Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an <em>In Vitro</em> Blood Brain Barrier Model

Abstract

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3 . While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the Förster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid-4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8 . Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Unravelling the regulation of insulin transport across the brain endothelial cell

“…iBECs were isolated from Sprague Dawley rats using a previously established method [27] (see ESM Methods ‘Harvest of iBECs’). iBECs were 98% CD31+ by flow cytometry, expressed claudin-5, and had increased phosphorylated Akt in response to insulin stimulation (see ESM Methods ‘Flow cytometry’ and ‘Immunofluorescence’, ESM Fig.…”

In Vitro Models of CNS Barriers

“…In an interesting breakthrough, Förster et al ( 2005 ) returned to the earlier cell transduction technology used for bEND3 and bEND5 to generate mouse cEND cells, which uniquely among immortalized brain endothelial cell lines can produce tight monolayers, with reported TEER up to >800 Ω cm 2 . The details of the immortalization method have been published and the cells have been used for studies on the involvement of glucocorticoids on tight junction regulation, and on hypoxia and multiple sclerosis (Burek et al 2012 ).…”

Primer Koyun Serebral Mikrodamar Endotelyal ve Astrosit Hücrelerinin Birlikte Kültüre Edilmesiyle Oluşturulmuş In vitro Kan Beyin Bariyeri Modeli

“…The depths of the media above and below the Transwell chamber were measured again after culturing for 4 h at 37°C under 5% CO 2 condition. If the difference between the liquids remained at 0.4-0.5 cm, this showed that the cells had formed a positive sense barrier [24][25][26][27] .…”