Cryopreservation of human endothelial cells for vascular tissue engineering

To investigate the influence of cryopreservation on endothelial cell growth, morphology, and function human umbilical vein endothelial cells (HUVECs) were frozen following a standard protocol. Cell suspensions were exposed to 10% dimethyl sulfoxide in a high-potassium solution, cooled to −80 °C at 1 °C/min and stored in liquid nitrogen for 7-36 days. Samples were thawed in a 37 °C water bath and the cryoprotectant was removed by serial dilution. The growth of cell suspensions was assayed by culturing 7300 cells/cm2 for 3-5 days in order to determine the cell multiplication factor. Fresh and cryopreserved/thawed cells were analyzed for their growth, and their anti-inflammatory and anti-coagulant function by using cellular ELISA. Cryopreservation resulted in a retrieval of 66 ± 5% and a viability of 79 ± 3%. Cryopreserved/thawed and fresh cells showed identical doubling times and identical cell counts in the confluent monolayers. However, the lag phase of thawed HUVECs was approximately 36 h longer, resulting in significant differences in the cell multiplication factor at 3 and 5 days after seeding. After expansion to a sufficient cell count the lag phases were identical. Fresh and cryopreserved/thawed cells showed comparable anti-inflammatory and anti-coagulant activity, as judged by the basal and TNF-induced VCAM-1, ICAM-1, E-selectin, and thrombomodulin expression. Cryopreserved/thawed and recultivated endothelial cells are suitable for endothelialization of autologous allograft veins. Such tissue-engineered grafts will offer the necessary clinical safety for those patients who lack autologous material.