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Abstract:The influence of basement membrane proteins on cellular barrier properties of primary cultures of porcine brain capillary endothelial cells grown on permeable filter inserts has been investigated. Measurements of transcellular electrical resistance (TEA) by impedance spectroscopy were performed with cells cultured on type IV collagen, fibronectin, laminin, and one-to-one mixtures of these proteins. Moreover, a one-to-one combination of type IV collagen and SPARC (secreted protein acidic and rich in cysteine) has been studied. Rat tail collagen has been used as a reference substratum. If TERs of cells from a given preparation were low (-~350Q x cm 2) on the reference substratum, type IV collagen, fibronectin, and laminin as well as one-to-one combinations of these proteins elevated transcellular resistances significantly (2.3-to 2.9-fold) compared with rat tail collagen. TER of cells exhibiting a high reference level (~-~1 000~l x cm2) could, by contrast, be increased only 1.1-to 1.2-fold. The type IV collagen/SPARC mixture did not elevate TER. Our findings suggest that type IV collagen, fibronectin, and laminin are involved in tight junction formation between cerebral capillary endothelial cells. The differential effects observed for individual preparations probably reflect more or less dedifferentiated states of the endothelium, in which basement membrane proteins can influence cellular differentiation more or less strongly. However, our results indicate that type IV collagen, fibronectin, and laminin enhance the reliability and suitability of primary microvascular endothelial cell cultures as an in vitro model of the blood-brain barrier. Key Words: Bloodbrain barrier-Primary cell culture-Transcellular electrical resistance-Basement membrane proteins-Extracellular matrix.

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 Ω· cm2 in the presence of serum to 1,500 Ω· 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.