Porcine Choroid plexus epithelial cells in culture: regulation of barrier properties and transport processes.

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.

The polarity of choroid plexus epithelial cells in vitro is improved in serum-free medium.

The influence of culture conditions on the development of normal characteristics of the choroid plexus epithelium has been investigated in vitro with respect to polarity, barrier properties, transport, and secretory activity. Withdrawal of serum supplement in the culture medium of cells grown on filters caused morphologically visible changes by an increased trimming of microvilli at the apical membrane side, which is accompanied by an increased expression of the Na+,K+-ATPase. Moreover cells under serum-free conditions exhibit structural changes in tight junctional zonula occludens protein-1 (ZO-1) organization, a reduced permeability, and a drastically increased electrical resistance from 150 ohms x cm2 in the presence of serum to 1,500 ohms x cm2 after serum withdrawal. Under these conditions, cell monolayers are able to build up a transcellular proton gradient and to secrete fluid into the upper (apical) filter compartment, which is accompanied by a polarized secretion of proteins like transthyretin. Active transport of the dyes fluorescein and phenol red by the organic anion transporter is found to be driven by the Na+,K+-ATPase. We come to the conclusion that removal of serum favors the differentiation process of the plexus epithelium in vitro, which brings the cell culture model closer to the physiological situation in vivo. We present preliminary evidence that epidermal growth factor may be one component in serum preventing the proper in vitro differentiation.

Active transport properties of porcine choroid plexus cells in culture.

We have investigated the transport properties of cultured porcine choroid plexus cells grown on permeable membranes and in serum-free medium. Withdrawal of serum yielded cell cultures with permeabilities low enough to establish and maintain a pH-gradient between the two compartments of the filter system and to allow apical fluid secretion. This became possible because of ten-fold increased electrical resistance of 1700 Omega cm2 in the absence of serum. These plexus epithelial cells transported phenol red, fluorescein, riboflavin and penicillin G from the apical to the basolateral side. KM values and vmax were determined and come close to in vivo values. Competitive inhibition with probenicid showed that the organic anion transporter is involved. Riboflavin transport however was not completely inhibited and did not respond quantitatively to the stilben derivate SITS that blocks the Cl-/HCO3--exchanger. We assume that an additional transport system exists for riboflavin. Ascorbic acid and myo-inositol were transported from the basolateral to the apical side in vitro which strongly resembles the in vivo transport from the blood to the cerebrospinal fluid. Again the experimental in vitro KM values come close to the in vivo values. The established epithelial cell culture model thus closely mimics the blood-CSF-barrier and may be a useful tool to further elucidate transport to and from the brain.