To begin, submerge a vial of cryopreserved HUVECs in a 37 degree water bath for two minutes. Then rinse the vial with 70%ethanol to prevent contamination. Pipette five milliliters of pre-warmed ECGM into a 15 milliliter conical tube.
With a micropipette, carefully transfer the thawed cells from the vial into the conical tube. Then add one milliliter of fresh pre-warmed medium to the cryovial to rinse the inside and transfer any remaining cells into the conical tube. After discarding the supernatant, resuspend the cells in 10 milliliters of fresh medium.
Then transfer the cells to a T75 flask. Incubate the flask in a humidified carbon dioxide incubator at 37 degrees Celsius. Rinse the 90%confluent HUVEC cells in a T75 flask with 10 milliliters of DPBS.
Then add one milliliter of 0.25%TE to the flask. After incubation, gently tap the sides of the flask and add five milliliters of trypsin neutralizing solution. Then transfer the cells to a 15 milliliter conical tube.
Collect the remaining cells with five milliliters of fresh ECGM and transfer them into the conical tube. Now centrifuge the conical tube at 250 x g for three minutes to collect the cell pellet. After discarding the supernatant, resuspend the cell pellet in one milliliter of fresh ECGM and count the cells.
After centrifuging again, discard the supernatant and resuspend the cells in 90 microliters of fresh ECGM. Next, briefly vacuum suction the channels of the printed vessels-on-a-plate to clear the lumen. With a micropipette, gently load the channel with 15 microliters of the HUVEC cell suspension.
Place the plate flat inside an incubator for two hours. Subsequently, invert the plate to 180 degrees, maintaining it in a flat position for the next two hours. After four hours, wash the channel with DPBS to remove non-adherent and dead cells.
Then pipette two milliliters of fresh ECGM into each well. Place the dynamic culture on a rocker at a 10 degree tilt angle and five RPM. The HUVEC cells rapidly attached and proliferated to cover the luminal surface of the vascular channels of the VOP.
The maturation of the endothelium was verified by immunostaining for CD31 5 days post cell seeding, which demonstrated the localization of CD31 at the tight junctions.
