Improved Method for the Establishment of an <em>In Vitro</em> Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Abstract

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 ± 80 Ω cm, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10 ± 0.13 10 cm sec (mean ± SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Characterization of basigin monoclonal antibodies for receptor-mediated drug delivery to the brain

“…The cross-reactivity eases test of the mAbs in preclinical models and progression of the lead candidates into clinical studies. The porcine cross-reactivity is convenient for in vitro modeling of the transcytosis using the robust porcine BBB model as well as a good human pharmacokinetic prediction using pigs for the in vivo experiments 36,37 .…”

CoQ10 Deficient Endothelial Cell Culture Model for the Investigation of CoQ10 Blood–Brain Barrier Transport

“…Primary porcine brain endothelial cells (PBEC) were isolated and cultured as previously published [ 22 ]. Briefly, cells were seeded at 10 × 10 4 cells/cm 2 on collagen and fibronectin-coated polycarbonate Transwell filters (Corning 0.4 µm pore size) in 12-well plates, and grown in low glucose DMEM (Sigma D5546) supplemented with bovine plasma-derived serum (10% v/v ; BPDS, First Link UK), glutamine (2 mM), heparin (125 µg/mL), penicillin (100 U/mL) and streptomycin (100 µg/mL), at 37 °C in a 5% CO 2 incubator.…”

Transport of a Peptide from Bovine αs1-Casein across Models of the Intestinal and Blood–Brain Barriers

“…Astrocyte conditioned medium was collected every third day from primary rat astrocytes, sterile filtered and frozen until use. Isolation of primary rat astrocytes and preparation of astrocyte conditioned media were carried out as described in [ 20 ].…”

“…The tight BBB can be modelled in vitro using primary porcine brain capillary endothelial cells (PBECs). This well-established PBEC model is highly comparable to the human BBB with regard to high transendothelial electrical resistance (TEER) values, restricted passive permeability and expression of tight junction proteins and transporters specific for brain endothelium [ 19 , 20 , 21 ]. Based on the TEER values, the model is superior to models based on immortalized human cell lines, and is accepted as being a highly translational model for human studies.…”