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).
Highlights
Brain endothelial cells (BECs) denote the blood-brain barrier (BBB) and form a major physical restraint on the transport into the brain of several molecules present in blood plasma for transport
The aim of the present study was to establish a triple culture based entirely on porcine cells i.e. porcine brain endothelial cells (PBECs), astrocytes and pericytes and to determine if this preferable cellular combination for BBB formation would compare to PBECs co-cultured with rat astrocytes and pericytes isolated from newborn rat pups
The results show that primary porcine astrocytes and pericytes are useable for triple culture with PBECs instead of primary rat astrocytes and pericytes, as high trans-endothelial electric resistance (TEER) values, low passive permeability and expression of hallmarks of BECs can be observed
Summary
Brain endothelial cells (BECs) denote the blood-brain barrier (BBB) and form a major physical restraint on the transport into the brain of several molecules present in blood plasma for transport. Pericytes and end-feet of astrocytes form close contact with the BECs and participate in the formation, regulation and maintenance of the integrity of the BBB [1,5,7,8,9,10,11]. The BBB in vitro forms many characteristics of the in vivo conditions, e.g. robust tight junction expression and luminal to abluminal molecular transport indicative of defined polarity. Both primary and immortalized cells are being used for in vitro studies of the BBB. Primary BECs have been isolated and cultured from most mammals with the foremost originating from rats, mice, pigs, cows, and even humans (e.g.[12,13,14,15])
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