title: Cryopreservation of human endothelial cells for vascular tissue engineering.
abstract: 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 degrees C at 1 degrees C/min and stored in liquid nitrogen for 7-36 days. Samples were thawed in a 37 degrees 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.
authors.affiliation: Clinic of Cardiothoracic Surgery, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93042 Regensburg, Germany. Karla.Lehle@klinik.uni-regensburg.de
authors.forename: Karla
authors.initials: K
authors.lastname: Lehle
authors.forename: Markus
authors.initials: M
authors.lastname: Hoenicka
authors.forename: Volker R
authors.initials: VR
authors.lastname: Jacobs
authors.forename: Franz X
authors.initials: FX
authors.lastname: Schmid
authors.forename: Dietrich E
authors.initials: DE
authors.lastname: Birnbaum
mesh_terms: D015815:Cell Adhesion Molecules / Q000096:biosynthesis; D049109:Cell Proliferation; D015925:Cryopreservation / Q000379:methods*; D004730:Endothelium, Vascular / Q000166:cytology*; D006801:Humans; D008928:Mitochondria / Q000201:enzymology; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D018180:Thrombomodulin / Q000096:biosynthesis; D023822:Tissue Engineering / Q000379:methods*; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D015815:Cell Adhesion Molecules; D013778:Tetrazolium Salts; D013844:Thiazoles; D018180:Thrombomodulin; C070380:3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium