Mesenchymal stem/stromal cell quality control: validation of mixed lymphocyte reaction assay using flow cytometry according to ICH Q2(R1)
Mesenchymal stem/stromal cells (MSC) have immunomodulatory properties, studied in a wide range of diseases. Validated Quality Controls must be able to confirm this activity in the context of clinical trials. This study presents the validation of a method, quantifying the ability of MSC to inhibit T cell proliferation according to the ICH Q2 standard. Peripheral blood mononuclear cells (PBMC) from a bank of ten donors, labeled with CellTrace Violet (CTV), were co-cultured with MSC at seven different ratios for seven days. Flow cytometry analysis was used to obtain the percentage of division (PD) of T cells. Two parameters were calculated: the percentage of inhibition (PI) of T cell proliferation, for each ratio X, determined using the following formula PI ratio X = (PD control – PD ratio X) / PD control, and the corresponding area under the curve (AUC). The validation of two different CTV-PBMC banks did not show any statistical difference and demonstrated stability over 509 days of storage. Analysis of repeatability and reproducibility showed a standard deviation (SD) of 6.1% and 4.6%, respectively. Robustness analysis, corresponding to the ability of a method to resist small but deliberate variations in its parameters, for PBMC, and MSC, did not present any difference. The assay was linear on the exploited range and permitted to distinguish MSC presenting different ranges of inhibition activity. This quantification method of MSC immunomodulatory activity displayed low analytical variability, sufficient robustness, and no inter-bank variability of PBMC. Therefore, it could be used for MSC manufacturing batch qualification.