A rapid and high-throughput T cell immunophenotyping assay for cellular therapy bioprocess using the Cellaca PLX image cytometer

Abstract

Abstract Cellular therapies such as chimeric antigen receptor (CAR) T or NK cells undergo phenotypic characterization during discovery and development of novel therapies, biomanufacturing, and upstream patient cell processing for clinical use. Samples are routinely analyzed via flow cytometry for T cell, NK cell, B cell, and monocyte surface marker expression. Image cytometry systems perform cell-based assays that can be used as a convenient alternative to flow cytometry. Recently, a new high-throughput image cytometer, the Cellaca PLX system (Nexcelom, Lawrence, MA) was developed for immunophenotyping, transfection/transduction efficiency, and cell health assays. This new instrument can assess several critical quality attributes (CQAs) such as cell identity, viability, and other relevant biological functions recommended by the International Organization for Standardization using the ISO Cell Characterization that focuses on cellular therapeutic products. Here we demonstrate a rapid and high-throughput immunophenotyping and viability detection in peripheral blood mononuclear cells (PBMCs) using the Cellaca PLX. PBMCs underwent red blood cell (RBC) lysis and CD3 enrichment, were stained with Hoechst/CD3/CD4/CD8 or Hoechst/CD3/CD8/RubyDead surface marker kits and measured on the Cellaca PLX and three different flow cytometers for comparison. Results showed highly comparable cell populations between the flow cytometers and the Cellaca PLX suggesting that the image cytometer may provide a rapid and convenient alternative method for immunophenotyping. The proposed method can streamline workflows from sample staining to data analysis, quickly moving precious patient samples into downstream processes.