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.
Highlights
The cerebrospinal fluid (CSF) is produced by the choroid plexus (CP), a highly perfused structure located in the ventricles of the brain
We demonstrate that PCP-R form tight junctions and display important characteristics of a functional blood-CSF barrier (BCSFB) in vitro model, i.e. the development of a high transendothelial electrical resistance (TEER) concomitant with a low permeability for macromolecules when grown on cell culture inserts
These include the rat CP epithelial cell lines Z310 and TRCSFB3, which have been established using transfection with the simian virus 40 large T-antigen [11,12,13], as well as the human cell line CPC-2 derived from CP carcinoma [14]
Summary
The cerebrospinal fluid (CSF) is produced by the choroid plexus (CP), a highly perfused structure located in the ventricles of the brain. Several CP cell lines originating from murine [11,12,13] or human [14,15] systems have been described These cell lines differ in expression of tight junction proteins, transport proteins and the development of TEER. Based on PCPEC cultures [8,9] we have established a porcine choroid plexus epithelial cell line (PCP-R) and analyzed the expression of tight junction proteins, transport proteins and the development of TEER as well as the permeability of macromolecules. We demonstrate that PCP-R form tight junctions and display important characteristics of a functional BCSFB in vitro model, i.e. the development of a high TEER concomitant with a low permeability for macromolecules when grown on cell culture inserts
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