Standardized Isolation and Culture of Murine Liver Sinusoidal Endothelial Cells

Rajkumar Cheluvappa1

1 Department of Medicine, St. George Clinical School, University of New South Wales, Sydney, New South Wales
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 2.9
DOI:  10.1002/0471143030.cb0209s65
Online Posting Date:  December, 2014
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Abstract

Isolation of murine liver sinusoidal endothelial cells (LSECs) is an exacting and finicky procedure. After exhaustive standardization, we were able to devise an easily reproducible protocol which produced consistent results. Moreover, we scripted a protocol which clarifies even the smallest of steps, following which isolation of LSECs is made significantly easier. Using the standardized LSEC isolation protocol herein, we demonstrated that the bacterial toxin pyocyanin (from Pseudomonas aeruginosa) induced a significant dose‚Äźdependent reduction in LSEC porosity, this being preventable by the enzyme catalase, but not by the enzyme superoxide dismutase. ¬© 2014 by John Wiley & Sons, Inc.

Keywords: liver sinusoidal endothelial cells; LSECs; hepatocytes; portal; cell culture; pyocyanin; Pseudomonas aeruginosa

     
 
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Table of Contents

  • Basic Protocol 1: Liver Sinusoidal Endothelial Cell (LSEC) Isolation
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Liver Sinusoidal Endothelial Cell (LSEC) Isolation

  Materials
  • Sprague‐Dawley rats (male, 250 to 350 g)
  • Ketamine
  • Xylazil
  • Normal saline (0.9% w/v aqueous NaCl)
  • Heparin
  • Hank's balanced salt solution (HBSS, no Ca2+; for initial rat liver perfusion; Sigma‐Aldrich, cat. no. 55025C)
  • Collagenase A solution (see recipe)
  • Collagenase B solution (see recipe)
  • PBS (see appendix 2A)
  • Percoll gradient (see recipe)
  • Complete RPMI‐1640 medium (see recipe)
  • Collagen‐coated coverslips (see recipe)
  • Trypan blue solution (Sigma‐Aldrich, cat. no. T8154)
  • Sterile Bag 1: 2 straight forceps
  • Sterile Bag 2:
  •  5 mosquito artery forceps
  •  1 broad‐end scissors
  •  1 sharp‐end scissors
  •  2 forceps
  • Insulin syringe
  • Infusion sets
  • Catheter (18‐G)
  • Centrifuge
  • Warm water bath
  • Petri dishes
  • Nylon gauze (100 mesh)
  • Neubauer chamber, appropriate coverslips, and microscope
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Figures

Videos

Literature Cited

Literature Cited
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  Cheluvappa, R., Hilmer, S.N., Kwun, S.Y., Jamieson, H.A., O'Reilly, J. N., Muller, M., Cogger, V.C., and Le Couteur, D.G. 2007b. The effect of old age on liver oxygenation and the hepatic expression of VEGF and VEGFR2. Exp. Gerontol. 42:1012‐1019.
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