Accuracy of spiked cell counting methods for designing a pre-clinical tumorigenicity study model
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 aime...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2020-07-01
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| Series: | Heliyon |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405844020312676 |
| _version_ | 1811292218506346496 |
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| author | Hiroaki Osada Masahide Kawatou Masafumi Takeda Jun-ichiro Jo Takashi Murakami Yasuhiko Tabata Kenji Minatoya Jun K. Yamashita Hidetoshi Masumoto |
| author_facet | Hiroaki Osada Masahide Kawatou Masafumi Takeda Jun-ichiro Jo Takashi Murakami Yasuhiko Tabata Kenji Minatoya Jun K. Yamashita Hidetoshi Masumoto |
| author_sort | Hiroaki Osada |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T04:42:06Z |
| format | Article |
| id | doaj.art-1fdd8de63a0542dd9a6f7d9de62341c3 |
| institution | Directory Open Access Journal |
| issn | 2405-8440 |
| language | English |
| last_indexed | 2024-04-13T04:42:06Z |
| publishDate | 2020-07-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Heliyon |
| spelling | doaj.art-1fdd8de63a0542dd9a6f7d9de62341c32022-12-22T03:01:57ZengElsevierHeliyon2405-84402020-07-0167e04423Accuracy of spiked cell counting methods for designing a pre-clinical tumorigenicity study modelHiroaki Osada0Masahide Kawatou1Masafumi Takeda2Jun-ichiro Jo3Takashi Murakami4Yasuhiko Tabata5Kenji Minatoya6Jun K. Yamashita7Hidetoshi Masumoto8Department 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, JapanDepartment 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, JapanDepartment 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, JapanLaboratory of Biomaterials, Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, JapanDepartment of Microbiology, Saitama Medical University, Faculty of Medicine, Saitama, JapanLaboratory of Biomaterials, Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, JapanDepartment of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, Kyoto, JapanDepartment of Cell Growth and Differentiation, Center for iPS Cell Research and Application, Kyoto University, Kyoto, JapanDepartment 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.http://www.sciencedirect.com/science/article/pii/S2405844020312676BiochemistryCell biologyTissue engineeringCell cultureStem cells researchBiomedical engineering |
| spellingShingle | Hiroaki Osada Masahide Kawatou Masafumi Takeda Jun-ichiro Jo Takashi Murakami Yasuhiko Tabata Kenji Minatoya Jun K. Yamashita Hidetoshi Masumoto Accuracy of spiked cell counting methods for designing a pre-clinical tumorigenicity study model Heliyon Biochemistry Cell biology Tissue engineering Cell culture Stem cells research Biomedical engineering |
| title | Accuracy of spiked cell counting methods for designing a pre-clinical tumorigenicity study model |
| title_full | Accuracy of spiked cell counting methods for designing a pre-clinical tumorigenicity study model |
| title_fullStr | Accuracy of spiked cell counting methods for designing a pre-clinical tumorigenicity study model |
| title_full_unstemmed | Accuracy of spiked cell counting methods for designing a pre-clinical tumorigenicity study model |
| title_short | Accuracy of spiked cell counting methods for designing a pre-clinical tumorigenicity study model |
| title_sort | accuracy of spiked cell counting methods for designing a pre clinical tumorigenicity study model |
| topic | Biochemistry Cell biology Tissue engineering Cell culture Stem cells research Biomedical engineering |
| url | http://www.sciencedirect.com/science/article/pii/S2405844020312676 |
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