Comparison of chimeric antigen receptor‐T cell‐mediated cytotoxicity assays with suspension tumor cells using plate‐based image cytometry method

Abstract

In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. Image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance. In this work, we employed the Celigo Image Cytometer to evaluate and compare two CAR-T cell-mediated cytotoxicity assays using GFP-expressing or fluorescent dye-labeled myeloma and plasmacytoma cells. The GFP-based method demonstrated higher sensitivity in detecting CAR-T cell-mediated cytotoxicity when compared to the CMFDA/DAPI viability method. We have established the criteria and considerations for the selection of cytotoxicity assays that are fit-for-purpose to ensure the results produced are meaningful for the specific testing conditions.

Quantum pBac: An effective, high-capacitypiggyBac-based gene integration vector system for unlocking gene therapy potential

“…CD19 + (K562 CD19-tdTomato), CD20 + (K562 CD20-GFP), CD19 + CD20 + (Raji-GFP) or non-relevant K562 BCMA-GFP target cells were seeded at 5 × 10 3 cells per well in 96-well culture plates (Corning), and control non-transfected Pan-T or CAR-T cells were added at an E:T ratio of 5:1, 1:1, 1:3 or 1:10. Cytotoxicity of CAR-T cells on target cells was then assessed as previously described 54 . Briefly, a Celigo imaging cytometer was used to measure the percentage of live target cells at 0, 24, 48, 72 and 96 h of co-culture.…”

QuantumpBac: An effective, high‐capacity piggyBac‐based gene integration vector system for unlocking gene therapy potential

“…CD19 + (K562 CD19‐tdTomato), CD20 + (K562 CD20‐GFP), CD19 + CD20 + (Raji‐GFP) or non‐relevant K562 BCMA‐GFP target cells were seeded at 5 × 10 3 cells per well in 96‐well culture plates (Corning), and control non‐transfected Pan‐T or CAR‐T cells were added at an E:T ratio of 5:1, 1:1, 1:3 or 1:10. Cytotoxicity of CAR‐T cells on target cells was then assessed as previously described 16 . Briefly, a Celigo imaging cytometer was used to measure the percentage of live target cells at 0, 24, 48, 72 and 96 h of co‐culture.…”

“…Cytotoxicity of CAR-T cells on target cells was then assessed as previously described. 16 Briefly, a Celigo imaging cytometer was used to measure the percentage of live target cells at 0, 24, 48, 72 and 96 h of co-culture. Cell aggregates were separated by pipetting prior to Celigo imaging.…”