DataSheet_1_Optimizing an immunomodulatory potency assay for Mesenchymal Stromal Cell.docx

The expeditious progress of Mesenchymal Stromal Cells (MSC) for therapeutic intervention calls for means to compare differences in potency of cell products. The differences may be attributed to innumerable sources including tissue origin, production methods, or even between batches. While the immunomodulatory potential of MSC is recognized and well-documented by an expansive body of evidence, the methodologies and findings vary markedly. In this study, we utilized flowcytometric analysis of lymphocyte proliferation based on cryopreserved peripheral blood mononuclear cells for quantification of the inhibitory effect of MSC. Technical aspects of fluorescent staining and cryopreservation of peripheral blood mononuclear cells were evaluated to obtain optimal results and increase feasibility. A range of common specific and unspecific mitogens was titrated to identify the conditions, in which the effects of Adipose tissue-derived Stromal Cells (ASC; a type of MSC) were most pronounced. Specific stimulation by antibody-mediated activation of CD3 and CD28 via TransAct and Dynabeads lead to substantial proliferation of lymphocytes, which was inhibited by ASC. These results were closely mirrored when applying unspecific stimulation in form of phytohemagglutinin (PHA), but not concanavalin A or pokeweed mitogen. The mixed lymphocyte reaction is a common assay which exploits alloreactivity between donors. While arguably more physiologic, the output of the assay often varies substantially, and the extent of proliferation is limited since the frequency of alloreactive cells is low, as opposed to the mitogens. To heighten the proliferative response and robustness, combinations of 2-5 donors were tested. Maximum proliferation was observed when combining 4 or more donors, which was efficiently suppressed by ASC. Several desirable and unfavorable traits can be attributed to the tested stimuli in the form of keywords. The importance of these traits should be scored on a laboratory-level to identify the ideal mitogen. In our case the ranking listed PHA as the most suited candidate. Developing robust assays is no trivial feat. By disclosing the full methodological framework in the present study, we hope to aid others in establishing functional metrics on the road to potency assays.

间充质基质细胞（Mesenchymal Stromal Cells, MSC）用于治疗干预的研究进展迅猛，亟需建立可比较不同细胞制品效价差异的方法。这类差异可由众多因素导致，包括组织来源、生产工艺，甚至不同批次之间的差异。尽管MSC的免疫调节潜能已得到大量研究证据的证实与详实记载，但相关研究方法与结果仍存在显著差异。
本研究采用基于冷冻保存外周血单个核细胞的淋巴细胞增殖流式细胞术分析，量化MSC的免疫抑制效应。研究对荧光染色及外周血单个核细胞冷冻保存的技术细节进行了评估优化，以获得最佳实验结果并提升实验可行性。我们对一系列常见的特异性与非特异性有丝分裂原进行浓度梯度滴定，以确定脂肪来源基质细胞（Adipose tissue-derived Stromal Cells, ASC；属于MSC的一类）的抑制效应最为显著的实验条件。
通过TransAct与Dynabeads介导CD3与CD28的抗体激活，可诱导淋巴细胞发生显著增殖，该增殖过程可被ASC显著抑制。采用植物血凝素（phytohemagglutinin, PHA）进行非特异性刺激时，也得到了高度相似的结果，但刀豆蛋白A与商陆有丝分裂原则未出现类似效应。
混合淋巴细胞反应是一种利用供者间同种异体反应性的经典检测方法，尽管该方法更贴近生理状态，但检测结果往往大幅波动，且由于同种反应性细胞的频率较低，与有丝分裂原刺激相比，其增殖程度相对有限。为提升增殖反应强度与实验稳定性，我们测试了2-5名供者的外周血单个核细胞混合体系，结果显示，当混合4名及以上供者的细胞时可获得最大增殖幅度，且该增殖效应可被ASC有效抑制。
我们可通过关键词归纳上述受试刺激物的优劣特性，并在实验室层面对这些特性进行评分，以筛选出理想的有丝分裂原。在本研究中，PHA被列为最适合的候选刺激物。
建立稳定可靠的功能检测方法并非易事。本研究公开了完整的实验方法框架，以期为其他研究者建立效价检测所需的功能学指标提供参考与帮助。