Mechanical stress regulates transport in a compliant 3D model of the blood-brain barrier (original) (raw)
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Pulsatility and high shear stress deteriorate barrier phenotype in brain microvascular endothelium
Journal of Cerebral Blood Flow & Metabolism, 2016
Microvascular endothelial cells at the blood–brain barrier exhibit a protective phenotype, which is highly induced by biochemical and biomechanical stimuli. Amongst them, shear stress enhances junctional tightness and limits transport at capillary-like levels. Abnormal flow patterns can reduce functional features of macrovascular endothelium. We now examine if this is true in brain microvascular endothelial cells. We suggest in this paper a complex response of endothelial cells to aberrant forces under different flow domains. Human brain microvascular endothelial cells were exposed to physiological or abnormal flow patterns. Physiologic shear (10–20 dyn/cm2) upregulates expression of tight junction markers Zona Occludens 1 (1.7-fold) and Claudin-5 (more than 2-fold). High shear stress (40 dyn/cm2) and/or pulsatility decreased their expression to basal levels and altered junctional morphology. We exposed cells to pathological shear stress patterns followed by capillary-like condition...
Brain Research, 2007
The blood-brain barrier (BBB) is a structural and functional barrier that regulates the passage of molecules into and out of the brain to maintain the neural microenvironment. We have previously developed the in vitro BBB model with human brain microvascular endothelial cells (HBMEC). However, in vivo HBMEC are shown to interact with astrocytes and also exposed to shear stress through blood flow. In an attempt to develop the BBB model to mimic the in vivo condition we constructed the flow-based in vitro BBB model using HBMEC and human fetal astrocytes (HFA). We also examined the effect of astrocyte conditioned medium (ACM) in lieu of HFA to study the role of secreted factor(s) on the BBB properties. The tightness of HBMEC monolayer was assessed by the permeability of dextran and propidium iodide as well as by measuring the transendothelial electrical resistance (TEER). We showed that the HBMEC permeability was reduced and TEER was increased by non-contact, co-cultivation with HFA and ACM. The exposure of HBMEC to shear stress also exhibited decreased permeability. Moreover, HFA/ACM and shear flow exhibited additive effect of decreasing the permeability of HBMEC monolayer. In addition, we showed that the HBMEC expression of ZO-1 (tight junction protein) was increased by co-cultivation with ACM and in response to shear stress. These findings suggest that the non-contact co-cultivation with HFA helps maintain the barrier properties of HBMEC by secreting factor(s) into the medium. Our in vitro flow model system with the cells of human origin should be useful for studying the interactions between endothelial cells, glial cells, and secreted factor(s) as well as the role of shear stress in the barrier property of HBMEC.
Biomaterials, 2018
The blood-brain barrier (BBB) regulates molecular trafficking, protects against pathogens, and prevents efficient drug delivery to the brain. Models to date failed to reproduce the human anatomical complexity of brain barriers, contributing to misleading results in clinical trials. To overcome these limitations, a novel 3-dimensional BBB microvascular network model was developed via vasculogenesis to accurately replicate the in vivo neurovascular organization. This microfluidic system includes human induced pluripotent stem cell-derived endothelial cells, brain pericytes, and astrocytes as self-assembled vascular networks in fibrin gel. Gene expression of membrane transporters, tight junction and extracellular matrix proteins, was consistent with computational analysis of geometrical structures and quantitative immunocytochemistry, indicating BBB maturation and microenvironment remodelling. Confocal microscopy validated microvessel-pericyte/astrocyte dynamic contact-interactions. Th...
Annals of Biomedical Engineering, 2010
The blood-brain barrier (BBB) is a major obstacle for drug delivery to the brain. To seek for in vitro BBB models that are more accessible than animals for investigating drug transport across the BBB, we compared four in vitro cultured cell models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and primary rat astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. The expression of the BBB tight junction proteins in these in vitro models was assessed using RT-PCR and immunofluorescence. We also quantified the hydraulic conductivity (L p ), transendothelial electrical resistance (TER) and diffusive solute permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran 70K. Our results show that L p and P of the endothelial monoculture and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels, P of the endothelial monoculture and coculture models are not significantly different from in vivo data for Dextran 70K, but they are 2-4 times higher for TAMRA and Dextran 10K. This suggests that the endothelial monoculture and all of the coculture models are fairly good models for studying the transport of relatively large solutes across the BBB.
Brain Research, 2002
Blood-brain barrier endothelial cells are characterized by the presence of tight intercellular junctions, the absence of fenestrations, and a paucity of pinocytotic vesicles. The in vitro study of the BBB has progressed rapidly over the past several years as new cell culture techniques and improved technologies to monitor BBB function became available. Studies carried out on viable in vitro models are set to accelerate the design of drugs that selectively and aggressively can target the CNS. Several systems in vitro attempt to reproduce the physical and biochemical behavior of intact BBB, but most fail to reproduce the three-dimensional nature of the in vivo barrier and do not allow concomitant exposure of endothelial cells to abluminal (glia) and lumenal (flow) influences. For this purpose, we have developed a new dynamic in vitro BBB model (NDIV-BBB) designed to allow for extensive pharmacological, morphological and physiological studies. Bovine aortic endothelial cells (BAEC) developed robust growth and differentiation when co-cultured alone. In the presence of glial cells, BAEC developed elevated Trans-Endothelial Electrical Resistance (TEER). Excision of individual capillaries proportionally decreased TEER; the remaining bundles were populated with healthy cells. Flow played an essential role in EC differentiation by decreasing cell division. In conclusion, this new dynamic model of the BBB allows for longitudinal studies of the effects of flow and co-culture in a controlled and fully recyclable environment that also permits visual inspection of the abluminal compartment and manipulation of individual capillaries.
Brain microvascular endothelial cells resist elongation due to curvature and shear stress
Scientific Reports, 2014
The highly specialized endothelial cells in brain capillaries are a key component of the blood-brain barrier, forming a network of tight junctions that almost completely block paracellular transport. In contrast to vascular endothelial cells in other organs, we show that brain microvascular endothelial cells resist elongation in response to curvature and shear stress. Since the tight junction network is defined by endothelial cell morphology, these results suggest that there may be an evolutionary advantage to resisting elongation by minimizing the total length of cell-cell junctions per unit length of vessel. T he diameter of blood vessels in humans ranges from about 8 mm in capillaries to more than 1 cm in large elastic arteries, a range of more than four orders of magnitude 1. In larger vessels there are hundreds of cells around the perimeter, whereas in a capillary a single endothelial cell may wrap around to form a junction with itself as well as its upstream and downstream neighbors 2-6. Since vessel diameters, and hence curvatures (k 5 1/r where r is the vessel radius), span such a large range, we consider the question: does curvature play a role in dictating endothelial cell morphology (Figure 1a). Curvature is a fundamental physical property that influences a wide range of everyday processes. For endothelial cells in vessels, if curvature is energetically unfavorable, then its effects can be minimized by elongating along the length of the vessel to avoid wrapping around in the radial direction. Conversely, if curvature is energetically favorable then cells may elongate in the radial direction to wrap around the vessel and contract in the axial direction (Figure S1 in Supplementary Information). How a cell responds to curvature and shear stress is important since junctional networks are defined by endothelial cell morphology. For example, for a fixed projected cell area and vessel diameter, elongation increases the number of cells around the perimeter and results in an increase in the total length of cell-cell junctions per unit length of vessel. Since tight junctions in brain capillaries are responsible for preventing paracellular transport, we hypothesize that cell morphology may play an important role in the structure and function of the blood-brain barrier. Previous studies of the influence of curvature on cell behavior have focused on the motility of isolated cells in the context of tumor cell invasion 7-12. Isolated fibroblasts seeded on small diameter glass rods (,200 mm) were shown to exhibit preferential elongation and alignment 7-9 , and preferential migration along the cylinder axis, leading to the concept of contact guidance as a possible mechanism for tumor cell invasion 7. These studies suggest that curvature may play a role in regulating the morphology and function of endothelial cells in confluent monolayers. While the influence of curvature has been relatively unexplored, the role of shear stress on endothelial cell morphology and function has been more widely studied. Blood flow results in a frictional drag, or shear stress, on the vessel wall parallel to the endothelium in the direction of flow. These stresses play an important role in regulating endothelial cell morphology and function, and in mediating a wide range of signaling and transport processes between the vascular system and surrounding tissue 13-18. Endothelial cells in blood vessels in sections away from branch points show elongation and axial alignment 19,20. In cell culture, a physiological shear stress results in a transition from a cobblestone-like morphology to an elongated spindle-like morphology and alignment in the direction of flow 21-25 , very similar to the morphology observed in large resected vessels.
Interstitial flow regulates in vitro three-dimensional self-organized brain micro-vessels
Biochemical and Biophysical Research Communications, 2020
Cell culture under medium flow has been shown to favor human brain microvascular endothelial cells function and maturation. Here a three-dimensional in vitro model of the 2 human brain microvasculature, comprising brain microvascular endothelial cells but also astrocytes, pericytes and a collagen type I microfiber-fibrin based matrix, was cultured under continuous medium flow in a pressure driven microphysiological system (10 kPa, in 60-30 s cycles). The cells self-organized in micro-vessels perpendicular to the shear flow. Comparison with static culture showed that the resulting interstitial flow enhanced a more defined micro-vasculature network, with slightly more numerous lumens, and a higher expression of transporters, carriers and tight junction genes and proteins, essential to the blood-brain barrier functions.
Unique features of the arterial Blood-Brain Barrier
CNS vasculature differs from vascular networks of peripheral organs by its ability to tightly control selective material exchange across capillary barriers. Capillary permeability is mostly defined by unique cellular components of the endothelium. While capillaries are extensively investigated, the barrier properties of larger vessels are understudied. Here, we investigate barrier properties of CNS arterial walls. Using tracer challenges and various imaging modalities, we discovered that at the mouse cortex, the arterial barrier does not reside at the classical level of the endothelium. The arterial wall’s unique permeability acts bi-directionally; CSF substances travel along the glymphatic path and can penetrate from the peri-vascular space through arteriolar walls towards the lumen. We found that caveolae vesicles in arteriole endothelial and smooth-muscle cells are functional transcytosis machinery components, and that a similar mechanism is evident in the human brain. Our discov...
Modeling Nanocarrier Transport across a 3D In Vitro Human Blood‐Brain–Barrier Microvasculature
Advanced Healthcare Materials
The blood-brain-barrier (BBB) forms a highly selective barrier between the microvascular blood stream and brain tissue; it is composed of endothelial cells (ECs), which form the microvasculature, surrounded by pericytes and astrocytes, [1,2] and regulates the passage of substances between the blood and brain, maintaining brain homeostasis, while providing a barrier against pathogens and neurotoxins. [2] Due to its high selectivity, the BBB also represents a major obstacle to the efficient delivery of large molecules for the treatment of brain diseases. [3-5] Polymer nanoparticles (NPs), by virtue of their small size and tunable properties, have been explored for delivering diagnostic agents, [6] nucleic acids, [7,8] proteins, [9] and traditional small molecule medicines to the brain. [10] Specifically, their chemical properties can be tuned to achieve target stability, drug encapsulation, and release Polymer nanoparticles (NPs), due to their small size and surface functionalization potential have demonstrated effective drug transport across the blood-brain-barrier (BBB). Currently, the lack of in vitro BBB models that closely recapitulate complex human brain microenvironments contributes to high failure rates of neuropharmaceutical clinical trials. In this work, a previously established microfluidic 3D in vitro human BBB model, formed by the self-assembly of human-induced pluripotent stem cell-derived endothelial cells, primary brain pericytes, and astrocytes in triculture within a 3D fibrin hydrogel is exploited to quantify polymer NP permeability, as a function of size and surface chemistry. Microvasculature are perfused with commercially available 100-400 nm fluorescent polystyrene (PS) NPs, and newly synthesized 100 nm rhodamine-labeled polyurethane (PU) NPs. Confocal images are taken at different timepoints and computationally analyzed to quantify fluorescence intensity inside/outside the microvasculature, to determine NP spatial distribution and permeability in 3D. Results show similar permeability of PS and PU NPs, which increases after surface-functionalization with brainassociated ligand holo-transferrin. Compared to conventional transwell models, the method enables rapid analysis of NP permeability in a physiologically relevant human BBB setup. Therefore, this work demonstrates a new methodology to preclinically assess NP ability to cross the human BBB.