Protocol for Cryopreservation of Endothelial Monolayers: An Update.

Understanding the cryobiological responses of endothelial cells is important to ensure the successful cryopreservation of vascularized tissues and organs. The endothelium, a monolayer of endothelial cells comprising the inner lining of blood vessels and other tissues such as the cornea, is sensitive to cryoinjury. In this chapter, we update our published cryopreservation protocol, which we have since validated for several more cell types. Our optimized protocol involves culturing cells on Rinzl plastic coverslips, using a combination of a permeating cryoprotectant (5% dimethyl sulfoxide) and a non-permeating cryoprotectant (6% hydroxyethyl starch), addition of 2% chondroitin sulfate, controlled-rate slow cooling, storage in liquid nitrogen, and removal of cryoprotectants immediately after thaw. Our post-thaw assessments include membrane integrity and a functional assay immediately after thaw and after extended incubation in culture conditions.