Name: improved method for the establishment of an in vitro blood-brain barrier model based on porcine brain endothelial cells
Uploaded: 2017-09-24T14:00:00
Description: 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

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.

Porcine brains were obtained as byproducts of the Danish food industry. Danish Slaughterhouses are under strict supervision and observation by the Danish Ministry of Environment and Food.
Rats used for isolation of astrocytes were bred and group-housed in the local animal facility at an ambient temperature of 22 &#176;C-23 &#176;C and on a 12/12 h dark/light cycle under inspection of the veterinarian and according to Danish regulations for lab animals. The rats were euthanized before they were...

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>ddH2O</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 &#956;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 &#956;m filters</td>
			<td>MERCK</td>
			<td>NY4H04700</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;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&#181;l/ml final working conc</td>
		</tr>
		<tr>
			<td>Lucifer Yellow</td>
			<td>Sigma</td>
			<td>L0144</td>
			<td>10 &#181;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) ...

The overall goal of this protocol is to purify and isolate primary porcine brain endothelial cells in order to set up a tight in vitro blood-brain barrier model. This method can help answer key questions in the blood-brain barrier field, such as drug delivery, permeability, receptor trafficking, as well as the tightness and polarity of the BB.The main advantage of this model is the high yield of primary brain endothelial cells that can be used to create an in vitro blood-brain barrier model which has high TEER and low permeability. To begin this procedure, place the brains in a sterile flow bench and gently wash them with one liter of PBS in a beaker placed on ice.Next, carefully remove the meninges from each brain using fine-tip forceps. Using a scalpel, scrape off gray matter from the brains one at at time and transfer the isolated material to Petri dishes containing 20 milliliters of DMEM/F-12 placed on ice. For initial fragmentation, run the gray matter material through a 50-milliliter syringe without the needle.Repeat the procedure for all brains and pool the collected gray matter material together. Transfer the isolated gray matter material with DMEM/F-12 medium to the grinder tube of a handheld tissue homogenizer. Homogenize the material by making eight up-and-down strokes with a loose pestle, followed by eight up-and-down strokes with a tight pestle.Next, isolate the capillaries by filtering the homogenate using a 500-milliliter blue cap bottle with filter holder and 140-micron filter mesh. Place the capillary-containing filters in Petri dishes with a digestion solution. Subsequently, incubate the Petri dishes at 37 degrees Celsius for one hour on an orbital shaker at 180 RPM, or stir them gently every 10 minutes.After one hour, wash off the capillaries from the filters with suspension from the Petri dish. Then, split the suspension from the three dishes into two 50-milliliter tubes and stop the digestion by adding 10 milliliters of DMEM/F-12 to each 50-milliliter tube. Centrifuge the cell suspensions at 250 times g, four degrees Celsius for five minutes.Afterward, aspirate the supernatants and resuspend each pellet in 10 milliliters of DMEM/F-12. Then, add a further 20 milliliters of DMEM/F-12 to each tube. To optimize the conditions for the attachment of PBECs, add coating solution to the T-75 flask, resulting in the final concentrations of collagen four at 150 micrograms per milliliter and fibronectin at 50 micrograms per milliliter and by distilled water, making sure that the solution covers the whole surface.Next, thaw the capillaries in a 37-degree Celsius water bath. To remove freezing medium, spin the capillaries down at 250 times g, four degrees Celsius, for seven minutes. After that, carefully aspirate the supernatant and resuspend the pellet in one to two milliliters of the 10-milliliter aliquot.Following that, transfer the capillary suspension to the coated T-75 flask, and gently tilt the flask to ensure equal distribution over the surface. Place the T-75 flask at 37 degrees Celsius and 5%carbon dioxide. After overnight incubation, prepare 10 milliliters of PBEC growth medium, supplemented with puromycin at four micrograms per milliliter for one T-75 flask and perform a medium change.Then, prepare the permeable membrane inserts for PBEC seeding by coating them with collagen IV at 500 micrograms per milliliter, and fibronectin at 100 micrograms per milliliter, and by distilled water. Trypsin-ize the cells by adding two milliliters of trypsin-EDTA solution to the T-75 flask, and place it at 37 degrees Celsius, 5%carbon dioxide, for five to seven minutes. To detach the brain endothelial cells, gently tap the side of the T-75 flask with fingertips.Subsequently, transfer the cell solution to a 15-milliliter tube, and spin down the brain endothelial cells by centrifugation at 250 times g, four degrees Celsius, for seven minutes. Afterward, carefully remove the supernatant, and resuspend the pellet in one milliliter of medium. Then, add a further two milliliters of medium to bring the total volume to three milliliters.Count the number of cells manually using a cell-counting chamber or an automatic cell-counting system. For non-contact coculture, transfer inserts to the wells that have astrocytes, which were refreshed with astrocyte growth medium the day before. The next day, replace the medium on the permeable membrane inserts containing PBECs.Then, carefully aspirate the medium from the wells and add 750 microliters of differentiation medium to each well. Following that, carefully aspirate medium from the inserts and add 500 microliters of differentiation medium per insert. Then, add the remaining 750 microliters of differentiation medium to each astrocyte well.After that, incubate the cells with differentiation medium at 37 degrees Celsius, 5%carbon dioxide, until the next day. On the day after stimulating the cells with differentiation medium, prepare the tissue-resistance measurement chamber system for TEER measurements by rinsing the chamber two times with bi-distilled water, one time with 70%ethanol for five minutes and two more times with bi-distilled water. Next, connect the chamber to the system, add four milliliters of DMEM/F-12 to the tissue-resistance measurement chamber, and let it stay at room temperature for approximately 30 minutes.After that, perform TEER measurements by carefully placing the inserts in the tissue-resistance measurement chamber. Here are the phase phase-contrast microscopy images of PBECs, cultures, and astrocytes viewed over five days. Purified capillary fragments on the first day of the initial culture showed presence of both capillaries and contaminating cells, which after medium change on day two and an additional day of growth, showed selection for PBECs, starting their growth from the capillary fragments.Confluent monolayers of PBECs were usually achieved on day four through six, at which time PBECs showed spindle-shaped morphology and were longitudinally-aligned. Rat astrocytes seeded in the bottom wells normally proliferated to confluent layers within five days, and were used for non-contact coculture models after two weeks of growth. Once mastered, this technique can be done in seven hours.After its development, this technique paved the way for researchers to explore drug delivery and receptor trafficking in tight and polarized brain endothelial cells. After watching this video, you should have a good understanding how to create an in vitro blood-brain barrier model that can be used for new studies that cannot carry out in vivo.

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Medicine

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

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 ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

An Improved Method for Rapid Intubation of the Trachea in Mice

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity is a commonly used model to study both acute lung injury and fibrosis, and multiple methods have been developed for administering bleomycin (and other toxic agents) into the lungs. However, many of these approaches, such as transtracheal instillation, have inherent drawbacks, including the need for strong anesthetics and survival surgery. This paper reports a quick, reproducible method of intratracheal intubation that involves mild inhaled anesthesia, visualization of the trachea, and the use of a surrogate spirometer to confirm exposure. As a proof of concept, 8-12 week old C57BL/6 mice were administered either 2.0 U/kg of bleomycin or an equivalent volume of PBS, and both damage and fibrotic endpoints were measured post-exposure. This procedure allows researchers to treat a large cohort of mice in a relatively short period with little expense and minimal post-procedure care.

This article presents a rapid and simple method for administering bleomycin directly into the mouse trachea via intubation. Key advantages of this method are that it is highly reproducible, easy to master, and does not require specialized equipment or lengthy recovery times.

Despite some anatomical and physiological differences, mouse models continue to be an essential tool for studying human lung disease. Bleomycin toxicity ...

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle screws after spinal surgery, which is a serious concern. To address this problem, diverse types of pedicle screws have been examined to identify those with good fixation strength and osseointegration in spine bone. The porcine spine is a good alternative for the human spine in the evaluation of pedicle screws due to the anatomical size, mechanical characteristics, and cost. Although several studies have reported that pedicle screws are efficient in the porcine model, no study has described detailed protocols for the evaluation of a pedicle screw using the porcine model. Here, we describe a detailed method for evaluating transpedicular screws using an in vivo porcine lumbar spine model. The technical details for anesthesia, spine surgery, and harvest provided here will facilitate with the evaluation of the transpedicular screw fixation model.

An Anesthesia, Surgery, and Harvest Method for the Evaluation of Transpedicular Screws Using an In Vivo Porcine Lumbar Spine Model

Here, we introduce a method to evaluate transpedicular screws by using an in vivo porcine lumbar spine model.

Pedicle screw fixation is the gold standard for the treatment of spinal diseases. However, many studies have reported the issue of loosening pedicle ...

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...