This work was supported by NIH grants (R01 EY028100, EY024963, and EY031765) to JC. ZW was supported by a Knights Templar Eye Foundation Career Starter Grant.

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Boston Children&#39;s Hospital for the generation of light microscopy and EM images (Figure 3). Protocols for the in vivo studies can be obtained from Wang et al.24. All experiments involving human retinal microvascular endothelial cells (HRMECs) were approved by the Institutional Biosafety Committee (IBC) at Boston Children&#39;s Hospital.
1. Preparation of Reagents
<ol>
	<li>Solution for coating tissue culture dish: Prepare 0.1% gelatin solution by dissolving 1 mL of gelatin stock solution (40%-50%) in 500 mL of sterile tissue culture grade 1x PBS (pH = 7.4). Filter the solution through a 0.22 &#181;m filter. Store 0.1% gelatin solution at 2-8&#176;C. It can be stored at the said temperature for an indefinite time in a sterile condition.</li>
	<li>Growth medium: Prepare 500 mL of endothelial cell growth complete medium (EGM) by dissolving EGM supplements into the endothelial cell growth basal medium (EBM) according to the manufacturer&#39;s instructions (Table of Materials). Aliquot growth medium into 50 mL tubes and store the tubes at 4 &#176;C. The shelf life of the growth medium is 12 months at 4 &#176;C.</li>
	<li>Trypsin solution: For 0.25% trypsin-EDTA solution, aliquot from the stock vial into 50 mL tubes and store at 4 &#176;C (up to 2 weeks) or &#8722;20 &#176;C (up to 24 months).</li>
</ol>
2. Culturing HRMECs
<ol>
	<li>Initial cell culture
	<ol>
		<li>Coat cell culture Petri dishes (100 mm diameter x 20 mm height) with 5 mL of 0.1% gelatin solution under a laminar flow hood and keep them undisturbed in the hood for 30 min at room temperature (RT) for uniform coating. 
		NOTE: Gelatin coating of dishes enhances cell attachment. Alternatively, gelatin-coated T75 culture flasks can also be used.</li>
		<li>Before seeding the cells, aspirate the gelatin solution using a vacuum pump. The vacuum pressure of the aspirator used was up to 724 mmHg. 
		NOTE: Aspiration must be done immediately before seeding cells to prevent drying out of the coating solution.</li>
		<li>Thaw a frozen vial of HRMECs (from storage in liquid nitrogen) either by using a water bath at 37 &#176;C or by adding a warm growth medium into the vial. Once thawed, transfer the cell suspension to 9 mL of growth medium (assuming thawed vial of HRMECs is 1 mL). 
		NOTE: The HRMECs were commercially obtained (Table of Materials). The growth medium added to the cells should always be prewarmed to 37 &#176;C before use.</li>
		<li>Spin the cells at 200 x g for 5 min at RT and carefully aspirate the supernatant to avoid removing the cell pellet.</li>
		<li>Resuspend the cell pellet in 10 mL of growth medium and transfer the resultant suspension onto a gelatin-coated Petri dish. Keep the cells in the incubator at 37 &#176;C and 5% CO2. Typically, HRMECs are 70%-80% confluent after 72 h. 
		NOTE: The growth medium is changed every other day.</li>
	</ol>
	</li>
	<li>Subculture of HRMECs on permeable membrane inserts
	<ol>
		<li>Prepare cell culture filter inserts (6.5 mm inserts with 0.4 &#181;m pore size polycarbonate membrane, placed in a 24-well plate) for seeding HRMECs by coating each insert (apical chamber) with 200 &#181;L of 0.1% gelatin solution for 30 min under a laminar flow hood. Ensure the solution covers the entire bottom surface of the filter insert. 
		NOTE: Each well has an apical and basolateral chamber separated by a porous membrane.</li>
		<li>Take out the Petri dish with cultured HRMECs (step 2.1.5.) from the incubator and aspirate the growth media. Gently rinse the cells 2x with 10 mL of 1x PBS under a laminar flow hood to get rid of potential floating/dead cells.</li>
		<li>Dissociate the cells with 0.5-1 mL of 0.25% trypsin-EDTA solution and place the Petri dish in the incubator (37 &#176;C and 5% CO2)&#160;for 5 min.</li>
		<li>Quench the trypsin activity by adding 4.5-9 mL of growth media and transfer the cell suspension into a 15 mL tube using a 10 mL pipette.</li>
		<li>Spin the cells at 200 x g for 5 min at RT, carefully remove the supernatant, and resuspend the pellet in 3 mL of growth media.</li>
		<li>Count the number of cells using a manual hemocytometer or an automated cell counter and seed at a density of 4 x 104 cells per filter insert (i.e., 1.25 x 105 cells/cm2). The volume of cell suspension for each insert is 250 &#181;L.</li>
		<li>Aspirate the coating solution from the wells containing permeable inserts and transfer 250 &#181;L of the cell suspension per insert (apical chamber). Add only medium (i.e., without cells) to one of the inserts, which will be used as a &#34;blank control&#34; for TEER measurement. Simultaneously, also add 750 &#181;L of medium per well, in the basolateral chambers.</li>
		<li>Keep the 24-well plate with permeable inserts in the incubator (37 &#176;C and 5% CO2) for 7-12 days until the cultured cells become fully confluent and the desired TEER value of ~20 &#8486;&#183;cm2 is achieved.</li>
		<li>Change the growth medium every other day. During media change, carefully aspirate the media to minimize disruption of the cell monolayer and add 250 &#181;L and 750 &#181;L of fresh media per well to the apical and basolateral chambers, respectively. 
		&#8203;NOTE: Growth medium is changed for both the apical and basolateral chambers of the wells.</li>
	</ol>
	</li>
</ol>
3. TEER measurements (Figure 4)
<ol>
	<li>On day 14 post cell seeding (step 2.2.9.), measure the TEER for HRMECs using an epithelial volt-ohm meter (EVOM) electrical resistance (ER) system (Table of Materials) as follows.</li>
	<li>Pre-charge the ER system and check the meter functionality using the STX04 test electrode. Calibrate, if required.</li>
	<li>Connect the electrode to the meter and equilibrate the electrode by first soaking it in 70% ethanol for 15-20 min and then immersing it briefly in the cell culture EGM growth medium.</li>
	<li>In the meantime, keep the cell-containing 24-well plate in the laminar flow hood at RT for 15-20 min for temperature equilibration. 
	NOTE: TEER measurements are affected by temperature, so the equilibration step is essential.</li>
	<li>For TEER measurement, add fresh growth medium to both the apical (250 &#181;L) and basolateral (750 &#181;L) chambers of the wells.</li>
	<li>Perform TEER measurement by carefully immersing the electrode such that the shorter tip is in the insert and the longer tip touches the bottom of the well. Measure the resistance across the blank control first. For each insert, measure TEER in triplicates. 
	NOTE: For rinsing the electrode in between the measurements, culture media is used. Ensure the electrode is held at a 90&#176; angle to the bottom of the insert for stable readings.</li>
	<li>Calculate electrical resistance (in &#8486;&#183;cm2) across the monolayer using the formula, TEER = net resistance (&#8486;) x surface area of filter insert (cm2); here, net resistance is the difference between the resistance of each well (growth medium with cells) and the blank well (only growth medium).</li>
	<li>Carry out further treatment of the cells only after the TEER value reaches ~20 &#8486;&#183;cm2. If the desired TEER level is not reached, keep the plate in the incubator and measure the TEER the following day. 
	&#8203;NOTE: HRMEC confluency can also be validated by morphological examination of cell shape (under a microscope) with typical cobblestone morphology and/or by the presence of cell junction proteins with immunohistochemistry staining separately.</li>
</ol>
4. Transcytosis assay
<ol>
	<li>Clathrin-mediated in vitro transcytosis assay using Cy3-tagged transferrin (Figure 5)
	<ol>
		<li>Upon reaching confluency with TEER values around 20 &#8486;&#183;cm2, serum deprive the cells for 24 h at 37 &#176;C and 5% CO2 using 0.5% FBS in EBM (serum-reduced medium) in both chambers (apical chamber: 250 &#181;L and basolateral chamber: 750 &#181;L) prior to treatment with the ligand. Serum-reduced EBM was used throughout the assay.</li>
		<li>Incubate the cells (using the serum-reduced medium in step 4.1.1.) in the apical chamber with fluorescent (cyanine 3)-tagged transferrin ligand (Cy3-Tf) (final concentration of 40 &#181;g/mL) for 60 min at 37 &#176;C. 
		NOTE: Plates containing Cy3-Tf should be protected from light to avoid photobleaching of Cy3-Tf by wrapping in them aluminum foil and performing the experiment in a cell culture hood with the lights turned off.</li>
		<li>After 1 h, place the plate on ice and wash the monolayer apically and basolaterally 4x (3-5 min per wash) with the serum-reduced medium at RT to remove the free unbound Cy3-Tf. 
		NOTE: Washing is essential to remove free tracer molecules and allow accurate reading of transcytosis without potential leakage from the paracellular route.</li>
		<li>Add fresh serum-reduced medium (as in step 4.1.1.) to the thoroughly washed filter inserts containing cells and transfer the inserts to fresh wells of the 24-well plate containing pre-warmed serum-reduced medium.</li>
		<li>Incubate the cells for another 90 min in the incubator (37 &#176;C and 5% CO2), and then collect the medium from the basolateral chamber.</li>
		<li>Record the fluorescence intensity of the solution from the basolateral chamber using a fluorescence detector. The levels of fluorescence intensity, measured as relative fluorescent units (RFU), indicate the amount of Cy3-Tf complex transcytosed across the HRMEC monolayer via clathrin-dependent transcytosis.</li>
	</ol>
	</li>
	<li>Caveolae-mediated in vitro transcytosis assay using HRP (Figure 6)
	<ol>
		<li>Upon reaching full confluency with TEER values around 20 &#8486;&#183;cm2, serum deprive the cells for 24 h at 37 &#176;C and 5% CO2 using 0.5% FBS-containing EBM medium (serum-reduced medium) before treatment (as in step 4.1.1.). Serum-reduced EBM medium was used throughout the entire assay.</li>
		<li>Treat the cells in the apical chamber with the desired treatments and vehicle controls. 
		NOTE: Here, we have used Wnt modulators as an example to demonstrate the regulation of caveolae-mediated transcytosis by the Wnt signaling pathway in HRMECs: human recombinant Norrin and Wnt inhibitor XAV939. In a typical experiment, cells were treated for 24 h in an incubator (37 &#176;C and 5% CO2) with the following concentrations: Norrin (125 ng/mL), Norrin (125 ng/mL) + XAV939 (10 &#181;M), and vehicle control solution. In addition, Wnt3a-conditioned medium (produced from L Wnt-3A cells) was also used.</li>
		<li>Incubate the cells in the apical chamber with HRP (5 mg/mL) for 15 min at 37 &#176;C.</li>
		<li>Afterward, place the 24-well plate on ice and wash the apical and basolateral chambers intensively 6x with P buffer (10 mM HEPES pH = 7.4, 1 mM sodium pyruvate, 10 mM glucose, 3 mM CaCl2, 145 mM NaCl) in order to remove the free extracellular HRP. 
		NOTE: Washing is essential to remove free tracer molecules and allow accurate reading of transcytosis without potential leakage from the paracellular route.</li>
		<li>Add fresh serum-reduced medium (as in step 4.1.1.) to the apical chamber and transfer the inserts to a fresh well containing pre-warmed media.</li>
		<li>Incubate the monolayer for an additional 90 min in the incubator at 37 &#176;C and 5% CO2 before collecting the medium from the basolateral chamber.</li>
		<li>To the collected medium, add 100 &#181;L of HRP fluorogenic peroxidase substrate (Table of Materials) as per the manufacturer&#39;s instructions, and incubate at RT for 10 min before stopping the reaction with 100 &#181;L of Stop solution.</li>
		<li>Detect the levels of HRP substrate reaction product in the media using a fluorescence plate reader. Measure the fluorescence intensity at 420 nm emission wavelength (with 325 nm excitation) and as relative fluorescent units (RFU). The values indicate the level of HRP transcytosed across the HRMEC layer through caveolae-mediated transcytosis. 
		NOTE: The fluorescent product used here (Table of Materials) does not photobleach. Light protection against photobleaching is not needed.</li>
	</ol>
	</li>
</ol>

The authors have no conflicts of interest or financial interest to disclose.

BRB plays an essential role in retinal health and disease. In vitro techniques assessing vascular permeability have proven to be crucial tools in studies concerning barrier (BRB/BBB) development and function. The procedure described here could be utilized to study the molecular mechanisms underlying EC transcytosis or evaluate related molecular modulators affecting BRB permeability. In vitro EC transcytosis assays have multiple advantages over in vivo assays or techniques used for evaluating va...

The human retina is one of the highest energy-demanding tissues in the body. Proper functioning of the neural retina requires an efficient supply of oxygen and nutrients along with a restricted flux of other potentially harmful molecules to protect the retinal environment, which is mediated via the blood-retinal barrier (BRB)1. Similar to the blood-brain barrier (BBB) in the central nervous system, the BRB acts as a selective barrier in the eye, regulating the movement of ions, water, amino acids, and sugar in and out of the retina. BRB also maintains retinal homeostasis and its immune privilege by preventing exposure to circulatory factors such as immune cells, antibodies, and harmful pathogens2. BRB dysfunction contributes to the pathophysiology of several vascular eye diseases, such as diabetic retinopathy, age-related macular degeneration (AMD), retinopathy of prematurity (ROP), retinal vein occlusion, and uveitis, resulting in vasogenic edema and subsequent vision loss3,4,5.
The BRB consists of two separate barriers for two distinct ocular vascular networks, respectively: the retinal vasculature and the fenestrated choriocapillaris beneath the retina. The inner BRB (iBRB) is primarily composed of retinal microvascular endothelial cells (RMECs) lining the retinal microvasculature, which nourishes the inner retinal neuronal layers. On the other hand, the retinal pigment epithelium forms the major component of the outer BRB, which lies between the neurosensory retina and choriocapillaris2. For the iBRB, molecular transport across RMECs takes place through both paracellular and transcellular routes (Figure 1). The high degree of substance selectivity across the iBRB relies upon (i) the presence of junctional protein complexes that restrict paracellular transport between adjacent endothelial cells (ECs), and (ii) low expression levels of caveolae mediators, transporters, and receptors within the endothelial cells that maintain low rates of transcellular transport1,6,7,8. Junctional complexes regulating paracellular flux are composed of tight junctions (claudins, occludins), adherens junctions (VE cadherins), and gap junctions (connexins), allowing the passage of water and small water-soluble compounds. While small lipophilic molecules passively diffuse across the interior of RMECs, the movement of larger lipophilic and hydrophilic molecules is regulated by ATP-driven trans-endothelial pathways including vesicular transport and membrane transporters5,9.
Vesicular transcytosis may be categorized as caveolin-mediated caveolar transcytosis, clathrin-dependent (and receptor-mediated) transcytosis, and clathrin-independent macropinocytosis (Figure 2). These vesicular transport processes involve different-sized vesicles, with macropinosomes being the largest (ranging from 200-500 nm) and caveolae being the smallest (averaging 50-100 nm), while clathrin-coated vesicles range from 70-150 nm10. Caveolae are flask-shaped lipid-rich plasma membrane invaginations with a protein coat, primarily composed of caveolin-1 that binds lipid membrane cholesterol and other structural and signaling proteins via their caveolin-scaffolding domain11. Caveolins work together with peripherally attached cavin to promote caveolae stabilization at the plasma membrane12. Caveolar membranes also may carry receptors for other molecules such as insulin, albumin, and circulating lipoproteins including high-density lipoprotein (HDL) and low-density lipoprotein (LDL) to assist their movement across endothelial cells13. During development, the formation of functional BRB depends on the suppression of EC transcytosis8. Mature retinal endothelium, hence, has relatively low levels of caveolae, caveolin-1, and albumin receptors with respect to other endothelial cells under physiological conditions, contributing to its barrier properties4,9.
Because iBRB breakdown is a major hallmark of many pathological eye conditions, it is essential to develop methods to assess retinal vascular permeability in vivo and in vitro. These methods help provide probable insights into the mechanisms of compromised BRB integrity and assess the efficacy of potential therapeutic targets. Current in vivo imaging or quantitative vascular leakage assays typically employ fluorescent (sodium fluorescein and dextran), colorimetric (Evans Blue dye and horseradish peroxidase [HRP] substrate), or radioactive tracers14 to detect extravasation from the vasculature into surrounding retinal tissues with microscope imaging or in isolated tissue lysate. An ideal tracer for quantifying vascular integrity should be inert and large enough to freely permeate compromised vessels while confined within healthy and intact capillaries. Methods employing sodium fluorescein or fluorescein isothiocyanate-conjugated dextran (FITC-dextran) in live fundus fluorescein angiography (FFA) or isolated retinal flat mounts are widely used for quantitating retinal extravasation in vivo or ex vivo. FITC-dextran has the advantage of being available in different molecular weights ranging from 4-70 kDa for size-selective studies15,16,17. FITC-albumin (~68 kDa) is an alternative large-sized protein tracer of biological relevance for vascular leakage studies18. Evans Blue dye, injected intracardially19, retro-orbitally, or through the tail vein20, also relies on its binding with endogenous albumin to form a large molecule that can be quantified by mostly spectrophotometric detection or, less commonly, fluorescence microscopy in flat mounts20,21. These quantitative or light imaging methodologies, however, often do not distinguish paracellular transport from trans-endothelial transport. For the specific analysis of transcytosis with ultrastructural visualization of transcytosed vesicles, tracer molecules such as HRP are typically used to locate transcytosed vesicles within endothelial cells that can be observed under an electron microscope22,23,24 (Figure 3A-C).
The development and use of in vitro iBRB models to evaluate the endothelial cell permeability could provide robust and high throughput assessment to complement in vivo experiments and aid the investigation of molecular regulators of vascular leakage. Commonly used assays to assess the paracellular transport and integrity of tight junctions include trans-endothelial electrical resistance (TEER), a measure of ionic conductance (Figure 4)2,25, and in vitro vascular leakage assay using small molecular weight fluorescent tracers26. In addition, transferrin-based transcytosis assays modeling BBB have been utilized to explore clathrin-dependent transcytosis27. Despite this, assays to evaluate BRB and, more specifically, retinal EC caveolar transcytosis in vitro are limited.
In this study, we describe an EC transcytosis assay using human retinal microvascular endothelial cells (HRMECs) as an in vitro model to determine iBRB permeability and EC transcytosis. This assay relies on the ability of HRMECs to transport transferrin or HRP via the receptor-mediated or caveolae-dependent transcytosis pathways, respectively (Figure 2). HRMECs cultured to full confluency in the apical chamber (i.e., filter insert) were incubated with fluorescent-conjugated transferrin (Cy3-Tf) or HRP to measure the fluorescence intensity corresponding to the levels of transferrin or HRP transferred to the bottom chamber through EC transcytosis solely. Confluency of the cell monolayer can be confirmed by measuring TEER, indicating the tight junction integrity25. To demonstrate the TEER and transcytosis assay technique, known molecular modulators of vascular permeability and EC transcytosis were used, including vascular endothelial growth factor (VEGF)28 and those in Wnt signaling (Wnt ligands: Wnt3a and Norrin)29.

<table><tbody><tr><td>Biological Safety Cabinet&amp;nbsp;</td><td>Thermo Electron Corporation, Thermo Fisher Scientific</td><td>1286</td><td></td></tr><tr><td>Cell culture petridish&amp;nbsp;</td><td>Nest Biotechnology</td><td>704001</td><td></td></tr><tr><td>Centrifuge&amp;nbsp;</td><td>Eppendorf</td><td>5702</td><td></td></tr><tr><td>Centrifuge tubes (15 mL)</td><td>Corning Inc.</td><td>352097</td><td></td></tr><tr><td>Centrifuge tubes (50 mL)</td><td>Denville Scientific Inc.</td><td>C1062-P</td><td></td></tr><tr><td>Cyanine 3-human Transferrin&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>AB_2337082</td><td></td></tr><tr><td>Endothelial Cell Basal Medium-2 (EBM-2)</td><td>Lonza Bioscience</td><td>CC-3156</td><td></td></tr><tr><td>Endothelial Cell Growth Medium-2 (EGM-2) SingleQuots supplements</td><td>Lonza Bioscience</td><td>CC-4176</td><td></td></tr><tr><td>EVOM Millicell Electrical Resistance System-2 (ERS-2)</td><td>Millipore</td><td>MERS00002</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Lonza Bioscience</td><td>CC-4102B</td><td></td></tr><tr><td>Gelatin</td><td>Sigma-Aldrich</td><td>G7765</td><td></td></tr><tr><td>Hemocytometer (2-chip)</td><td>Bulldog Bio</td><td>DHC-N002</td><td></td></tr><tr><td>Horseradish Peroxidase (HRP)</td><td>Sigma-Aldrich</td><td>P8250</td><td></td></tr><tr><td>Human retinal microvascular endothelial cells (HRMEC)</td><td>Cell Systems</td><td>ACBRI 181</td><td></td></tr><tr><td>Incubator</td><td>Thermo Electron Corporation, Thermo Fisher Scientific</td><td>3110</td><td></td></tr><tr><td>L cells (for Control-conditioned medium)</td><td>ATCC</td><td>CRL-2648</td><td></td></tr><tr><td>L Wnt-3A cells (for Wnt3A-conditioned medium)</td><td>ATCC</td><td>CRL-2647</td><td></td></tr><tr><td>Light microscope</td><td>Leica</td><td>DMi1</td><td></td></tr><tr><td>Multimode Plate Reader</td><td>EnSight, PerkinElmer</td><td></td><td></td></tr><tr><td>Phosphate-buffered saline (PBS) buffer (1x)</td><td>GIBCO</td><td>10010-023</td><td></td></tr><tr><td>QuantaBlu Fluorogenic Peroxidase Substrate kit</td><td>Thermo Fisher Scientific</td><td>15169</td><td></td></tr><tr><td>Recombinant human Norrin (rhNorrin)</td><td>R&amp;amp;D Systems</td><td>3014-NR</td><td></td></tr><tr><td>Recombinant human Vascular endothelial growth factor (rhVEGF)</td><td>R&amp;amp;D Systems</td><td>293-VE</td><td></td></tr><tr><td>Syringe filter (0.22 &amp;micro;m)</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>Transwell inserts (6.5 mm transwell, 0.4 &amp;micro;m pore polyester membrane insert)</td><td>Corning Inc.</td><td>CLS3470-48EA</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%) (1x)</td><td>GIBCO</td><td>25-200-072</td><td></td></tr><tr><td>Water bath</td><td>Precision, Thermo Fisher Scientific</td><td>51221060</td><td></td></tr><tr><td>XAV939 (Wnt/&amp;beta;-catenin Inhibitor)</td><td>Selleckchem</td><td>S1180</td><td></td></tr></tbody></table>

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.

EM images of retinal vascular endothelium show transcytotic vesicular transport and caveolar vesicles in endothelial cells in vivo. 
EC transcytosis can be visualized in vivo within retinal cross-sections with dark brown precipitate reflecting HRP-containing blood vessels under a light microscope (Figure 3A) and as electron-dense precipitate indicative of HRP-containing transcytotic vesicles (Figur...

Watch this Scientific Journal Video about Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability at JoVE.com

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.

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 ...

endothelial-cell-transcytosis-assay-as-an-vitro-model-to-evaluate

Research

JoVE Journal

Medicine

4.7K Views.  Harvard Medical School. 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. 

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

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Bioengineering

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

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 ...

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.

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.

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

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

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

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

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

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

Immunology and Infection

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

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 ...

Neuroscience

Transendothelial Resistance Measurement to Assess Cell Barrier Integrity

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating the interaction between restricted transit of harmful substances, nutrient absorption, and metabolite clearance in the brain, the BBB is essential in preserving central nervous system homeostasis. Building in vitro models of the BBB is a valuable tool for exploring the pathophysiology of neurological disorders and creating pharmacological treatments. This study describes a procedure for creating an in vitro monolayer BBB cell model by seeding bEnd.3 cells into the upper chamber of a 24-well plate. To assess the integrity of cell barrier function, the conventional epithelial cell voltmeter was used to record the transmembrane electrical resistance of normal cells and CoCl2-induced hypoxic cells in real-time. We anticipate that the above experiments will provide effective ideas for the creation of in vitro models of BBB and drugs to treat disorders of central nervous system diseases.

Author Spotlight: Applications of TEER Detection to Assess Cell Barrier Integrity 

This protocol describes a dependable and efficient in vitro model of the brain blood barrier. The method uses mouse cerebral vascular endothelial cells bEnd.3 and measures transmembrane electrical resistance.

The blood-brain barrier (BBB) is a dynamic physiological structure composed of microvascular endothelial cells, astrocytes, and pericytes. By coordinating ...

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/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

Cancer Research

Cancers of the Nervous System

In vitro studies

Assessing Transendothelial Electrical Resistance in an In Vitro Blood-Brain Barrier Model

Source: Kuzmanov, I., et al. <a href="https://app.jove.com/v/54592">An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction.</a> J. Vis. Exp. (2016)
This video demonstrates a method for assessing transendothelial electrical resistance (TEER) in mouse brain microvascular endothelial cells in vitro. A high TEER value indicates intact endothelial cell junctions, while the addition of activated T cells lowers the TEER value, reflecting disruption of the junctional barrier.

Source: Kuzmanov, I., et al. An In Vitro Model of the Blood-brain Barrier Using Impedance Spectroscopy: A Focus on T Cell-endothelial Cell Interaction. J. ...

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

Summary

Explore More Videos

Chapters in this video

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

An in vivo Assay to Test Blood Vessel Permeability

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

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

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

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

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

Barrier Functional Integrity Recording on bEnd.3 Vascular Endothelial Cells via Transendothelial Electrical Resistance Detection

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

Assessing Transendothelial Electrical Resistance in an In Vitro Blood-Brain Barrier Model