Characterization of CAR T cell expansion and cytotoxic potential during Ex Vivo manufacturing using image-based cytometry

J Immunol Methods. 2020 Sep-Oct:484-485:112830. doi: 10.1016/j.jim.2020.112830. Epub 2020 Aug 1.


Since the FDA approval of two Chimeric Antigen Receptor (CAR) T cell therapies against CD19+ malignancies, there has been significant interest in adapting CAR technology to other diseases. As such, the ability to simultaneously monitor manufacturing criteria and functional characteristics of multiple CAR T cell products by a single instrument would likely accelerate the development of candidate therapies. Here, we demonstrate that image-based cytometry yields high-throughput measurements of CAR T cell proliferation and size, and captures the kinetics of in vitro antigen-specific CAR T cell-mediated killing. The data acquired and analyzed by the image cytometer are congruent with results derived from conventional technologies when tested contemporaneously. Moreover, the use of bright-field and fluorescence microscopy by the image cytometer provides kinetic measurements and rapid data acquisition, which are direct advantages over industry standard instruments. Together, image cytometry enables fast, reproducible measurements of CAR T cell manufacturing criteria and effector function, which can greatly facilitate the evaluation of novel CARs with therapeutic potential.

Keywords: Bioluminescence (BLI)-based cytotoxicity assay; CAR T cell manufacturing; Cell-mediated cytotoxicity; Chimeric antigen receptor (CAR) T cells; Image cytometry.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antigens, CD / genetics
  • Antigens, CD / immunology*
  • Antigens, CD / metabolism
  • Cell Proliferation*
  • Coculture Techniques
  • Cytotoxicity, Immunologic*
  • Flow Cytometry*
  • Humans
  • Immunotherapy, Adoptive*
  • K562 Cells
  • Kinetics
  • Leukemia, Myeloid / immunology
  • Leukemia, Myeloid / metabolism
  • Leukemia, Myeloid / therapy*
  • Microscopy, Fluorescence*
  • Receptors, Chimeric Antigen / genetics
  • Receptors, Chimeric Antigen / immunology*
  • Receptors, Chimeric Antigen / metabolism
  • T-Lymphocytes / immunology*
  • T-Lymphocytes / metabolism
  • Workflow


  • Antigens, CD
  • Receptors, Chimeric Antigen