目的:人细胞处理的治疗产品(hCTP)的给药与由于转化的细胞污染物引起的肿瘤发生的风险有关。为了减轻这种风险,这些杂质应使用灵敏和经过验证的测定法进行检测。数字软琼脂集落形成(D-SAC)测定法是一种超灵敏的体外测试,用于检测hCTP中的致瘤性转化细胞。
方法:在本研究中,我们首先根据之前报道的来自多个实验室的D-SAC检测方案(方案I),评估了阳性对照样品中致瘤性参考细胞的集落形成效率(CFE)精确度.然而,CFE在实验室之间差异很大。因此,我们将试验方案改进和优化为方案II,以减少致瘤参考细胞CFE的变异性.随后,改进的方案在多个地点得到验证.人骨髓间充质基质细胞(hMSCs)作为模型细胞,和通过用HeLa细胞掺入它们来制备阳性对照样品。
结果:根据先前报道的协议,使用多板中超低浓度(0.0001%)的阳性对照样品估算CFE.接下来,我们改进了方案以降低CFE变异性.根据CFE的结果,我们将样本量估计为孔数(方案II),并评估了hMSCs中0.0001%HeLa细胞的可检测性,以在多个位点验证方案.使用方案I产生低CFE(平均值:30%)和实验室之间的高变异性(再现性变异系数[CV]:72%)。相比之下,第二号议定书,在阳性对照样品中掺入相对高浓度(0.002%)的HeLa细胞,导致更高的CFE值(平均值:63%)和更低的变异性(重复性CV:18%)。此外,根据实验室特定的CFE(42-76%),将用于测试的样本量估计为每个实验室的孔数(314-570孔).在这些条件下,所有实验室在预定数量的孔中hMSCs中达到0.0001%HeLa细胞的检测限.此外,在单独接种hMSC的孔中未观察到集落形成。
结论:D-SAC测定法是一种用于检测hCTP中作为杂质的恶性细胞的高度灵敏和可靠的测试。此外,建立了最佳测定条件,以高灵敏度和任意假阴性率检测hCTP中的致瘤杂质。
OBJECTIVE: The administration of human cell-processed therapeutic products (hCTPs) is associated with a risk of tumorigenesis due to the transformed cellular contaminants. To mitigate this risk, these impurities should be detected using sensitive and validated assays. The digital soft agar colony formation (D-SAC) assay is an ultrasensitive in vitro test for detecting tumorigenic transformed cells in hCTPs.
METHODS: In this study, we first evaluated the colony formation efficiency (CFE) precision of tumorigenic reference cells in positive control samples according to a previously reported D-SAC assay protocol (Protocol I) from multiple laboratories. However, the CFE varied widely among laboratories. Thus, we improved and optimized the test protocol as Protocol II to reduce variability in the CFE of tumorigenic reference cells. Subsequently, the improved protocol was validated at multiple sites. Human mesenchymal stromal cells (hMSCs) were used as model cells, and positive control samples were prepared by spiking them with HeLa cells.
RESULTS: Based on the previously reported protocol, the CFE was estimated using an ultra-low concentration (0.0001%) of positive control samples in multiple plates. Next, we improved the protocol to reduce the CFE variability. Based on the CFE results, we estimated the sample size as the number of wells (Protocol II) and assessed the detectability of 0.0001% HeLa cells in hMSCs to validate the protocol at multiple sites. Using Protocol I yielded low CFEs (mean: 30%) and high variability between laboratories (reproducibility coefficient of variance [CV]: 72%). In contrast, Protocol II, which incorporated a relatively high concentration (0.002%) of HeLa cells in the positive control samples, resulted in higher CFE values (mean: 63%) and lower variability (reproducibility CV: 18%). Moreover, the sample sizes for testing were estimated as the number of wells per laboratory (314-570 wells) based on the laboratory-specific CFE (42-76%). Under these conditions, all laboratories achieved a detection limit of 0.0001% HeLa cells in hMSCs in a predetermined number of wells. Moreover, colony formation was not observed in the wells seeded with hMSCs alone.
CONCLUSIONS: The D-SAC assay is a highly sensitive and robust test for detecting malignant cells as impurities in hCTPs. In addition, optimal assay conditions were established to test tumorigenic impurities in hCTPs with high sensitivity and an arbitrary false negative rate.