Development of an In Vitro Blood‐Brain Barrier

Damir Janigro1, Kathe A. Stanness1, Carl Soderland2, Gerald A. Grant3

1 Cleveland Clinic Foundation, Cleveland, Ohio, 2 Cell Systems, Kirkland, Washington, 3 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 12.2
DOI:  10.1002/0471140856.tx1202s03
Online Posting Date:  May, 2001
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Abstract

The blood‐brain barrier represents a significant protective barrier for the passage of pathogens and toxicants, but it is difficult to study in vivo. This unit describes an in vitro system that can be used as a model for the blood‐brain barrier to study its functions in an accessible format.

     
 
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Table of Contents

  • Basic Protocol 1: Establishing a Dynamic Blood‐Brain Barrier
  • Support Protocol 1: Monitoring Cell Growth
  • Support Protocol 2: Measurement of Transendothelial Permeability
  • Support Protocol 3: Functional Assessment of Blood‐Brain Barrier Integrity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Establishing a Dynamic Blood‐Brain Barrier

  Materials
  • Human endothelial cells (Cell Systems)
  • Human glial cells: normal human astrocytes (NHA; Clonetics)
  • CS‐C Complete Medium (Cell Systems) containing 10% (v/v) FBS
  • Gentamycin (Sigma)
  • Fungizone (Bio‐Whittaker)
  • AGM BulletKit medium (Clonetics) containing 5% (v/v) FBS
  • Attachment factor (Cell Systems)
  • Versene (EDTA) and trypsin/versene (Bio‐Whittaker)
  • DPBS, Ca2+‐ and Mg2+‐free (see recipe)
  • 75‐cm2 tissue culture flasks (Corning Costar), uncoated for glial cells and coated with 50 µg/ml attachment factor for endothelial cells
  • Cartridge/hollow fiber culturing system (see Fig. ; Spectrum)
  • 1‐, 3‐, 5‐, and 10‐ml syringes, sterile
  • 18‐G needles, blunt ended and sterile
  • 22‐G needles, sterile
  • Additional reagents and equipment for determining glucose levels (see protocol 2)

Support Protocol 1: Monitoring Cell Growth

  • [14C]sucrose (Amersham)
  • 3H‐labeled drug under investigation
  • Additional reagents and equipment for determining glucose consumption and lactate production (see protocol 2)
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and the surroundings. Carry out the experiment and dispose of wastes in appropriately designated areas, following the guidelines provided by the local radiation safety officer (also see appendix 1A).
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Figures

Videos

Literature Cited

Literature Cited
   Grant, G.A., Abbott, N.J., and Janigro, D. 1998. Understanding the physiology of the blood‐brain barrier: The role of in vitro models. News Physiol. Sci 13:287‐293.
   Janigro, D., Leaman, S., and Stanness, K.A. 1999. Dynamic in vitro modeling of the BBB: A novel tool for the studies of drug delivery to the brain. Pharm. Sci. Technol. 2:7‐12.
   Pekny, M., Stanness, K.A., Eliasson, C., Betsholtz, C., and Janigro, D. 1997. Impaired induction of blood‐brain barrier properties in aortic endothelial cells by astrocytes from GFAP‐deficient mice. Glia 22:1‐11.
   Stanness, K.A., Guatteo, E., and Janigro, D.A. 1996. A dynamic model of the blood‐brain barrier “in vitro.” Neurotoxicology 17:481‐496.
   Stanness, K.A., Westrum, L.E., Mascagni, P., Fornaciari, E., Nelson, J.A., Stenglein, S.G., and Janigro, D. 1997. Morphological and functional characterization of an in vitro blood‐brain barrier model. Brain Res 771:329‐342.
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