Home › Pharmacology › A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells
Pharmacology JoVE (Open Access) Citable · DOI

A Real-time Potency Assay for Chimeric Antigen Receptor T Cells Targeting Solid and Hematological Cancer Cells

DOI: 10.3791/59033-v
What you'll learn
  • ✓ Generate CAR T cells encoding lentiviral constructs targeting tumor antigens
  • ✓ Perform real-time cytolysis assay using impedance-based potency measurement
  • ✓ Quantify CAR T cell killing efficacy against solid and liquid tumors
  • ✓ Assess CAR expression and validate immune effector cell potency
Protocol

We describe a quantitative real-time in vitro cytolysis assay system to evaluate the potency of chimeric antigen receptor T cells targeting liquid and solid tumor cells. This protocol can be extended to assess other immune effector cells, as well as combination treatments.

Difficulty
advanced
Total time
~7-10 days (lentivirus generation and CAR T expansion) + ~24 hrs (potency assay per sample)
Model organism
Human T cells, tumor cell lines (hematological and solid)
Biosafety
BSL-2

Steps

1
Generate chimeric antigen receptor-encoding lentivirus

Produce lentiviral particles containing CAR transgene constructs for transduction of T cells. This foundational step enables CAR expression in target T lymphocytes.

▶ 00:30
2
Generate and expand CAR T cells

Transduce primary human T cells with lentiviral CAR constructs and expand cells in culture to generate sufficient quantities for potency testing.

▶ 02:29
3
Detect CAR expression on T cells

Verify CAR surface expression using flow cytometry or immunofluorescence to confirm successful transduction and validate CAR T cell populations before potency assessment.

▶ 03:16
4
Perform real-time cytolysis potency assay

Execute impedance-based real-time cellular analysis (RTCA) to quantitatively measure CAR T cell-mediated killing of target tumor cells in real time.

▶ 04:55
5
Analyze CAR T cell target killing efficacy

Interpret RTCA data to determine killing kinetics and potency metrics comparing CAR T cell activity against solid and hematological tumor targets.

▶ 06:48
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