.g., after seeding or transferring cells) before starting the next measurement step (before step 3.2.5) helps minimize initial deflection caused by temperature or pH fluctuations in the growth curves.

4. Round 2 : Tumor cell seeding (Day 3)

1. Prepare a new 96-well xCELLigence E-Plate as described in steps 1.1 and 1.4.
2. Seed fresh tumor cells as described in step 2.
3. Incubate the E-plate overnight to allow cell adherence.

5. Round 2 : CAR T cell transfer to second E-plate (Day 4)

1. Transfer the contents of each well into a corresponding well of a U-bottom 96-well plate using a multichannel pipette. Be careful not to touch the electrodes at the bottom of the E-plate.
2. Centrifuge at room temperature, 400 × g for 5 min.
3. Carefully aspirate the supernatant and retain it for enzyme-linked immunosorbent assay (ELISA) if needed.
4. Resuspend the cell pellet in 100 µL of fresh medium without expansion cytokines.
   NOTE (optional): T cells can now be split (e.g., 50 µL = 1:2 split) and used for further analysis, such as flow cytometry. Splitting T cells adjusts for their proliferation during the xCELLigence killing assay. It helps to reduce the necessary number of cycles until exhaustion becomes apparent. Testing splitting ratios between 2 and 4 is recommended.
5. Add 100 µL/well T cells to the second-round tumor E-plate (50 µL if 1:2 split), ensuring medium and, if applicable, cytokine/compound are fully replenished.

   

6. Continuation of serial killing assay (Days 5 - 10 and beyond)

1. Repeat Steps 4 and 5 for additional rounds of killing as needed with 48 h intervals.
2. Monitor killing activity and T cell persistence at each stage using xCELLigence readouts and supplementary assays such as ELISA or flow cytometry.

7. Data collection and E-plate handling

1. After the desired measurement duration, press STOP in the software.
2. Export data normalized to the time point of effector-cell addition and non-averaged to enable accurate mean ± SEM plotting in the analysis software (R, GraphPad Prism, etc.). Export the averaged dataset if desired.
3. Unlock and remove the E-plate. Release the experiment in the software.
4. Clean or store the E-plate in the incubator for additional analyses (e.g., supernatant for ELISA or cell harvest for flow cytometry).

8. Optional: E-plate washing for reuse

NOTE: This optional step allows reuse of xCELLigence E-plates to reduce experimental costs. Reuse is only recommended if the electrical properties of the E-plate remain stable across experiments and until individual wells fall within the acceptable baseline range. Follow all safety procedures, especially when handling sodium hydroxide (NaOH). Visually inspect E-plates after washing to detect potential faults in the electrodes.

1. Discard all contents of the E-plate without touching the bottom of the E-plate.
2. Wash each well three times with 200 µL deionized water.
3. Add 200 µL of 0.1 M NaOH to each well.
   CAUTION: NaOH is corrosive. Ensure that local safety guidelines are followed. Wear gloves, coat and safety goggles. Handle 1 M NaOH in a safety cabinet. Discard NaOH waste down the sink with water running to ensure proper dilution. Never mix concentrated acids and bases. Do not add water to acid; only acid to water.
4. Incubate the E-plate at room temperature for 5 min.
5. Wash each well at least three times with deionized water, ensuring all residual NaOH is removed.
6. Dry the E-plate by gently tapping it on a tissue, followed by air drying.
7. Sterilize the E-plate under UV light inside a biosafety cabinet for at least 1 h or a UV irradiation device.
   NOTE: Position the E-plate so that the wells face directly upward. UV light does not penetrate plastic well bottoms.

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