Cryopreservation of human umbilical vein and porcine corneal endothelial cell monolayers

Cryopreservation of endothelium 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 (Me2SO) 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 endothelial monolayers. The addition of 2% chondroitin sulfate to 5% Me2SO 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. The optimized cryopreservation protocol was applied to monolayers of primary porcine corneal endothelial cells, and resulted in high post-thaw viability (95.9 ± 3.7% membrane integrity) with metabolic activity 12 h post-thaw comparable to unfrozen control.