Abstract: Cryopreservation of endothelial cell monolayers is one of the major challenges in the cryopreservation of complex tissues. Human umbilical vein endothelial cells (HUVECs) in suspension are available commercially and recently their post-thaw cell membrane integrity was significantly improved by cryopreservation in 5% dimethyl sulfoxide (DMSO) and 6% hydroxyethyl starch (HES). However, cryopreservation of cells in monolayers has been elusive. The exact mechanisms of damage during cell monolayer cryopreservation are still under investigation. Here, we show that a combination of different factors contribute to significant progress in cryopreservation of cell monolayers. The addition of 2% chondroitin sulfate to 5% DMSO and 6% HES and cooling at 0.2 or 1° C./min led to high membrane integrity (97.3±3.2%) immediately after thaw when HUVECs were cultured on a substrate with a coefficient of thermal expansion similar to that of ice.