The authors acknowledge Assessorato alla Sanit&#224;, Regione Lombardia and Sacco Hospital (NanoMeDia Project) for research funding.

1. Establishing the BBB Model
Note: For establishing the BBB model we suggest using commercially available primary RBMECs and RCAs. All steps must be performed with sterile reagents and disposables, handled in a laminar flow hood.&#160;
<ol>
	<li>Cell Culture
	<ol>
		<li>Coat cell culture flasks with poly-L-lysine 100 &#181;g/ml (1 hr at RT) or fibronectin 50 &#181;g/ml (1 hr at 37 &#176;C) to promote the attachment of RCAs or RBMECs, respectively. Then, thaw 1 x 106 RCAs and 5 x 105 RBMECs in Endothelial Cell Medium, supplemented with 5% fetal bovine serum, 1% endothelial cell growth supplement and 1% penicillin/streptomycin (sECM). Seed RCAs in a T175 flask in 20 ml sECM and RBMECs in a T75 flask in 10 ml sECM. 
		Note: The thawing and seeding passages of RCAs and RBMECs must be adjusted, in terms of cell density and time in culture, according to the number of experimental conditions to be tested with the BBB model. By starting with 1 vial of 1 x 106 RCAs and 1 vial of 5 x 105 RBMECs, it is possible to obtain up to 20 BBB-bearing inserts.</li>
		<li>Maintain the cells at 37 &#176;C and 5% CO2 in a humidified atmosphere for approximately 6 days, until about 80% confluence for RCAs and over 90% confluence for RBMECs. Detach cells using trypsin-EDTA solution (trypsin 1:250) for 5 min (1 ml trypsin for T75 flasks and 2 ml for T175 flasks). Stop trypsin activity with sECM (2:1), centrifuge cell suspensions at 750 &#215; g for 5 min and resuspend the pellets in 60 ml sECM.</li>
		<li>Split the RBMECs into 3 T175 flasks and culture in sECM for another 3 days before seeding onto inserts. Count the total number of living RCAs, by observing a cell suspension diluted 1:1 with trypan blue under an optical microscope in a Burker chamber.</li>
	</ol>
	</li>
	<li>Cell Seeding onto Inserts
	<ol>
		<li>Treat one side of the Polyethylene terephthalate (PET) membrane of 6 multi-well transparent inserts with poly-L-lysine 100 &#181;g/ml and the other side with fibronectin 50 &#181;g/ml, to allow attachment of RCAs and BMECs. Handle the inserts with tweezers in order to avoid contact with the PET membrane.
		<ol>
			<li>Place the inserts into a 6 well plate and add fibronectin solution (minimum 500 &#181;l) into the upper chamber. After 1 hr of incubation at 37 &#176;C, remove the fibronectin solution, take the inserts off the multi-well plate and put them upside down on the bottom of a 150 cm2 Petri dish, which is used here as a sterile support for the already coated inserts.</li>
			<li>Gently add 800 &#181;l of poly-L-lysine onto the bottom side of the insert (as shown in Figure 1A; referred to as RCAs seeding), and keep the solution on the inserts for 1 hr at RT.</li>
			<li>Aspirate the solution and let the inserts dry at RT for 15-30 min. The inserts are now ready for cell seeding, but can also be stored in the multi-well plate for several days at 4 &#176;C, before proceeding with the BBB construction. 
			Note: Remember to keep at least 3 coated inserts free from cells, to be used as controls to validate BBB establishment by FD40 flux measurements.</li>
		</ol>
		</li>
		<li>Seed RCAs (35,000/cm2) onto the bottom side of the poly-L-lysine-coated inserts, by dropping 800 &#181;l of cell suspension onto the upside down insert (Figure 1A). Leave the RCA suspension on the inserts for 4 hr at RT, in order to allow efficient attachment of the cells to the membrane.</li>
		<li>Aspirate the residual solution, place the inserts into wells containing 2 ml of sECM and maintain the multi-well plate at 37 &#176;C and 5% CO2 in a humidified atmosphere, changing the sECM every 2 days.</li>
		<li>After 3 days, when the RCAs have coated the lower face of the insert, seed RBMECs onto the upper side of the insert, following these steps:
		<ol>
			<li>Detach RBMECs from T175 flasks and count the total living cells, as reported in steps 1.1.2 and 1.1.3.</li>
			<li>Seed RBMECs (60,000/cm2) onto the upper surface of the insert in sECM (1,000 &#181;l) (Figure 1B), leaving 3 inserts with the astrocytes only, to be used as TEER background. Place the multi-well plate in standard culture conditions. Maintain the system to culture for at least 3 days, changing the sECM in the inner and lower chamber every 2 days.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. BBB Validation
<ol>
	<li>TEER Measurement
	<ol>
		<li>On the 3rd day of co-culture, check the TEER by inserting the BBB-bearing inserts into an appropriate chamber(that has a cap and where both chamber and cap contain a pair of voltage-sensing and current electrodes), filled with 4 ml of sECM. Connect to an epithelial tissue volt/ohmmeter.</li>
		<li>Together with the TEER measurements of the cell BBB-systems, record the TEER values of the 3 inserts bearing the RCAs that will be subtracted from the values obtained with the BBBs. Multiply the resulting TEER values by the surface of the insert (4.2 cm2) in order to express the results as &#8486; x cm2.</li>
		<li>From the 3rd day of co-culture, measure the TEER every day. Note: After an initial period where the TEER increases, the recorded values should remain stable for at least two consecutive days (usually between the 5th and the 7th day of co-culture). At that point the BBB is ready for the second step of validation (section 2.2) and/or for the trans-BBB experiments. 
		Note: The procedure described in section 2.1 can be performed on the same BBB-systems devoted to the following permeability experiments (section 3). Operate under sterile conditions.</li>
	</ol>
	</li>
	<li>Trans-BBB Flux of FITC-dextran 40 (FD40)
	<ol>
		<li>Measure the FD40 flux from the upper to the lower chamber of the BBB models compared to that across 3 empty inserts (see the note of section 1.2.1) according to the following steps:
		<ol>
			<li>Add 1 mg/ml FD40 (diluted in sECM) into the upper compartment of the BBBs, and after 1, 2 and 3 hr withdraw 200 &#181;l sECM from the lower chamber and measure the fluorescence intensity by spectrofluorimeter (&#955;ex 488 nm, &#955;em 515 nm, slit 5).</li>
			<li>Obtain the values for sECM background fluorescenceby measuring 500 &#181;l of virgin medium using the same analytical parameters. Subtract the sECM background fluorescence from all FD40 fluorescence values.</li>
			<li>Determine the amount of permeated FD40 by comparison of the observed fluorescence values with a calibration curve produced with known concentrations (i.e. 0.312, 0.625, 1.25, 2.5 &#181;g/ml) of the fluorescent tracer dissolved in 500 &#181;l of sECM.</li>
			<li>Calculate the apparent permeability coefficient (Papp) from the mean flux values according to: 
			Papp = J/AC 
			where J is the flux of the molecule (moles/sec), A is the permeation area (cm2) and C is the concentration of the molecule in the upper compartment (moles/cm3). 
			Note: Three or more BBB-systems that have reached suitable TEER values, must be exclusively devoted to this validation procedure, and should not be employed for the following permeability experiments (section 3). Sterility is not mandatory.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Trans-BBB Permeation of FITC-loaded Ferritins (FnN)
Note: A recombinant variant of human ferritin (Fn), produced in Escherichia coli and assembled in nanocages (FnN) for the encapsulation of different fluorescent molecules, is available from the NanoBioLab of Prof. Prosperi (University of Milan-Bicocca, Italy). FnN are loaded with FITC, according to a previously described protocol13 and the concentrations of both Fn and the loaded molecules are accurately determined.&#160;
<ol>
	<li>Trans-BBB Flux of FITC and FITC-FnN
	<ol>
		<li>Measure the FITC-FnN flux from the upper to the lower chamber of validated BBB models (usually at the 5th&#160;- 7th day of co-culture), compared to that of the free dye after 7 and 24 hr of incubation, according to the following steps:
		<ol>
			<li>Add FITC-FnN (50 &#181;g/ml FnN, 1.1 &#181;M FITC) or an equal amount of free FITC into the upper chamber of at least 6 BBB systems for each formulation, in order to withdraw 2 ml of sECM solution from the lower chamber of at least 3 inserts after 7 hr, and from the lower chamber of other 3 inserts after 24 hr.</li>
			<li>Measure the fluorescence intensity of 500 &#181;l of the collected samples by spectrofluorimeter (&#955;ex 488 nm, &#955;em 515 nm, slit 5).</li>
			<li>Obtain the sECM background fluorescence by measuring 500 &#181;l of medium using the same analytical parameters. Subtract the sECM background fluorescence from all FITC or FITC-FnN fluorescence values.</li>
			<li>Determine the concentration of permeated FITC or FITC-FnN by comparing the obtained fluorescence values with two different calibration curves produced with known concentrations (i.e. 6.87, 13.75, 27.5, 55 nM) of the free or nanoformulated dye dissolved in 500 &#181;l sECM.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>FITC-FnN Localization in RBMECs
	<ol>
		<li>Remove sECM from the upper chamber of the BBB systems, wash the inserts with PBS and fix the RBMECs on at least two inserts for each experimental group by adding 500 &#181;l paraformaldehyde (4% in phosphate buffer saline-PBS) in the upper compartment for 10 min at RT.</li>
		<li>Wash the inserts three times with PBS to remove residual paraformaldehyde. 
		Note: From this point on, sterility is not necessary.</li>
		<li>Cut suitable pieces of the PET membrane, and proceed with the immunodecoration of the cells according the following steps:
		<ol>
			<li>Permeabilize RBMECs with 0.1% Triton X-100 in PBS for 10 minutes. Perform a blocking step for 1 hr at RT with a solution containing 2% bovine serum albumin (BSA), 2% goat serum in PBS. Incubate the samples with an anti-Von Willebrand Factor (VWF) at 1:20 dilution, for 2 hr at RT.</li>
			<li>After washing three times with PBS, expose the cells for 2 hr at RT to a 2% BSA, 2% goat serum solution containing a suitable secondary antibody at a 1:300 dilution in order to reveal the anti-VWR, and DAPI at a 1:1,500 dilution for nuclei detection.</li>
		</ol>
		</li>
		<li>Mount the insert pieces onto microscope slides using an antifade mounting solution (one drop per insert fragment), then close the sample with a cover slip. Analyze by confocal microscopy using an oil immersion lens at 40X&#160;magnification, 1.5 zoom and 1,024 x 1,024 pixel resolution.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

The in vitro method described here represents a useful validated approach to study the trans-BBB delivery of fluorescent molecules upon nanoformulation with nanoparticles. Here we use FnN, which represents a good candidate to study the translocation of cargo molecules across the BBB. FnN is considered the gold nanovector for trans-BBB delivery of drug/agents since it is specifically recognized by the TfR1 receptor, which is expressed on the luminal membrane of BMECs and mediates the nanoparticle uptake using a receptor-mediated internalization pathway. Moreover, FnN is a natural nanoparticle and it has therefore a good biocompatibility profile. Finally, FnN is able to encapsulate low dimension hydrosoluble molecules by a simple and efficient mechanism. The trans-BBB permeation of other different types of organic or inorganic nanoparticles could be also assessed with this protocol, according to few considerations regarding nanoparticles features. First prerequisite to study the permeation of nanoformulated drugs/agents is the safety of the void nanoparticle. Another crucial issue is the interaction of the investigated nanoparticle with the PET filter. This aspect, has to be evaluated preliminarily in order to exclude a significant retention into the membrane pores and avoid alterations of their permeation across the BBB. Our group has recently employed the proposed strategy to study a polymer-coated iron oxide nanoparticle as a trans-BBB delivery system for fluorescence-labeled antiretroviral drugs14. In that case, we associated electron microscopy localization of nanoformulations in RBMECs to the fluorescence detection. Moreover, other highly sensitive diagnostic methods, such as Inductively Coupled Plasma (ICP)-MS could be employed to evaluate the trans-BBB delivery of inorganic nanosystems.
The BBB in vitro model used in this protocol is based on co-culture of rat BMECs and astrocytes. In the wide scenario of the BBB models10,11,12, some of which have been accurately described in literature with a well-detailed protocol15, our model represents a good quality alternative. Indeed, even though the recorded TEER was below the best standards suggested in the literature (150-200 &#8486; cm2)10, the trans-BBB permeability of the high dimension tracer Dextran 40 was in total agreement with the formation of a tight barrier.
This in vitro model, obtained with commercially available rat BMECs and astrocytes, has a few advantages: (1) optimal growth efficiency of the endothelial cells, (2) a suitable period of time for the production of the final BBB model (no longer than 13 days), (3) the compliance with ethical issues, avoiding the use of cells from human brain tissue (autopsy materials, surgical specimens, and fetal tissue) and 4) saving of time and costs related to animal housing, and primary cells extraction. Nevertheless, a limitation of the present model is related to the high costs of non-immortalized primary RBMECs, which must be purchased (frozen at passage one) for each experimental setting. Indeed, our preliminary studies of BBB production have demonstrated that the ability of endothelial cells to produce a tight BBB is strictly associated with the number of passages in culture of the RBMECs after the first post-purchase thawing. Further implementation of the BBB model described here with endothelial supplements, such as cAMP and hydrocortisone, could be considered in order to improve endothelial tightness and increase TEER values.10,11,16
The present technique is related to the possibility to take advantage of a single BBB model for different assays, thus providing several data sets from each experimental setting. With a single BBB system exposed to free or nanocomplexed fluorescent molecules, it is possible: (1) to measure the trans-barrier flux of the molecule by analyzing the fluorescence intensity of sECM aliquots collected from the lower chamber at different time points of incubation; (2) to investigate the nano-mediated internalization of the molecules in RBMECs and their intracellular trafficking by confocal microscopy analysis of the cells on inserts; (3) to get an indication of the status of the BBB cells upon exposure to the nanoformulations, by measuring TEER at the end of the experiment or by analyzing endothelium integrity by electron microscopy.
In conclusion, here we described a protocol for the study of the permeation of nanocomplexed fluorescent molecules across a high-quality BBB in vitro model. We consider this methodology a useful tool for investigating the effect of nanoformulation on drug delivery across the BBB.

The resistance of central nervous system (CNS) diseases (i.e. cancer, epilepsy, depression, schizophrenia and HIV-associated neurological disorder) to pharmacological therapies is due to various different mechanisms, including arduous drug permeation across the blood-brain barrier (BBB). The BBB is the boundary that isolates brain tissues from the substances circulating in the blood. Within this barrier, a layer of brain microvascular endothelial cells (BMECs), supported by pericytes and astrocytes endfeet, is responsible for the high selectivity of the BBB to those hydrosoluble drugs with a molecular weight higher than 400 Da1. Another drug-related resistance mechanism is linked to the presence on BMECs of drug efflux transporters (P-glycoprotein and multidrug resistance proteins), which co-operate to reduce drug penetration into the CNS and facilitate their extrusion from the brain2.
In the last decade, a large number of nanotechnological approaches have been developed to meet the clinical and biological challenge of delivering drugs across the BBB3-6. In this context, ferritin nanospheres (FnN) represent a completely innovative and promising solution. FnN are 12 nm spheres of 24 self-assembling ferritin (Fn) monomers, which are arranged in a hollow spherical structure of 8 nm inner diameter. Ferritin subunits can be disassembled at acidic pH and reassembled in a shape-memory fashion by bringing the pH to neutrality, allowing various organic molecules to be encapsulated. Therefore, FnN represent an interesting model for the development of multifunctional drug delivery systems7,8. Moreover, FnN may interact with BMECs thanks to the specific recognition of Transferrin Receptor (TfR) 1, which is expressed on the luminal membrane of these cells9.
So far, different in vitro models of the BBB have been developed in order to elucidate trans-BBB permeability to various drugs, toxicity toward the BBB, or the interaction of molecules with efflux transporters. Indeed, these models are considered valid in vitro approaches for a rapid screening of active molecules before proceeding with in vivo studies. These models consist of a single endothelial layer of BMECs or co-cultured BMECs and astrocytes (more rarely pericytes), obtained from animal (rat, mouse, pig and bovine) or human cell lines10,11,12. The TransEndothelial Electrical Resistance (TEER) and the apparent permeability (Papp) of tracers with a defined molecular weight represent two critical parameters that are used to determine the quality of the in vitro model. Here we describe the employment of a BBB in vitro model, based on a co-culture of rat BMECs (RBMECs) and rat cortical astrocytes (RCAs) to study the trans-BBB permeation of ferritin nanocages encapsulating fluorescein isothiocyanate (FITC).

<table><tbody><tr><td>Rat Brain Microvascular Endothelial Cells&amp;nbsp;</td><td>Innoprot</td><td>P10308</td><td>isolated from Sprague Dawley rat brain tissue, cryopreserved at passage one and delivered frozen</td></tr><tr><td>Cortical Astrocytes&amp;nbsp;</td><td>Innoprot</td><td>P10202</td><td>isolated from 2 days rat brain tissue, cryopreserved at passage one and delivered frozen.</td></tr><tr><td>Endothelial Cell Medium kit</td><td>Innoprot</td><td>P60104</td><td>ECM (500 ml) and fetal bovin serum (25 ml), endothelial cell growth supplement (5 ml) and penicillin/streptomycin (5 ml). Warm in 37 &amp;deg;C water bath before use and protect from light</td></tr><tr><td>Trypsin-EDTA without Phenol Red</td><td>EuroClone</td><td>ECM0920D</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Fluorescein isothiocyanate-dextran 40,000</td><td>Sigma</td><td>FD40S</td><td>protect from light</td></tr><tr><td>paraformaldehyde&amp;nbsp;</td><td>Sigma</td><td>158127</td><td>diluition in chemical hood</td></tr><tr><td>Dulbecco&#039;s phosphate buffer saline w/o Ca and Mg</td><td>EuroClone</td><td>ECB4004L</td><td></td></tr><tr><td>Triton&amp;nbsp;X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>bovine serum albumin&amp;nbsp;</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>goat serum&amp;nbsp;</td><td>EuroClone</td><td>ECS0200D</td><td></td></tr><tr><td>mouse monoclonal anti-Von Willebrand Factor</td><td>Dako</td><td>M0616</td><td></td></tr><tr><td>AlexaFluor 546-conjugated antibody against mouse IgGs</td><td>ThermoFischer Scientific</td><td>A-11003</td><td>protect from light</td></tr><tr><td>DAPI (4&amp;rsquo; ,6-diamidino-2-phenylindole)&amp;nbsp;</td><td>ThermoFischer Scientific</td><td>D1306</td><td>protect from light</td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>ThermoFischer Scientific</td><td>P36934</td><td></td></tr><tr><td>Poly-L-lysine Hydrobromide</td><td>Sigma</td><td>P1274</td><td>the same solution can be used several times</td></tr><tr><td>fibronectin from bovine plasma&amp;nbsp;</td><td>Sigma</td><td>F1141</td><td>the same solution can be used several times</td></tr><tr><td>Polyethylene terephthalate (PET) inserts</td><td>Falcon</td><td>F3090</td><td>Transparent Polyethylene terephthalate (PET) membranes; surface area: 4.2 cm&lt;sup&gt;2&lt;/sup&gt;; pore size 0.4 &amp;micro;m/surface area</td></tr><tr><td>T75 Primo TC flask</td><td>EuroClone</td><td>ET7076</td><td></td></tr><tr><td>T175 Primo TC flask</td><td>EuroClone</td><td>ET7181</td><td></td></tr><tr><td>EVOM2 Epithelial Tissue Volt/Ohmmeter&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td>World Precision Instruments Germany</td><td>EVOM2</td><td></td></tr><tr><td>Endohm- 24SNAP cup</td><td>World Precision Instruments Germany</td><td>ENDOHM-24SNAP</td><td></td></tr><tr><td>Light/fluorescence microscope with camera</td><td>Leica Microsystems</td><td>DM IL LED Fluo/ ICC50 W Camera Module</td><td>&amp;nbsp;inverted microscope for live cells with camera&amp;nbsp;</td></tr><tr><td>Confocal Microscope</td><td>Leica Microsystems</td><td>TCS SPE</td><td></td></tr><tr><td>Spectrofluorimeter</td><td>Jasco</td><td>FP-8000</td><td></td></tr></tbody></table>

in vitro permeation of fitc-loaded ferritins across a rat blood-brain barrier: a model to study the delivery of nanoformulated molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

During the establishment of the BBB model, cell attachment and growth on the inserts can be monitored using a light microscope thanks to the transparent nature of the PET membranes. RCAs, seeded at a density of 35,000 cells/cm2, attach efficiently to the bottom side of the insert after 4 hr of incubation at RT (Figure 2A) and grow to cover the membrane surface in 3 days, taking a spindle-shaped morphology (Figure 2B). RBMECs, seeded at a density of 60,000 cells/cm2, are visibly attached to the upper face of the PET membrane after about 3 hr of incubation at 37 &#176;C (Figure 2C). The developing RBMEC layer can be difficult to visualize over the following days with an optical microscope, because of the overlap with the underlying RCA layer (Figure 2D).
A correct validation of the BBB model always requires TEER measurement and this can be confirmed by evaluating the trans-BBB Papp of a low permeability tracer, such as FD40.
The TEER values, recorded over the co-culture period, represent the first clear indication of the correct formation of the endothelial barrier. Three days after RBMEC seeding, the recorded TEER, subtracted from the TEER of the astrocytes-bearing inserts, is mainly due to the contribution of the non-electrogenic layer of RCAs and the developing layer of RBMECs. At this time point, our BBB gives values ranging between 20 and 40 &#8486; x cm2. During the following days, usually between the 4th and the 5th day of co-culture, the TEER values increase because of the formation of tight junctions between adjacent endothelial cells14, reaching values usually between 55 and 110 &#8486; x cm2, and in exceptional cases higher values14. The values measured at the 4th/5th day of co-culture remain stable at least until the 7th&#160;- 8th day before they start to decrease; therefore, there is a very narrow time window available for undertaking the trans-BBB flux experiments.
Between the 5th and the 7th day of co-culture, the integrity of the experimental models can be confirmed by evaluating the FD40 trans-BBB permeability. Figure 3 shows an example of the trans-BBB flux of FD40 (1 mg/ml), compared to the flux across empty inserts, over 3 hr of incubation; the BBBs are at the 6th day of co-culture and the recorded TEER is 55.6 &#177; 15.8 &#8486; cm2 (mean &#177; SE, n = 3). The flux is linear between 1 and 3 hr of incubation and the mean Papp calculated between 1 and 2 hr, and between 2 and 3 hr of incubation is 0.12 &#177; 0.01 x 10-6 cm sec-1 (&#177; SE, n = 6).
Once at least three consecutive successful BBBs, in terms of TEER and FD40 Papp, have been obtained in independent experiments the measurement of tracer permeability can be avoided; however, the TEER always needs to be recorded for each experiment.
The trans-BBB permeability of fluorescent molecules and the effect of the nanocomplex on their delivery can be investigated using the rat BBB models described above. Figure 4 shows the permeation of the model dye FITC upon encapsulation in FnN across BBBs with TEER of 100.4 &#177; 3.5 &#8486; cm2 (n = 16) at the 7th day of co-culture. The histograms, representing FITC concentration in the lower chamber after 7 and 24 hr from the addition of free or nanoformulated dye in the upper compartment, indicate that FnN is able to significantly increase the delivery of FITC across the BBB.
Confocal microscope images of the upper side of the insert after 7 and 24 hr of incubation with FITC-FnN (Figure 5A, C) or FITC (Figure 5B, D) show that while free FITC is not internalized by the RBMECs, its loading into the FnN allows it to enter the cells.
Before processing the inserts for confocal microscopy, a further check of the TEER is necessary to ensure there are no FnN-mediated effects upon BBB integrity.
<img alt="Figure 1" src="/files/ftp_upload/54279/54279fig1.jpg" /> 
Figure 1: Cell Seeding onto Inserts. RCA (A) and RBMEC (B) seeding procedure onto the two opposite sides of the multi-well plate inserts. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54279/54279fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54279/54279fig2.jpg" /> 
Figure 2: Optical Microscope Images of RCAs and RBMVECs on Inserts. Inserts seeded with RCAs and RBMECs are observed with a light microscope (20X&#160;optical zoom). (A) Four hr after seeding, round and translucent RCAs are visibly attached to the bottom surface of the insert; (B) Spindle-shaped RCAs are visible on the 3rd day of culture; (C) Round and translucent RBMECs are attached on the upper side of the insert after 3 hr of incubation; (D) On the 4th day of co-culture both the surfaces of the insert are completely covered with cells, and the two layers are not easily distinguishable. Scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54279/54279fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54279/54279fig3.jpg" /> 
Figure 3: FD40 Flux across the BBB. Flux of FD40 (1 mg/ml) from the upper to the lower side of the BBB in vitro system, compared to that across the empty insert. The amount of FD40 in lower chamber has been measured at 60, 120 and 180 min post the addition of the dye to the upper chamber. Means &#177; SE; n&#176; inserts = 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54279/54279fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54279/54279fig4.jpg" /> 
Figure 4: Effect of FnN Encapsulation on FITC Permeation Across the BBB. Concentration of FITC in the lower chamber of the BBB in vitro system calculated at 7 and 24 hr after the addition of FITC or FITC-FnN into the upper chamber. Mean &#177; SE of 4-5 replicates; ****P &#60;0.0005, FITC-FnN vs. FITC (Student&#39;s t-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54279/54279fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54279/54279fig5.jpg" /> 
Figure 5: Confocal Microscopy of RBMECs on Inserts. Confocal laser-scanning micrographs (single optical sections) of RBMECs after 7 hr (A, B) or 24 hr (C, D) of incubation with free FITC (B, C) or FITC-FnN (A, C). FITC is green; endothelial cells are immunodecorated with anti-VWF (red) and DAPI (blue). Panels represent, from left to right, merged images of blue and red channels, green channel images, and merged images of all channels. Scale bar = 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54279/54279fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules at JoVE.com

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs ...

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7.8K Views.  Università di Milano. A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

Video: In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Predicting In Vivo Payloads Delivery using a Blood-brain Tumor-barrier in a Dish

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of brain tumors, the molecular selectivity of BBB also shields the neoplastic cells by blocking the delivery of peripherally administered chemotherapies. The development of novel drugs (including nanoparticles) targeting malignant brain tumors ideally requires the use of preclinical animal models to study the drug&#8217;s transcytosis and antitumor efficacy. In order to comply with the 3R principle (refine, reduce, and replace) to reduce the number of laboratory animals in experimental setup and perform the high-throughput screening of a large library of antitumor agents, we developed a reproducible in vitro human and murine mimic of the blood-brain tumor-barrier (BBTB) using three-layered cultures of endothelial cells, astrocytes, and patient-derived glioblastoma spheres. For higher scalability and reproducibility, commercial cell lines or immortalized cells have been used in tailored conditions to allow the formation of a barrier resembling the actual BBB. Here we describe a protocol to obtain a BBTB mimic by culturing endothelial cells in contact with astrocytes at specific cell densities on inserts. This BBTB mimic can be used, for instance, for the quantification and confocal imaging of the nanoparticle passage through the endothelial and astrocytic barriers, in addition to the evaluation of the tumor cell targeting within the same assay. Moreover, we show that the obtained data can be used to predict the behavior of nanoparticles in preclinical animal models. In a broader perspective, this in vitro model could be adapted to other neurodegenerative diseases for the determination of the passage of new therapeutic molecules through the BBB and/or be supplemented with brain organoids to directly evaluate the efficacy of drugs.

Drug targeting to central nervous system tumors is a major challenge. Here we describe a protocol to produce an in vitro mimic of the blood-brain tumor-barrier using murine and/or human cells and discuss their relevance for the predictability of central nervous system tumor targeting in vivo.

Highly selective by nature, the blood-brain barrier (BBB) is essential for the brain homeostasis in physiological conditions. However, in the context of ...

Cancer Research

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 ...

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 ...

Medicine

Assessment of Blood-brain Barrier Permeability by Intravenous Infusion of FITC-labeled Albumin in a Mouse Model of Neurodegenerative Disease

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay between perturbed BBB homeostasis and the pathogenesis of brain disorders needs further investigation, the development and validation of a reliable procedure to accurately detect BBB alterations may be crucial and represent a useful tool for potentially predicting disease progression and developing targeted therapeutic strategies.
Here, we present an easy and efficient procedure for evaluating BBB leakage in a neurodegenerative condition like that occurring in a preclinical mouse model of Huntington disease, in which defects in the permeability of BBB are clearly detectable precociously in the disease. Specifically, the high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin, which is able to cross the BBB only when the latter is impaired, is acutely infused into a mouse jugular vein and its distribution in the vascular or parenchymal districts is then determined by fluorescence microscopy.
Accumulation of green fluorescent-albumin in the brain parenchyma functions as an index of aberrant BBB permeability and, when quantitated by using Image J processing software, is reported as Green Fluorescence Intensity.

In this study, we present an easy and efficient procedure for evaluating the disruption of the blood-brain barrier under neurodegenerative conditions. To achieve our objective, we infused high molecular weight fluorescein isothiocyanate labelled (FITC)-albumin into mouse jugular vein and evaluated its leakage into the brain parenchyma by fluorescence microscopy.

Disruption of blood-brain barrier (BBB) integrity is a common feature for different neurological and neurodegenerative diseases. Although the interplay ...

Neuroscience

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

Blood-Brain Barrier Mimic Permeability Assay: An In Vitro Assay to Assess the Permeability of BBB Mimic to Tracer Molecules

Source: Le Joncour, V. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59384/predicting-vivo-payloads-delivery-using-blood-brain-tumor-barrier">Predicting In Vivo Payloads Delivery using a Blood-Brain Tumor-Barrier in a Dish.</a> J. Vis. Exp. (2019)
This video describes a permeability assay to evaluate the permeability of in vitro blood-brain barrier using sodium fluorescein molecules.

Source: Le Joncour, V. et al. Predicting In Vivo Payloads Delivery using a Blood-Brain Tumor-Barrier in a Dish. J. Vis. Exp. (2019)
This video describes a ...

JoVE Encyclopedia of Experiments

Cancers of the Nervous System

In vitro studies

Studying the Permeation of FITC and FITC-Ferritin through an In Vitro Blood-Brain Barrier Model

Source: Fiandra, L., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54279">In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules.</a> J. Vis. Exp. (2016).
This video demonstrates a method to study permeation through an in vitro blood-brain barrier (BBB) model using astrocytes and endothelial cells on a membrane insert, with free FITC and FITC-loaded ferritin. FITC-ferritin bound to receptors on endothelial cells and crossed the membrane barrier to the lower chamber while free FITC showed restricted permeation and remained in the upper chamber.

In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

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