scholarly journals Hypoxia-enhanced Blood-Brain Barrier Chip recapitulates human barrier function, drug penetration, and antibody shuttling properties

2018 ◽  
Author(s):  
Tae-Eun Park ◽  
Nur Mustafaoglu ◽  
Anna Herland ◽  
Ryan Hasselkus ◽  
Robert Mannix ◽  
...  

The highly specialized human brain microvascular endothelium forms a selective blood-brain barrier (BBB) with adjacent pericytes and astrocytes that restricts delivery of many pharmaceuticals and therapeutic antibodies to the central nervous system. Here, we describe an in vitro microfluidic ‘organ-on-a-chip’ (Organ Chip) model of the BBB lined by induced pluripotent stem cell-derived human brain microvascular endothelium (iPS-BMVEC) interfaced with primary human brain astrocytes and pericytes that recapitulates the high level of barrier function of the in vivo human BBB for at least one week in culture. The endothelium expresses high levels of tight junction proteins, multiple functional efflux pumps, and displays selective transcytosis of peptides and anti-transferrin receptor antibodies previously observed in vivo. This increased level of barrier functionality was accomplished using a developmentally-inspired induction protocol that includes a period of differentiation under hypoxic conditions. This enhanced BBB Chip may therefore represent a new in vitro tool for development and validation of delivery systems that transport drugs and therapeutic antibodies across the human BBB.The human blood-brain barrier (BBB) is a unique and selective physiological barrier that controls transport between the blood and the central nervous system (CNS) to maintain homeostasis for optimal brain function. The BBB is composed of brain microvascular endothelial cells (BMVECs) that line the capillaries as well as surrounding extracellular matrix (ECM), pericytes, and astrocytes, which create a microenvironment that is crucial to BBB function1. The brain microvascular endothelium differs from that found in peripheral capillaries based on its complex tight junctions, which restrict paracellular transit and instead, require that transcytosis be used to transport molecules from the blood through the endothelium and into the CNS2. BMVECs also express multiple broad-spectrum efflux pumps on their luminal surface that inhibit uptake of lipophilic molecules, including many drugs, into the brain3,4. The astrocytes and pericytes provide signals that are required for differentiation of the BMVECs5,6, and all three cell types are needed to maintain BBB integrity in vivo as well as in vitro7–9. The BBB is also of major clinical relevance because dysfunction of the BBB associated is observed in many neurological diseases, and the efficacy of drugs designed to treat neurological disorders is often limited by their inability to cross the BBB10. Unfortunately, neither animal models of the BBB nor in vitro cultures of primary or immortalized human BMVECs alone effectively mimic the barrier and transporter functions of the BBB observed in humans11–14. Thus, there is a great need for a human BBB model that could be used to develop new and more effective CNS-targeting therapeutics and delivery technologies as well as advance fundamental and translational research8,9.Development of human induced pluripotent stem (iPS) cell technology has enabled differentiation of brain-like microvascular endothelial cells (iPS-BMVECs) that exhibit many properties of the human BBB, including well-organized tight junctions, expression of nutrient transporters and polarized efflux transporter activity15,16. The trans-endothelial electrical resistance (TEER) values exhibited by the permeability barrier generated by these human iPS-BMVECs reach physiological levels (∼3000-5000 Ω·cm2) within 24-48 h when cultured in Transwell inserts or within a microfluidic organ-on-a-chip (Organ Chip) device15,17–19, a level that is more than an order of magnitude higher than TEER values previously reported in other in vitro human BBB models6,17,20.However, the usefulness of these iPS-BMVEC models for studies on targeted delivery to the CNS is limited because they can only maintain these high TEER levels for ∼2 days, and the expression of efflux pumps in these iPS-BMVECs does not fully mimic those of human brain endothelium in vivo21. Here, we describe the development of an enhanced human BBB model created with microfluidic Organ Chip culture technology22,23 that contains human iPS-BMVECs interfaced with primary human pericytes and astrocytes, and that uses a developmentally-inspired differentiation protocol24–26. The resulting human BBB Chip exhibits physiologically relevant levels of human BBB function for at least one week in vitro, including low barrier permeability and expression of multiple efflux pumps and transporter functions that are required for analysis of drug and therapeutic antibody transport.

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Gwenaëlle Le Roux ◽  
Rafika Jarray ◽  
Anne-Cécile Guyot ◽  
Serena Pavoni ◽  
Narciso Costa ◽  
...  

Abstract The development of effective central nervous system (CNS) drugs has been hampered by the lack of robust strategies to mimic the blood-brain barrier (BBB) and cerebrovascular impairments in vitro. Recent technological advancements in BBB modeling using induced pluripotent stem cells (iPSCs) allowed to overcome some of these obstacles, nonetheless the pertinence for their use in drug permeation study remains to be established. This mandatory information requires a cross comparison of in vitro and in vivo pharmacokinetic data in the same species to avoid failure in late clinical drug development. Here, we measured the BBB permeabilities of 8 clinical positron emission tomography (PET) radioligands with known pharmacokinetic parameters in human brain in vivo with a newly developed in vitro iPSC-based human BBB (iPSC-hBBB) model. Our findings showed a good correlation between in vitro and in vivo drug brain permeability (R2 = 0.83; P = 0.008) which contrasted with the limited correlation between in vitro apparent permeability for a set of 18 CNS/non-CNS compounds using the in vitro iPSCs-hBBB model and drug physicochemical properties. Our data suggest that the iPSC-hBBB model can be integrated in a flow scheme of CNS drug screening and potentially used to study species differences in BBB permeation.


Pharmaceutics ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 65 ◽  
Author(s):  
Elisabetta Muntoni ◽  
Katia Martina ◽  
Elisabetta Marini ◽  
Marta Giorgis ◽  
Loretta Lazzarato ◽  
...  

Glioblastoma is the most common and invasive primary tumor of the central nervous system and normally has a negative prognosis. Biodistribution in healthy animal models is an important preliminary study aimed at investigating the efficacy of chemotherapy, as it is mainly addressed towards residual cells after surgery in a region with an intact blood–brain barrier. Nanoparticles have emerged as versatile vectors that can overcome the blood–brain barrier. In this experimental work, solid lipid nanoparticles, prepared using fatty acid coacervation, have been loaded with an active lipophilic ester of cytotoxic drug methotrexate, and functionalized with either transferrin or insulin, two proteins whose receptors are abundantly expressed on the blood–brain barrier. Functionalization has been achieved by grafting a maleimide moiety onto the nanoparticle’s surface and exploiting its reactivity towards thiolated proteins. The nanoparticles have been tested in vitro on a blood–brain barrier cellular model and in vivo for biodistribution in Wistar rats. Drug metabolites, in particular 7-hydroxymethotrexate, have also been investigated in the animal model. The data obtained indicate that the functionalization of the nanoparticles improved their ability to overcome the blood–brain barrier when a PEG spacer between the proteins and the nanoparticle’s surface was used. This is probably because this method provided improved ligand–receptor interactions and selectivity for the target tissue.


2019 ◽  
Author(s):  
Tyler M. Lu ◽  
David Redmond ◽  
Tarig Magdeldin ◽  
Duc-Huy T. Nguyen ◽  
Amanda Snead ◽  
...  

AbstractBrain microvascular endothelial cells (BMECs) possess unique properties underlying the blood-brain-barrier (BBB), that are crucial for homeostatic brain functions and interactions with the immune system. Modulation of BBB function is essential for treatment of neurological diseases and effective tumor targeting. Studies to-date have been hampered by the lack of physiological models using cultivated human BMECs that sustain BBB properties. Recently, differentiation of induced pluripotent stem cells (iPSCs) into cells with BBB-like properties has been reported, providing a robust in vitro model for drug screening and mechanistic understanding of neurological diseases. However, the precise identity of these iBMECs remains unclear. Employing single-cell RNA sequencing, bioinformatic analysis and immunofluorescence for several pathways, transcription factors (TFs), and surface markers, we examined the molecular and functional properties of iBMECs differentiated either in the absence or presence of retinoic acid. We found that iBMECs lack both endothelial-lineage genes and ETS TFs that are essential for the establishment and maintenance of EC identity. Moreover, iBMECs fail to respond to angiogenic stimuli and form lumenized vessels in vivo. We demonstrate that human iBMECs are not barrier-forming ECs but rather EpCAM+ neuroectodermal epithelial cells (NE-EpiCs) that form tight junctions resembling those present in BBB-forming BMECs. Finally, overexpression of ETS TFs (ETV2, FLI1, and ERG) reprograms NE-EpiCs to become more like the BBB-forming ECs. Thus, although directed differentiation of human iBMECs primarily gives rise to epithelial cells, overexpression of several ETS TFs can divert them toward a vascular BBB in vitro.


2020 ◽  
Vol 3 ◽  
Author(s):  
Dustin Parsons ◽  
Jason Hughes ◽  
Scott Canfield

Background and Hypothesis:  Propofol is an anesthetic commonly used to induce general anesthesia for a myriad of medical procedures. However, a growing corpus of evidence suggests that propofol-induced increases in VEGF may contribute to blood-brain barrier (BBB) leakiness in varying animal models. The BBB is a neurovascular structure which protects the central nervous system from pathogens, toxins, and other deleterious metabolites; therefore, considerations regarding BBB integrity in humans are indispensable to the practice of anesthesia. We hypothesize that propofol-induced BBB dysfunction in human models is partially mediated by an increase in VEGF expression.    Methods:  We utilized human induced pluripotent stem cells (hiPSC) to derive brain microvascular endothelial cells (BMECs)—the barrier forming cell type of the BBB. BMECs were then subjected to clinically relevant doses of propofol for 3 hours, and barrier integrity was monitored via transendothelial electrical resistance (TEER) and para-cellular permeability for up to 72 hours. Propofol-induced VEGF levels were determined with an ELISA assay. Axitinib, a VEGF receptor blocker, was further utilized to assess the role of VEGF in propofol-induced BBB breakdown.    Results:  Prior works, including this study, have shown that propofol induces BBB damage, as demonstrated by decreases in TEER; here, preliminary work with ELISA assays further suggest that BMECs treated with propofol demonstrate an upregulation of VEGF. Pretreatment of BMECs with Axitinib before the addition of propofol partially rescues TEER and thus attenuates the propofol-mediated diminution of TEER. These observations thereby implicate VEGF as a damage mediator after propofol treatment.    Conclusion and Potential Impact:  This study utilized an in vitro model to demonstrate that propofol may mediate, in part, damage to blood- brain barrier endothelium via a VEGF dependent mechanism; thus, this work may guide future investigations to facilitate the development of safer anesthetic alternatives, or towards additional pharmacologic interventions that counteract propofol-mediated damage during anesthetic induction. 


2019 ◽  
Vol 18 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Fengjin Hao ◽  
Yueqin Feng ◽  
Yifu Guan

Objective: Botulinum toxin has many applications in the treatment of central diseases, as biological macromolecules, it is difficult to pass through the blood-brain barrier which greatly limits their application. In this paper, we verified whether the botulinum toxin heavy chain HCS has a specific neural guidance function. Methods: We have constructed a fusion protein with botulinum toxin heavy chain and a membrane penetrating peptide TAT (TAT-EGFP-HCS). Recombinant plasmid of botulinum toxin light chain (LC) and TAT were also constructed. The biological activity of HCS, LC, TAT-EGFP-HCS and TAT-EGFP-LC were measured by its ability to cleave protein SNAP-25. The intracellular expression efficiency was evaluated by detecting the fluorescence intensity of EGFP in the cells by fluorescence microscopy and FACS. In addition, we also determined the effect of the above plasmid expression on the apoptosis of PC12 cells. Finally, the tissue specificity of TAT-EGFP-HCS in vivo experiments was also examined. Results: In the present study, we have constructed a fusion protein with botulinum toxin heavy chain and a membrane penetrating peptide TAT which can lead the entire molecule through the blood-brain barrier and reach the central nervous system. Moreover, we also examined the biological activities of this recombinant biological macromolecule and its physiological effects on nerve cells in vitro and in vivo. Conclusion: TAT-EGFP-HSC expressed in vitro has neural guidance function and can carry large proteins across the cell membrane without influencing the biological activity.


Author(s):  
Marco Campisi ◽  
Sharon W. L. Lee ◽  
Tatsuya Osaki ◽  
Luca Possenti ◽  
Clara Mattu ◽  
...  

The blood-brain barrier (BBB) protects the brain from pathogens but also hinders drug delivery to the central nervous system. Most of the BBB models developed up to date failed to reproduce the human anatomical complexity of brain barriers, contributing to less predictive experimental platforms and poor patient outcomes. To overcome those limitations, the development of reliable in vitro models represents a crucial step towards more effective therapies. This contribution was focused on the development of an in vitro microfluidic model of the BBB able to replicate the human neurovascular organization. The microfluidic model included human induced pluripotent stem cell-derived endothelial cells, brain pericytes, and astrocytes as self-assembled microvascular networks in a 3-dimensional fibrin gel. As previously demonstrated, the BBB model exhibited perfusable and selective microvasculature, with permeability lower than conventional in vitro models and comparable with in vivo rat brain. Permeability of polystyrene nanoparticles (NPs) and synthesized polyurethane NP was measured across the BBB model as compared to conventional Transwell assays. This physiologically relevant BBB model offers an innovative and valuable platform to preclinically predict transport efficacy of drugs and carriers.


2005 ◽  
Vol 289 (5) ◽  
pp. H2012-H2019 ◽  
Author(s):  
Melissa A. Fleegal ◽  
Sharon Hom ◽  
Lindsay K. Borg ◽  
Thomas P. Davis

The blood-brain barrier (BBB) is a metabolic and physiological barrier important for maintaining brain homeostasis. The aim of this study was to determine the role of PKC activation in BBB paracellular permeability changes induced by hypoxia and posthypoxic reoxygenation using in vitro and in vivo BBB models. In rat brain microvessel endothelial cells (RMECs) exposed to hypoxia (1% O2-99% N2; 24 h), a significant increase in total PKC activity was observed, and this was reduced by posthypoxic reoxygenation (95% room air-5% CO2) for 2 h. The expression of PKC-βII, PKC-γ, PKC-η, PKC-μ, and PKC-λ also increased following hypoxia (1% O2-99% N2; 24 h), and these protein levels remained elevated following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Increases in the expression of PKC-ε and PKC-ζ were also observed following posthypoxic reoxygenation (95% room air-5% CO2; 2 h). Moreover, inhibition of PKC with chelerythrine chloride (10 μM) attenuated the hypoxia-induced increases in [14C]sucrose permeability. Similar to what was observed in RMECs, total PKC activity was also stimulated in cerebral microvessels isolated from rats exposed to hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min). In contrast, hypoxia (6% O2-94% N2; 1 h) and posthypoxic reoxygenation (room air; 10 min) significantly increased the expression levels of only PKC-γ and PKC-θ in the in vivo hypoxia model. These data demonstrate that hypoxia-induced BBB paracellular permeability changes occur via a PKC-dependent mechanism, possibly by differentially regulating the protein expression of the 11 PKC isozymes.


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