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

Thomsen LB, Burkhart A, Moos T - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neurobiology, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.

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).

No MeSH data available.


Related in: MedlinePlus

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).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4524625&req=5

pone.0134765.g004: 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).

Mentions: The mRNA expression of claudin-5, occludin, transferrin receptor, P-gp and BCRP was confirmed by RT-qPCR (Fig 4). These molecules are all relevant for the BBB by means of being tight junction proteins (claudin-5, occludin), nutrient transporter (transferrin receptor) or drug/scavenger efflux transporters (P-gp, BCRP) and their expression indicates maintenance of important BBB features in the PBECs in culture. No significant differences were found in the relative mRNA expression of all the genes between PBECs grown in triple culture with porcine astrocytes and pericytes and PBECs cultured in triple culture with rat astrocytes and pericytes. Therefore, a triple culture model consisting entirely of porcine cells is preferred based on relative gene expression, when considering costs and ethics.


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

Thomsen LB, Burkhart A, Moos T - PLoS ONE (2015)

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).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4524625&req=5

pone.0134765.g004: 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).
Mentions: The mRNA expression of claudin-5, occludin, transferrin receptor, P-gp and BCRP was confirmed by RT-qPCR (Fig 4). These molecules are all relevant for the BBB by means of being tight junction proteins (claudin-5, occludin), nutrient transporter (transferrin receptor) or drug/scavenger efflux transporters (P-gp, BCRP) and their expression indicates maintenance of important BBB features in the PBECs in culture. No significant differences were found in the relative mRNA expression of all the genes between PBECs grown in triple culture with porcine astrocytes and pericytes and PBECs cultured in triple culture with rat astrocytes and pericytes. Therefore, a triple culture model consisting entirely of porcine cells is preferred based on relative gene expression, when considering costs and ethics.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neurobiology, Department of Health Science and Technology, Aalborg University, Aalborg, Denmark.

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).

No MeSH data available.


Related in: MedlinePlus