Accuracy of spiked cell counting methods for designing a pre-clinical tumorigenicity study model
Hiroaki Osada,
Masahide Kawatou,
Masafumi Takeda,
Jun-ichiro Jo,
Takashi Murakami,
Yasuhiko Tabata,
Kenji Minatoya,
Jun K. Yamashita,
Hidetoshi Masumoto
Affiliations
Hiroaki Osada
Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
Masahide Kawatou
Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan; Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Institute for Advancement of Clinical and Translational Science, Kyoto University Hospital, Kyoto, Japan
Masafumi Takeda
Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; Institute for Advancement of Clinical and Translational Science, Kyoto University Hospital, Kyoto, Japan
Jun-ichiro Jo
Laboratory of Biomaterials, Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
Takashi Murakami
Department of Microbiology, Saitama Medical University, Faculty of Medicine, Saitama, Japan
Yasuhiko Tabata
Laboratory of Biomaterials, Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
Kenji Minatoya
Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan
Jun K. Yamashita
Department of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
Hidetoshi Masumoto
Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan; Clinical Translational Research Program, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan; Corresponding author.
Background: Evaluations for the tumorigenicity of transplantation of stem cell products is mandatory for clinical application. It is of importance to establish a system to accurately quantify contaminated tumorigenic cells regardless of the format of stem cell product. In the present report, we aimed to examine the accuracy of the quantification of tumorigenic cell numbers with commonly used 2 methods, quantitative polymerase chain reaction (qPCR) and flow cytometry (FCM) using experimental models of stem cell products spiked with tumorigenic cells. Methods: Human mesenchymal stem cells (hMSCs) and melanoma Mewo-Luc cells constitutively expressing luciferase were used. We stained Mewo-Luc cells with a cell linker then spiked onto hMSC suspensions and hMSC sheets. We validated the accuracy of 10-fold serial dilution technique for Mewo-Luc cell suspension using a Coulter counter. The samples spiked with Mewo-Luc cells were subjected to qPCR and FCM analyses, respectively for the quantification of Mewo-Luc cells. Results: Ten-fold serial dilutions of Mewo-Luc cells were performed accurately with small deviation. In samples spiked with or less than 100 cells in hMSC suspensions, and samples spiked with or less than 1,000 cells in hMSC sheets showed significantly higher cell numbers in calculations by FCM, respectively (suspensions; qPCR vs FCM: 100 cells: 59 ± 25 vs 232 ± 35 cells, p = 0.022/10 cells: 21 ± 7 vs 114 ± 27 cells, p = 0.030, sheets; qPCR vs FCM: 1,000 cells: 1723 ± 258 vs 5810 ± 878 cells, p = 0.012/100 cells: 110 ± 18 vs 973 ± 232 cells, p = 0.012/10 cells: 20 ± 6 vs 141 ± 36 cells, p = 0.030). Conclusion: Differences in accuracy between quantification methods should be considered in designing a tumorigenicity study model.
