Abstract：
Ensuring the safety of parenteral drugs before injection into patients is of utmost importance. New regulations around the globe and the need to refrain from using animals however, have highlighted the need for new cell sources to be used in next-generation bioassays to detect the entire spectrum of possible contaminating pyrogens. Given the current drawbacks of the Monocyte-Activation-Test (MAT) with respect to the use of primary peripheral blood mono-nuclear cells or the use of monocytic cell lines, we here demonstrate the manufacturing of sensor monocytes/macrophages from human induced pluripotent stem cells (iMonoMac), which are fully defined and superior to current cell products. Using a modern and scalable manufacturing platform, iMonoMac showed typical macrophage-like morphology and stained positive for several Toll like receptor (TLRs) such as TLR-2, TLR-5, TLR-4. Furthermore, iMonoMac derived from the same donor were sensitive to endotoxins, non-endotoxins, and process related pyrogens at a high dynamic range and across different cellular densities. Of note, iMonoMac showed increased sensitivity and reactivity to a broad range of pyrogens, demonstrated by the detection of interleukin-6 at low concentrations of LPS and MALP-2 which could not be reached using the current MAT cell sources. To further advance the system, iMonoMac or genetically engineered iMonoMac with NF-κB-luciferase reporter cassette could reveal a specific activation response while correlating to the classical detection method employing enzyme-linked immunosorbent assay to measure cytokine secretion. Thus, we present a valuable cellular tool to assess parenteral drugs safety, facilitating the future acceptance and design of regulatory-approved bioassays.
摘要：
在注射给患者之前确保胃肠外药物的安全性是至关重要的。然而，全球各地的新法规以及避免使用动物的必要性，强调了需要在下一代生物测定中使用新的细胞来源来检测可能的污染热原的整个光谱。鉴于目前单核细胞活化试验（MAT）在使用原代外周血单核细胞（PBMC）或使用单核细胞系方面的缺点，我们在这里证明了从人类诱导多能干细胞（iMonoMac）制造传感器单核细胞/巨噬细胞，完全定义，优于当前的电池产品。使用现代可扩展的制造平台，iMonoMac显示典型的巨噬细胞样形态,并且对几种toll样受体(TLR)如TLR-2、TLR-5、TLR-4染色为阳性。此外，来自同一供体的iMonoMacs对内毒素敏感，非内毒素，并在高动态范围和不同细胞密度下处理相关的热原。值得注意的是，iMonoMac对多种热原显示出更高的敏感性和反应性，通过在低浓度的WHOLPS和MALP-2下检测IL-6证明，使用当前的MAT细胞来源无法达到。为了进一步推进该系统，iMonoMac或带有NF-κB-荧光素酶报告盒的基因工程iMonoMac可以揭示特定的激活反应，同时与采用ELISA测量细胞因子分泌的经典检测方法相关。因此，我们提出了一种有价值的细胞工具来评估肠胃外药物的安全性，促进未来接受和设计监管批准的生物测定。
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