Validation of an ICH Q2 Compliant Flow Cytometry-Based Assay for the Assessment of the Inhibitory Potential of Mesenchymal Stromal Cells on T Cell Proliferation
Natascha Piede, Melanie Bremm, Anne Farken, Lisa‐Marie Pfeffermann, Claudia Cappel, Halvard Bönig, Theres Fingerhut, Laura Puth, Kathrin Vogelsang, Andreas Peinelt, Rolf Marschalek, Matthias Müller, Peter Bader, Zyrafete Kuçi, Selim Kuçi, Sabine Huenecke
Abstract
Mesenchymal stromal cells (MSCs) have the potential to suppress pathological activation of immune cells and have therefore been considered for the treatment of Graft-versus-Host-Disease. The clinical application of MSCs requires a process validation to ensure consistent quality. A flow cytometry-based mixed lymphocyte reaction (MLR) was developed to analyse the inhibitory effect of MSCs on T cell proliferation. Monoclonal antibodies were used to stimulate T cell expansion and determine the effect of MSCs after four days of co-culture based on proliferation tracking with the violet proliferation dye VPD450. Following the guidelines of the International Council for Harmonisation (ICH) Q2 (R1), the performance of n = 30 peripheral blood mononuclear cell (PBMC) donor pairs was assessed. The specific inhibition of T cells by viable MSCs was determined and precision values of <10% variation for repeatability and <15% for intermediate precision were found. Compared to a non-compendial reference method, a linear correlation of r = 0.9021 was shown. Serial dilution experiments demonstrated a linear range for PBMC:MSC ratios from 1:1 to 1:0.01. The assay was unaffected by PBMC inter-donor variability. In conclusion, the presented MLR can be used as part of quality control tests for the validation of MSCs as a clinical product.