Comparative studies between the murine immortalized brain endothelial cell line (bEnd.3) and induced pluripotent stem cell-derived human brain endothelial cells for paracellular transport

Abstract
Brain microvascular endothelial cells, forming the anatomical site of the blood-brain barrier (BBB), are widely used as in vitro complements to in vivo BBB studies. Among the immortalized cells used as in vitro BBB models, the murine-derived bEnd.3 cells offer culturing consistency and low cost and are well characterized for functional and transport assays, but result in low transendothelial electrical resistance (TEER). Human-induced pluripotent stem cells differentiated into brain microvascular endothelial cells (ihBMECs) have superior barrier properties, but the process of differentiation is time-consuming and can result in mixed endothelial-epithelial gene expression. Here we performed a side-by-side comparison of the ihBMECs and bEnd.3 cells for key paracellular diffusional transport characteristics. The TEER across the ihBMECs was 45- to 68-fold higher than the bEnd.3 monolayer. The ihBMECs had significantly lower tracer permeability than the bEnd.3 cells. Both, however, could discriminate between the paracellular permeabilities of two tracers: sodium fluorescein (MW: 376 Da) and fluorescein isothiocyanate (FITC)–dextran (MW: 70 kDa). FITC-dextran permeability was a strong inverse-correlate of TEER in the bEnd.3 cells, whereas sodium fluorescein permeability was a strong inverse-correlate of TEER in the ihBMECs. Both bEnd.3 cells and ihBMECs showed the typical cobblestone morphology with robust uptake of acetylated LDL and strong immuno-positivity for vWF. Both models showed strong claudin-5 expression, albeit with differences in expression location. We further confirmed the vascular endothelial- (CD31 and tube-like formation) and erythrophagocytic-phenotypes and the response to inflammatory stimuli of ihBMECs. Overall, both bEnd.3 cells and ihBMECs express key brain endothelial phenotypic markers, and despite differential TEER measurements, these in vitro models can discriminate between the passage of different molecular weight tracers. Our results highlight the need to corroborate TEER measurements with different molecular weight tracers and that the bEnd.3 cells may be suitable for large molecule transport studies despite their low TEER.

Introduction
The brain microvascular endothelial cells form the anatomical site of the blood-brain barrier (BBB) [1]. Experimental evidence showing the presence of the BBB dates to more than 100 years ago, wherein simple vital dyes were used to show the presence of a barrier separating the peripheral circulation and the central nervous system [2]. The primary site of the barrier is the endothelial layer lining the cerebral microvasculature, which is further supported by the presence of a basement membrane lining the endothelial cells and the pericytes, and the astrocyte foot-processes that envelope 99% of the cerebral microvasculature [3]. The key features of the brain microvascular endothelial cells that make the BBB an impermeable interface are the continuous arrangement of tight-junction proteins, lack of fenestrae, low pinocytic activity, and the resultant high transendothelial electrical resistance (TEER). As a result, even small molecule transport, which is restricted to molecules < 400 Da that have <3 hydrogen bond donors and < 7 hydrogen bond acceptors, is limited at the BBB [4–6]. Other features that make the BBB a highly selective physical barrier include high mitochondrial content and polarized expression of transporters and metabolic enzymes [7, 8]. Additionally, the brain microvascular endothelial cells respond to inflammatory stimuli through the expression of adhesion molecules and exhibit vascular endothelial characteristics [9]. These peculiar characteristics are required for the optimal functioning of the BBB to maintain a stable brain microenvironment.

Methods
Murine brain microvascular endothelial cell culture
bEnd.3 cells (American Type Culture Collection, Manassas, VA, USA; Catalog no. CRL-2299) were cultured in Dulbecco’s Modified Eagle’s Medium (American Type Culture Collection, Manassas, VA, USA) supplemented with 10% fetal bovine serum (R&D systems, Minneapolis, MN, USA), and 100 μg/mL penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at standard cell culture conditions (5% CO2, 95% air). Cells between passages 24 and 30 were seeded onto multi-well plates (Corning Inc., Corning, NY, USA), 0.2% gelatin-coated glass coverslips, or Transwells with 0.4 μm pore polyester membrane inserts of 24-well plates (Corning Inc., Corning, NY, USA) at a density of 1x105 to 1x106 cells/cm2, unless otherwise stated.

Results
TEER measurement across the bEnd.3 and ihBMEC monolayer
To compare the paracellular passage across the brain endothelial monolayers, we performed TEER measurements starting 24 h after seeding cells on Transwells. The average TEER of the cell-free inserts was 41 ± 0.5 ohm•cm2. TEER values of cell-free inserts were subtracted from the TEER values of inserts with endothelial cells and are shown in Fig 1A. As shown in Fig 1A, over the five days, the TEER values across the bEnd.3 monolayer ranged between 15 ± 0.8 ohm•cm2 (day 1) and 28.1 ± 2.3 ohm•cm2 (day 5). The TEER measurements across the ihBMECs were at least 42-fold higher than those across the bEnd.3 monolayer and ranged between 928 ± 81 ohm•cm2 (day 1) and 1169 ± 200 ohm•cm2 (day 2) (Fig 1A). Many studies using bEnd.3 cells have reported TEER measurements for 6–8 days [34–37]. However, our pilot studies showed that TEER measurements across the ihBMECs begin to decline five days after replating on Transwells inserts (TEER values on day 6 were: 796 ± 46 ohm cm2). Therefore, TEER measurements were performed for five days in the current study.

Discussion
In the current study, we compared two popular in vitro BBB models: the ihBMECs, which are iPSC-derived human brain microvascular endothelial cells, and the bEnd.3 cells, which are immortalized murine brain microvascular endothelial cells. The ihBMECs displayed superior barrier properties (higher TEER and significantly lower tracer permeabilities). However, both the bEnd.3 cells and the ihBMECs could discriminate between the passage of the two different molecular weight markers (sodium fluorescein and FITC-dextran), showing paracellular passage selectivity of the two monolayers. FITC-dextran permeability paralleled TEER measurements across the bEnd.3 monolayer, while sodium fluorescein permeability paralleled TEER measurements across the ihBMEC monolayer. Evaluation of key endothelial phenotypic markers confirmed the expression of vWF and uptake of acetylated LDL and the vascular endothelial marker, CD31, for both the cells. Claudin-5 expression pattern differed between these two models. We observed distinct junctional claudin-5 expression in the ihBMECs, whereas junctional claudin-5 expression was less distinct and a more diffuse cytoplasmic expression of claudin-5 was observed in the bEnd.3 cells.

Conclusions
In conclusion, both the murine immortalized cell line, bEnd.3 cells, and the iPSC-derived human brain endothelial cells, ihBMECs, express key endothelial phenotypic markers, show red blood cell adhesion and phagocytic features, and express vascular endothelial markers, making them useful models for BBB mechanistic studies. The major difference between these two in vitro BBB models was in their paracellular barrier characteristics, with the ihBMECs having significantly higher TEER and restrictive permeability to the small molecular weight tracer (sodium fluorescein). However, despite the many-fold higher TEER measurements across the ihBMECs, the bEnd.3 monolayer restricted > 80% passage of free FITC-dextran. Our results, therefore, suggest that the bEnd.3 cells may be a suitable model for transport studies of large molecules and for cell interaction and migration studies despite their low TEER [27]. Modification of the culture conditions, e.g., serum-free conditions and co-cultures, can be adapted to further enhance bEnd.3 barrier properties [42]. Our results also suggest the need to corroborate TEER measurements with different molecular weight tracers as a more reliable measure of barrier properties for an in vitro BBB model designed for paracellular transport studies. Although the choice of the in vitro BBB model selected will ultimately be based on the nature of the investigation, the choice will also be driven by other factors such as batch-to-batch variability, time and cost of culturing, and questions about the endothelial lineage with the ihBMECs [18, 20], versus the ease of culturing, consistency between passages and commercial availability of the bEnd.3 cells.

Citation: Sun J, Ou W, Han D, Paganini-Hill A, Fisher MJ, Sumbria RK (2022) Comparative studies between the murine immortalized brain endothelial cell line (bEnd.3) and induced pluripotent stem cell-derived human brain endothelial cells for paracellular transport. PLoS ONE 17(5): e0268860. https://doi.org/10.1371/journal.pone.0268860

Editor: Mária A. Deli, Eötvös Loránd Research Network Biological Research Centre, HUNGARY

Received: January 25, 2022; Accepted: May 9, 2022; Published: May 25, 2022

Copyright: © 2022 Sun et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: Research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke under award number R01NS20989 (MJF), and by the National Institute of Aging under award number R01AG072896 (RKS and DH), of the National Institutes of Health (https://www.nih.gov/). Approximately, $250k (100%) of federal funds supported this project. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0268860#abstract0