A Novel Porcine In Vitro Model of the Blood-Cerebrospinal Fluid Barrier with Strong Barrier Function

Abstract

Epithelial cells of the plexus choroideus form the structural basis of the blood-cerebrospinal fluid barrier (BCSFB). In vitro models of the BCSFB presenting characteristics of a functional barrier are of significant scientific interest as tools for examination of BCSFB function. Due to a lack of suitable cell lines as in vitro models, primary porcine plexus epithelial cells were subjected to a series of selective cultivation steps until a stable continuous subcultivatable epithelial cell line (PCP-R) was established. PCP-R cells grow in a regular polygonal pattern with a doubling time of 28–36 h. At a cell number of 1.5×105 in a 24-well plate confluence is reached in 56–72 h. Cells are cytokeratin positive and chromosomal analysis revealed 56 chromosomes at peak (84th subculture). Employing reverse transcription PCR mRNA expression of several transporters and components of cell junctions could be detected. The latter includes tight junction components like Claudin-1 and -3, ZO-1, and Occludin, and the adherens junction protein E-cadherin. Cellular localization studies of ZO-1, Occludin and Claudin-1 by immunofluorescence and morphological analysis by electron microscopy demonstrated formation of a dense tight junction structure. Importantly, when grown on cell culture inserts PCP-R developed typical characteristics of a functional BCSFB including high transepithelial electrical resistance above 600 Ω×cm2 as well as low permeability for macromolecules. In summary, our data suggest the PCP-R cell line as a suitable in vitro model of the porcine BCSFB.

Culture models to study leukocyte trafficking across the choroid plexus

“…Of note, we also established a novel porcine choroid plexus epithelial cell line (PCP-R), which displays important features of a functional BCSFB in vitro model, i.e. the formation of a strong barrier function as demonstrated by a high TEER and a low permeability for macromolecules [33]. In preliminary experiments, this cell line seems also to be suitable for the establishment of an inverted cell culture system.…”

Pathogens Penetrating the Central Nervous System: Infection Pathways and the Cellular and Molecular Mechanisms of Invasion

“…Choroid plexus epithelial cell lines that display the characteristic properties of the BCSFB have been described (140,141); however, the majority of in vitro microbial invasion studies have been performed using monocultures of brain microvascular endothelial cells.…”

In Vitro Models of CNS Barriers

“…Using a VO device Stazielle and Ghersi-Egea ( 1999 ) recorded 187 Ω cm 2 in primary rat CPE, while Baehr et al ( 2006 ) reported 100-150 Ω cm 2 in pig choroid plexus, and commented this would be equivalent to ~600 Ω cm 2 in an impedance system. Using a VO system with a stable continuous subcultivatable porcine CPE cell line, Schroten et al ( 2012 ) reported TEER >600 Ω cm 2 . In general the values up to ~600 Ω cm 2 fi t better with evidence for leakier tight junctions in CPE than BBB (Bouldin and Krigman 1975 ), but it is clear that more "side-by-side" comparisons of VO and IS systems using a particular in vitro model would be helpful to clarify the situation.…”

“…6.1.4.3 ), some models are tight enough for demonstration of CSF secretion, and the models have been used for a variety of studies of transport, metabolism and leukocyte traffi c (Redzic 2013 ;Strazielle and GhersiEgea 2013 ;Monot and Zheng 2013 ). A stable continuous sub-cultivatable porcine cell line (PCP-R) (Schroten et al 2012 ) and some immortalized cell lines (human Z310, Monot and Zheng 2013 ; rat TR-CSFB3, Terasaki and Hosoya 2001 ) have been introduced. The models have generally not been used for drug permeability screening.…”