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Primary hepatocyte culture is a crucial tool for investigations of liver function and for evaluating the toxic effects of drugs. In addition, chromosomal analysis of hepatocytes could also prove useful for understanding the mechanisms of hepatocarcinogenesis. However, cultivation of primary hepatocytes for chromosome analysis has been hampered by the specific equipment and skill required to perform the in situ perfusion step necessary for isolation of primary hepatocytes. In the present study, we aimed to establish a simple and efficient method of isolating hepatocytes suitable for chromosome analysis. We performed hepatocyte isolation without using collagenase perfusion, instead digesting liver tissues using collagenase in tubes. In addition, we examined hepatocyte and bone marrow cell (BMC) co-culture and cultivation of hepatocytes with medium containing BMC culture medium supernatants. We found that hepatocyte viability and attachment rate were significantly improved, both by co-culture with BMCs and medium containing BMC culture media supernatants, with the latter also significantly increasing the mitotic index. Using this simple method of isolation and cultivation, we could successfully perform chromosomal analysis of mouse primary hepatocytes. This method has the potential to help understand the mechanisms underlying chromosomal instability-mediated hepatocarcinogenesis.

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The authors would like to thank I. Asari and other laboratory members for technical and secretarial assistance and Dr. Shimada at the National Institute of Radiological Sciences for technical assistance and Dr. BJ Blyth for helpful discussion and critical reading of the manuscript.

All animal experiments were conducted according to the Hirosaki University guidelines for animal experimentation, and the research protocol was approved in advance by the Ethics Committee.

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Correspondence to Kentaro Ariyoshi.

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Ariyoshi, K., Fujishima, Y., Miura, T. et al. Rapid isolation of murine primary hepatocytes for chromosomal analysis. In Vitro Cell.Dev.Biol.-Animal 53, 474–478 (2017).

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