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

This protocol provides a useful cell-based model for studying the function and permeability of selective vascular barriers such as blood-retinal or blood-brain barrier with a specific focus on endothelial transcytosis. This assay is easy to set up and use requiring no live animals. It can be utilized for investigating various molecular regulators of endothelial cell permeability and transcytosis affecting inner blood-retinal barrier integrity.

This assay can provide insights into vascular biology and eye and brain research, allowing the better understanding of molecule mechanisms underlying BRB BBB control and exploring potential drug delivery across the barriers. To begin, prepare the cell culture filter inserts for seeding human retinal microvascular endothelial cells or HRMECs, by coating each insert with 200 microliters of 0.1%gelatin solution for 30 minutes under a laminar flow hood. Ensure the solution covers the entire bottom surface of the filter insert.

Take a Petri dish with cultured HRMECs incubated at 37 degrees Celsius and 5%carbon dioxide, and aspirate the growth media. Gently rinse the cells twice with 10 milliliters of 1X PBS under the laminar flow hood to get rid of the potential floating or dead cells. Dissociate the cells with 0.5 to 1 milliliter of 0.25%trypsin-EDTA solution and place the Petri dish in the incubator at 37 degrees Celsius and 5%carbon dioxide for 5 minutes.

Quench the trypsin activity by adding 4.5 to 9 milliliters of growth media and transfer the cell suspension into a 15-milliliter tube using a 10-milliliter pipette. Spin the cells at 200 times g for 5 minutes at room temperature. Carefully remove the supernatant and resuspend the pellet in 3 milliliters of growth media.

Count the number of cells using a manual hemocytometer or an automated cell counter and seed at a density of 40, 000 cells per filter insert. The volume of cell suspension for each insert is 250 microliters. Aspirate the coating solution from the wells containing permeable inserts and transfer 250 microliters of the cell suspension per insert, then add only medium to one of the inserts, which will be used as a blank control for transendothelial electrical resistance, or TEER, measurement.

Simultaneously, also add 750 microliters of medium per well in the basolateral chambers. Keep the 24-well plate with permeable inserts in the incubator at 37 degrees Celsius and 5%carbon dioxide for 7 to 12 days until the cultured cells become fully confluent and the desired TEER value of around 20 ohms per square centimeter is achieved. Before TEER measurements, place the cell-containing 24-well plate in the laminar flow hood at room temperature for 15 to 20 minutes for temperature equilibration.

To measure the TEER for HRMECs, using an epithelial volt/ohm meter electrical resistance system, connect the electrode to the meter, and equilibrate the electrode by first soaking it in 70%ethanol for 15 to 20 minutes and then immersing it briefly in the cell culture EGM growth medium. 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, then for each insert, measure TEER in triplicates.

Upon reaching confluency with TEER values around 20 ohm square centimeters, serum-deprive the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide using 0.5%FBS in EBM in both the chambers prior to treatment with the ligand. Serum-reduced EBM was used throughout the assay. Incubate the cells using the serum-reduced medium in the apical chamber with fluorescent cyanine-3-tag to transferrin ligand for 60 minutes at 37 degrees Celsius.

After washing the monolayer apically and basolaterally 4 times with the serum-reduced medium, add fresh medium to the filter inserts containing the cells and transfer the inserts to fresh wells of the 24-well plate containing prewarmed serum-reduced medium. Incubate the cells for another 90 minutes in the incubator and then collect the medium from the basolateral chamber. Record the fluorescence intensity of the solution from the basolateral chamber using a fluorescence detector.

Upon reaching confluency with TEER values around 20 ohm square centimeter, serum-deprive the cells for 24 hours at 37 degrees Celsius and 5%carbon dioxide using 0.5%FBS in EBM in both the chambers prior to treatment with the ligand. Serum-reduced EBM was used throughout the assay. Next, treat the cells in the apical chamber with the desired treatments and vehicle controls.

Add 5 milligrams per milliliter of horseradish peroxidase, or HRP, to the cells, and incubate for 15 minutes at 37 degrees Celsius. Add fresh serum-reduced medium to the apical chamber and transfer the inserts to a fresh well containing prewarmed media. Incubate the monolayer for an additional 90 minutes in the incubator.

After collecting medium from the basolateral chamber, add 100 microliters of HRP fluorogenic peroxidase substrate to the collected medium and incubate at room temperature for 10 minutes. Stop the reaction with 100 microliters of stop solution and detect the levels of HRP substrate reaction product in the media using a fluorescence plate reader. Before demonstrating the in vitro transcytosis assays, visualization of endothelial cell transcytosis in the retina is shown as background information.

Here, the light microscope image shows an HRP-filled blood vessel lumen from a 3-month-old wild-type mouse retinal section stained with DAB. HRP was retro-orbitally injected. HRP-filled retinal vessels can be seen as dark brown precipitate under the light microscope.

Transmission electron microscope ultra thin section shows HRP-filled transcytotic vessels with RMECs depicting EC transcytosis across the inner blood-retinal barrier. Here, the large vesicle potentially reflects macropinosome and a red blood cell on the luminol side. TeM image shows HRP-filled small transcytotic vesicles, likely caveola vesicles, within the RMEC.

Immunohistochemistry staining of CAV-1 antibody and isolectin B4 demonstrates the localization of CAV-1, A marker of caveola vesicles in retinal blood vessels, in the 3-month-old wild-type mouse retina. TeM image of immunogold-labeled CAV-1 within the retinal endothelial cells is shown here with a magnified image of CAV-1 positive caveola vesicle. For clathrin-mediated EC transcytosis assay in HRMECs using transferrin, here, a fluorescence microscope image shows endocytosed Cy3 transferrin in red color within HRMECs stained with DAPI in blue.

For caveolae-mediated EC transcytosis using HRP, Wnt signaling here is used as an example to demonstrate the assay. Wnt signaling was found recently to regulate caveolae-mediated EC transcytosis. Cells were treated with Wnt pathway activator Wnt3a-conditioned medium and recombinant Norrin, with or without Wnt signaling inhibitor XAV939 and their reactive controls.

Wnt activators reduced the level of the HRP-based transcytosis, which were reversed by XAV939. Correct TEER measurement is crucial to determine monolayer integrity. It is greatly affected by various factors such as improper handling, temperature fluctuations, culture medium, culture duration, et cetera.

This assay could be modified as co-culture or 3D organotypic culture systems mimicking in vivo conditions.