Cytokinesis-block micronucleus assay performed in 0 and 2 Gy irradiated whole blood and isolated PBMCs in a 6-well transwell co-culture system
Creators: name: Goh, Valerie Swee Ting
name: Takebayashi, Kai
name: Nakayama, Ryo
name: Fujishima, Yohei
name: Yoshida, Mitsuaki A.
name: Kasai, Kosuke
name: Ariyoshi, Kentaro
name: Miura, Tomisato
Publisher: name: Taylor & Francis
Year: 2021
Subjects: subject: 29999 Physical Sciences not elsewhere classified
subject: FOS: Physical sciences
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Molecular Biology
subject: Physiology
subject: 59999 Environmental Sciences not elsewhere classified
subject: FOS: Earth and related environmental sciences
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: 69999 Biological Sciences not elsewhere classified
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: 60506 Virology

description: 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. 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. 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. 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.