AP-HP, Hôpital Saint-Louis, Unité de Thérapie Cellulaire, 75010, Paris, France.
CHRU de Nancy, Unité de Thérapie Cellulaire et banque de tissus, 54500, Vandœuvre-lès-Nancy, France.
Stem Cell Res Ther. 2020 Oct 1;11(1):426. doi: 10.1186/s13287-020-01947-6.
Mesenchymal stem/stromal cells (MSC) have immunomodulatory properties, studied in a wide range of diseases. Validated quality controls must confirm this activity in the context of clinical trials. This study presents a method's validation, assessing MSC's ability to inhibit lymphocyte proliferation, according to the ICH Q2 standard.
MSC were co-cultured with CellTrace™ Violet-labeled Peripheral blood mononuclear cells (PBMC) coming from a bank of ten donors, at seven different ratios for 7 days. Cell trace violet PBMC bank was validated in parallel. Flow cytometry analysis was used to obtain the division percentage of T cells. The percentage of inhibition of lymphocyte proliferation by MSC, for each ratio X, was calculated using the formula: Ratio × percentage of inhibition = (control percentage of division-ratio × percentage of division)/control percentage of division. The inhibition percentage of lymphocyte proliferation function of co-culture ratios was represented in a line graph. The corresponding area under the curve was calculated, representing MSC's ability to inhibit lymphocyte proliferation.
Two cell trace violet PBMC banks were compared for bank validation. When compared using four different MSC samples coming each from a different donor, their area under the curve did not show any statistical differences and were correlated. Moreover, the stability of one cell trace violet PBMC bank was confirmed up to 509 days of storage. Analytical parameters were investigated for method validation. Analysis of repeatability and reproducibility respectively showed a standard deviation of 6.1% and 4.6%. The assay was robust regarding PBMC, as no statistical differences were found between inhibitory activities when testing three adjacent concentrations of PBMC. Still, attention is needed on MSC quantity as it can influence results. Linearity was evaluated: the percentage of inhibition of lymphocyte proliferation function of co-culture ratios was linear on the exploited range. Finally, the assay measurement range allowed to differentiate MSC presenting different inhibition activities.
This quantification method displayed low analytical variability and no inter-bank variability of PBMC. However, MSC quantification should be checked before co-culture to reduce variability. Therefore, it could be used for the qualification of MSC batches' immunomodulatory activity.
间充质干细胞（MSC）具有免疫调节特性，已在广泛的疾病中进行了研究。在临床试验中，必须通过经过验证的质量控制来确认这种活性。本研究根据 ICH Q2 标准，介绍了一种评估 MSC 抑制淋巴细胞增殖能力的方法验证。
MSC 与来自十个供体的 CellTrace™ Violet 标记外周血单核细胞（PBMC）共培养，共培养 7 天，共七种不同比例。同时验证 CellTrace 紫 PBMC 库。使用流式细胞术分析获得 T 细胞的分裂百分比。通过公式计算 MSC 对淋巴细胞增殖的抑制百分比：Ratio×抑制百分比=(对照分裂百分比-Ratio×分裂百分比)/对照分裂百分比。将共培养比例的淋巴细胞增殖抑制百分比表示在折线图中。计算相应的曲线下面积，代表 MSC 抑制淋巴细胞增殖的能力。
对两个 CellTrace 紫 PBMC 库进行了库验证比较。当使用来自四个不同供体的四个不同 MSC 样本进行比较时，它们的曲线下面积没有显示出任何统计学差异，并且是相关的。此外，一个 CellTrace 紫 PBMC 库的稳定性在储存 509 天内得到了确认。对方法验证进行了分析参数研究。重复性和重现性分析分别显示出 6.1%和 4.6%的标准偏差。该测定对 PBMC 具有稳健性，因为在测试三个相邻浓度的 PBMC 时，抑制活性没有发现统计学差异。然而，仍需要注意 MSC 的数量，因为它会影响结果。对线性度进行了评估：共培养比例的淋巴细胞增殖抑制功能百分比在线性范围内。最后，该测定的测量范围可以区分具有不同抑制活性的 MSC。
该定量方法显示出低分析变异性和 PBMC 之间无批次间变异性。然而，在共培养之前，应该检查 MSC 的定量情况，以减少变异性。因此，它可以用于 MSC 批次免疫调节活性的鉴定。
