establishing blood brain barrier monoculture in &#181;sim device for immunocytochemistry staining and confocal analysis

Analysis of Blood-Brain Barrier Models via Immunofluorescence Staining

Living tissues are compartmentalized by specialized cells that create and maintain barriers and regulate which cells and molecules are transported from one compartment to another. The improper regulation of barrier functions can be the source of both acute illnesses and chronic disease. The blood-brain barrier (BBB) is the most restrictive tissue barrier in the human body1. Dysfunction of the BBB underlies a wide range of diseases of the central nervous system (CNS), including Alzheimer&#39;s disease2, Parkinson&#39;s disease3,4, and multiple sclerosis5,6. Injury to the BBB is also linked to long-term cognitive impairment as an outcome of acute disorders, such as sepsis7, COVID-198, and postoperative delirium9. The development of drugs with the potential to treat brain disorders has been frustratingly difficult because of the challenge of intentionally breaching the BBB to deliver bioactive molecules to targets in the brain10. For these reasons, methods to study BBB function in vitro are of paramount importance to the understanding and treatment of diseases of the CNS.
The basic methods for measuring barrier function in vitro involve the establishment of a monolayer or co-culture on a semi-permeable membrane and measuring the resistance imparted by cells to either small molecule diffusion or small electrical currents11,12,13,14. While the emergence of microphysiological systems (MPS) has produced an abundance of choices for modeling the BBB in 3D15,16, the diversity of system geometries makes it difficult to compare permeability measurements between MPS or to established literature values. The establishment of trusted baseline values is particularly important in BBB research where, because of the extensive barrier regulation by brain vascular endothelial cells, in vitro permeability values are highly scrutinized12,17,18. For these reasons, permeability measurements across monolayers established on 2D membranes will remain a staple in BBB studies for years to come. This holds true for other tissue barriers, including epithelial barriers, where absolute values of baseline permeability are used to validate and compare in vitro models19,20,21.
With the goal of establishing a valuable new tool for the BBB research community, we have introduced22 and advanced23,24,25,26 the microdevice featuring a silicon membrane (&#181;SiM) platform for use in barrier tissue modeling over the last 5 years. The enabling feature of the platform is an ultrathin (&#60;100 nm thick) membrane with hundreds of millions of nanopores27,28 or a mix of nanopores and micropores29. The free-standing membrane chips are produced on a 300 &#181;m silicon &#39;chip&#39; that stabilizes the ultrathin structures30 and allows them to be handled by tweezers for device assembly. Because of their ultrathin nature, the membranes have a permeability that is two orders of magnitude higher than that of conventional track-etched membranes used in commercial membrane culture devices31,32. In practice, this means that the membrane&#39;s hindrance to the diffusion of molecules smaller than the nanopores (&#60;60 nm) is negligible33. Thus, for cellular barriers, only the cells and the matrices they deposit will determine the rate of small molecule transport from the apical to basal compartments that are separated by the membrane34. The device design and the ultrathin nature of the membranes also provide many advantages for optical microscopy. These include 1) the ability to follow live cultures using phase contrast or bright field imaging, 2) the ability to fluorescently stain and image in situ without the need to extract and transfer the membrane to a cover glass, and 3) the fact that the membranes are thinner than confocal &#39;slices&#39; so that direct co-cultures have a more natural spacing between cell types than can be achieved with 6-10 &#181;m-thick track-etched membranes.
Most recently, we advanced the platform to a modular format to facilitate rapid assembly34 and customization35,36. We leveraged the modular format to distribute device components between our bioengineering and collaborating brain barrier laboratories. We then jointly developed protocols for device assembly, monoculture and co-culture, immunofluorescent staining, and small molecule permeability and showed that these methods were reproducible between labs. Using these protocols, we also showed that the modular platform supports a validated BBB developed using the extended endothelial culture method (EECM) for creating brain microvascular-like endothelial cells (BMEC) from human induced pluripotent stem cells (hiPSCs)37. The purpose of the current report is to review these methods in greater detail and, with the aid of the accompanying video, facilitate&#160;broader adoption of the platform in the BBB community.

Author Spotlight: Advancing the Use of Tissue Chip Technology for Studying Human Tissues 

Use of the MicroSiM (&#181;SiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

We are developing a tissue GI model of the blood-brain barrier to understand the impact of sepsis on the brain and the contribution of blood-borne factors. Using human-induced pluripotent stem cells. We plan on evaluating the contribution of genetic factors to the susceptibility of certain patient populations.Current experimental challenges within the tissue chip community are the reproducibility of results and the adoption of techniques by outside labs. Unique tissue chip designs, small volumes within tissue chips, and lack of standard functional assays all contribute to these challenges and often limit users to fluorescent based assays as their primary output. Micro simm devices offer modularity for versatile in vitro modeling, a glass light surface ideal for high resolution lifestyle imaging and ultra syn 100 nanometer, highly produced nano membrane, ensuring seamless soluble factor crosstalk and precise permeability assays.With the micro simm, we have seen that specific cell types tend to produce more of the matrix proteins that provide a supporting scaffold around brain blood vessels. Now we can ask whether inflammation causes these same cells to strengthen or break down their matrices, thereby altering barrier integrity. In the future, we will add another compartment to the model with green cells such as astrocytes and microglia to study how those cells interact with the blood-brain barrier in cases of systemic inflammation like sepsis.We also plan to investigate additional causes of systemic inflammation like postoperative delirium, superimposed on dementia.

Watch this Scientific Journal Video about Establishing Blood Brain Barrier Monoculture in µSiM Device for Immunocytochemistry Staining and Confocal Analysis at JoVE.com

Establishing Blood Brain Barrier Monoculture in &#181;SiM Device for Immunocytochemistry Staining and Confocal Analysis  

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Research

JoVE Journal

Bioengineering

359 visualizações.  University of Rochester. Para começar, coloque o dispositivo micro sim montado em abraçadeiras de mangueira estéreis. Cultivar o dispositivo em uma grande placa de Petri estéril. Para umidade extra, coloque uma tampa de tubo cônico de 50 mililitros cheia de água estéril.Para preparar o dispositivo para cultivo celular, enxágue a câmara superior com 100 microlitros de água. Em seguida, prepare uma solução de revestimento misturando colágeno quatro, fibronectina bovina e água estéril. Retire a ág...