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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Abstract

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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Materials

Basic Protocol 1: Isolating Hepatocytes From Mice

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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

  Materials
  • 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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Figures

Videos

Literature Cited

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Internet Resources
  http://mouselivercells.com/
  Excellent Web site with detailed protocols, images, and information on isolating mouse hepatocytes and additional assays.
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