Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

This in vitro assay demonstrates angiogenic sprouting in a high throughput and scalable platform. This could be used to find new targets in angiogenesis. This angiogenesis assay combines complex cellular micro environments that includes gradients and perfusion with a platform that's compatible with high throughput screening.

Angiogenesis plays an important role in both health and disease. Understanding the mechanisms that occur during angiogenesis could help us to understand how cancers grow and how tissues repair. This method could give insight in the mechanisms during cardiovascular diseases and tissue regeneration.

In both, angiogenesis plays an important role. For the first-time use of this microfluidic platform, you need to be aware of the law of volumes and how to pipette. You can confirm the steps by looking under a microscope or flipping the plate upside down.

Some steps are difficult to get a feel for when just reading the method, for example how small the microfluidic channels are or how to hold the plate. Demonstrating these procedures will be Wendy Stam, a technician from our laboratory. To begin, transfer the microfludic 384-well plate to a sterile laminar flow hood.

Remove the lid and add 50 microliters of water or phosphate buffered saline to each of the 40 observation wells using a multi-channel pipette. Then add 1.5 microliters of four milligrams per milliliter collagen one solution to the gel inlet of each microfluidic unit. Make sure the droplet of gel is placed in the middle of each well in order for the gel to enter the channel.

After filling five microfluidic units, flip the plate upside down to visually inspect correct filling of the gel channels. Place the microfludic plate in an incubator at 37 degrees Celsius and 5%carbon dioxide for 10 minutes to polymerize collagen one. Then take the plate out of the incubator and transfer to a sterile laminar flow hood.

Add 50 microliters of 10 micrograms per milliliter fibronectin coding solution to the outlet well of the top perfusion channel of each microfluidic unit. Press the pipette tip against the side of the well for correct filling of the well without trapping air bubbles. The channel fills and the liquid pans on the outlet without filling the outlet well.

Then place the plate in the incubator for at least two days. Now, rapidly thaw the frozen IPSC-ECs for less than one minute in a 37 degree Celsius water bath. Then transfer the cells to a 15 milliliter tube and slowly add 10 milliliters of basal medium to dilute.

Draw 10 microliters of the solution and count the cells on a cell counter. Centrifuge the tube at 100 times g for five minutes. Aspirate the supernatant without disturbing the cell pellet and resuspend in basal medium to yield a concentration of two times 10 to the seventh cells per milliliter.

Next, transfer the microfluidic 384-well plate from the incubator to a sterile laminar flow hood. Aspirate the fibronectin coding solution from the perfusion outlet and add 25 microliters of basal medium in the outlet wells. Add a one microliter droplet of cell suspension to each top perfusion inlet well.

The droplet flattens in a few seconds. Check under a microscope whether the seeding is homogenous within a single channel and between channels. Incubate the microfluidic well plate for one to 1.5 hours at 37 degrees Celsius and 5%carbon dioxide.

After that, check the cells to confirm they have adhered. Remove the basal medium from the top perfusion outlet wells. Add warm vessel culture medium in the top perfusion inlet and outlet.

Place the plate on a rocker platform which is set on a seven degree angle and eight minutes rocking interval in the incubator. At day one and two post-seeding, image the plate using a Brightfield microscope with automated stage to confirm cell viability. After two days, a confluent monolayer has formed against the collagen one scaffold.

First, prepare 4.5 milliliters of angiogenic sprouting medium by supplementing 4.5 milliliters of basal medium with 4.5 microliters of VEGF stock, 4.5 microliters of PMA stock, and 2.25 microliters of S1P stock. Prepare 8.5 milliliters of vessel growth medium by supplementing basal medium with 5.1 microliters of VEGF and 3.4 microliters of BFGF. Aspirate medium from the wells and add 50 microliters of fresh vessel culture medium in the top perfusion inlet and outlet wells and gel inlet and outlet wells.

Then add 50 microliters of angiogenic sprout mixture to each of the bottom perfusion channel inlet and outlet wells. Place the plate back into the incubator on the rocker platform in order to form a gradient of angiogenic growth factors. One day and two days after addition of the angiogenic growth factors, use a Brightfield microscope to image the wells on an automated stage.

To study anastomosis and sprout stabilization, add one microliter of 0.5 milligrams per milliliter fluorescently labeled albumin to the top perfusion inlet. Then mix the solution with a 50 microliter pipette. Transfer the plate to a fluorescent microscope with automated stage and incubator set at 37 degrees Celsius.

Set the microscope at the 10X objective and correct the exposure settings. Acquire timelapse images every minute for 10 minutes. Remove the plate from the microscope and transfer the plate to a sterile laminar flow hood.

Remove all medium from the wells and replace both the vessel culture medium and angiogenic sprouting medium in the corresponding wells. Place the microfluidic plate back into the incubator to continue angiogenic sprouting. On day six, repeat the imaging to study the permeability.

To fix the cell culture, aspirate all culture media from the wells. Add 25 microliters of 4%PFA in PBS to all the perfusion inlet and outlet wells. Then place one side of the microfluidic plate on the lid at a slight angle of five degrees to induce flow.

Incubate for 10 minutes at room temperature. After that, aspirate the PFA from the wells. Wash all the perfusion inlets and outlets twice with 50 microliters of HBSS.

Then aspirate the HBSS from the wells. Paraformaldehyde is a hazardous chemical which should be handled wearing gloves and only opened in a fume hood. Next, add 50 microliters of 0.2%non-ionic surfactant to all the perfusion inlets and outlets to permeabilize at room temperature for 10 minutes and place the microfluidic plate on the lid at a slight angle of five degrees.

Aspirate the non-ionic surfactant from the wells and wash the perfusion channels twice by adding 50 microliters of HBSS to all perfusion inlet and outlet wells. Aspirate the HBSS from the wells. Prepare Hoechst 1, 000 to 2, 000 for staining the nuclei and prepare Phalloidin 100 to 200 in HBSS for staining F-actin.

Combine six milliliters of HBSS, 30 microliters of F-actin, and three microliters of Hoechst in a Falcon tube. Then add 25 microliters to each perfusion inlet and outlet well. Place the plate under a slight angle and incubate at room temperature for at least 30 minutes.

Wash twice with 50 microliters of HBSS in all the perfusion inlets and outlets. Then directly image using a fluorescent microscope with automated stage or store the plate protected from light at four degrees Celsius for later use. In this protocol, the micro vessels were continuously perfused and exposed to a gradient of angiogenic growth factors.

Seeding the IPSC-ECs using the passive pumping method resulted in homogenous seeding densities. The timelapse of a droplet that is placed on top of the inlet of the microfluidic channel shows the droplet right after addition, the droplet on top of the inlet trunk after addition, until the droplet meniscus was pinned by the inlet which resulted in a flow towards the outlet. Exposure to a gradient of angiogenic factors resulted in directional angiogenic sprouting of the micro vessels within the patterned collagen one gel.

Clear tip cell formation and invasion into the collagen one gel was visible 24 hours after addition of the angiogenic gradient while stock cells including lumen formation were visible after 48 hours. After fixation and staining, the shape and length of these sprouts were identified. However, without addition of growth factors, no invasion into the collagen one gel was observed.

With confocal imaging, the sprout diameter was determined and the lumen formation was confirmed. This method could be used to screen for new angiogenic inhibitors. This could help to find new treatments against diabetic retinopathy, rheumatoid arthritis and cancer.

We are currently exploring how we can create co-cultures including a model of the blood-brain barrier and vascularized organoids.