Abstract
Primary cell isolation from the central nervous system (CNS) has allowed fundamental understanding of blood-brain barrier (BBB) properties. However, poorly described isolation techniques or suboptimal cellular purity has been a weak point of some published scientific articles. Here, we describe in detail how to isolate and enrich, using a common approach, endothelial cells (ECs) from adult mouse brains, as well as pericytes (PCs) and astrocytes (ACs) from newborn mouse brains. Our approach allowed the isolation of these three brain cell types with purities of around 90%. Furthermore, using our protocols, around 3 times more PCs and 2 times more ACs could be grown in culture, as compared to previously published protocols. The cells were identified and characterized using flow cytometry and confocal microscopy. The ability of ECs to form a tight monolayer was assessed for passages 0 to 3. The expression of claudin-5, occludin, zonula occludens-1, P-glycoprotein-1 and breast cancer resistance protein by ECs, as well as the ability of the cells to respond to cytokine stimuli (TNF-α, IFN-γ) was also investigated by q-PCR. The transcellular permeability of ECs was evaluated in the presence of pericytes or astrocytes in a Transwell® model by measuring the transendothelial electrical resistance (TEER), dextran-FITC and sodium fluorescein permeability. Overall, ECs at passages 0 and 1 featured the best properties valued in a BBB model. Furthermore, pericytes did not increase tightness of EC monolayers, whereas astrocytes did regardless of their seeding location. Finally, ECs resuspended in fetal bovine serum (FBS) and dimethyl sulfoxide (DMSO) could be cryopreserved in liquid nitrogen without affecting their phenotype nor their capacity to form a tight monolayer, thus allowing these primary cells to be used for various longitudinal in vitro studies of the blood-brain barrier.
Highlights
The blood-brain barrier (BBB) is composed of specialized endothelial cells (ECs) surrounded by two basement membranes, pericytes (PCs) and astrocytes (ACs) [1]
Two other adhesion proteins previously characterized on ECs, namely activated leukocyte cell adhesion molecule (ALCAM) and intercellular adhesion molecule-1 (ICAM1), were observed by confocal microscopy (S3 Fig)
Yields and purities can vary widely depending on the method performed and the cost associated with some techniques can become prohibitive to specific applications
Summary
The blood-brain barrier (BBB) is composed of specialized endothelial cells (ECs) surrounded by two basement membranes, pericytes (PCs) and astrocytes (ACs) [1]. The presence of very few pinocytotic vesicles and a high concentration of efflux transporters has been previously described on bloodbrain barrier forming ECs [3, 4] Together, those characteristics generate a physically sealed barrier allowing brain capillaries to control the passage of compounds from the blood into the central nervous system (CNS). Pardrige highlighted the necessity to improve our knowledge on the fundamental properties of the BBB [5] and since extensive studies have led to a better understanding of molecules, pathways and cells able to generate and maintain the BBB [6] These efforts have been complemented by the design of several in vitro models and systems to evaluate the BBB in healthy and pathological conditions. Careful interpretation of previously published results is warranted due to the use of contaminated cell lines by other cell types and in some cases, the misidentification of the original cells used to generate the cell lines [8, 9]
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