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

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

Comparison of mRNA expression levels of genes encoding for transporter proteins and junctional proteins in PCP-R and porcine CP tissue.

<p>The mRNA expression of all analyzed genes was normalized that of β-actin in the same sample. The expression in porcine CP tissue was arbitrarily set as 100%. Data represent mean ± SD (n = 3).</p>*<p>p<0.05.</p>**<p>p<0.01.</p>***<p>p<0.001 as compared to controls.</p

Generation of a stable subcultivatable porcine choroid plexus cell line.

<p>(A) Light microscopic depiction of primary porcine choroid plexus epithelial cells. (B) Subculture 43 (day 4): pure population of epithelial choroid plexus cells. These cells were continuously subcultivatable. (C) Immunocytochemistry with a mouse-anti human cytokeratin monoclonal antibody. PCP-R express the epithelial cell marker cytokeratin. (D) Immunofluorescence analysis of PCP-R shows expression of cytokeratin (green). The nuclei are counterstained with propidium iodide (red). Subculture 40 was analyzed in (C) and (D).</p

PCP-R displayed continuous tight junction strands.

<p>The cells, grown on coverslips, were stained for detection of ZO1 (A), Occludin (B) and Claudin-1 (C). Secondary antibodies were labeled with Alexa Fluor 594 (Alexa-594, red). Pictures presented are Apotome-generated images; <i>bottom</i> of each panel is an <i>xy</i> en face view of a cell culture monolayer shown in a maximum-intensity projection through the z-axis; <i>top and side</i> of each panel is a cross section through the z-plane of multiple optical slices. The basolateral side of PCP-R is oriented towards the top or right side, respectively, of the top and right images of each panel. In (A) and (B) the actin cytoskeleton was in parallel stained with phalloidin Alexa Fluor 488 (green). Since Claudin-1 samples were fixed with methanol we could not observe a qualitatively sufficient actin staining. In all samples nuclei were stained with DAPI (blue). The images shown are representative examples of multiple stainings.</p

Chromosomes of PCP-R.

<p>Chromosome analysis revealed aneuploidy with 56 chromosomes at peak.</p

Scheme for treatment of the cell cultures with Ara-C

<p>Ara-C, cytosine arabinoside.</p

PCP-R display barrier functions when grown on cell culture inserts (high TEER values correlate with low FITC-inulin flux through PCP-R-layers).

<p>1.5×10⁴ (white bars) or 2.5×10⁴ (grey bars) PCP-R cells were seeded on cell culture inserts; TEER values (A) and permeability coefficients for FITC-inulin (B) were measured over time. Shown is the mean ± SD of at least three experiments performed in duplicates.</p

Synthetic oligonucleotides used for RT- PCR and QPCR

<p>ACTNB, β-actin; ATP7A, ATPase 7A; CDH1; E-cadherin; CLDN, claudin; DMT1, divalent metal transporter-1; LEPR, leptin receptor; OCLN, Occludin; TFRC, transferrin receptor; ZnT1, zinc transporter-1; ZO-1, tight junction protein ZO-1.</p