The authors would like to acknowledge Elisabeth Helena Bruun, Sarah Christine Christensen, and Niels M. Kristiansen for technical assistance, and the Lundbeck Foundation grant number R155-2013-14113.

The authors report no conflict of interest.

Purification and Proliferation of pBEC
During the purification procedure, critical steps include rapid and effective removal of meninges and separation of white and grey matter, which is important for the purification yield and purity and for the proper establishment of the model. For the presented in vitro BBB model using pBECs, we have improved and simplified a purification procedure based on mechanical homogenization of isolated grey matter, size-selective filtering for isolation of m...

Cellular Structure and Function of the Blood-Brain Barrier
At the interface of the circulatory and central nervous system (CNS), the BBB acts as a key regulatory site for homoeostatic control of the CNS microenvironment, which is essential for proper function and protection of the nervous system. The site of the BBB is the endothelial cells lining the blood vessel lumen. In brain capillaries, endothelial cells form complex intercellular tight junctions and strongly polarized expression patterns of particular influx and efflux transporters ensure highly specific molecular transport between the blood and the brain 1. The structural components of the tight junction complexes include proteins from the occludin and claudin family, zonula occludens (ZO) proteins, cingulin, and associated junctional adhesion molecules (JAMs). Claudin 5 is particularly important in the paracellular junctional restriction. Induction and maintenance of this characteristic BBB endothelial phenotype involve dynamic interactions with surrounding cells, including pericytes, astrocytes, neurons and the basement membranes, which together with brain endothelial cells form the neurovascular unit (NVU)2,3. The mechanisms involved in these interactions are not yet fully understood, but include exchange of chemical signals between cells, which allows modulation of BBB permeability in the short term and induces long-term BBB features4. Astrocytes especially are known to contribute to the brain endothelial cell phenotype and are a source of regulatory factors such as glial-derived neurotrophic factor (affecting intracellular cAMP)5, basic fibroblast growth factor6, hydrocortisone7, and transforming growth factor &#946; (TGF-&#946;)8. The effect of TGF-&#946;, however, has been debated9.
From In Vivo to In Vitro BBB
In vivo studies continue to provide valuable information on BBB biology. However, cell culture models can provide additional insights and constitute useful tools for understanding detailed molecular and functional aspects of the BBB in both health and disease. Although the complex interactions between the cell types and constituents of the BBB are difficult to fully achieve in in vitro models, there has been, since the first purification of brain endothelial cells and application of these in mono-cultures10,11,12, extensive development of the purification procedures and growth conditions of the BBB cell culture models, resulting in greater resemblance to the in vivo barrier. The commonly used in vitro BBB models are based on primary cells of rodent, porcine, and bovine origin, and on immortalized cell lines. Each model has different advantages and drawbacks. For comparison and model choice, validation markers such as expression of BBB enzymes, transporters, receptors, and structural proteins are used to create overviews of the current established models1.
Aim of the Protocol
One important feature of the BBB is barrier tightness and high TEER, yet a large number of the available models do not reflect well the in vivo levels. Incorporating development and optimization contributions from several laboratories, the aim of this protocol is to present a method for establishing a high TEER in vitro BBB model based on primary pBECs in MC with or without ACM, or in NCC with primary astrocytes of rat or porcine origin. The applied procedures and establishment of the model include efforts to eliminate contaminating cells and to improve differentiation of pBECs into a BBB phenotype. This work has resulted in the establishment of reliable, high TEER models with low paracellular permeability and good functional expression of key tight junctional proteins, transporters, and receptors. However, as the astrocytes are a contributing factor to the brain endothelial cell phenotype, the three different conditions of culture represent three different phenotypes of brain endothelial cells. The NCC model is specifically useful for studies of certain specialized mechanisms involved in drug discovery, transport studies and intracellular trafficking, as well as for investigation of cell-cell interactions where maximal expression of BBB features is advantageous.
Origin and History of the Protocol
The pBEC model described here is largely based on the porcine model developed at Eisai Laboratories (London) by Dr. Louise Morgan and colleagues, whichis based on a successful earlier bovine brain endothelial cell model13. The original method of cell preparation was a two-stage filtration using nylon meshes to catch the microvessels, followed by a subculturing step to improve purity. In the earlier development of the method, optimal BBB phenotype and barrier tightness were achieved by growth in supplemented medium, including ACM. Further modifications to the method were made by R. Skinner in Prof N. Rothwell&#39;s lab in Manchester UK14,15. The method was adopted by the Abbott laboratory, KCL London, where Patabendige made it significantly simpler to prepare by avoiding the use of astrocytes or ACM and by eliminating contaminating cells such as pericytes with puromycin. The first papers confirmed that the MC model preserved several important features of the in vivo BBB, including effective tight junctions, membrane transport systems and receptor-mediated transcytosis16,17,18,19,20. Later S. Yusof again tested astrocyte co-culture and found it significantly improved TEER, so this is the preferred variant currently used in the Abbott lab21. The model has now been successfully transferred to the M. Nielsen lab in Aarhus, where further modifications have been introduced (this protocol), including simplifying grey matter extraction, using only one mesh filtration step, and a single filter coating step combining collagen and fibronectin. The applied procedure for isolation of porcine astrocytes (this protocol) was based on protocols developed by the T. Moos laboratory in Aalborg, described by Thomsen et al22. The TEER and other properties of the model generated in London and Aarhus are similar, which lends confidence to the notion that the model is readily transferred between labs and responds well to careful observation and rationalization of method steps. Indeed, S. Yusof has now established the MC model in a tropical country (Malaysia)23, which involved further adjustment for local conditions and tissue sources.
Advantages over Alternative Methods and Currently Established Models
Compared to brain endothelial cells of bovine and rodent origin, pBECs provide the advantage of having a lower rate of loss of the in vivo BBB phenotype following isolation24. Furthermore, pBECs are capable of forming relatively tight endothelial barriers, even when grown in MC (800 &#937; cm2)16 as compared to the commonly reported levels for monolayers of cell lines such as bEND.5 and bEND.3 (50 &#937; cm2)25,26,27, cEND (300-800 &#937; cm2)28,29,30, and cerebEND (500 &#937; cm2)29,31,32, and primary brain endothelial cells of mouse (100-300 &#937; cm2)33,34,35,36 and rat (100-300 &#937; cm2)37,38. However, the TEER has shown dependency on the purification and culture procedures. In most cases, the addition of ACM or co-culture with astrocytes shows differentiating effects on the endothelial cells and an increase in tightness of the endothelial layers1. Nevertheless, with efforts to optimize the culturing conditions, only the bovine-based models have shown TEER values comparable to the porcine-based models (averages of 800 &#937; cm2 in MC, up to 2500 &#937; cm2 in astrocyte co-culture)13,39,40,41,42,43,44,45. As the models based on primary bovine brain endothelial cells have shown large variations, both between and within laboratories14,45,46,47,48, reproducibility could be an issue. In the pBEC model reported here, the contributing laboratories have achieved very similar TEER and paracellular permeability values with low variability, both in and between laboratories. Hence, it should be possible for other laboratories to establish a robust model with low variability using the method presented here. In addition to forming tight endothelial layers, models with pBECs have previously been validated by expression of tight junction proteins, functional BBB transporters, receptors and enzymes, and demonstrated suitability for a range of studies15,16,17,19,20,22,49,50,51,52,53,54,55,56,57,58,59. Furthermore, unpublished transcriptome data on the co-cultures of pBECs shows an expected profile of BBB transporters and receptors (unpublished results, Nielsen et al.).
The porcine-based BBB model has a further advantage as the genome, anatomy, physiology, and disease progression of the pig reflect the human biology to a higher degree than other established models60, which are favorable features for the pharmaceutical industry. As porcine brains are a common by-product of the meat industry, they constitute an easily accessible source of brain endothelial cells, minimizing the number of animals needed for the experiments, and providing a high purification yield from one porcine brain. Although purification and cultivation of primary cells is somewhat time-consuming and requires expertise for standardization in setting up the model, primary cells generate the most reliable BBB models. Immortalized cell lines cannot be a substitute, as important properties such as barrier tightness, transporter expression profiles, and microenvironment regulation do not reflect the experimental findings in vivo61,62. In vitro models provide the advantage of live-cell imaging with higher resolution, making visualization of intracellular processes possible by allowing a close proximity approach to the sampled or observed cells, using objectives with higher magnification and better optical quality63. This is not the case for the use of two-photon microscopy in living animals. Furthermore, in vitro models provide the ability to transfect cells, allowing visualization of tagged proteins and investigation of their trafficking.
Applications of the Model
The function of the BBB is not fixed and can be dynamically modulated in both physiology and pathology. In many neurological diseases, including neurodegenerative, inflammatory and infectious diseases, disruption and increased permeability of the BBB is observed64,65,66,67. In order to reduce and prevent disease progression and subsequent damage, identification and characterization of the molecular mechanisms underlying the modulation of the BBB are of major importance. In this context, reliable in vitro models are in high demand by the pharmaceutical industry, and furthermore play important roles in predicting BBB permeability of drugs to the CNS. Any in vitro model serving as a permeability screen should display a restrictive paracellular pathway, a physiologically realistic cell architecture and functional expression of transporter mechanisms68. Demonstrated in previous studies16,17,57, and by paracellular permeability and expression of TJ and AJ proteins here, the presented model meets all these criteria and is suitable for a range of BBB studies in both normal physiology and in pathology. The strengths of the presented purification and cultivation method include a combination of simplicity and reproducibility and the ability to include astrocytic influence with a resulting robust and reliable high TEER in vitro BBB model. For this purpose, astrocytes of porcine and rat origin have been shown to augment the BBB phenotype of pBECs in a similar way22.

<table><tbody><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>F1141</td><td></td></tr><tr><td>Collagen IV</td><td>Sigma-Aldrich</td><td>C5533</td><td></td></tr><tr><td>Poly-L-lysine</td><td>Sigma-Aldrich</td><td>P1524</td><td></td></tr><tr><td>DMEM/F-12</td><td>Lonza</td><td>BE12-719F</td><td></td></tr><tr><td>DMEM/Low Glucose</td><td>Sigma-Aldrich</td><td>D6046</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco Invitrogen</td><td>15140</td><td></td></tr><tr><td>Plasma derived serum (PDS)</td><td>First Link UK Ltd.</td><td>60-00-89</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco Invitrogen</td><td>10-270-106</td><td></td></tr><tr><td>Trypsin/EDTA</td><td>Gibco Invitrogen</td><td>15090-046</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3393</td><td></td></tr><tr><td>Puromycin</td><td>Sigma-Aldrich</td><td>P8833</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H4001</td><td></td></tr><tr><td>8-CPT-cAMP</td><td>Biolog</td><td>C010</td><td></td></tr><tr><td>RO 20-1724</td><td>Sigma-Aldrich</td><td>B8279</td><td></td></tr><tr><td>Gentamicin Sulfate</td><td>Lonza</td><td>17-518Z</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>34896</td><td></td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>D8537</td><td></td></tr><tr><td>EtOH</td><td>VWR</td><td>20,824,296</td><td>Mix the 70 % solution from the 96 % EtOH</td></tr><tr><td>DNAse 1</td><td>Sigma-Aldrich</td><td>D4513</td><td></td></tr><tr><td>Collagenase CLS2</td><td>Sigma-Aldrich</td><td>C6885</td><td></td></tr><tr><td>ddH&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>Made with Elga System</td></tr><tr><td>T75 flasks</td><td>Thermo Scientific</td><td>156499</td><td></td></tr><tr><td>Costar Transwell inserts (Cell permeable membrane inserts)</td><td>Costar</td><td>CLS3401</td><td>12-well plate, 12 mm diameter, 0.4 &amp;mu;m polycarbonate membrane</td></tr><tr><td>15 ml centrifuge tubes</td><td>Cellstar</td><td>188271</td><td></td></tr><tr><td>50 ml centrifuge tubes</td><td>Cellstar</td><td>227261</td><td></td></tr><tr><td>Petri dishes</td><td>Thermo Scientific</td><td>150350</td><td></td></tr><tr><td>Cryo vials</td><td>Thermo Scientific</td><td>377224</td><td></td></tr><tr><td>500 ml bottle</td><td>Thermo Scientific</td><td>159910/159920</td><td></td></tr><tr><td>Scalpels</td><td>Swann-Morten</td><td>REF0211</td><td>Type 24</td></tr><tr><td>Tissue homogenizer</td><td>Sigma</td><td>D9188</td><td></td></tr><tr><td>140 &amp;mu;m filters</td><td>MERCK</td><td>NY4H04700</td><td></td></tr><tr><td>40 &amp;mu;m filters</td><td>Corning</td><td>431750</td><td></td></tr><tr><td>EndOhm chamber system</td><td>World Precision Instruments</td><td>ENDOHM-12</td><td>EndOhm chamber for 12mm Culture Cups</td></tr><tr><td>EVOM2 electrode system</td><td>World Precision Instruments</td><td>300523+STX100C</td><td>TEER measurement system with rigid STX-100C electrode pair</td></tr><tr><td>Long needle</td><td>Sigma</td><td></td><td>Attach to a syringe</td></tr><tr><td>Fine-tip curved forceps</td><td>KLS Martin</td><td>12-409-12-07</td><td></td></tr><tr><td>Broad tip forceps</td><td>VWR</td><td>82027-390</td><td></td></tr><tr><td>Filter holder</td><td>MERCK Milipore</td><td>Swinnex-47</td><td></td></tr><tr><td>50 ml syringe</td><td>Braun</td><td>4617509F</td><td></td></tr><tr><td>10 ml syringe</td><td>Terumo</td><td>SSt20ESI</td><td></td></tr><tr><td>Anti-Occludin antibody</td><td>Abcam</td><td>ab31721</td><td>1:100</td></tr><tr><td>Anti-p120 Catenin antibody</td><td>BD Transduction laboratories</td><td>610133</td><td>1:200</td></tr><tr><td>Anti-ZO-1 antibody</td><td>Invitrogen</td><td>61-7300</td><td>1:200</td></tr><tr><td>Anti-Claudin 5 antibody</td><td>Sigma-Aldrich</td><td>SAB4502981</td><td>1:100</td></tr><tr><td>Donkey anti rabbit IgG conjugated with Alexa Flour 568</td><td>Thermo Scientific</td><td>A10042</td><td>1:500</td></tr><tr><td>Donkey anti mouse IgG conjugated with Alexa Flour 488</td><td>Thermo Scientific</td><td>A21202</td><td>1:500</td></tr><tr><td>Sucrose</td><td>Perkin Elmer</td><td>NEC100X250UC</td><td>0.15&amp;micro;l/ml final working conc</td></tr><tr><td>Lucifer Yellow</td><td>Sigma</td><td>L0144</td><td>10 &amp;micro;g/ml final working conc</td></tr></tbody></table>

improved method for the establishment of an in vitro blood-brain barrier model based on porcine brain endothelial cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Establishment of the BBB In Vitro Models
In the presented, optimized method, cultivation of pBECs and the establishment of the permeable membrane insert system with MC or without ACM or NCC with astrocytes (Figure 1) was carried out for a period of 9-11 days (Figure 2). For selection of endothelial cells, an initial culture of purified capillary fragments was comb...

Watch this Scientific Journal Video about Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells at JoVE.com

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

improved-method-for-establishment-an-vitro-blood-brain-barrier-model

Research

JoVE Journal

Medicine

14.7K Views.  Aarhus University. The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery. 

Video: Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Bioengineering

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1). Human tissue samples are available only in a restricted number of laboratories or companies 2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.
Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the F&ouml;rster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8. Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

This method describes how to isolate and immortalize microvascular endothelial cells from mouse brain. We describe a step-by-step protocol starting from the homogenization of brain tissue, digestion steps, seeding and immortalization of the cells. Usually, it takes about five weeks to obtain a homogenous, immortalized microvascular endothelial cell line.

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The ...

Neuroscience

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Developmental Biology

Isolation of Primary Murine Brain Microvascular Endothelial Cells

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. 
Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies.
This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

Brain microvascular endothelial cells (BMEC) are interconnected by specific junctional proteins forming a highly regulated barrier separating blood and the central nervous system (CNS), the so-called blood-brain-barrier (BBB). The isolation of primary murine brain microvascular endothelial cells, as discussed in this protocol, enables detailed in vitro studies of the BBB.

The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional ...

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). This process is characterized by the transmigration of activated T cells across brain endothelial cells (ECs), the main constituents of the BBB. However, the consequences on brain EC function upon interaction with such T cells are largely unknown. Here we describe an assay that allows for the evaluation of primary mouse brain microvascular EC (MBMEC) function and barrier integrity during the interaction with T cells over time. The assay makes use of impedance cell spectroscopy, a powerful tool for studying EC monolayer integrity and permeability, by measuring changes in transendothelial electrical resistance (TEER) and cell layer capacitance (Ccl). In direct contact with ECs, stimulated but not na&#239;ve T cells are capable of inducing EC monolayer dysfunction, as visualized by a decrease in TEER and an increase in Ccl. The assay records changes in EC monolayer integrity in a continuous and automated fashion. It is sensitive enough to distinguish between different strengths of stimuli and levels of T cell activation and it enables the investigation of the consequences of a targeted modulation of T cell-EC interaction using a wide range of substances such as antibodies, pharmacological reagents and cytokines. The technique can also be used as a quality control for EC integrity in in vitro T-cell transmigration assays. These applications make it a versatile tool for studying BBB properties under physiological and pathophysiological conditions.

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Here, we describe an in vitro murine model of the blood-brain barrier that makes use of impedance cell spectroscopy, with a focus on the consequences on endothelial cell integrity and permeability upon interaction with activated T cells.

Breakdown of the blood-brain barrier (BBB) is a critical step in the development of autoimmune diseases such as multiple sclerosis (MS) and its animal ...

Immunology and Infection

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

A 3D Blood-Brain Barrier Model Derived from Human Pluripotent Stem-Cells

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting the brain from pathogens. The inherent barrier properties of the BBB pose a challenge for therapeutic drug delivery into the CNS to treat neurological diseases. Impaired BBB function has been related to neurological disease. Cerebral amyloid angiopathy (CAA), the deposition of amyloid in the cerebral vasculature leading to a compromised BBB, is a co-morbidity in most cases of Alzheimer&#39;s disease (AD), suggesting that BBB dysfunction or breakdown may be involved in neurodegeneration. Due to limited access to human BBB tissue, the mechanisms that contribute to proper BBB function and BBB degeneration remain unknown. To address these limitations, we have developed a human pluripotent stem cell-derived BBB (iBBB) by incorporating endothelial cells, pericytes, and astrocytes in a 3D matrix. The iBBB self-assembles to recapitulate the anatomy and cellular interactions present in the BBB. Seeding iBBBs with amyloid captures key aspects of CAA. Additionally, the iBBB offers a flexible platform to modulate genetic and environmental factors implicated in cerebrovascular disease and neurodegeneration, to investigate how genetics and lifestyle affect disease risk. Finally, the iBBB can be used for drug screening and medicinal chemistry studies to optimize therapeutic&#160;delivery to the CNS. In this protocol, we describe the differentiation of the three types of cells (endothelial cells, pericytes, and astrocytes) arising from human pluripotent stem cells, how to assemble the differentiated cells into the iBBB, and how to model CAA in vitro using exogenous amyloid. This model overcomes the challenge of studying live human brain tissue with a system that has both biological fidelity and experimental flexibility, and enables the interrogation of the human BBB and its role in neurodegeneration.

Author Spotlight: A Personalized Approach Towards Investigating Alzheimer's Disease Using an In Vitro Blood-Brain Barrier Model 

The blood-brain barrier (BBB) has a crucial role in sustaining a stable and healthy brain environment. BBB dysfunction is associated with many neurological diseases. We have developed a 3D, stem-cell-derived model of the BBB to investigate cerebrovascular pathology, BBB integrity, and how the BBB is altered by genetics and disease.

The blood-brain barrier (BBB) is a key physiological component of the central nervous system (CNS), maintaining nutrients, clearing waste, and protecting ...

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls and restrict access to the brain parenchyma. However, with the development of nanotechnologies, large panels of new nanomaterials were developed to improve drug delivery, highlighting the need for reliable in vitro microsystems to predict brain penetration in the frame of preclinical assays. Here is a straightforward method to set up a microphysiological system to model the BBB using solely human cells. In its configuration, the model consists of a triple culture including brain-like endothelial cells (BLECs), pericytes, and astrocytes, the three main BBB cellular actors necessary to induce and regulate the BBB properties in a more physiological manner without the requirement of tightening compounds. The model developed in a 12-well plate format, ready after 6 days of triple culture, is characterized in physical properties, gene, and protein expressions and used for polymeric nanogel transport measurement. The model can be used for an extensive range of experiments in healthy and pathological conditions and represents a valuable tool for preclinical assessments of molecule and particle transport, as well as inter-and intracellular trafficking.

This protocol describes a method to establish a human blood-brain barrier (BBB) in vitro model. The endothelial cells and pericytes are seeded on each side of an insert filter (blood compartment), and astrocytes are seeded in the bottom well (brain compartment). The model characterized was used for nanoparticles transport experiments.

The delivery of drugs to the brain remains a challenge due to the blood-brain barrier's (BBB) highly specific and restrictive properties, which controls ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

Summary

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Chapters in this video

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

Isolation of Primary Murine Brain Microvascular Endothelial Cells

An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

Reconstruction of the Blood-Brain Barrier In Vitro to Model and Therapeutically Target Neurological Disease

A Triple Culture Cell System Modeling the Human Blood-Brain Barrier