Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

Our protocol allows researchers to study the vessel network formation process in a clear and easily trackable fashion, opening the doors to new studies and shedding light on blood vessel behavior. This technique presents the possibility of creating highly organized and repeatable vascular networks with respond to the surrounding environment. Endothelial cells from patients suffering from a vascular disease can be retrieved in using this system, making it possible to recreate the vascular disease and find an appropriate treatment.

The proposed method can be extended for studying vascular network behavior when paired with different cell types, allowing to observe the interaction of developing vessels with a specific tissue type. Begin by submerging the scaffolds in 70%ethanol for a minimum of 15 minutes, then wash them twice in PBS. Mix 1.5 microliters of fibronectin stock solution with 28.5 microliters of PBS per scaffold to be seeded.

Prepare one fibronectin dilution to be used for all scaffolds to avoid pipetting errors. Place the scaffolds sparsely on top of a hydrophobic surface and cover each scaffold with 30 microliters of the fibronectin dilution. Replace the plate's lid and put the fibronectin-covered scaffolds into an incubator set to 37 degrees Celsius and 100%humidity for a minimum of one hour.

After incubation, lightly rinse the scaffolds in PBS to remove fibronectin remnants. The scaffolds can be kept in PBS at four degrees Celsius for up to a week. Prepare endothelial cell or EC medium by mixing the basal medium with the corresponding medium kit components, including an antibiotic solution, FBS, and endothelial cell growth supplements as indicated by the manufacturer.

Make the human adipose microvascular endothelial cell suspension in EC medium with a concentration of four million cells per milliliter. Using forceps, place one fibronectin-coated scaffold per well in a non-TC 24-well plate. Cover each scaffold with 25 microliter droplets of the cell suspension, making sure not to let the suspension flow away from the scaffold.

Put the lid on the plate and place it in an incubator at 37 degrees Celsius, 5%carbon dioxide and 100%humidity for 60 to 90 minutes. After the incubation, fill each well with 700 microliters of EC medium. Incubate the endothelialized scaffolds until EC confluence can be observed using fluorescent microscopy or for three days.

Change the medium every other day. Prepare DPSC medium by mixing 500 milliliters of low glucose DMEM, 57.5 milliliters of FBS, 5.75 milliliters of non-essential amino acids, 5.75 milliliters of GlutaMAX, and 5.75 milliliters of penicillin-streptomycin-nystatin solution. Transfer the endothelialized scaffolds into a new non-TC 24-well plate.

Discard all media from the current plate using a pipette or vacuum suction, taking care not to apply vacuum directly to the scaffold. Place one scaffold into the center of each well, then dry the surrounding area with light vacuum. Dilute thrombin and fibrinogen stock solutions with PBS to a final concentration of five units per milliliter and 15 milligrams per milliliter respectively.

Prepare an eight million DPSC per milliliter suspension in the thrombin dilution and distribute 12.5 microliters of the suspension into individual microtubes per scaffold to be seeded. Set a five to 50 microliter pipette to 12.5 microliters and fill it with fibrinogen solution. Without removing the tip, set the pipette to 25 microliters.

The material in the tip should rise and leave an empty volume. Slowly press the plunger button until the liquid reaches the tip opening, but does not leak out. Hold the plunger in this position and put the tip into one of the microcentrifuge tubes containing the cells in thrombin suspension, making sure the tip is in contact with the liquid.

Gently release the plunger button and draw the cell suspension into the tip. Thoroughly mix both materials, avoiding bubble formation. Quickly dispense the mixed materials on top of an endothelialized scaffold.

Repeat the previous steps for each scaffold, making sure to change tips between uses to avoid unexpected fibrin gel formation within the tip. Replace the plate lid and incubate the scaffolds at 37 degrees Celsius, 5%carbon dioxide and 100%humidity for 30 minutes. After incubation, fill each well with one milliliter of one-to-one DPSC and EC medium.

Culture for one week, changing the medium every other day. At different time points during culture, remove the medium from the well and image the constructs using a confocal microscope to study the vascular development or any other parameter of interest. This protocol allows for the fabrication of tessellated scaffolds made of SU-8 photoresist.

Scaffolds with distinct compartment geometries and highly accurate and repeatable features were obtained. With traditional simultaneous seeding of both endothelial cells and support cells, the cells were homogeneously distributed over the scaffold resulting in unpredictable and disorganized vascular networks. Contrarily, stepwise cell seeding resulted in highly organized vascular networks.

When using fluorescent endothelial cells, the vessels can be imaged in real time. Red fluorescent protein expressing endothelial cells were cultured on hexagonal scaffolds and imaged. The support cells were added at day one and the vascular networks were imaged every other day to quantify vessel development.

For each time point, wide images of the whole scaffold were taken. Vessel growth was quantified as total vessel length and area. A confocal imaging time-lapse was performed to allow single vessel tracking using multi-colored endothelial cells.

The vessel path was generated from the time-lapse, making it possible to observe vessel migration. Vessel maturation was observed by the presence of smooth muscle actin and support cells. For the circular, hexagonal, and squared compartments, the cells multiply and organize into structures.

By day seven, all shapes showed a rich and complex vascular network. Higher magnification images revealed a denser smooth muscle actin and support cell presence co-localized with formed vessels, evidencing support cell recruitment and differentiation surrounding vascular structures. This technique can be used to study the behavior of three-dimensional vascular networks when exposed to different conditions, such as specific cell inhibitors or in the presence of additional cell types.