在再生医学中，细胞治疗产品（CTPs）中细胞的致瘤能力是应用于患者的主要问题。本研究提出了一种方法——使用聚合酶链式反应（PCR）的软琼脂成瘤性检测法来评估致瘤性。将受到HeLa细胞污染的MRC-5细胞在软琼脂培养基中培养了最长4周。细胞增殖相关的mRNAs，Ki-67和cyclin B，可在5天的培养后检测到0.01％的HeLa细胞中，而在2周后可以检测到cyclin-dependent kinase 1（CDK1）。另一方面，即使是经过4周的培养，CDK2，增殖细胞核抗原（PCNA）和小染色体维护蛋白7（MCM7）也无法检测到HeLa细胞。在0.01％的HeLa细胞中，癌干细胞（CSC）标记物醛脱氢酶1（ALDH1）和CD133分别可以在培养2周和4周后检测到。然而，另一个CSC标记物CD44没有用处，因为它的表达也被检测到MRC-5细胞中。本研究表明，将PCR方法应用于软琼脂成瘤性检测法中，不仅可以评估短期内的致瘤性，还可以表征成瘤体，最终提高CTPs的安全性。©2023. 作者。

In regenerative medicine, the tumorigenic potency of cells in cellular therapy products (CTPs) is a major concern for their application to patients. This study presents a method-the soft agar colony formation assay using polymerase chain reaction (PCR)-to evaluate tumorigenicity. MRC-5 cells, contaminated with HeLa cells, were cultured for up to 4 weeks in soft agar medium. Cell-proliferation-related mRNAs, Ki-67 and cyclin B, could be detected in 0.01% of HeLa cells after 5 days of culture, whereas cyclin-dependent kinase 1 (CDK1) could be detected after 2 weeks. On the other hand, CDK2, proliferating cell nuclear antigen (PCNA), and minichromosome maintenance protein 7 (MCM7) were not useful to detect HeLa cells even after 4 weeks of culture. The cancer stem cell (CSC) markers, aldehyde dehydrogenase 1 (ALDH1) and CD133 in 0.01% of HeLa cells, could be detected 2 and 4 weeks after culture, respectively. However, another CSC marker CD44 was not useful because its expression was also detected in MRC-5 cells alone. This study suggests that the application of the PCR method to the soft agar colony formation assay could evaluate not only the tumorigenic potency in the short-term but also characterize the colonies, eventually improving the safety of CTPs.© 2023. The Author(s).