Table 1.
The table displays the reference sequence numbers and primer sequences of the six primers used in this study.
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.
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.
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.
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).