Porcine choroid plexus epithelial cells in culture: Regulation of barrier properties and transport processes

Abstract

The epithelial cells of the choroid plexus are the structural basis of the blood-cerebrospinal fluid (CSF)-barrier. Here we summarise our recent efforts to culture those cells mainly on permeable supports in vitro. Isolated from porcine brains, we report a simple protocol for the primary culture using cytosine arabinoside as an additive that is cytotoxic for other cells except the plexus epithelial cells. Enhanced barrier properties are obtained by withdrawal of serum from the culture medium after confluency is reached. Cells improve their polarity, permeability for hydrophilic substrates is lowered, electrical resistance is increased tenfold, and a pH-gradient is built up across the cell monolayer. Polarised secretion of proteins and most importantly fluid secretion into the apical filter compartment was attained and proven to be dependent on the Na(+),K(+)-ATPase activity. Active transport processes (penicillin G, riboflavin, myo-inositol, ascorbic acid) were studied and clearly showed the involvement of the organic anion transporter. The permeability of the barrier was found to be regulated by cyclic adenosine monophosphate (cAMP). Moreover, we report that cell proliferation and differentiation is controlled by components of the extracellular matrix. The present culture model could now be used as an in vitro system to quantify drug transport across the blood-CSF-barrier.

Impact of Manganese on and Transfer across Blood-Brain and Blood-Cerebrospinal Fluid Barrier in Vitro

“…The applied, well established in vitro cell culture model of the blood-CSF barrier is built up by PCPECs (20), shows several features of physiological activity, and mimics the in vivo situation quite closely (21,24). In this in vitro model, the apical side of the PCPEC layer mimics the ventricular compartment, whereas the basolateral side represents the blood side in vivo.…”

“…On day DIV8, cells reached confluence, and the medium was replaced by serum-free medium (SFM-PCPEC) (DMEM/Ham's F 12 (1:1) supplemented with 4 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 5 g/ml insulin) to allow full cell differentiation. The cells were further supplemented with SFM on DIV11, and barrier integrity and transport studies were started on day DIV13, when the cells were fully differentiated and built up their respective functions (21,24). PCPECs started to actively pump medium from the basolateral into the apical chamber, a process that is comparable with the secretion of cerebrospinal fluid in vivo, and transported phenol red to the basolateral chamber.…”

Matrix Metalloprotease 8-Dependent Extracellular Matrix Cleavage at the Blood-CSF Barrier Contributes to Lethality during Systemic Inflammatory Diseases

“…Not much is known about the composition and function of the ECM deposited along the outer surface of the basal side of the CP epithelial cells. Based on in vitro and in vivo analyses, several matrix proteins, such as thrombospondin, fibronectin, laminin, and collagen types I, III, IV, and XVIII, are believed to be present (Gabrion et al, 1998;Wei et al, 2000;Haselbach et al, 2001;Utriainen et al, 2004;Thouvenot et al, 2006;Thanos et al, 2011). Because MMP8 has collagenolytic activity, we speculated that MMP8 modulates the CP epithelial BM by degradating collagen of the basal lamina.…”

How Does the Benzamide Antipsychotic Amisulpride get into the Brain?—An In Vitro Approach Comparing Amisulpride with Clozapine

“…PCEC from porcine choroid plexus were obtained by a preparation basically described by Crook et al (1981), slightly modified by Gath et al (1997), and described in detail by Hakvoort et al (1998) and Haselbach et al (2001). In brief, porcine choroid plexus tissue was digested by trypsin, released cells were centrifuged and thereafter resuspended in DME/HAM's F12 medium supplemented with 10% fetal bovine serum (Biochrom), 4 mM L-glutamin, 5 mg/ml insulin (Sigma), 20 mM cytosine arabinoside (Sigma), and 100 mg/ml antibiotics (Sigma).…”