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) ...

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