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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Biological Safety Cabinet&#160;</td>
			<td>Thermo Electron Corporation, Thermo Fisher Scientific</td>
			<td>1286</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture petridish&#160;</td>
			<td>Nest Biotechnology</td>
			<td>704001</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</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&#160;</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&#38;D Systems</td>
			<td>3014-NR</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human Vascular endothelial growth factor (rhVEGF)</td>
			<td>R&#38;D Systems</td>
			<td>293-VE</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter (0.22 &#181;m)</td>
			<td>Millipore</td>
			<td>SLGP033RS</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell inserts (6.5 mm transwell, 0.4 &#181;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/&#946;-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...

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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 ...

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4.5K 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

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

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 ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

An 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 ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research

The goal of this protocol is to demonstrate the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The technique can be utilized as an in vitro screening tool in hearing research.

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

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

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

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

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

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