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Title: Cytokinesis-block micronucleus assay performed in 0 and 2 Gy irradiated whole blood and isolated PBMCs in a 6-well transwell co-culture system
Kai Takebayashi
Ryo Nakayama
Yohei Fujishima
Mitsuaki A. Yoshida
Kosuke Kasai
Kentaro Ariyoshi
Tomisato Miura
Keywords: cytokinesis-block micronucleus assay
cytogenetic biodosimetry
human peripheral blood mononuclear cells
radiation-induced bystander effect
whole blood
Issue Date: 23-Sep-2021
Publisher: Taylor & Francis
Citation: GOH SWEE TING VALERIE, Kai Takebayashi, Ryo Nakayama, Yohei Fujishima, Mitsuaki A. Yoshida, Kosuke Kasai, Kentaro Ariyoshi, Tomisato Miura (2021-09-23). Cytokinesis-block micronucleus assay performed in 0 and 2 Gy irradiated whole blood and isolated PBMCs in a 6-well transwell co-culture system. International Journal of Radiation Biology : 1-28. ScholarBank@NUS Repository.
Rights: Attribution-NonCommercial-NoDerivatives 4.0 International
Abstract: Purpose: Cytokinesis-block micronucleus (CBMN) assay in cytogenetic biodosimetry uses micronucleus (MN) frequency scored in binucleated cells (BNC) for dose estimation. Cell-cycle progression parameters of nuclear division index (NDI) and percentage of BNC (% BNC) are also evaluated. Whole blood (WB) or peripheral mononuclear cells (PBMCs) isolated from WB can be used for lymphocyte culture. Previously, 2 Gy PBMCs showed higher NDI and lower MN frequency than WB in 15 ml polypropylene tube single cultures. In this follow-up study, we wanted to assess if soluble factors present in WB but absent in PBMCs could increase MN frequency or decrease NDI in PBMCs co-cultured with WB. Materials and methods: Peripheral blood from four healthy donors (2 males: 25, 51; 2 females: 23, 26 years old) was irradiated with X-rays at 1 Gy/min. CBMN assay was performed with different combinations of 0 and 2 Gy WB and PBMC (WB, WB-IR, PBMC, PBMC-IR) mono- and co-cultures in a polystyrene 6-well plate. Co-cultures were separated by 0.4 µm transwell inserts. Log2 fold changes and values of NDI, % BNC and MN frequency analyzed by 3 scorers were obtained. Results: As upper and lower wells of the same culture condition showed some significant differences, wells of the same level were compared. NDI of PBMCs increased when PBMC or PBMC-IR were co-cultured with WB or WB-IR, respectively, as compared to mono-cultures. There was no increase in PBMC-IR's MN frequency when co-cultured with WB or WB-IR. MN frequency was consistently higher in WB-IR than PBMC-IR in both mono- and co-cultures. NDI, % BNC and MN frequency were similar when WB or PBMC were co-cultured with PBMC-IR or WB-IR, respectively. Significantly lower NDI and % BNC, and higher MN frequency were also seen in some conditions of 15 ml cultures than 6-well mono-cultures. Conclusion: Instead of the hypothesized decrease in NDI and increase in MN frequency, our co-culture set-up showed that in the absence of direct cell-cell interaction, soluble factors in WB increased NDI but not MN frequency in PBMCs. Moreover, radiation-induced bystander effects could not be observed. As the type of cell culture (WB, PBMC) and culture vessels could influence NDI and MN frequency, CBMN culture protocols should be kept consistent for dose-response calibration curve construction and dose estimation.
Source Title: International Journal of Radiation Biology
ISSN: 09553002
DOI: 10.1080/09553002.2021.1981555
Rights: Attribution-NonCommercial-NoDerivatives 4.0 International
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