Establishment of C-myc-immortalized Kupffer Cell Line from a C57BL/6 Mouse Strain

Overview

Journal Results Immunol

Publisher Elsevier

Specialty Allergy & Immunology

Date 2014 Nov 8

PMID 25379377

Citations 14

Authors

Hiroshi Kitani

Chisato Sakuma

Takato Takenouchi

Mitsuru Sato

Miyako Yoshioka

Noriko Yamanaka

Affiliations

Soon will be listed here.

Abstract

We recently demonstrated in several mammalian species, a novel procedure to obtain liver-macrophages (Kupffer cells) in sufficient numbers and purity using a mixed primary culture of hepatocytes. In this study, we applied this method to the C57BL/6 mouse liver and established an immortalized Kupffer cell line from this mouse strain. The hepatocytes from the C57BL/6 adult mouse liver were isolated by a two-step collagenase perfusion method and cultured in T25 culture flasks. Similar to our previous studies, the mouse hepatocytes progressively changed their morphology into a fibroblastic appearance after a few days of culture. After 7-10 days of culture, Kupffer-like cells, which were contaminants in the hepatocyte fraction at the start of the culture, actively proliferated on the mixed fibroblastic cell sheet. At this stage, a retroviral vector containing the human c-myc oncogene and neomycin resistance gene was introduced into the mixed culture. Gentle shaking of the culture flask, followed by the transfer and brief incubation of the culture supernatant, resulted in a quick and selective adhesion of Kupffer cells to a plastic dish surface. After selection with G418 and cloning by limiting dilutions, a clonal cell line (KUP5) was established. KUP5 cells displayed typical macrophage morphology and were stably passaged at 4-5 days intervals for more than 5 months, with a population doubling time of 19 h. KUP5 cells are immunocytochemically positive for mouse macrophage markers, such as Mac-1, F4/80. KUP5 cells exhibited substantial phagocytosis of polystyrene microbeads and the release of inflammatory cytokines upon lipopolysaccharide stimulation. Taken together, KUP5 cells provide a useful means to study the function of Kupffer cells in vitro.

Citing Articles

Comparative in vitro efficacy of antibiotics against the intracellular reservoir of Staphylococcus aureus.

Beadell B, Yamauchi J, Wong-Beringer A J Antimicrob Chemother. 2024; 79(10):2471-2478.

PMID: 39073778 PMC: 11441993. DOI: 10.1093/jac/dkae241.

Isolation and immortalization of macrophages derived from fetal porcine small intestine and their susceptibility to porcine viral pathogen infections.

Takenouchi T, Masujin K, Miyazaki A, Suzuki S, Takagi M, Kokuho T Front Vet Sci. 2022; 9:919077.

PMID: 35923820 PMC: 9339801. DOI: 10.3389/fvets.2022.919077.

Generation and characterization of genome-modified chondrocyte-like cells from the zebra finch cell line immortalized by c-MYC expression.

Jung K, Kim Y, Yoo E, Han J Front Zool. 2022; 19(1):18.

PMID: 35690812 PMC: 9188209. DOI: 10.1186/s12983-022-00464-x.

Downregulation of miR-122-5p Activates Glycolysis via PKM2 in Kupffer Cells of Rat and Mouse Models of Non-Alcoholic Steatohepatitis.

Inomata Y, Oh J, Taniguchi K, Sugito N, Kawaguchi N, Hirokawa F Int J Mol Sci. 2022; 23(9).

PMID: 35563621 PMC: 9101520. DOI: 10.3390/ijms23095230.

Distinguishing functional exosomes and other extracellular vesicles as a nucleic acid cargo by the anion-exchange method.

Seo N, Nakamura J, Kaneda T, Tateno H, Shimoda A, Ichiki T J Extracell Vesicles. 2022; 11(3):e12205.

PMID: 35289089 PMC: 8920962. DOI: 10.1002/jev2.12205.

References

McNally A, Anderson J . Macrophage fusion and multinucleated giant cells of inflammation. Adv Exp Med Biol. 2011; 713:97-111. DOI: 10.1007/978-94-007-0763-4_7. View

Takenouchi T, Ogihara K, Sato M, Kitani H . Inhibitory effects of U73122 and U73343 on Ca2+ influx and pore formation induced by the activation of P2X7 nucleotide receptors in mouse microglial cell line. Biochim Biophys Acta. 2005; 1726(2):177-86. DOI: 10.1016/j.bbagen.2005.08.001. View

Kitani H, Yoshioka M, Takenouchi T, Sato M, Yamanaka N . Characterization of the liver-macrophages isolated from a mixed primary culture of neonatal swine hepatocytes. Results Immunol. 2014; 4:1-7. PMC: 3973824. DOI: 10.1016/j.rinim.2014.01.001. View

Bilzer M, Roggel F, Gerbes A . Role of Kupffer cells in host defense and liver disease. Liver Int. 2006; 26(10):1175-86. DOI: 10.1111/j.1478-3231.2006.01342.x. View

Baffy G . Kupffer cells in non-alcoholic fatty liver disease: the emerging view. J Hepatol. 2009; 51(1):212-23. PMC: 2694233. DOI: 10.1016/j.jhep.2009.03.008. View

Lemaire I, Falzoni S, Adinolfi E . Purinergic signaling in giant cell formation. Front Biosci (Elite Ed). 2011; 4(1):41-55. DOI: 10.2741/359. View

Peng Y, Murr M . Establishment of immortalized rat Kupffer cell lines. Cytokine. 2007; 37(3):185-91. DOI: 10.1016/j.cyto.2007.03.003. View

Iwamaru Y, Takenouchi T, Ogihara K, Hoshino M, Takata M, Imamura M . Microglial cell line established from prion protein-overexpressing mice is susceptible to various murine prion strains. J Virol. 2006; 81(3):1524-7. PMC: 1797525. DOI: 10.1128/JVI.01379-06. View

Hendriks H, Brouwer A, Knook D . Isolation, purification, and characterization of liver cell types. Methods Enzymol. 1990; 190:49-58. DOI: 10.1016/0076-6879(90)90008-o. View

10.

Heymann F, Trautwein C, Tacke F . Monocytes and macrophages as cellular targets in liver fibrosis. Inflamm Allergy Drug Targets. 2009; 8(4):307-18. DOI: 10.2174/187152809789352230. View

11.

Imajo K, Fujita K, Yoneda M, Nozaki Y, Ogawa Y, Shinohara Y . Hyperresponsivity to low-dose endotoxin during progression to nonalcoholic steatohepatitis is regulated by leptin-mediated signaling. Cell Metab. 2012; 16(1):44-54. DOI: 10.1016/j.cmet.2012.05.012. View

12.

Dinarello C . The IL-1 family and inflammatory diseases. Clin Exp Rheumatol. 2004; 20(5 Suppl 27):S1-13. View

13.

Takenouchi T, Iwamaru Y, Sato M, Yokoyama T, Shinagawa M, Kitani H . Establishment and characterization of SV40 large T antigen-immortalized cell lines derived from fetal bovine brain tissues after prolonged cryopreservation. Cell Biol Int. 2006; 31(1):57-64. DOI: 10.1016/j.cellbi.2006.09.006. View

14.

Kitani H, Takenouchi T, Sato M, Yoshioka M, Yamanaka N . A novel isolation method for macrophage-like cells from mixed primary cultures of adult rat liver cells. J Immunol Methods. 2010; 360(1-2):47-55. DOI: 10.1016/j.jim.2010.06.004. View

15.

Yoshioka M, Nakajima Y, Ito T, Mikami O, Tanaka S, Miyazaki S . Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells. Vet Immunol Immunopathol. 1997; 58(2):155-63. DOI: 10.1016/s0165-2427(97)00030-5. View

16.

Roberts R, Ganey P, Ju C, Kamendulis L, Rusyn I, Klaunig J . Role of the Kupffer cell in mediating hepatic toxicity and carcinogenesis. Toxicol Sci. 2006; 96(1):2-15. DOI: 10.1093/toxsci/kfl173. View

17.

Ishimaru M, Yusuke N, Tsukimoto M, Harada H, Takenouchi T, Kitani H . Purinergic signaling via P2Y receptors up-mediates IL-6 production by liver macrophages/Kupffer cells. J Toxicol Sci. 2014; 39(3):413-23. DOI: 10.2131/jts.39.413. View

18.

Olynyk J, Britton R, Stephenson A, Leicester K, ONeill R, Bacon B . An in vitro model for the study of phagocytosis of damaged hepatocytes by rat Kupffer cells. Liver. 1999; 19(5):418-22. DOI: 10.1111/j.1478-3231.1999.tb00071.x. View

19.

Knolle P, Schlaak J, Uhrig A, Kempf P, Meyer zum Buschenfelde K, Gerken G . Human Kupffer cells secrete IL-10 in response to lipopolysaccharide (LPS) challenge. J Hepatol. 1995; 22(2):226-9. DOI: 10.1016/0168-8278(95)80433-1. View

20.

Clark J, Pateman J . A study of cultured fetal and SV40-transformed Chinese hamster Kupffer cells. Exp Cell Res. 1978; 114(1):47-56. DOI: 10.1016/0014-4827(78)90034-4. View