Primary Porcine Brain Microvessel Endothelial Cell Isolation and Culture

Adjanie Patabendige1, N. Joan Abbott2

1 Institute of Infection and Global Health, University of Liverpool, Liverpool, 2 Institute of Pharmaceutical Science, King's College London, London
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 3.27
DOI:  10.1002/0471142301.ns0327s69
Online Posting Date:  October, 2014
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Abstract

ABSTRACT

Cell culture models of the blood‐brain barrier (BBB) are useful tools to study the functionality of the BBB in health and disease. Several good in vitro BBB models are available from different species. However, most brain endothelial cells lose some of their in vivo BBB phenotype in culture. Porcine brain endothelial cells (PBECs) tend to retain most of their in vivo BBB characteristics and usually give higher transendothelial electrical resistance (TEER, representing functional well‐developed tight junctions) compared to brain endothelial cells from other species. The protocol described in this unit gives detailed instructions for isolation and culture of PBECs from fresh porcine brains. This porcine BBB model generates high TEER without the need for co‐culture with astrocytes. However, astrocyte‐derived factors can be introduced to the system through the use of astrocyte‐conditioned medium or co‐culture with astrocytes, which may be necessary for further enhancing the BBB phenotype for certain complex studies. Curr. Protoc. Neurosci. 69:3.27.1‐3.27.17. © 2014 by John Wiley & Sons, Inc.

Keywords: blood‐brain barrier; porcine brain endothelial cells; transendothelial electrical resistance; cell culture; astrocyte‐conditioned medium

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Culture of Porcine Brain Microvessel Endothelial Cells
  • Alternate Protocol 1: Using Astrocyte‐Conditioned Medium to Enhance BBB Phenotype
  • Support Protocol 1: Measuring Transendothelial Electrical Resistance
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation and Culture of Porcine Brain Microvessel Endothelial Cells

  Materials
  • Fresh porcine brains (Cheale Meats Ltd.)
  • 70% ethanol
  • Ice
  • Transport solution (see recipe)
  • Phosphate buffered saline (PBS) solution (see recipe)
  • Isolation solution (see recipe)
  • Enzyme solution (see recipe)
  • Freezing solution (see recipe)
  • Rat‐tail collagen Type I solution (see recipe)
  • Hanks’ balanced salt solution (HBSS) without Ca2+, Mg2+ (see appendix 2A)
  • Human fibronectin solution (see recipe)
  • Microvessel culture medium (see recipe)
  • Trypsin‐EDTA solution (Sigma, cat. no. T4299)
  • PBEC culture medium (see recipe)
  • Differentiation medium (see recipe)
  • Class II vertical laminar‐flow biological safety cabinet
  • Sterile 1000‐ml containers
  • Large polystyrene box
  • Sterile gauze
  • 150‐mm diameter Petri dishes
  • Sterile dissecting instrument set
    • Fine curved forceps
    • Coarse curved forceps
    • Sterile scalpels and blades (No. 10)
  • 50‐ml syringes
  • 40‐ml Dounce glass homogenizer
  • T175 flasks
  • Nalgene reusable filter holder unit for 47‐mm diameter membranes with 500‐ml receiver
  • 60‐μm and 150‐μm nylon mesh, cut into circles (6 of each)
  • Gilson pipets (P20, P200, and P1000) and tips
  • 15‐ml and 50‐ml Falcon centrifuge tubes
  • Refrigerated centrifuge
  • Stripettes (10 and 20 ml)
  • Cryovials
  • T75 flasks
  • Inverted microscope
  • Hemacytometer
  • Corning “clear” Transwell inserts (12‐mm diameter, 0.4‐μm pore) with 12‐well plates

Alternate Protocol 1: Using Astrocyte‐Conditioned Medium to Enhance BBB Phenotype

  Materials
  • Poly‐D‐lysine solution (see recipe)
  • Astrocyte culture medium (see recipe)
  • Primary human astrocytes (1800, ScienCell)
  • Trypsin‐EDTA solution (Sigma‐Aldrich, cat. no. T4299)
  • PBS without Ca2+, Mg2+ (see appendix 2A)
  • Class II vertical laminar‐flow biological safety cabinet
  • T75 flasks
  • Inverted microscope
  • 15‐ and 50‐ml Falcon tubes
  • 10‐ml stripette

Support Protocol 1: Measuring Transendothelial Electrical Resistance

  Materials
  • 70% ethanol
  • Differentiation medium (see recipe)
  • Class II vertical laminar‐flow biological safety cabinet
  • 30‐ml universal tubes
  • STX100C or STX2 electrodes (World Precision Instruments)
  • EVOM2 epithelial voltohmmeter (World Precision Instruments)
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Figures

Videos

Literature Cited

Literature Cited
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