Characterization of human fetal brain endothelial cells reveals barrier properties suitable for in vitro modeling of the BBB with syngenic co-cultures

Abstract

Endothelial cells (ECs) form the basis of the blood-brain barrier (BBB), a physical barrier that selectively restricts transport into the brain. In vitro models can provide significant insight into BBB physiology, mechanisms of human disease pathology, toxicology, and drug delivery. Given the limited availability of primary human adult brain microvascular ECs ( aBMVECs), human fetal tissue offers a plausible alternative source for multiple donors and the opportunity to build syngenic tri-cultures from the same host. Previous efforts to culture fetal brain microvascular ECs ( fBMVECs) have not been successful in establishing mature barrier properties. Using optimal gestational age for isolation and flow cytometry cell sorting, we show for the first time that fBMVECs demonstrate mature barrier properties. fBMVECs exhibited similar functional phenotypes when compared to aBMVECs for barrier integrity, endothelial activation, and gene/protein expression of tight junction proteins and transporters. Importantly, we show that tissue used to culture fBMVECs can also be used to generate a syngenic co-culture, creating a microfluidic BBB on a chip. The findings presented provide a means to overcome previous challenges that limited successful barrier formation by fBMVECs. Furthermore, the source is advantageous for autologous reconstitution of the neurovascular unit for next generation in vitro BBB modeling.

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in-vitro models of the human blood–brain barrier

“…Primary human brain microvascular endothelial cells (hBMVECs) were isolated from fetal brain tissue as described( Andrews et al, 2018 ). Healthy tissue was provided (under informed consent) by the Laboratory of Developmental Biology (University of Washington, Seattle, WA) with approval granted by Temple University's (Philadelphia, PA) Institutional Review Board and in full compliance by the National Institutes of Health's (NIH) ethical guidelines.…”

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in vitro models of the human blood–brain barrier

“…Healthy tissue was provided (under informed consent) by the Laboratory of Developmental Biology (University of Washington, Seattle, WA) with approval granted by Temple University's (Philadelphia, PA) Institutional Review Board and in full compliance by the National Institutes of Health's (NIH) ethical guidelines. Fetal human brain microvascular endothelial cells (hBMVEC) were isolated as described 32 . Cells were grown on rat-tail collagen I coated flasks (BD Biosciences) in EBM-2 medium supplemented with EGM-2MV SingleQuots (Lonza, Cat No CC-3156 and CC-4147) in an incubator set to 37 o C, 5 % CO2, and 100 % humidity.…”

In vitro modeling of blood–brain barrier and interface functions in neuroimmune communication

“…Primary BECs from humans are difficult to obtain in sufficient quality and quantity, and usually require isolation from fresh, surgically resected tissues from living patients to reliably develop high TEER [2]. Most of these BECs are from donors receiving temporal lobe resective surgery for intractable epilepsy, but advances in surgical techniques such as gamma knife radiosurgery may diminish availability of tissue for primary BEC isolation [26]. Although many BBB properties have been extensively evaluated in surgically-derived human BECs [27,28], there is some question whether BECs derived from healthy brain tissues of epileptic patients would perform similarly to those of healthy humans [29], and this issue may also vary by donor.…”