iHCPEnC express the endothelial markers PECAM1 and VWF and retained important PLVAP-based morphological characteristics of primary CP endothelial cells, such as caveolae and fenestrae. upon request. Summary The choroid plexus (CP) is usually a highly vascularized structure made up of endothelial and epithelial cells located in the ventricular system of the central nervous system (CNS). The role of the fenestrated CP endothelium is usually under-researched and requires the generation of an immortalized CP endothelial cell collection with preserved features. Transduction of main human CP endothelial cells (HCPEnC) with the human telomerase reverse transcriptase (hTERT) resulted in immortalized HCPEnC (iHCPEnC), which grew as monolayer with contact inhibition, created capillary-like tubes in Matrigel, and showed no colony growth in soft agar. iHCPEnC expressed pan-endothelial markers and offered characteristic plasmalemma vesicle-associated protein-containing structures. Cultivation of iHCPEnC and human epithelial CP papilloma (HIBCPP) cells on reverse sides of cell culture filter inserts generated an model with a consistently enhanced barrier function specifically by iHCPEnC. Overall, iHCPEnC present a tool that will contribute to the understanding of CP organ functions, especially endothelial-epithelial interplay. models based on main cells isolated from different species or mostly animal cell lines of tumor origin (Strazielle and Ghersi-Egea, 2011; Tenenbaum et?al., 2013; Schwerk et?al., 2015). Noteworthy, many of the functions of the CP are related to the ability of this organ to generate a barrier between the blood and the CSF. Beside animal models, functional CP cultures, based on the barrier properties and restricted paracellular permeability, have been established with CP epithelial cells that are cultured on microporous inserts. These include human epithelial CP papilloma Eprinomectin (HIBCPP) cells that at confluence develop polarity and high (500C800 cm2) transepithelial electrical resistance (TEER), making them useful for studying therapeutic drug transport as well as pathogen and immune cell passage (Strazielle and Ghersi-Egea, 2011; Tenenbaum et?al., 2013; Dinner et?al., 2016). It is assumed that an important role in maintaining organ functions is usually played by organ specific endothelial cells (Augustin and Koh, 2017), arguing for any potential significance of the CP endothelium at the BCSFB. Even though HIBCPP cells present a functional human cell line of the CP epithelium (Ishiwata et?al., 2008; Schwerk et?al., 2012), a cell line of human CP endothelial cells (HCPEnC) is still missing with the consequence of highly underrepresented research around the CP endothelium. In addition, coculture systems employing both epithelial and endothelial cells of the CP are required. However, the limited amount of cellular passages of main cells and the inevitable Eprinomectin variability between different Eprinomectin main cell cultures led to the necessity to generate a cell collection presenting characteristic endothelial markers as well as reproducible morphology and gene expression. The endothelial cells of the CP are fenestrated and the tight junctional proteins Claudin 1 (CLDN1), Claudin 5 (CLDN5), Occludin (OCLN), and Zonula occludens 1 (ZO1) are detected in rats (Lippoldt et?al., 2000). The proteinaceous substrate of endothelial fenestration is the plasmalemma vesicle-associated protein (PLVAP) that assembles into stomatal and fenestral diaphragms covering caveolae, transendothelial channels (TEC), and fenestrae (Stan et?al., 1997, 1999) and is expressed in the CP endothelium of mice (Dani et?al., 2021). The main function of PLVAP is usually associated with regulation of Eprinomectin cell layer permeability and transendothelial extravasation of immune cells (Keuschnigg et?al., 2009; Itgbl1 Bosma et?al., 2018). The limited cell division of main human endothelial cells can be overcome by ectopic expression of human telomerase reverse transcriptase (hTERT), which induces immortalization of cells (Harley et?al., 1990; Bodnar et?al., 1998). The ectopic expression of the catalytic domain name of hTERT, alone or in combination with a viral oncogene (simian computer virus 40 (SV40) large T antigen), was efficient in immortalization of human fibroblasts, retinal pigment epithelial cells, and brain microvascular endothelial cells (Jiang et?al., 1999; Weksler et?al., 2005). The immortalized microvascular endothelial cell lines express common markers as the platelet endothelial cell adhesion molecule (PECAM1), vascular endothelial cadherin (CDH5), and von Willebrand factor (VWF) (Weksler et?al., 2005). We describe here the generation and.