Projection Microstereolithographic Microbial Bioprinting for Engineered Biofilms.

Microbes are critical drivers of all ecosystems and many biogeochemical processes, yet little is known about how the three-dimensional (3D) organization of these dynamic organisms contributes to their overall function. To probe how biofilm structure affects microbial activity, we developed a technique for patterning microbes in 3D geometries using projection stereolithography to bioprint microbes within hydrogel architectures. Bacteria were printed and monitored for biomass accumulation, demonstrating postprint viability of cells using this technique. We verified our ability to integrate biological and geometric complexity by fabricating a printed biofilm with two E. coli strains expressing different fluorescence. Finally, we examined the target application of microbial absorption of metal ions to investigate geometric effects on both the metal sequestration efficiency and the uranium sensing capability of patterned engineered Caulobacter crescentus strains. This work represents the first demonstration of the stereolithographic printing of microbials and presents opportunities for future work of engineered biofilms and other complex 3D structured cultures.

Performance of three-dimensional printed nasopharyngeal swabs for COVID-19 testing.

ABSTRACT: At the start of the COVID-19 pandemic, the US faced nationwide shortages of nasopharyngeal swabs due to both overwhelmed supply chains and an increase in demand. To address this shortfall, multiple 3D printed swabs were ultimately produced and sold for COVID-19 testing. In this work, we present a framework for mechanical and functional bench-testing of nasopharyngeal swabs using standard and widely available material testing equipment. Using this framework, we offer a comprehensive, quantitative comparison of the 3D printed swabs to benchmark their performance against traditional flocked swabs. The test protocols were designed to emulate the clinical use of the nasopharyngeal swabs and to evaluate potential failure modes. Overall, the 3D printed swabs performed comparably to, or outperformed, the traditional swabs in all mechanical tests. While traditional swabs outperformed some of the new 3D printed swabs in terms of sample uptake and retention, similar amounts of RNA were recovered from both 3D printed and traditional swabs.

Percutaneous Inserted Venous Catheter via Femoral Vein in Extremely Low-Birth-Weight Infants: A Single-Center Experience.

OBJECTIVE: This study aimed to assess the applicability of the insertion of small diameter catheters through the femoral vein in extremely low-birth-weight (ELBW) infants. STUDY DESIGN: All femoral small diameter catheters (Silastic or femoral arterial catheter [FAC]) inserted in ELBW infants in a tertiary level neonatal intensive care unit were retrospectively reviewed. Success rate, dwelling time, and percutaneously inserted central venous catheter-related complications were recorded. RESULTS: Thirteen small diameter catheters were inserted in seven ELBW infants. Mean gestational age at birth was 25+3 weeks (standard deviation [SD] ± 2.12) and mean birth weight was 686 g (SD ± 204.9). Mean weight at the first time of insertion was 1,044 g (SD ± 376.3). In two occasions, a FAC was used instead of a Silastic. In most cases (11/13, 84.6%), the patient was intubated prior to the procedure. The mean dwelling time was 16.7 days (SD ± 9.8). Most of the inserted small diameter catheters were removed electively (8/12, 66.7%), except for one episode of clinical sepsis from coagulase-negative Staphylococcus and three cases of accidental line extravasation. No other complications were reported. The success rate was 92.3%. CONCLUSION: Femoral venous catheterization using small diameter catheters in ELBW infants may be promising when other routes have been exhausted. Our results support that it is a feasible technique that can be performed at the bedside with successful results when conducted by experienced personnel.

Thoracoscopic esophagoesophagostomy for a refractory stricture in a patient with esophageal atresia.

Anastomosis stricture is a well-known complication after esophageal atresia repair. Endoscopic dilatation is the gold standard treatment for esophageal stenosis. However, surgical interventions are indicated for refractory cases. We present a 2-year-old girl with esophageal stricture refractory to regular endoscopic dilatation after esophageal atresia repair that underwent thoracoscopic stricture resection and reanastomosis. Although thoracoscopic approach is widely used for esophageal atresia repair, this approach has not been used before for the treatment of anastomosis stricture.

Vascular dysfunction in hemorrhagic viral fevers: opportunities for organotypic modeling.

The hemorrhagic fever viruses (HFVs) cause severe or fatal infections in humans. Named after their common symptom hemorrhage, these viruses induce significant vascular dysfunction by affecting endothelial cells, altering immunity, and disrupting the clotting system. Despite advances in treatments, such as cytokine blocking therapies, disease modifying treatment for this class of pathogen remains elusive. Improved understanding of the pathogenesis of these infections could provide new avenues to treatment. While animal models and traditional 2D cell cultures have contributed insight into the mechanisms by which these pathogens affect the vasculature, these models fall short in replicatingin vivohuman vascular dynamics. The emergence of microphysiological systems (MPSs) offers promising avenues for modeling these complex interactions. These MPS or 'organ-on-chip' models present opportunities to better mimic human vascular responses and thus aid in treatment development. In this review, we explore the impact of HFV on the vasculature by causing endothelial dysfunction, blood clotting irregularities, and immune dysregulation. We highlight how existing MPS have elucidated features of HFV pathogenesis as well as discuss existing knowledge gaps and the challenges in modeling these interactions using MPS. Understanding the intricate mechanisms of vascular dysfunction caused by HFV is crucial in developing therapies not only for these infections, but also for other vasculotropic conditions like sepsis.

FGF-1 delivery from multilayer alginate microbeads stimulates a rapid and persistent increase in vascular density.

In recent years, great advances have been made in the use of islet transplantation as a treatment for type I diabetes. Indeed, it is possible that stimulation of local neovascularization upon transplantation could improve functional graft outcomes. In the present study, we investigate the use of multilayered alginate microbeads to provide a sustained delivery of FGF-1, and whether this results in increased neovascularization in vivo. Multilayered alginate microbeads, loaded with either 150ng or 600ng of FGF-1 in the outer layer, were surgically implanted into rats using an omentum pouch model and compared to empty microbead implants. Rats were sacrificed at 4days, 1week, and 6weeks. Staining for CD31 showed that both conditions of FGF-1 loaded microbeads resulted in a significantly higher vessel density at all time points studied. Moreover, at 6weeks, alginate microbeads containing 600ng FGF-1 provided a greater vascular density compared to both the control group and the microbeads loaded with 150ng FGF-1. Omenta analyzed via staining for smooth muscle alpha actin showed no variation in mural cell density at either 4days or 1week. At 6weeks, however, omenta exposed to microbeads loaded with 600ng FGF-1 showed an increase in mural cell staining compared to controls. These results suggest that the sustained delivery of FGF-1 from multilayered alginate microbeads results in a rapid and persistent vascular response. An increase in the local blood supply could reduce the number of islets required for transplantation in order to achieve clinical efficacy.

A perfused multi-well bioreactor platform to assess tumor organoid response to a chemotherapeutic gradient.

There is an urgent need to develop new therapies for colorectal cancer that has metastasized to the liver and, more fundamentally, to develop improved preclinical platforms of colorectal cancer liver metastases (CRCLM) to screen therapies for efficacy. To this end, we developed a multi-well perfusable bioreactor capable of monitoring CRCLM patient-derived organoid response to a chemotherapeutic gradient. CRCLM patient-derived organoids were cultured in the multi-well bioreactor for 7 days and the subsequently established gradient in 5-fluorouracil (5-FU) concentration resulted in a lower IC50 in the region near the perfusion channel versus the region far from the channel. We compared behaviour of organoids in this platform to two commonly used PDO culture models: organoids in media and organoids in a static (no perfusion) hydrogel. The bioreactor IC50 values were significantly higher than IC50 values for organoids cultured in media whereas only the IC50 for organoids far from the channel were significantly different than organoids cultured in the static hydrogel condition. Using finite element simulations, we showed that the total dose delivered, calculated using area under the curve (AUC) was similar between platforms, however normalized viability was lower for the organoid in media condition than in the static gel and bioreactor. Our results highlight the utility of our multi-well bioreactor for studying organoid response to chemical gradients and demonstrate that comparing drug response across these different platforms is nontrivial.

Stability of alginate microbead properties in vitro.

Alginate microbeads have been investigated clinically for a number of therapeutic interventions, including drug delivery for treatment of ischemic tissues, cell delivery for tissue regeneration, and islet encapsulation as a therapy for type I diabetes. The physical properties of the microbeads play an important role in regulating cell behavior, protein release, and biological response following implantation. In this research alginate microbeads were synthesized, varying composition (mannuronic acid to guluronic acid ratio), concentration of alginate and needle gauge size. Following synthesis, the size, volume fraction, and morphometry of the beads were quantified. In addition, these properties were monitored over time in vitro in the presence of varying calcium levels in the microenvironment. The initial volume available for solute diffusion increased with alginate concentration and mannuronic (M) acid content, and bead diameter decreased with M content but increased with needle diameter. Interestingly, microbeads eroded completely in saline in less than 3 weeks regardless of synthesis conditions much faster than what has been observed in vivo. However, microbead stability was increased by the addition of calcium in the culture medium. Beads synthesized with low alginate concentration and high G content exhibited a more rapid change in physical properties even in the presence of calcium. These data suggest that temporal variations in the physical characteristics of alginate microbeads can occur in vitro depending on synthesis conditions and microbead environment. The results presented here will assist in optimizing the design of the materials for clinical application in drug delivery and cell therapy.

Extracellular matrix modulates T cell clearance of malignant cells in vitro.

Despite the success of T cell checkpoint therapies, breast cancers rarely express these immunotherapy markers and are believed to be largely "immune cold" with limited inflammation and immune activation. The reason for this limited immune activation remains poorly understood. We sought to determine whether extracellular matrix substrate could contribute to this limited immune activation. Specifically, we asked whether extracellular matrix could alter T cell cytotoxicity against malignant mammary gland carcinoma cells (MCC) in a setup designed to promote maximal T cell efficacy (i.e., rich media with abundant IL2, high ratio of T cells to MCC). We observed that T cell clearance of MCC varied from 0% in collagen 4 or 6 conditions to almost 100% in fibronectin or vitronectin. Transcriptomics revealed that T cell function was defective in MCC/T cell cocultures on collagen 4 (Col4), potentially corresponding to greater expression of cytokines MCC cultured in this environment. In contrast, transcriptomics revealed an effective, exhausted phenotype on vitronectin. The observation that Col4 induces T cell suppression suggests that targeting tumor-ECM interactions may permit new approaches for utilizing immunotherapy in tumors which do not provoke a strong immune response.

Go with the flow: modeling unique biological flows in engineered in vitro platforms.

Interest in recapitulating in vivo phenomena in vitro using organ-on-a-chip technology has grown rapidly and with it, attention to the types of fluid flow experienced in the body has followed suit. These platforms offer distinct advantages over in vivo models with regards to human relevance, cost, and control of inputs (e.g., controlled manipulation of biomechanical cues from fluid perfusion). Given the critical role biophysical forces play in several tissues and organs, it is therefore imperative that engineered in vitro platforms capture the complex, unique flow profiles experienced in the body that are intimately tied with organ function. In this review, we outline the complex and unique flow regimes experienced by three different organ systems: blood vasculature, lymphatic vasculature, and the intestinal system. We highlight current state-of-the-art platforms that strive to replicate physiological flows within engineered tissues while introducing potential limitations in current approaches.

Collagen glycation alters neovascularization in vitro and in vivo.

Microvascular network formation is required for the success of many therapies in regenerative medicine. The process of vessel assembly is fundamentally altered, however, in many people within the potential patient population, including the elderly and people with diabetes. Significant research has been performed to determine how cellular dysfunction contributes to this inadequate neovascularization, but alterations in the extracellular matrix (ECM) may also influence this process. Glycation of ECM proteins, specifically type I collagen, increases as people age and is accelerated due to uncontrolled diabetes. This glycation results in increased ECM stiffness and resistance to degradation. The goal of this research is to determine whether collagen glycation consistent with changes in aged (defined as people older than 80 years old) and diabetic individuals influences neovascularization. Collagen gels that were incubated in glucose-6-phopshate (G6P) for varying times exhibited cross-linking (26.2+/-8.1% and 31.3+/-5.6% for incubation in 375 mM G6P for 5 and 8 days, respectively), autofluorescence, and advanced glycation end product levels (666+/-481 and 2122+/-501 pmol/mg protein for 5 and 8 days of 375 mM G6P, respectively) consistent with aged and diabetic populations. Three-dimensional culture models showed that sprouting angiogenesis was delayed in collagen gels with high levels of glycation. When implanted in vivo, glycated gels were degraded (44.4+/-4.2% and 49.5+/-11.7% nondegraded gel remaining for gels incubated for 5 and 8 days in 375 mM G6P, respectively) and vascularized (75.5+/-32.0 and 73.7+/-23.6 vessels/mm(2)) more slowly than controls (22.3+/-9.9% gel remaining and 133.3+/-31.0 vessels/mm(2)). These results suggest that glycation of collagen can alter neovascularization and may contribute to alterations in vessel assembly observed as people age and due to diabetes.

Fibroblast growth factor-1 (FGF-1) loaded microbeads enhance local capillary neovascularization.

BACKGROUND: Growth of new blood vessels (neovascularization) occurs naturally in the body, but the slow rate of the process may not be sufficient for survival of engineered tissues and transplanted cells, such as pancreatic islets. For transplanted islets, it is crucial that the transplantation site has sufficient vasculature to support the needs of the islets. Therefore, the specific aim of this research was quantify the effect of FGF-1 incorporation into alginate microbeads on neovascularization of such capsules in an in vivo rat transplant model. MATERIALS AND METHODS: Microbeads loaded with FGF-1 or control beads (beads without FGF-1) were implanted in the rat omental pouch model. Animals were sacrificed 7 d post-implantation. RESULTS: Microbeads loaded with FGF-1 stimulated a significant increase in vascular density compared with control rats implanted with control beads. CONCLUSIONS: These results indicate that alginate microbeads loaded with FGF-1 enhance local neovascularization around implanted microbeads. These data provide a compelling impetus for experimental pursuit of FGF-loaded alginate microcapsules for vascularization of transplanted islets.

Evaluation of inflammatory biomarkers associated with oxidative stress and histological assessment of low-level laser therapy in experimental myopathy.

UNLABELLED: The objective of the present work was to study the effect of helium-neon (He-Ne) and gallium-arsenide (Ga-As) laser upon inflammatory biomarkers associated with oxidative stress: fibrinogen, nitric oxide (NO), L-citrulline, and superoxide dismutase (SOD). These were evaluated through histological assessment, in rats with experimental myopathy. MATERIALS AND METHODS: The groups studied were: (A) control, (B) injured, (C) injured and treated with He-Ne laser, (D) injured and treated with Ga-As laser, (E) irradiated with He-Ne; and (F) irradiated with Ga-As laser. Myopathy was induced by injecting 0.05 mg/rat/day of adrenaline in the left posterior limb muscle at the same point on 5 consecutive days, in groups B, C, and D. Low-level laser therapy (LLLT) was applied with 9.5 J/cm(2) daily for 7 consecutive days with each laser. The determination of the biomarkers was made by spectrophotometry. The muscles (5/8, single blinded) were stained with Gomori Trichrome and examined by optic microscopy. The quantitative variables were statistically analyzed by the Fisher's test and categorical data by the Axionvision 4.8 program. Pearson's chi-squared test was applied, setting significant difference at P < 0.05 for all cases. RESULTS: In group B, the biomarkers were significantly increased compared to the other groups (P < 0.001), except for NO which in group B decreased significantly (P < 0.001). In group B, there was a higher inflammatory infiltration level (80.67%) in relation to destroyed fibers. CONCLUSIONS: LLLT caused significant changes in inflammatory biomarkers and oxidative stress: decreased levels of fibrinogen, L-citrulline and SOD as opposed to the increase of NO in rats with experimental myopathies and significant muscle recovery.

The impact of a telehealth web-based solution on neurosurgery triage and consultation.

INTRODUCTION: To enhance the quality of neurosurgery consultations, triage, and transport decisions between a Level I trauma service neurosurgery program at the University of New Mexico Hospital and referring hospitals, a secure Health Insurance Portability and Accountability Act (HIPAA)-compliant Web-based system was developed, to which digital neurological images could be sent for review by a neurosurgeon for consultation or patient transfer. Based upon prior experience of neurosurgery, it was predicted that 25% of transfer requests would be avoided if the neurosurgeons reviewed the computerized tomography scans at the time of a transfer request. In addition, it was predicted in 25% of the case that changes in management recommendations would take place independent of the transfer decision. METHODS: The program was designed to allow referring hospitals to transmit digital images to the Web site, providing consulting doctors with additional patient information. This project analyzed the neurosurgeons' responses to questions designed to determine if transport or management decisions were altered when using this telehealth program in response to a request for consultation or transfer from a rural facility. RESULTS: Analysis of the responses of the consulting neurosurgeons revealed that, after viewing the images, 44% of the potential transfers were avoided and 44% of consulted cases resulted in management recommendation changes independent of the transfer decision. CONCLUSIONS: Use of the system resulted in improved triage and changes in transfer or management recommendations. A significant number of potential transfers were avoided, resulting in transport cost avoidance, more effective use of resources, and more appropriate use of the neurosurgery service as well as improved patient preparation.

Investigating the Interaction Between Circulating Tumor Cells and Local Hydrodynamics via Experiment and Simulations.

INTRODUCTION: The biological and mechanical properties of circulating tumor cells (CTCs) in combination with the hemodynamics affect the preference of metastatic sites in the vasculature. Despite the extensive literature on the effects of biological properties on cell adhesion, the effects of hydrodynamic forces on primary attachment remains an active area of research. Using simulations in conjunction with experimentation, we provide new insight into the interplay of CTCs dynamics and local hydrodynamics. METHODS: A flow experiment of CTC attachment was performed within a bioprinted, double branching endothelialized vessel. Simulations of fluid flow and CTC transport in the reconstructed and idealized bifurcated vessel were respectively performed by HARVEY, our in-house massively parallel computational fluid dynamics solver. HARVEY is based on the lattice Boltzmann and finite element methods to model the fluid and cells dynamics. The immersed boundary method is employed for resolving the fluid-structure interaction. RESULTS: CTC attachment was quantified experimentally at all regions of the complex vessel. The results demonstrate a clear preference for CTCs to attach at the branch points. To elucidate the effect of the vessel topology on the location of attachment, a fluid-only simulation was performed assessing the differences in the hydrodynamics along the vessel. CTC transport in idealized bifurcated vessels was subsequently studied to examine the effects of cell deformability on the local hydrodynamics patterns and, thus, the preference of attachment sites. CONCLUSIONS: The current work provides evidence on the correlation of the hydrodynamics forces arising from the vessel topology and CTC properties on the attachment regions.

Macromolecular gelatin properties affect fibrin microarchitecture and tumor spheroid behavior in fibrin-gelatin gels.

The biophysical properties of extracellular matrices (ECM) are known to regulate cell behavior, however decoupling cell behavior changes due to the relative contributions of material microstructure versus biomechanics or nutrient permeability remains challenging, especially within complex, multi-material matrices. We developed four gelatin-fibrin interpenetrating network (IPN) formulations which are identical in composition but possess variable gelatin molecular weight distributions, and display differences in microstructure, biomechanics, and diffusivity. In this work we interrogate the response of multicellular tumor spheroids to these IPN formulations and found that a high stiffness, gelatin-network dominated IPNs impeded remodeling and invasion of multicellular tumor spheroids; whereas relatively lower stiffness, fibrin-network dominated IPNs permitted protease-dependent remodeling and spheroid invasion. Cell proliferation correlated to nutrient diffusivity across tested IPN formulations. These findings demonstrate the complexity of ECM IPNs, relative to single polymer matrices, and highlight that cell response does not derive from a single aspect of the ECM, but rather from the interplay of multiple biomechanical properties. The methodology developed here represents a framework for future studies which aim to characterize cellular phenotypic responses to biophysical cues present within complex, multi-material matrices.

Three-dimensional bioprinting of aneurysm-bearing tissue structure for endovascular deployment of embolization coils.

Various types of embolization devices have been developed for the treatment of cerebral aneurysms. However, it is challenging to properly evaluate device performance and train medical personnel for device deployment without the aid of functionally relevant models. Currentin vitroaneurysm models suffer from a lack of key functional and morphological features of brain vasculature that limit their applicability for these purposes. These features include the physiologically relevant mechanical properties and the dynamic cellular environment of blood vessels subjected to constant fluid flow. Herein, we developed three-dimensionally (3D) printed aneurysm-bearing vascularized tissue structures using gelatin-fibrin hydrogel of which the inner vessel walls were seeded with human cerebral microvascular endothelial cells (hCMECs). The hCMECs readily exhibited cellular attachment, spreading, and confluency all around the vessel walls, including the aneurysm walls. Additionally, thein vitroplatform was directly amenable to flow measurements via particle image velocimetry, enabling the direct assessment of the vascular flow dynamics for comparison to a 3D computational fluid dynamics model. Detachable coils were delivered into the printed aneurysm sac through the vessel using a microcatheter and static blood plasma clotting was monitored inside the aneurysm sac and around the coils. This biomimeticin vitroaneurysm model is a promising method for examining the biocompatibility and hemostatic efficiency of embolization devices and for providing hemodynamic information which would aid in predicting aneurysm rupture or healing response after treatment.

Comparative Molecular Analysis of Cancer Behavior Cultured In Vitro, In Vivo, and Ex Vivo.

Current pre-clinical models of cancer fail to recapitulate the cancer cell behavior in primary tumors primarily because of the lack of a deeper understanding of the effects that the microenvironment has on cancer cell phenotype. Transcriptomic profiling of 4T1 murine mammary carcinoma cells from 2D and 3D cultures, subcutaneous or orthotopic allografts (from immunocompetent or immunodeficient mice), as well as ex vivo tumoroids, revealed differences in molecular signatures including altered expression of genes involved in cell cycle progression, cell signaling and extracellular matrix remodeling. The 3D culture platforms had more in vivo-like transcriptional profiles than 2D cultures. In vivo tumors had more cells undergoing epithelial-to-mesenchymal transition (EMT) while in vitro cultures had cells residing primarily in an epithelial or mesenchymal state. Ex vivo tumoroids incorporated aspects of in vivo and in vitro culturing, retaining higher abundance of cells undergoing EMT while shifting cancer cell fate towards a more mesenchymal state. Cellular heterogeneity surveyed by scRNA-seq revealed that ex vivo tumoroids, while rapidly expanding cancer and fibroblast populations, lose a significant proportion of immune components. This study emphasizes the need to improve in vitro culture systems and preserve syngeneic-like tumor composition by maintaining similar EMT heterogeneity as well as inclusion of stromal subpopulations.

Optimizing cell encapsulation condition in ECM-Collagen I hydrogels to support 3D neuronal cultures.

BACKGROUND: The emergence of three-dimensional (3D) cell culture in neural tissue engineering has significantly elevated the complexity and relevance of in vitro systems. This is due in large part to the incorporation of biomaterials to impart structural dimensionality on the neuronal cultures. However, a comprehensive understanding of how key seeding parameters affect changes in cell distribution and viability remain unreported. NEW METHOD: In this study, we systematically evaluated permutations in seeding conditions (i.e., cell concentration and atmospheric CO2 levels) to understand how these affect key parameters in 3D culture characterization (i.e., cell health and distribution). Primary rat cortical neurons (i.e., 2 × 106, 4 × 106, and 1 × 107 cells/mL) were entrapped in collagen blended with ECM proteins (ECM-Collagen) and exposed to atmospheric CO2 (i.e., 0 vs 5% CO2) during fibrillogenesis. RESULTS: At 14 days in vitro (DIV), cell distribution within the hydrogel was dependent on cell concentration and atmospheric CO2 during fibrillogenesis. A uniform distribution of cells was observed in cultures with 2 × 106 and 4 × 106 cells/mL in the presence of 5% CO2, while a heterogeneous distribution was observed in cultures with 1 × 107 cells/mL or in the absence of CO2. Furthermore, increased cell concentration was proportional to the rise in cell death at 14 DIV, although cells remain viable >30 DIV. COMPARISON WITH EXISTING METHODS: ECM-Collagen gels have been shown to increase cell viability of neurons long-term. CONCLUSION: In using ECM-collagen gels, we highlight the importance of optimizing seeding parameters and thorough 3D culture characterization to understand the neurophysiological responses of these 3D systems.

A Reconfigurable In Vitro Model for Studying the Blood-Brain Barrier.

Much of what is currently known about the role of the blood-brain barrier (BBB) in regulating the passage of chemicals from the blood stream to the central nervous system (CNS) comes from animal in vivo models (requiring extrapolation to human relevance) and 2D static in vitro systems, which fail to capture the rich cell-cell and cell-matrix interactions of the dynamic 3D in vivo tissue microenvironment. In this work we have developed a BBB platform that allows for a high degree of customization in cellular composition, cellular orientation, and physiologically-relevant fluid dynamics. The system characterized and presented in this study reproduces key characteristics of a BBB model (e.g. tight junctions, efflux pumps) allowing for the formation of a selective and functional barrier. We demonstrate that our in vitro BBB is responsive to both biochemical and mechanical cues. This model further allows for culture of a CNS-like space around the BBB. The design of this platform is a valuable tool for studying BBB function as well as for screening of novel therapeutics.