Rapid isolation of murine primary hepatocytes for chromosomal analysis.
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.
Institute of Radiation Emergency Medicine, Hirosaki University, 66-1 Hon-cho, Hirosaki, 036-8564, Japan. ariyoshi@hirosaki-u.ac.jp.
Kentaro
K
Ariyoshi
Department of Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, 036-8564, Japan.
Yohei
Y
Fujishima
Department of Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, 036-8564, Japan.
Tomisato
T
Miura
Department of Radiation Effects Research, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology|, 4-9-1, Anagawa, Inage-ku, Chiba, 263-8555, Japan.
Yi
Y
Shang
Department of Radiation Effects Research, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology|, 4-9-1, Anagawa, Inage-ku, Chiba, 263-8555, Japan.
Shizuko
S
Kakinuma
Department of Radiation Effects Research, National Institute of Radiological Sciences, National Institutes for Quantum and Radiological Science and Technology|, 4-9-1, Anagawa, Inage-ku, Chiba, 263-8555, Japan.
Shimada
S
Yoshiya
Department of Biomedical Sciences, Hirosaki University Graduate School of Health Sciences, 66-1 Hon-cho, Hirosaki, 036-8564, Japan.
Kosuke
K
Kasai
Department of Basic Pharmacy, Hokkaido Pharmaceutical University School of Pharmacy, 7-1 Katsuraoka-cho, Otaru, Hokkaido, 047-0264, Japan.
Akifumi
A
Nakata
Department of Biological Sciences, College of Science, Ibaraki University, Bunkyo 2-1-1, Mito, Ibaraki, 310-8512, Japan.
Akira
A
Tachibana
Institute of Radiation Emergency Medicine, Hirosaki University, 66-1 Hon-cho, Hirosaki, 036-8564, Japan.
Mitsuaki A
MA
Yoshida
D000818:Animals; D018920:Coculture Techniques; D003470:Culture Media; D022781:Hepatocytes / Q000166:cytology*; D059785:Karyotype; D008099:Liver / Q000166:cytology* / Q000254:growth & development; D051379:Mice; D061251:Primary Cell Culture
D003470:Culture Media
Chromosome analysis; Co-culture; Hepatocyte