	Print Version
BioSafety Level	II
Organism	Neonatal mouse (WT 129 Sv strain)
Source Organ	Cerebral cortex
Growth Properties	Adherent
Morphology	spindle shaped
Recommended Seeding Density	Recommended split ratio: no greater than 1:4
Markers	Claudin-5, Occuldin, Glut-1, VE-Cadherin
Applications	For Research Use Only
Immortalization Method	Transformation with oncoprotein of murine Polyomavirus, Polyoma middle T antigen (PymT)
Description	Homeostasis of the central nervous system (CNS) is maintained by the blood brain barrier (BBB) and disruptions to the BBB are linked to many disorders of the CNS. The Immortalized Mouse Cerebral Capillary Endothelial Cell Line (cEND) provides a useful model for studies involving the differentiation and regulation of BBB as it shares principal features of the BBB in vivo, specifically in that 1) it retains the endothelial markers VE-Cadherin and PECAM-1; 2) it displays tight junction associated markers such as occludin, claudin-3, -5, -12, 3) it expresses the BBB marker protein Glut-1 and 4) it shows high electrical resistance, representing barrier properties. This cell line is also responsive to glucocorticoid, estrogen-treatment and pro-inflammatory mediator such as TNFα, making this cell line valuable in elucidating cellular responses of endothelial cells to different stimuli. Together with the Immortalized Mouse Cerebellar Capillary Endothelial Cell Line (cerebEND), the two cell lines represent important in vitro model system for these different brain regions.
Procedure Overview
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Propagation	Use of PriCoat? T25 Flasks (G299) or Applied Cell Extracellular Matrix (G422) is required for cell adhesion to the culture vessels. Grow cells in ECM-coated culture vessels with the following conditions. The base medium for this cell line is Prigrow III medium available from ABM (TM003). To make the completed growth medium, add the following components to the base medium: heat-inactivated fetal bovine serum (TM999) to final concentration of 10% and 1% Penicillin/Streptomycin (G255). Atmosphere: air: 95%, CO?: 5%; Temperature: 37.0°C. The Differentiation Medium is composed of 2% heat-inactivated fetal bovine serum (TM999) instead of 10%. Culturing cEND cells in serum-reduced medium leads to an increase in TER (from 150 Ωcm2in the presence of 10% serum to 500 Ωcm2in the presence of 2% serum), as well as change in cell morphology (from spindle-shaped to cobble-stone like). TER can be further increased by the addition of 110nM hydrocortisone (800 Ωcm2) or 1μM insulin (1000 Ωcm2) into the differentiation medium.
Preservation	1. Freeze Medium: Complete growth medium with 20% FBS and 10% DMSO. 2. Storage Temperature: Liquid nitrogen vapour phase.
Quality Control	1) Endothelial marker VE-Cadherin and PECAM-1 assessed by immunostainning; 2) Tight junction protein Claudin-5 assessed by immunostainning and claudin-1, -3 and -12 detected in mRNA level; 2) BBB marker protein Glut-1 and tight junction-associated protein occludin measured by western blot analysis. The cell line's response to pro-inflammatory stimuli was measured by 1) TER, 2) western blot and 3) gene expression analysis using RT-PCR.
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