Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Aug 4;10(8):e0134765.
doi: 10.1371/journal.pone.0134765. eCollection 2015.

A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes

Louiza Bohn Thomsen  1 , Annette Burkhart  1 , Torben Moos  1
Affiliations

A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes

Louiza Bohn Thomsen et al. PLoS One. .

Abstract

In vitro blood-brain barrier (BBB) models based on primary brain endothelial cells (BECs) cultured as monoculture or in co-culture with primary astrocytes and pericytes are useful for studying many properties of the BBB. The BECs retain their expression of tight junction proteins and efflux transporters leading to high trans-endothelial electric resistance (TEER) and low passive paracellular permeability. The BECs, astrocytes and pericytes are often isolated from small rodents. Larger species as cows and pigs however, reveal a higher yield, are readily available and have a closer resemblance to humans, which make them favorable high-throughput sources for cellular isolation. The aim of the present study has been to determine if the preferable combination of purely porcine cells isolated from the 6 months old domestic pigs, i.e. porcine brain endothelial cells (PBECs) in co-culture with porcine astrocytes and pericytes, would compare with PBECs co-cultured with astrocytes and pericytes isolated from newborn rats with respect to TEER value and low passive permeability. The astrocytes and pericytes were grown both as contact and non-contact co-cultures as well as in triple culture to examine their effects on the PBECs for barrier formation as revealed by TEER, passive permeability, and expression patterns of tight junction proteins, efflux transporters and the transferrin receptor. This syngenic porcine in vitro BBB model is comparable to triple cultures using PBECs, rat astrocytes and rat pericytes with respect to TEER formation, low passive permeability, and expression of hallmark proteins signifying the brain endothelium (tight junction proteins claudin 5 and occludin, the efflux transporters P-glycoprotein (PgP) and breast cancer related protein (BCRP), and the transferrin receptor).

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Characterization of primary cell cultures by immunocytochemistry.
PBECs express the tight junction proteins Claudin-5 (A, green) and Zonula occludens 1 (B, green) at the cell borders. Porcine mixed glial cells (C) mainly consist of astrocytes which express GFAP (green), and a few (5–10%) microglial cells which express tomato lectin (red). Rat astrocytes (D) express GFAP (green). Porcine pericytes (E) and rat pericytes (F) express alpha-smooth muscle actin (red). Porcine pericytes cultured in monoculture (G) and porcine pericytes cultured in a triple culture with porcine pericytes and PBECs in a triple culture (H) stain for PDGFR-beta (green) and alpha-smooth muscle actin (red). Only a few of the porcine pericytes cultured in triple culture express alpha-smooth muscle actin. Cell nuclei stained with DAPI (blue). Scale bar = 20μm.
Fig 2
Fig 2. Trans-endothelial electric resistance (TEER) measurement made across PBECs in thirteen co-culture combinations.
The mean TEER value of monocultures (n = 35) is significantly lower ($ $ $, P<0.001) than the mean TEER values for all other culture combinations (n = 13–31). Significant difference between the mean TEER value for PBECs cultured in contact co-culture with porcine pericytes (n = 24) compared to PBECs co-cultured in contact co-culture with rat pericytes (n = 23) (**, P<0.01)(n equals number of inserts).
Fig 3
Fig 3. Mannitol permeability measurements on PBECs in thirteen co-culture combinations as a function of their TEER.
TEER measured just before the permeability experiment was conducted. The Papp mannitol measured on n = 3 for each culture condition. Each point represents one hanging culture insert with PBECs.
Fig 4
Fig 4. Gene Expression of claudin-5, Occludin, P-gp, BCRP and transferrin in PBECs.
RT-qPCR performed on the PBECs from all thirteen different culture combinations. The relative gene expression of Claudin-5, Occludin, P-gp, BCRP and transferrin receptor-1 is shown for each culture combination. The results are given as relative expression normalized to beta-actin using the Pfaffl method (n = 3–6 replicates and one replicate consists of RNA from PBECs from 4–6 inserts).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Correale J, Villa A. Cellular elements of the BBB. Neurochem Res. 2009;34: 2067–2077. 10.1007/s11064-009-0081-y - DOI - PubMed
    1. Lichota J, Skjørringe T, Thomsen LB, Moos T. Macromolecular drug transport into the brain using targeted therapy. J Neurochem. 2010;113: 1–13. - PubMed
    1. Brightman MW, Reese TS. Junction between intimately apposed cell mambranes in the vertebrate brain. J Cell Biol. 1969;40: 648–677. - PMC - PubMed
    1. Bauer HC, Bauer H, Lametschwandtner A, Amberger A, Ruiz P, Steiner M. Neovascularization and the appearance of morphological characteristics of the blood-brain barrier in the embryonic mouse central nervous system. Dev Brain Res. 1993;75: 269–78. - PubMed
    1. Abbott NJ, Rönnbäck L, Hansson E. Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci. 2006;7: 41–53. - PubMed

Publication types

MeSH terms

Grants and funding

This work was partly supported by the Obel Family Foundation, Obel.com and the A.P. Moeller Foundation "Fonden til Lægevidenskabens Fremme", apmollerfonde.dk. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.