title: Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation.
abstract: 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.
authors.affiliation: Japan Agency for Medical Research and Development (AMED), Tokyo, Japan.|Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Tokyo, Japan.|Research and Development Center for Cell Therapy, Foundation for Biomedical Research and Innovation, Kobe, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Kusakawa
authors.affiliation: Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Tokyo, Japan.|Research and Development Center for Cell Therapy, Foundation for Biomedical Research and Innovation, Kobe, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yasuda
authors.affiliation: Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Tokyo, Japan.|Research and Development Center for Cell Therapy, Foundation for Biomedical Research and Innovation, Kobe, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Kuroda
authors.affiliation: Research and Development Center for Cell Therapy, Foundation for Biomedical Research and Innovation, Kobe, Japan.
authors.forename: Shin
authors.initials: S
authors.lastname: Kawamata
authors.affiliation: Division of Cell-Based Therapeutic Products, National Institute of Health Sciences, Tokyo, Japan.|Research and Development Center for Cell Therapy, Foundation for Biomedical Research and Innovation, Kobe, Japan.|Department of Quality Assurance Science for Pharmaceuticals, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan.|Department of Cellular and Gene Therapy Products, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan.|Department of Translational Pharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Yoji
authors.initials: Y
authors.lastname: Sato
mesh_terms: D002471:Cell Transformation, Neoplastic; D064987:Cell- and Tissue-Based Therapy / Q000009:adverse effects / Q000592:standards; D006367:HeLa Cells; D006801:Humans; D045164:Mesenchymal Stem Cell Transplantation / Q000009:adverse effects / Q000592:standards; D059630:Mesenchymal Stem Cells / Q000378:metabolism / Q000473:pathology; D008856:Microscopy, Fluorescence / Q000379:methods / Q000592:standards; D012680:Sensitivity and Specificity; D014410:Tumor Stem Cell Assay / Q000379:methods