Microvessels of the blood-brain barrier (BBB) regulate transport into the brain. The highly specialized brain microvascular endothelial cells, a major component of the BBB, express tight junctions and efflux transporters which regulate paracellular and transcellular permeability. However, most existing models of BBB microvessels fail to exhibit physiological barrier function. Here, using (iPSC)-derived human brain microvascular endothelial cells (dhBMECs) within templated type I collagen channels we mimic the cylindrical geometry, cell-extracellular matrix interactions, and shear flow typical of human brain post-capillary venules. We characterize the structure and barrier function in comparison to non-brain-specific microvessels, and show that dhBMEC microvessels recapitulate physiologically low solute permeability and quiescent endothelial cell behavior. Transcellular permeability is increased two-fold using a clinically relevant dose of a p-glycoprotein inhibitor tariquidar, while paracellular permeability is increased using a bolus dose of hyperosmolar agent mannitol. Lastly, we show that our human BBB microvessels are responsive to inflammatory cytokines via upregulation of surface adhesion molecules and increased leukocyte adhesion, but no changes in permeability. Human iPSC-derived blood-brain barrier microvessels support quantitative analysis of barrier function and endothelial cell dynamics in quiescence and in response to biologically- and clinicallyrelevant perturbations.

The blood–brain barrier (BBB) plays a key role in regulating transport into and out of the brain. With increasing interest in the role of the BBB in health and disease, there have been significant advances in the development of in vitro models. The value of these models to the research community is critically dependent on recapitulating characteristics of the BBB in humans or animal models. However, benchmarking in vitro models is surprisingly difficult since much of our knowledge of the structure and function of the BBB comes from in vitro studies. Here we describe a set of parameters that we consider a starting point for benchmarking and validation. These parameters are associated with structure (ultrastructure, wall shear stress, geometry), microenvironment (basement membrane and extracellular matrix), barrier function (transendothelial electrical resistance, permeability, efflux transport), cell function (expression of BBB markers, turnover), and co-culture with other cell types (astrocytes and pericytes). In suggesting benchmarks, we rely primarily on imaging or direct measurements in humans and animal models.

BackgroundTranswell-based models of the blood–brain barrier (BBB) incorporating monolayers of human brain microvascular endothelial cells (dhBMECs) derived from induced pluripotent stem cells show many of the key features of the BBB, including expression of transporters and efflux pumps, expression of tight junction proteins, and physiological values of transendothelial electrical resistance. The fabrication of 3D BBB models using dhBMECs has so far been unsuccessful due to the poor adhesion and survival of these cells on matrix materials commonly used in tissue engineering.MethodsTo address this issue, we systematically screened a wide range of matrix materials (collagen I, hyaluronic acid, and fibrin), compositions (laminin/entactin), protein coatings (fibronectin, laminin, collagen IV, perlecan, and agrin), and soluble factors (ROCK inhibitor and cyclic adenosine monophosphate) in 2D culture to assess cell adhesion, spreading, and barrier function.ResultsCell coverage increased with stiffness of collagen I gels coated with collagen IV and fibronectin. On 7 mg mL−1 collagen I gels coated with basement membrane proteins (fibronectin, collagen IV, and laminin), cell coverage was high but did not reliably reach confluence. The transendothelial electrical resistance (TEER) on collagen I gels coated with basement membrane proteins was lower than on coated transwell membranes. Agrin, a heparin sulfate proteoglycan found in basement membranes of the brain, promoted monolayer formation but resulted in a significant decrease in transendothelial electrical resistance (TEER). However, the addition of ROCK inhibitor, cAMP, or cross-linking the gels to increase stiffness, resulted in a significant improvement of TEER values and enabled the formation of confluent monolayers.ConclusionsHaving identified matrix compositions that promote monolayer formation and barrier function, we successfully fabricated dhBMEC microvessels in cross-linked collagen I gels coated with fibronectin and collagen IV, and treated with ROCK inhibitor and cAMP. We measured apparent permeability values for Lucifer yellow, comparable to values obtained in the transwell assay. During these experiments we observed no focal leaks, suggesting the formation of tight junctions that effectively block paracellular transport.Electronic supplementary materialThe online version of this article (10.1186/s12987-018-0092-7) contains supplementary material, which is available to authorized users.

Background Pericytes of the blood–brain barrier (BBB) are embedded within basement membrane between brain microvascular endothelial cells (BMECs) and astrocyte end-feet. Despite the direct cell–cell contact observed in vivo, most in vitro BBB models introduce an artificial membrane that separates pericytes from BMECs. In this study, we investigated the effects of pericytes on BMEC barrier function across a range of in vitro platforms with varied spatial orientations and levels of cell–cell contact. Methods We differentiated RFP-pericytes and GFP-BMECs from hiPSCs and monitored transendothelial electrical resistance (TEER) across BMECs on transwell inserts while pericytes were either directly co-cultured on the membrane, indirectly co-cultured in the basolateral chamber, or embedded in a collagen I gel formed on the transwell membrane. We then incorporated pericytes into a tissue-engineered microvessel model of the BBB and measured pericyte motility and microvessel permeability. Results We found that BMEC monolayers did not require co-culture with pericytes to achieve physiological TEER values (> 1500 Ω cm 2 ). However, under stressed conditions where TEER values for BMEC monolayers were reduced, indirectly co-cultured hiPSC-derived pericytes restored optimal TEER. Conversely, directly co-cultured pericytes resulted in a decrease in TEER by interfering with BMEC monolayer continuity. In the microvessel model, we observed direct pericyte-BMEC contact, abluminal pericyte localization, and physiologically-low Lucifer yellow permeability comparable to that of BMEC microvessels. In addition, pericyte motility decreased during the first 48 h of co-culture, suggesting progression towards pericyte stabilization. Conclusions We demonstrated that monocultured BMECs do not require co-culture to achieve physiological TEER, but that suboptimal TEER in stressed monolayers can be increased through co-culture with hiPSC-derived pericytes or conditioned media. We also developed the first BBB microvessel model using exclusively hiPSC-derived BMECs and pericytes, which could be used to examine vascular dysfunction in the human CNS. Electronic supplementary material The online version of this article (10.1186/s12987-019-0136-7) contains supplementary material, which is available to authorized users.

Three-dimensional (3D) tissue-engineered models of the blood-brain barrier (BBB) recapitulate in vivo shear stress, cylindrical geometry, and cell-ECM interactions. Here we address four issues associated with BBB models: cell source, barrier function, cryopreservation, and matrix stiffness. We reproduce a directed differentiation of brain microvascular endothelial cells (dhBMECs) from two fluorescently labeled human induced pluripotent stem cell lines (hiPSCs) and demonstrate physiological permeability of Lucifer yellow over six days. Microvessels formed from cryopreserved dhBMECs show expression of BBB markers and maintain physiological barrier function comparable to non-cryopreserved cells. Microvessels displaying physiological barrier function are formed in collagen I hydrogels with stiffness matching that of human brain. The dilation response of microvessels was linear with increasing transmural pressure and was dependent on matrix stiffness. Together these results advance capabilities for tissue-engineered BBB models.

In solid tumors, vascular structure and function varies from the core to the periphery. This structural heterogeneity has been proposed to influence the mechanisms by which tumor cells enter the circulation. Blood vessels exhibit regional defects in endothelial coverage, which can result in cancer cells directly exposed to flow and potentially promoting intravasation. Consistent with prior reports, we observed in human breast tumors and in a mouse model of breast cancer that approximately 6% of vessels consisted of both endothelial cells and tumor cells, so-called mosaic vessels. Due, in part, to the challenges associated with observing tumor-vessel interactions deep within tumors in real-time, the mechanisms by which mosaic vessels form remain incompletely understood. We developed a tissue-engineered model containing a physiologically realistic microvessel in coculture with mammary tumor organoids. This approach allows real-time and quantitative assessment of tumor-vessel interactions under conditions that recapitulate many in vivo features. Imaging revealed that tumor organoids integrate into the endothelial cell lining, resulting in mosaic vessels with gaps in the basement membrane. While mosaic vessel formation was the most frequently observed interaction, tumor organoids also actively constricted and displaced vessels. Furthermore, intravasation of cancer cell clusters was observed following the formation of a mosaic vessel. Taken together, our data reveal that cancer cells can rapidly reshape, destroy, or integrate into existing blood vessels, thereby affecting oxygenation, perfusion, and systemic dissemination. Our novel assay also enables future studies to identify targetable mechanisms of vascular recruitment and intravasation.Significance: A tissue-engineered microdevice that recapitulates the tumor-vascular microenvironment enables real-time imaging of the cellular mechanisms of mosaic vessel formation and vascular defect generation.

As the majority of therapeutic agents do not cross the blood–brain barrier (BBB), transient BBB opening (BBBO) is one strategy to enable delivery into the brain for effective treatment of CNS disease. Intra-arterial infusion of the hyperosmotic agent mannitol reversibly opens the BBB; however, widespread clinical use has been limited due to the variability in outcomes. The current model for mannitol-induced BBBO assumes a transient but homogeneous increase in permeability; however, the details are poorly understood. To elucidate the mechanism of hyperosmotic opening at the cellular level, we developed a tissue-engineered microvessel model using stem cell-derived human brain microvascular endothelial cells (BMECs) perturbed with clinically relevant mannitol doses. This model recapitulates physiological shear stress, barrier function, microvessel geometry, and cell-matrix interactions. Using live-cell imaging, we show that mannitol results in dose-dependent and spatially heterogeneous increases in paracellular permeability through the formation of transient focal leaks. Additionally, we find that the degree of BBB opening and subsequent recovery is modulated by treatment with basic fibroblast growth factor. These results show that tissue-engineered BBB models can provide insight into the mechanisms of BBBO and hence improve the reproducibility of hyperosmotic therapies for treatment of CNS disease.

Proper vascularization remains critical to the clinical application of engineered tissues. To engineer microvessels in vitro, we and others have delivered endothelial cells through preformed channels into patterned extracellular matrix-based gels. This approach has been limited by the size of endothelial cells in suspension, and results in plugging of channels below ~30 μm in diameter. Here, we examine physical and chemical signals that can augment direct seeding, with the aim of rapidly vascularizing capillary-scale channels. By studying tapered microchannels in type I collagen gels under various conditions, we establish that stiff scaffolds, forward pressure, and elevated cyclic AMP levels promote endothelial stability and that reverse pressure promotes endothelial migration. We applied these results to uniform 20-μm-diameter channels and optimized the magnitudes of pressure, flow, and shear stress to best support endothelial migration and vascular stability. This vascularization strategy is able to form millimeter-long perfusable capillaries within three days. Our results indicate how to manipulate the physical and chemical environment to promote rapid vascularization of capillary-scale channels within type I collagen gels.