Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation.
Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation.
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DOI：
10.1038/srep17892
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发表时间：
2015-12-08
影响因子：
4.6
通讯作者：
Sato Y
中科院分区：
文献类型：
--
作者：
Kusakawa S;Yasuda S;Kuroda T;Kawamata S;Sato Y
致瘤细胞杂质的污染是人类细胞加工治疗产品（hCTP）最紧迫的问题之一。软琼脂集落形成（SACF）试验是一种众所周知的用于检测恶性转化细胞的体外试验，适用于hCTP的质量评估。在这里，我们建立了一个基于图像的筛选系统的SACF试验使用高含量的细胞分析仪称为数字SACF试验。形成的集落的双重荧光染色和软琼脂的溶解导致用成像细胞仪准确检测转化细胞。将细胞样品分配到培养板的多个威尔斯孔中使得能够数字读出集落的存在并提高其检测的灵敏度。在实践中，数字SACF测定在30天内检测到低至hCTP的0.00001%的杂质水平，即10，000，000个人间充质干细胞中仅包含一个HeLa细胞。数字SACF测定节省时间，比体内致瘤性测试更敏感，并且将用于生产过程中的hCTP的质量控制。
Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process.