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1. Establishing the BBB Model
Note: For establishing the blood-brain barrier (BBB) model we suggest using commercially available primary rat brain microvascular endothelial cells (RBMECs) and rat cortical astrocytes (RCAs). All steps must be performed with sterile reagents and disposables handled in a laminar flow hood.
<ol>
	<li>Cell Culture
	<ol>
		<li>Coat cell culture flasks with poly-L-lysine 100 &#181;g/ml (1 hr at room temperature, 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-ethylenediamine tetraacetic acid (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 TransEndothelial Electrical Resistance (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 (with 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>
</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). FnN are loaded with fluorescein isothiocyanate (FITC), and the concentrations of both Fn and the loaded molecules are accurately determined.
<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 - 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 spectrofluorometer (&#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>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Rat Brain Microvascular Endothelial Cells</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</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 &#176;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 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate-dextran 40000</td>
			<td>Sigma</td>
			<td>FD40S</td>
			<td>protect from light</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffer saline w/o Ca and Mg</td>
			<td>EuroClone</td>
			<td>ECB4004L</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>EuroClone</td>
			<td>ECS0200D</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</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 cm2; pore size 0.4 &#181;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</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>inverted microscope for live cells with camera</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Leica Microsystems</td>
			<td>TCS SPE</td>
			<td></td>
		</tr>
	</tbody>
</table>

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/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.

<img alt="Figure 1" src="/files/ftp_upload/23096/23096_Figure1.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.

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

1. Establishing the BBB Model
Note: For establishing the blood-brain barrier (BBB) model we suggest using commercially available primary rat brain ...

Begin with a multiwell plate containing an in vitro blood-brain barrier or BBB model.
This model consists of endothelial cells adhered to the upper or blood side and astrocytes on the lower or brain side of a matrix-coated membrane insert.
Add fluorescein isothiocyanate or FITC, a fluorescent dye, to the upper chamber of a control well.
Conversely, add FITC-loaded ferritin, or FITC-ferritin, to the upper chamber of a sample well and then incubate.
In the control well, free FITC remains confined to the upper chamber, with minimal crossing through the BBB.
Meanwhile, in the sample well, FITC-ferritin complexes bind to receptors on the endothelial cells.
This allows receptor-mediated internalization and transport into the lower chamber.
Next, regularly collect medium from the lower chambers of both wells and measure fluorescence intensity.
The control exhibits lower fluorescence, while the sample displays higher fluorescence, confirming the differential BBB permeability for FITC and FITC-ferritin.

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43 Views. Fonte: Fiandra, L., et al. Permeazione in vitro di ferritine caricate con FITC attraverso una barriera emato-encefalica di ratto: un modello per studiare la consegna di molecole nanoformulate. J. Vis. Exp. (2016). Questo video mostra un metodo per studiare la permeazione attraverso un modello di barriera emato-encefalica (BBB) in vitro utilizzando astrociti e cellule endoteliali su un inserto di membrana, con FITC libero e ferritina caricata con FITC. FITC-ferritina si è legata ai recettori su...

Video: Studio della permeazione di FITC e FITC-ferritina attraverso un modello di barriera emato-encefalica in vitro - Concept

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.

Fornitura di payload predicting In Vivo utilizzando una tumore-barriera emato-encefalica in un piatto

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

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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.

Istituzione di un<em&gt; In Vitro</em&gt; Modello di Rat barriera emato-encefalica (BBB): A Focus on BBB impermeabilità e Trasporti recettore-mediata

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

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Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

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

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