Primary cell and tissue cultures of human choroid plexus epithelial cells.

Choroid plexus epithelial cells (CPEC) are implicated in cerebrospinal fluid (CSF) production and the site of the inner blood-cerebrospinal fluid barrier. CPEC are specialized ependymal cells with characteristic cell junctions, intermediate filaments, and transport protein expression, including aquaporins. We have recently found that, in addition to aquaporin-1, a subset of CPEC in the human brain express aquaporin-4 (AQP4), the main water channel in the brain that has been implicated in water homeostasis and glymphatic system function. We therefore aimed to establish primary cultures from human CP to study AQP4 expression. We collected CP tissue from human body donors post mortem to grow CPEC in vitro. We successfully established two primary culture models for human CP tissue. In an organ culture model, small pieces of CP tissue were explanted on PTFE membranes. In a second model, cells from CP were dissociated and seeded on either laminin or collagen substrate. We monitored cell and tissue growth and investigated protein expression by immunofluorescence. Tissue integrity of explant cultures was maintained throughout the cultivation period of several weeks. Immunofluorescence signals for zonula occludens protein-1 (ZO1), AQP1, and Na/K-ATPase were present although the expression patterns became increasingly irregular and non-polar over time. In dissociation cultures, cells attached and grew very slowly initially but reached confluency after several weeks. Cultures were passaged up to 6 times and could be cryopreserved. Application of cytosine arabinoside suppressed the growth of macrophages and most likely fibroblasts. These primary dissociation cultures were identified as epithelial by their morphology and positive immunofluorescence for transthyretin, vimentin. Na/K-ATPase, ZO-1, cytokeratin, and AQP1. In both culture models, cells expressed AQP4. In dissociation cultures, AQP4 appeared to colocalize with ZO-1 at cell-cell contact sites in many CPEC but showed separate localizations at high resolution. These studies show that primary organ and cell cultures can be generated from human post mortem CP tissue suitable to investigate regulation and localization of AQP4. Together, these culture models provide valuable tools to investigate the proteins involved in CSF production and glymphatic clearance, relevant for the pathogenesis of neurodegenerative diseases.