METHODS: The spike-in samples were produced from three independent experiments, each spiked with different PSC lines (PSC1, NH50191, and WA09 referred to as H9) into a background of primary fibroblasts (Hs68). These samples were then subjected to qPCR, ddPCR and RT-LAMP to determine their detection limit in measuring a commonly used PSC marker, LIN28A.
RESULTS: The results indicated that the three analytic methods all exhibited consistent results across different cell-line spiked samples, with ddPCR demonstrating the highest sensitivity of the three methods. The LIN28A ddPCR assay could confidently detect 10 residual PSCs in a million fibroblasts.
CONCLUSIONS: In our hand, ddPCR LIN28A assay demonstrated the highest sensitivity for detection of residual PSCs compared to the other two assays. Correlating such in vitro safety results with corresponding in vivo studies demonstrating the tumorigenicity profile of PSC-derived cell therapy could accelerate the safe clinical translation of cell therapy.
方法:加标样品由三个独立实验产生,每个都掺入不同的PSC系(PSC1,NH50191和WA09,称为H9)到原代成纤维细胞(Hs68)的背景中。然后对这些样品进行qPCR,ddPCR和RT-LAMP来确定它们在测量常用PSC标记时的检测限,LIN28A.
结果:结果表明,三种分析方法在不同细胞系加标样品中均显示出一致的结果,ddPCR证明了三种方法的最高灵敏度。LIN28A-ddPCR测定可以可靠地检测一百万个成纤维细胞中的10个残留PSC。
结论:在我们手中,与其他两种测定相比,ddPCRLIN28A测定显示出检测残余PSC的最高灵敏度。将这样的体外安全性结果与相应的体内研究相关联,证明PSC衍生的细胞疗法的致瘤性特征可以加速细胞疗法的安全临床转化。