Sawada Rumi, Kusakawa Shinji, Kusuhara Mika, Tanaka Kazusa, Miura Takumi, Yasuda Satoshi, Sato Yoji
Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki Ward, Kawasaki City, Kanagawa, 210-9501, Japan.
Division of Drugs, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki Ward, Kawasaki City, Kanagawa, 210-9501, Japan.
Regen Ther. 2025 Jan 9;28:321-332. doi: 10.1016/j.reth.2024.12.016. eCollection 2025 Mar.
The Quality by Design (QbD) approach for developing cell therapy products using mesenchymal stromal/stem cells (MSCs) is a promising method for designing manufacturing processes to improve the quality of MSC products. It is crucial to ensure the reproducibility and robustness of the test system for evaluating critical quality attributes (CQAs) in the QbD approach for manufacturing of pharmaceutical products. In this study, we explored the key factors involved in establishing a robust evaluation system for the immunosuppressive effect of MSCs, which can be an example of a CQA in developing and manufacturing therapeutic MSCs for treating graft-versus-host disease, , and we have identified method attributes to increase the robustness of a simple assay to assess the immunosuppressive effects of MSCs.
We evaluated the performance of an assay system to examine the proliferation of peripheral blood mononuclear cells (PBMCs) activated with the mitogen phytohemagglutinin (PHA) when co-cultured with MSCs, the so-called one-way mixed lymphocyte reaction (MLR) assay. The MLR assay was performed on the same MSCs using 10 PBMC lots from different donors. In addition, 13 cytokine production levels in PHA-stimulated PBMCs were assessed.
The PHA-stimulated proliferation response of PBMCs, the action of MSCs in the MLR test, and the cytokine release of the respective PBMCs significantly differed among the PBMC lots (p < 0.05). A correlation analysis between the amounts of cytokines released by PBMCs and the immunosuppressive potency of MSCs showed that IFNγ, TNFα, CXCL10, PD-L1, HGF, and CCL5 production in PBMCs was significantly correlated with the MSC-mediated inhibition of PBMC proliferation (p < 0.05). Therefore, we selected two PBMC lots with high PBMC proliferation and PHA-stimulated cytokine (such as IFNγ and TNFα) release for the subsequent one-way MLR assay. The robustness of the established test system was confirmed by repeating the assay several times on different days for the same MSCs (coefficient of variation <0.2).
To make robust the MSC immunosuppressive potency assay system, controlling the quality of PBMCs used for the assay is essential. Evaluating the inflammatory cytokine production capacity of PBMCs is effective in assessing the quality of the MLR assay system.
采用间充质基质/干细胞（MSC）开发细胞治疗产品的质量源于设计（QbD）方法，是一种用于设计制造工艺以提高MSC产品质量的有前景的方法。在药品生产的QbD方法中，确保用于评估关键质量属性（CQA）的测试系统的可重复性和稳健性至关重要。在本研究中，我们探索了建立用于评估MSC免疫抑制作用的稳健评估系统所涉及的关键因素，这可以作为开发和制造用于治疗移植物抗宿主病的治疗性MSC时CQA的一个示例，并且我们确定了方法属性以提高用于评估MSC免疫抑制作用的简单检测方法的稳健性。
我们评估了一种检测系统的性能，该系统用于检测在用丝裂原植物血凝素（PHA）激活后与MSC共培养时外周血单个核细胞（PBMC）的增殖情况，即所谓的单向混合淋巴细胞反应（MLR）检测。使用来自不同供体的10个PBMC批次，对相同的MSC进行MLR检测。此外，还评估了PHA刺激的PBMC中13种细胞因子的产生水平。
PBMC批次之间，PHA刺激的PBMC增殖反应、MLR检测中MSC的作用以及各个PBMC的细胞因子释放存在显著差异（p < 0.05）。PBMC释放的细胞因子量与MSC免疫抑制效力之间的相关性分析表明，PBMC中IFNγ、TNFα、CXCL10、PD-L1、HGF和CCL5的产生与MSC介导的PBMC增殖抑制显著相关（p < 0.05）。因此，我们选择了两个PBMC增殖率高且PHA刺激后细胞因子（如IFNγ和TNFα）释放量高的PBMC批次用于后续的单向MLR检测。通过在不同日期对相同的MSC重复进行多次检测（变异系数<0.2），证实了所建立检测系统的稳健性。
为使MSC免疫抑制效力检测系统稳健，控制检测所用PBMC的质量至关重要。评估PBMC的炎性细胞因子产生能力对于评估MLR检测系统的质量是有效的。
