Given the high level of product complexity and limited regulatory guidance, designing and implementing appropriate potency assays is often the most challenging part of establishing a quality control testing matrix for a cell-based medicinal product. Among the most elusive tasks are the selection of suitable read-out parameters, the development of assay designs that most closely model the pathophysiological conditions, and the validation of the methods. Here we describe these challenges and how they were addressed in developing an assay that measures the anti-inflammatory potency of mesenchymal stromal cells (MSCs) in an M1 macrophage-dominated inflammatory environment. An inflammation model was established by coculturing skin-derived ABCB5 MSCs with THP-1 monocyte-derived M1-polarized macrophages. Readout was the amount of interleukin 1 receptor antagonist (IL-1RA) secreted by the MSCs in the coculture, measured by an enzyme-linked immunosorbent assay. IL-1RA was quantified with guideline-concordant selectivity, accuracy and precision over a relevant concentration range. Consistent induction of the macrophage markers CD36 and CD80 indicated successful macrophage differentiation and M1 polarization of THP-1 cells, which was functionally confirmed by release of proinflammatory tumor necrosis factor α. Testing a wide range of MSC/macrophage ratios revealed the optimal ratio for near-maximal stimulation of MSCs to secrete IL-1RA, providing absolute maximum levels per individual MSC that can be used for future comparison with clinical efficacy. Batch release testing of 71 consecutively manufactured MSC batches showed a low overall failure rate and a high comparability between donors. We describe the systematic development and validation of a therapeutically relevant, straightforward, robust and reproducible potency assay to measure the immunomodulatory capacity of MSCs in M1 macrophage-driven inflammation. The insights into the challenges and how they were addressed may also be helpful to developers of potency assays related to other cellular functions and clinical indications.

中文翻译：

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预测细胞治疗产品对巨噬细胞驱动疾病的抗炎能力的效力测定：克服测定开发和验证的挑战

鉴于产品的高度复杂性和有限的监管指导，设计和实施适当的效力测定通常是为细胞医药产品建立质量控制测试矩阵中最具挑战性的部分。最难以捉摸的任务包括选择合适的读出参数、开发最接近地模拟病理生理条件的测定设计以及方法的验证。在这里，我们描述了这些挑战以及如何在开发一种测定方法来解决这些挑战，该测定方法可测量 M1 巨噬细胞主导的炎症环境中间充质基质细胞 (MSC) 的抗炎效力。通过将皮肤来源的 ABCB5 MSC 与 THP-1 单核细胞来源的 M1 极化巨噬细胞共培养建立炎症模型。读数是共培养物中 MSC 分泌的白细胞介素 1 受体拮抗剂 (IL-1RA) 的量，通过酶联免疫吸附测定进行测量。在相关浓度范围内，IL-1RA 的选择性、准确度和精密度符合指南要求。巨噬细胞标记物 CD36 和 CD80 的持续诱导表明 THP-1 细胞成功巨噬细胞分化和 M1 极化，促炎性肿瘤坏死因子 α 的释放在功能上证实了这一点。测试各种 MSC/巨噬细胞比例揭示了 MSC 接近最大刺激分泌 IL-1RA 的最佳比例，提供了每个个体 MSC 的绝对最大水平，可用于未来与临床疗效的比较。对 71 个连续生产的 MSC 批次进行的批次放行测试显示，总体失败率较低，供体之间具有较高的可比性。我们描述了一种治疗相关的、简单的、稳健的和可重复的效力测定的系统开发和验证，用于测量 MSC 在 M1 巨噬细胞驱动的炎症中的免疫调节能力。对挑战及其解决方法的深入了解也可能有助于与其他细胞功能和临床适应症相关的效力测定的开发人员。