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

卷:12

Article

关键词: VERSUS-HOST-DISEASE;    MIXED LEUKOCYTE REACTION;    STEM-CELLS;    DENDRITIC CELLS;    INTERNATIONAL-SOCIETY;    REMESTEMCEL-L;    IN-VITRO;    ANTIGEN;    THERAPY;    STIMULATION;   

DOI  :  10.3390/cells12060850

来源: SCIE

【 摘 要 】

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.