Academic literature on the topic 'Blood-brain-barrier-on-a-chip'

The blood–brain barrier is a dynamic and highly organized structure that strictly regulates the molecules allowed to cross the brain vasculature into the central nervous system. The blood–brain barrier pathology has been associated with a number of central nervous system diseases, including vascular malformations, stroke/vascular dementia, Alzheimer’s disease, multiple sclerosis, and various neurological tumors including glioblastoma multiforme. There is a compelling need for representative models of this critical interface. Current research relies heavily on animal models (mostly mice) or on two-dimensional (2D) in vitro models, neither of which fully capture the complexities of the human blood–brain barrier. Physiological differences between humans and mice make translation to the clinic problematic, while monolayer cultures cannot capture the inherently three-dimensional (3D) nature of the blood–brain barrier, which includes close association of the abluminal side of the endothelium with astrocyte foot-processes and pericytes. Here we discuss the central nervous system diseases associated with blood–brain barrier pathology, recent advances in the development of novel 3D blood–brain barrier -on-a-chip systems that better mimic the physiological complexity and structure of human blood–brain barrier, and provide an outlook on how these blood–brain barrier-on-a-chip systems can be used for central nervous system disease modeling. Impact statement The field of microphysiological systems is rapidly evolving as new technologies are introduced and our understanding of organ physiology develops. In this review, we focus on Blood–Brain Barrier (BBB) models, with a particular emphasis on how they relate to neurological disorders such as Alzheimer’s disease, multiple sclerosis, stroke, cancer, and vascular malformations. We emphasize the importance of capturing the three-dimensional nature of the brain and the unique architecture of the BBB – something that until recently had not been well modeled by in vitro systems. Our hope is that this review will provide a launch pad for new ideas and methodologies that can provide us with truly physiological BBB models capable of yielding new insights into the function of this critical interface.

Lab-on-a-chip (LOC) and organ-on-a-chip (OOC) devices are highly versatile platforms that enable miniaturization and advanced controlled laboratory functions (i.e., microfluidics, advanced optical or electrical recordings, high-throughput screening). The manufacturing advancements of LOCs/OOCs for biomedical applications and their current limitations are briefly discussed. Multiple studies have exploited the advantages of mimicking organs or tissues on a chip. Among these, we focused our attention on the brain-on-a-chip, blood–brain barrier (BBB)-on-a-chip, and neurovascular unit (NVU)-on-a-chip applications. Mainly, we review the latest developments of brain-on-a-chip, BBB-on-a-chip, and NVU-on-a-chip devices and their use as testing platforms for high-throughput pharmacological screening. In particular, we analyze the most important contributions of these studies in the field of neurodegenerative diseases and their relevance in translational personalized medicine.

Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2018.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 50-58).
With up to 40% of cancer patients showing metastatic lesions to the brain and a 30% five-year survival rate post-diagnosis, secondary tumors to the brain are a leading cause of cancer-related deaths. Understanding the mechanisms of tumor cell extravasation at the brain is therefore crucial to the development of therapeutic agents targeting this step in cancer metastasis, and to the overall improvement of cancer survival rates . Investigating the interactions between tumor cells and brain stroma is of particular interest due to the site's unique microenvironment. In fact, the interface between brain and blood, known as the blood-brain barrier (BBB), is the tightest endothelial barrier in humans. The presence of tight junctions between brain endothelial cells, coupled with the spatial organization of pericytes and astrocytes around the vasculature, restrict the entry of most solutes and cells into the brain. Yet, the brain constitutes a common metastatic site to many primary cancers originating from the lung, breast and skin. This suggests that tumor cells must employ specific mechanisms to cross the blood-brain barrier. While in vitro models aimed at replicating the human blood-brain barrier exist, most are limited in their physiological relevance. In fact, the majority of these platforms rely on a monolayer of human brain endothelial cells in contact with pencytes, astrocytes and neurons. While this approach focuses on incorporating the relevant cell types of the brain microenvironment, it fails to accurately replicate the geometry of brain capillaries, the barrier tightness of the BBB, and the juxtacrine and paracrine signaling events occurring between brain endothelial cells and stromal cells during vasculogenesis. To integrate these features into a physiologically relevant blood-brain barrier model, we designed an in vitro microvascular network platform formed via vasculogenesis, using endothelial cells derived from human induced pluripotent stem cells, primary human brain pericytes, and primary human brain astrocytes. The vasculatures formed with brain pericytes and astrocytes exhibit decreased cross-section areas, increased endothelial cell-cell tight junction expression and basement membrane deposition, as well as reduced and more physiologically relevant values of vessel permeability, compared to the vasculatures formed with endothelial cells alone. The addition of pericytes and astrocytes in the vascular system was also coupled with increased extravasation efficiencies of different tumor cell subpopulations, despite the lower permeability values measured in this BBB model. Moreover, an increase in the extravasation potential of metastasized breast tumor cells collected from the brain was recorded with the addition of pericytes and astrocytes, with respect to the parental breast tumor cell line. These results were not observed in metastasized breast tumor cells collected from the lung, thus validating our BBB model and providing useful insight into the role of pericytes and astrocytes in extravasation. Our microfluidic platform certainly provides advantages over the current state-of-the-art in vitro blood-brain barrier models. While being more physiologically relevant than most in vitro platforms when it comes to geometry, barrier function and juxtacrine/paracrine signaling between the relevant cell types, our model provides a robust platform to understand tumor cell-brain stromal cell interactions during extravasation.
by Cynthia Hajal.
S.M.

The blood-brain barrier (BBB) is crucial to maintain brainhomeostasis and prevent toxic substances from entering the brain.Endothelial cells (EC) are essential for the BBB and in this thesistwo different BBB-on-chips were designed for electricalcharacterization of immortalized mouse EC (bEnd3).Indium-Tin-Oxide (ITO) coated glass slides were etched, creating ITOelectrodes with increasing distance between them. The glass slideswere attached to a 3D-printed plastic well with UV-glue. The second prototype was an extension of the first prototype with acopper printed circuit board (PCB) attached to the ITO glass slidesusing silver epoxy to connect the ITO electrodes to the copperelectrodes. The aim with these two chips was to create chips withtransparent electrodes for live imaging of the cells with an opticalmicroscope. The chips were characterized with scanning electron microscopy (SEM) and a profilometer beforeseeding the cells inside the well. The absolute impedance wasmeasured across two parallel electrodes at a time. The impedance wasplotted against the distance between the electrodes. The method usedis called transmission line measurements (TLM) and is used to extractthe sheet impedance between the electrodes to evaluate the barriertightness of the cells. Only one chip from each prototype remained intact after thefabrication and sterilization, making it difficult to drawconclusions from the impedance measurements. However, based on thetwo chips, the TLM for the first prototype followed a linear trendwith a high R-square value whereas, the second prototype showed largevariations, causing the R-square value to decrease

Tese de mestrado integrado em Engenharia Biomédica e Biofísica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017
A barreira hemato-encefálica representa a mais complexa interface entre o sangue e o sistema nervoso central, sendo bastante importante na manutenção da homeostasia do cérebro e em proteger o mesmo de diversas substâncias tóxicas e patogénicas. Contudo, devido à baixa permeabilidade que advém das ligações formadas entre as diversas células endoteliais, esta barreira inibe também a entrada de vários agentes farmacêuticos no cérebro, constituindo assim um grande obstáculo ao desenvolvimento de novas terapias. Para além disto, qualquer pertubação no funcionamento desta estrutura pode originar diversas doenças neurodegenerativas. Várias têm sido as tentativas de desenvolver um modelo fidedigno da barreira hemato-encefálica, quer in vivo, ex-vivo, in silico e in vitro, que ajude a melhor compreender estes estados patológicos do cérebro e que possa fornecer novas perspectivas acerca do transporte de agentes terapêuticos através desta barreira. No que diz respeito aos modelos in vivo, estes não só requerem equipamento bastante específico e caro, como também são bastante morosos e dão origem a diversas questões éticas e morais. Os modelos ex-vivo, por outro lado, permitem analisar tecidos vivos, os quais podem ser fatias de um órgão ou o órgão completo, fora do seu contexto biológico, permitindo desta forma um maior controlo de todas as condições experimentais do que os modelos in vivo. No entanto, garantir que o ambiente artificial em que o tecido é analisado tenha exactamente as mesmas condições que o seu ambiente biológico pode tornar-se complicado, o que pode por sua vez causar a morte do tecido. No caso dos modelos in silico, estes são construídos com recurso a modelos computacionais baseados em dados obtidos em experiências realizadas in vivo, o que os torna bastante fidedignos em prever, por exemplo, a permeabilidade da barreira hemato-encefálica a um determinado medicamento. O facto destes modelos muitas vezes não terem em conta toda a complexidade da barreira hemato-encefálica é uma das suas limitações. No caso dos modelos in vitro, estes permitem o estudo das mais variadas estruturas biológicas fora do seu contexto natural. Para tal, células derivadas de tecidos cerebrais são cultivadas em modelos construídos propositadamente para o estudo em questão, o que permite um maior controlo sobre todas as condições experimentais. Deste modo, é perceptível que muitos esforços têm sido feitos para desenvolver um modelo in vitro que simule correctamente a barreira hemato-encefálica e que seja de fácil análise, reprodutível, e permita um maior rastreio (de agentes terapêuticos, por exemplo) que os modelos in vivo. O objectivo principal deste trabalho foi desenvolver e validar um chip microfluídico que pudesse ser usado como uma ferramenta para o estudo da barreira hemato-encefálica e que permitisse a criação de diferentes condições experimentais ao mesmo tempo, no mesmo chip. Para atingir este objectivo, foram construídos dois modelos diferentes: um bi-dimenisonal e um tri-dimensional. O primeiro modelo construído, o qual consistia em duas partes de dimetil polissiloxano com uma membrana entre elas, foi replicado a partir de um molde feito de silício, o qual por sua vez foi fabricado numa sala estéril com recurso a técnicas de microfabricação. Células endoteliais foram cultivadas neste modelo e as barreiras formadas pelas mesmas mantiveram-se viáveis, em todos os casos, durante pelo menos cinco dias de cultura celular, período após o qual o núcleo e o citoesqueleto das células foram coloridos de modo a verificar a integridade das barreiras formadas. Este modelo possibilitou ainda a monitorização da complexidade das barreiras de células endoteliais ao integrar funcionalidades que permitiam a análise da resistência eléctrica transendotelial e da permeabilidade das mesmas. Embora preliminares, os resultados obtidos em ambos os testes foram bastante encorajadores e os valores obtidos para a resistência eléctrica e para a permeabilidade das barreiras foram de 27.5 Ω·cm2 e 3.6·10-5 cm/s, respectivamente. Para além disto, foram também fabricadas membranas feitas de poliestireno, nitrato rico em silício e dimetil polissiloxano, com o intuito de substituir os suportes permeáveis feitos em policarbonato nos quais as células eram cultivadas. O segundo modelo, por outro lado, foi feito em acrílico. Para tal, um bloco de acrílico foi cortado a laser e as diferentes peças foram coladas umas às outras com recurso a um adesivo biocompatível. Depois de montado o chip, um gel de colagénio foi inserido em cada um dos compartimentos do mesmo e micro-agulhas foram posicionadas por entre os buracos das tampas do chip. Depois de solidificar o colagénio, as micro-agulhas foram cuidadosamente retiradas. Isto permitiu a criação, em colagénio, de estruturas tridimensionais em forma de lúmen nas quais as células foram cultivadas. Tal como no modelo bi-dimensional, as células endoteliais cultivadas no colagénio mantiveram-se viáveis durante pelo menos cinco dias de cultura celular. Não foram realizadas neste modelo quaisquer experiências que tivessem como intuito determinar a resistência eléctrica e a permeabilidade das barreiras de células endoteliais, embora estudos de permeabilidade pudessem ser feitos com o protocolo adequado. A capacidade de utilizar individualmente os diferentes compartimentos microfluídicos sem que ocorresse qualquer mistura entre os mesmos foi provada em ambas as plataformas. Em relação ao modelo bi-dimenional, foram realizadas, numa primeira fase, experiências com corantes, enquanto numa segunda fase tripsina, álcool e meio de cultura foram inseridos nos diferentes compartimentos microfluídicos para confirmar se ocorria alguma mistura das soluções que afectasse as diversas barreiras celulares. Para tal, os chips foram ligados a uma bomba microfluídica que puxou as diversas soluções em todos os compartimentos microfluídicos. No caso das experiências em que foram utilizados trisipsina, álcool e meio de cultura, foi realizada uma coloração para verificar a viabilidade das barreiras celulares no final da experiência, a qual revelou que os diferentes compartimentos podiam ser utilizados sem que houvesse qualquer contaminação entre os diferentes compartimentos que pudesse pôr em causa a integridade das barreiras celulares. No que diz respeito ao modelo tri-dimensional, a capacidade de utilizar individualmente os diversos compartimentos foi apenas provada ao introduzir os diferentes corantes nos mesmos, o que revelou que o método de fabricação do chip assegurava uma plataforma robusta na qual diversas experiências podiam ser realizadas sem qualquer risco de contaminação. Para além disto, foi também possível cultivar células endoteliais neste chip durante pelo menos cinco dias, embora a coloração das mesmas não tenha sido bem sucedida uma vez que os agentes fluorescentes se difundiram pelo colagénio. Isto fez com que fosse possível concluir que, embora diferentes, dois modelos igualmente relevantes em termos fisiológicos tinham sido desenvolvidos. Contudo, ambos os modelos necessitam de uma caracterização mais profunda. No caso do primeiro modelo, este beneficiaria caso melhorias fossem feitas no que diz respeito a medições de resistência eléctrica, de permeabilidade, e também de cultura dinâmica de células. Para tal, experiências futuras e protocolos adequados são necessários. No caso do segundo modelo, encontrar um agente que permita revestir as estruturas em acrílico de modo a que seja possível utilizar concentrações mais baixas de colagénio seria bastante benéfico. Para além disto, a criação de co-culturas de células endoteliais com células cerebrais que são conhecidas por aumentar a complexidade e a impermeabilidade da barreira hemato-encefálica, tais como astrócitos ou perócitos, devia também ser realizada em ambos os modelos de modo a verificar se as mesmas aumentariam a complexidade da barreira hemato-encefálica formada, como descrito na literatura. Por último, seria também de elevado interesse realizar testes de rastreio de diversos agentes farmacêuticos utilizados no tratamento de várias patologias, como por exemplo para as doenças de Alzheimer e de Parkinson, de modo a obter novas perspecivas no que à permeabilidade da barreira hemato-encefálica a estas substâncias diz respeito.
The blood-brain barrier (BBB) constitutes a complex interface between blood and the central nervous system (CNS), playing a vital role in maintaining brain homeostasis and protecting it from most toxic substances and pathogens. However, due to the extremely low permeability that arises from the tight junctions formed by the endothelial cells, the BBB also inhibits the brain uptake of many pharmaceuticals, therefore posing a major obstacle for drug development studies. Furthermore, the disturbance of the function of this unique structure can lead to many neurodegenerative disorders that are not yet fully understood, such as brain tumors or Alzheimer’s disease. There have been several attempts to establish reliable in vivo and in vitro models of the BBB that can increase the knowledge on such pathological states of the brain and provide useful insights on drug delivery across this barrier. However, as in vivo models require expensive and specific equipment, are labor intensive, and give rise to many ethical and moral concerns, much effort is being put into developing an in vitro model that truthfully resembles the BBB and is easy to analyze, reproducible, and allows higher throughput screening than the in vivo models. Therefore, the main goal of the present work was to design, fabricate and validate a microfluidic chip that could serve as a tool for the study of the blood-brain barrier and allowed the creation of several different experimental conditions at the same time in the same chip, thus increasing the throughput of the system. Two different models, a two-dimensional and a three-dimensional, were fabricated for this purpose. The first model that was built, which consisted of two poly(dimethylsiloxane) (PDMS) parts with a membrane in between, allowed the monitoring of the tightness of the endothelial cell layers by integrating on-chip transendothelial electrical resistance (TEER) and permeability analysis. Even though preliminary, the results obtained for both these assays were quite encouraging and TEER and permeability were found to be as high as 27.5 Ω·cm2 and as low as 3.6·10-5 cm/s, respectively. Moreover, polystyrene (PS), silicon-rich nitride (SiRN) and PDMS membranes were fabricated in order to improve the permeable supports on which cells were seeded inside this device. The second model, on the other hand, was made in plexiglas and allowed the creation of lumen-shaped three-dimensional structures within collagen on which cells were seeded. No experiments regarding TEER or permeability were performed in the second model, although permeability studies could be done with the appropriate protocol. The capability of individually addressing the microfluidic chambers without any mixing occurring between them was proven for both devices in experiments using dyes, trypsin and ethanol. Furthermore, confluent monolayers of endothelial cells were observed in the two models with both phase contrast and fluorescence microscopy, making it possible for us to conclude that two different yet equally physiologically relevant multiplexed devices that allow the individual addressing of their microfluidic chambers had been developed. However, further experimenting is required in order to fully characterize the devices, especially concerning TEER measurements, permeability assays and dynamic cell culturing. Moreover, finding a coating agent that would allow the use of lower concentrations of collagen to fabricate the hollow channels would make the second model more advantageous. Furthermore, the co-culture of endothelial cells with other cell types that are known to enhance the tightness of the BBB, such as astrocytes or pericytes, should also be looked into for both devices. Performing on-chip drug screening studies would also be of interest.