Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier

Abstract

We describe a method for generating primary cultures of human brain microvascular endothelial cells (HBMVEC). HBMVEC are derived from microvessels isolated from temporal tissue removed during operative treatment of epilepsy. The tissue is mechanically fragmented and size-filtered using polyester meshes. The resulting microvessel fragments are placed onto type-I collagen-coated flasks to allow HBMVEC to migrate and proliferate. The overall process takes under 3 h and does not require specialized equipment or enzymatic processes. HBMVEC are typically cultured for approximately 1 month until confluence. Cultures are highly pure (~97% endothelial cells; ~3% pericytes), reproducible, and display characteristic brain endothelial markers (von Willebrand factor, glucose transporter-1), robust expression of tight and adherens junction proteins, caveolin-1, and efflux protein P-glycoprotein. Monolayers of HBMVEC display characteristic high transendothelial electric resistance and have proven useful in multiple functional studies for in-vitro modeling of the human blood-brain barrier.

The TNF-α/Nf-κB Signaling Pathway has a Key Role in Methamphetamine–Induced Blood–Brain Barrier Dysfunction

“…We used lysophosphatidic acid (10 μmol/L) as a positive control since it produces a strong and transient opening of the ionic barrier. 20 Accordingly, TEER values of HBMVECs were decreased at 2 hours after drug exposure up until 24 hours ( Figure 1D). Noteworthy, the concentrations of METH used in the present study neither caused EC death nor decreased cell viability (Supplementary Figure 1).…”

“…Thus, to reach our goal we first took advantage of a wellestablished BBB in vitro model, 19 and we observed that METH increased ECs permeability at concentrations relevant to human abuse. Considering the differences between rodents and humans, we further used human ECs 20 to reproduce the results obtained with rat primary cultures. As it is also known that METH can induce death of various cell types 4,12 including ECs, 21 we also considered if METH-increased endothelial permeability was due to cell death.…”

A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

“…Similarly, in human primary brain endothelial cells, HBMVEC, we observed high P-gp expression ( Figure 1A). This mostly cytoplasmic P-gp localization in brain endothelial cells is reminiscent of a P-gp expression pattern previously reported in human primary brain endothelial cells (53). However, this expression pattern of P-gp localization is different from the expression pattern observed in drug-resistant cancer cells, which showed mostly surface expression (31).…”

Adverse effects of antipsychotics on micro-vascular endothelial cells of the human blood–brain barrier

“…This physical arrangement ensures that the endothelial cells act to restrict the movement of microscopic particles (e.g. bacteria) and hydrophilic molecules into the cerebrospinal fluid (CSF) (Bernas et al, 2010;Abbott 2013, Abbott et al 2010. Once this barrier is breached, the CNS is exposed to potential insult by hazardous solutes and pathogens circulating in the blood (Abbott et al 2010).…”

“…Since it is possible to isolate and culture the different cell types that constitute the BBB (Bernas et al, 2010) the primary aim of this study was to investigate the cellular and functional effects of two typical APs (CPZ and HAL) and two atypical APs: risperidone (RIS) and clozapine (CLZ), at the higher end of therapeutic levels, as well as those expected with over dosage, on a model system of the BBB. In particular, we wished to determine to what extent these drugs affect cell viability, programmed cell death, barrier permeability and to investigate putative mechanisms behind these processes.…”