A Mouse Primary Hepatocyte Culture Model for Studies of Circadian Oscillation

Penny C. Molyneux1, Lorna A. Pyle1, Martha Dillon1, Mary E. Harrington1

1 Neuroscience Program, Smith College, Northampton, Massachusetts
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo150101
Online Posting Date:  December, 2015
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Circadian rhythms regulate many aspects of behavior and physiological processes, and, through external signals, help an organism entrain to its environment. These rhythms are driven by circadian clocks in many cells and tissues within our bodies, and are synchronized by a central pacemaker in the brain, the suprachiasmatic nucleus. Peripheral oscillators include the liver, whose circadian clock controls persistent daily rhythms in gene expression and in liver‐specific functions such as metabolic homeostasis and drug metabolism. Chronic circadian clock disruption, as in rotating shiftwork, has been linked to disorders including obesity, diabetes, and cardiovascular disease. The mouse primary hepatocyte culture model allows the examination of circadian rhythms in these cells. This article describes a transgenic mouse model that uses a bioluminescent reporter to examine the circadian properties of a core clock gene Period2. Hepatocytes are isolated using a modified collagenase perfusion technique and cultured in a sandwich configuration, then sealed in a buffered medium containing luciferin for detection of whole‐culture or single‐cell bioluminescence. After synchronization by a medium change, cultures demonstrate coherent circadian period and phase measures of bioluminescence from the PERIOD2::LUCIFERASE reporter. © 2015 by John Wiley & Sons, Inc.

Keywords: primary hepatocyte; mouse hepatocyte; circadian rhythm; bioluminescence; Period2

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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolating Hepatocytes From Mice
  • Basic Protocol 2: Hepatocyte Culture: Primary Mouse Hepatocytes in Single or Mixed Cultures in Collagen Gel Sandwich Configuration
  • Support Protocol 1: Equipment Setup of Perfusion Apparatus for Hepatocyte Isolation
  • Support Protocol 2: Assess Hepatocyte Function by Measuring Gluconeogenesis From Glycerol
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Isolating Hepatocytes From Mice

  • Collagenase solution (see recipe)
  • EGTA solution (see recipe)
  • 70% (v/v) ethanol
  • Mouse
  • Ketamine/xylazine solution (400 mg/kg ketamine and 20 mg/kg xylazine)
  • WMS wash medium (see recipe)
  • WMS plating medium (see recipe)
  • 0.4% (w/v) trypan blue
  • Perfusion apparatus (see protocol 3)
  • Cannula, plastic wings removed (Fisher, cat. no. 02‐664‐7, or BD cat. no. 367297)
  • Dissection pan or stage
  • Tape
  • Dissecting tools:
    • Scissors with one rounded edge
    • Dumont forceps
    • Iris scissors
  • Cotton swabs
  • Sterile gauze pads
  • Sterile 35‐mm culture dish for transport of excised liver
  • 100‐mm culture dish
  • 100‐μm sterile cell strainer (Fisher, cat. no. 22363549)
  • 50‐ml sterile conical tubes (e.g., Corning Falcon)
  • Refrigerated centrifuge
  • Vacuum aspirator connection and sterile pipet
  • Hemacytometer and coverslip
  • Additional reagents and equipment for setup of perfusion apparatus ( protocol 3) and injection of mice (Donovan and Brown, )

Basic Protocol 2: Hepatocyte Culture: Primary Mouse Hepatocytes in Single or Mixed Cultures in Collagen Gel Sandwich Configuration

  • 1.25 mg/ml collagen, Type 1 (see recipe)
  • 10× DMEM (pH 7.3 to 7.4 at time of use; see recipe)
  • One or more freshly prepared hepatocyte cell suspensions (1 × 106 cells/ml), in WMS plating medium (see protocol 1)
  • WMS maintenance medium (see recipe), 37°C
  • WMS buffered maintenance medium (see recipe), 37°C
  • Luciferin potassium salt (Promega, cat. no. E1601)
  • 25‐ or 50‐ml flask
  • 35‐mm culture dishes

Support Protocol 1: Equipment Setup of Perfusion Apparatus for Hepatocyte Isolation

  • Water bath at 37°C
  • Peristaltic pump capable of 1 to 10 ml per min (e.g., Gilson Minipuls3)
  • Appropriate tubing for peristaltic pump (Gilson, cat. no. F117949)
  • 3‐way stopcock for tubing (Cole Parmer, cat. no. 30600‐02)
  • Bubble trap (Radnoti, cat. no. 130149)
  • Ringstand with clamp to attach bubble trap
  • Cannula, plastic wings removed (Fisher, cat. no. 02‐664‐7, or BD cat. no. 367297)
  • Timer
  • 10‐ml graduated cylinder to measure flow rate

Support Protocol 2: Assess Hepatocyte Function by Measuring Gluconeogenesis From Glycerol

  • Prepared hepatocyte cultures with top gel applied ( protocol 2), with known % viability
  • DMEM without glucose (see recipe)
  • GNG assay medium (see recipe)
  • Glucose standard curve (top standard = 10 μg/μl, to 0.3125 μg/μl in 2× serial‐dilution steps; see recipe)
  • GNG assay master mix (see recipe)
  • 96‐well plate
  • Spectrophotometer with microtiter plate reader, capable of measuring 405 nm absorbance
NOTE: All media are warmed to 37°C before application; all incubation steps longer than 2 min are carried out in a humidified 37°C 10% CO 2 incubator.
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Literature Cited

Literature Cited
  Bergmeyer, H.U., Gawehn, K., and Grassl, M., 1974. Methods of Enzymatic Analysis, Vol. I, 2nd ed., (H.U. Bergmeyer, ed.) pp. 457‐458, Academic Press, New York.
  Berthiaume, F., Moghe, P.V., Toner, M., and Yarmush, M.L. 1996. Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: Hepatocytes cultured in a sandwich configuration. FASEB J. 10:1471‐1484.
  Bianconi, E., Piovesan, A., Facchin, F., Beraudi, A., Casadei, R., Frabetti, F., Vitale, L., Pelleri, M.C., Tassani, S., Piva, F., Perez‐Amodio, S., Strippoli, P., and Canaider, S., 2013. An estimation of the number of cells in the human body. Ann. Hum. Biol. 40:463‐471. doi: 10.3109/03014460.2013.807878. Epub 2013 Jul 5. Erratum in: Ann. Hum. Biol. 2013;40:471.
  Dallmann, R., Viola, A.U., Tarokh, L., Cajochen, C., and Brown, S.A., 2012. The human circadian metabolome. Proc. Natl. Acad. Sci. U.S.A. 109:2625‐2629. doi: 10.1073/pnas.1114410109.
  DeBruyne, J.P., Weaver, D.R., and Dallmann, R., 2014. The hepatic circadian clock modulates xenobiotic metabolism in mice. J. Biol. Rhythms. 29:277‐287. doi: 10.1177/0748730414544740.
  Dibner, C., Schibler, U., and Albrecht, U., 2010. The mammalian circadian timing system: Organization and coordination of central and peripheral clocks. Annu. Rev. Physiol. 72:517‐549. doi: 10.1146/annurev-physiol-021909-135821.
  Donovan, J. and Brown, P. 2006. Parenteral injections. Curr. Protoc. Immunol. 73:1.6.1‐1.6.10. doi: 10.1002/0471142735.im0106s73.
  Dunn, J.C., Tompkins, R.G., and Yarmush, M.L., 1991. Long‐term in vitro function of adult hepatocytes in a collagen sandwich configuration. Biotechnol. Prog. 7:237‐245.
  Evans, J.A. and Davidson, A.J. 2013 Health consequences of circadian disruption in humans and animal models. Prog. Mol. Biol. Transl. Sci. 119: 283‐323. doi: 10.1016/B978-0-12-396971-2.00010-5.
  Guenthner, C.J., Luitje, M.E., Pyle, L.A., Molyneux, P.C., Yu, J.K., Li, A.S., Leise, T.L., and Harrington, M.E. 2014. Circadian rhythms of Per2::Luc in individual primary mouse hepatocytes and cultures. PLoS One 9:e87573. doi: 10.1371/journal.pone.0087573. eCollection 2014.
  Izumo, M., Pejchal, M., Schook, A.C., Lange, R.P., Walisser, J.A., Sato, T.R., Wang, X., Bradfield, C.A., and Takahashi, J.S. 2014. Differential effects of light and feeding on circadian organization of peripheral clocks in aforebrain Bmal1 mutant. Elife. 3. doi: 10.7554/eLife.04617.
  Klaunig, J.E., Goldblatt, P.J., Hinton, D.E., Lipsky, M.M., Chacko, J., and Trump, B.F. 1981. Mouse liver cell culture. I. Hepatocyte isolation. In Vitro 17:913‐925. doi: 10.1007/BF02618288.
  Lamia, K.A., Storch, K.F., and Weitz, C.J., 2008. Physiological significance of a peripheral tissue circadian clock. Proc. Natl. Acad. Sci. U.S.A. 105:15172‐15177. doi: 10.1073/pnas.0806717105. Epub 2008 Sep 8.
  LeCluyse, E.L., Audus, K.L., and Hochman, J.H., 1994. Formation of extensive canalicular networks by rat hepatocytes cultured in collagen‐sandwich configuration. Am. J. Physiol. 266:C1764‐1774.
  Li, W.C., Ralphs, K.L., and Tosh, D. 2010. Isolation and culture of adult mouse hepatocytes. Methods Mol. Biol. 633:185‐196. doi: 10.1007/978-1-59745-019-5_13.
  Pan, A., Schernhammer, E.S., Sun, Q., and Hu, F.B., 2011 Rotating night shift work and risk of type 2 diabetes: Two prospective cohort studies in women. PLoS Med. 8:e1001141. doi: 10.1371/journal.pmed.1001141. Epub 2011 Dec 6.
  Ramanathan, C., Khan, S.K., Kathale, N.D., Xu, H., and Liu, A.C. 2012. Monitoring cell‐autonomous circadian clock rhythms of gene expression using luciferase bioluminescence reporters. J. Vis. Exp. 67:e4234. doi:10.3791/4234.
  Ramanathan, C., Xu, H., Khan, S.K., Shen, Y., Gitis, P.J., Welsh, D.K., Hogenesch, J.B., and Liu, A.C. 2014. Cell type‐specific functions of period genes revealed by novel adipocyte and hepatocyte circadian clock models. PLoS Genet. 10:e1004244. doi: 10.1371/journal.pgen.1004244. eCollection 2014 Apr.
  Reddy, A.B. 2013. Genome‐wide analyses of circadian systems. Handb. Exp. Pharmacol. 217:379‐388. doi: 10.1007/978-3-642-25950-0_16.
  Riccalton‐Banks, L., Liew, C., Bhandari, R., Fry, J., and Shakesheff, K., 2003. Long‐term culture of functional liver tissue: Three‐dimensional coculture of primary hepatocytes and stellate cells. Tissue Eng. 9:401‐410. doi: 10.1089/107632703322066589.
  Saini, C., Liani, A., Curie, T., Gos, P., Kreppel, F., Emmenegger, Y., Bonacina, L., Wolf, J.P., Poget, Y.A., Franken, P., and Schibler, U. 2013. Real‐time recording of circadian liver gene expression in freely moving mice reveals the phase‐setting behavior of hepatocyte clocks. Genes Dev. 27:1526‐1536. doi: 10.1101/gad.221374.113.
  Scheer, F.A., Hilton, M.F., Mantzoros, C.S., and Shea, S.A., 2009. Adverse metabolic and cardiovascular consequences of circadian misalignment. Proc. Natl. Acad. Sci. U.S.A. 106:4453‐4458. doi: 10.1073/pnas.0808180106. Epub 2009 Mar 2.
  Seglen, P.O. 1976. Preparation of isolated rat liver cells. Methods Cell Biol. 13:29‐83. doi: 10.1016/S0091-679X(08)61797-5.
  Swift, B., Pfeifer, N.D., and Brouwer, K.L.R., 2010. Sandwich‐cultured hepatocytes: An in vitro model to evaluate hepatobiliary transporter‐based drug interactions and hepatotoxicity. Drug Metab. Rev. 42:446‐471. doi:10.3109/03602530903491881.
  Tahara, Y., Kuroda, H., Saito, K., Nakajima, Y., Kubo, Y., Ohnishi, N., Seo, Y., Otsuka, M., Fuse, Y., Ohura, Y., Komatsu, T., Moriya, Y., Okada, S., Furutani, N., Hirao, A., Horikawa, K., Kudo, T., and Shibata, S. 2012. In vivo monitoring of peripheral circadian clocks in the mouse. Curr. Biol. 22:1029‐1034. doi: 10.1016/j.cub.2012.04.009.
  van der Horst, G.T., Muijtjens, M., Kobayashi, K., Takano, R., Kanno, S., Takao, M., de Wit, J., Verkerk, A., Eker, A.P., van Leenen, D., Buijs, R., Bootsma, D., Hoeijmakers, J.H., and Yasui, A. 1999. Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature. 398:627‐630. doi: 10.1038/19323.
  Welsh, D.K., Imaizumi, T., and Kay, S.A., 2005. Real‐time reporting of circadian‐regulated gene expression by luciferase imaging in plants and mammalian cells. Methods Enzymol. 393:269‐288. doi: 10.1016/S0076-6879(05)93011-5.
  Welsh, D.K., Takahashi, J.S., and Kay, S.A., 2010. Suprachiasmatic nucleus: Cell autonomy and network properties. Annu. Rev. Physiol. 72:551‐577. doi:10.1146/annurev-physiol-021909-135919.
  Yoo, S.H., Yamazaki, S., Lowrey, P.L., Shimomura, K., Ko, C.H., Buhr, E.D., Siepka, S.M., Hong, H.K., Oh, W.J., Yoo, O.J., Menaker, M., and Takahashi, J.S. 2004. PERIOD2::LUCIFERASE real‐time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc. Natl. Acad. Sci. U.S.A. 101:5339‐5346. Epub 2004 Feb 12. doi: 10.1073/pnas.0308709101.
  Zhang, W. 2011. Development and Validation of a Primary Mouse Hepatocyte System in the Evaluation of Dioxin‐like PCB Exposure (Ph.D. Dissertation). University of Chicago. ProQuest/UMI 3460255.
  Zhang, W., Sargis, R.M., Volden, P.A., Carmean, C.M., Sun, X.J., and Brady, M.J. 2012. PCB 126 and other dioxin‐like PCBs specifically suppress hepatic PEPCK expression via the aryl hydrocarbon receptor. PLoS One. 7:e37103. doi: 10.1371/journal.pone.0037103. Epub 2012 May 16.
Internet Resources
  Excellent Web site with detailed protocols, images, and information on isolating mouse hepatocytes and additional assays.
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