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.

间充质干细胞/基质细胞（Mesenchymal stem/stromal cells，MSC）具有免疫调节特性，相关研究已在多种疾病领域展开。经验证的质量控制手段必须能够在临床试验场景下确认该细胞活性。本研究针对一种定量MSC抑制T细胞增殖能力的方法开展验证，该方法遵循ICH Q2指南标准。实验将来自10名供体来源细胞库的外周血单个核细胞（Peripheral blood mononuclear cells，PBMC）用CellTrace™ Violet（CTV）标记后，以七个不同的接种比例与MSC共培养7天。采用流式细胞术分析获取T细胞的增殖分裂百分比（PD）。本研究计算了两项参数：针对每个比例X的T细胞增殖抑制率（PI），其计算公式为PI_比例X = (PD对照组 – PD_比例X组)/PD对照组，以及对应的曲线下面积（AUC）。对两批不同的CTV标记PBMC库进行验证，结果未显示统计学差异，且在509天的储存期内表现出良好稳定性。重复性与重现性分析结果显示，其标准差（SD）分别为6.1%与4.6%。针对PBMC与MSC的稳健性分析（即方法抵抗参数小幅可控变化的能力）未发现显著差异。该检测方法在其应用范围内呈线性响应，且可区分具有不同抑制活性梯度的MSC。本定量检测方法针对MSC免疫调节活性，具有分析变异度低、稳健性充足且不存在PBMC库间差异的优势，因此可用于MSC生产批次的资格确认。