title: Cryopreservation of Endothelial Cells in Various Cryoprotective Agents and Media - Vitrification versus Slow Freezing Methods.
abstract: Vitrification of endothelial cells (MHECT-5) has not previously been compared with controlled slow freezing methods under standardized conditions. To identify the best cryopreservation technique, we evaluated vitrification and standardized controlled-rate -1°C/minute cell freezing in a -80°C freezer and tested four cryoprotective agents (CPA), namely dimethyl sulfoxide (DMSO), ethylene glycol (EG), propylene glycol (PG), and glycerol (GLY), and two media, namely Dulbecco's modified Eagle medium Ham's F-12 (DMEM)and K+-modified TiProtec (K+TiP), which is a high-potassium-containing medium. Numbers of viable cells in proliferation were evaluated by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega Corporation, Mannheim, Germany). To detect the exact frozen cell number per cryo vial, DNA content was measured by using Hoechst 33258 dye prior to analysis. Thus, results could be evaluated unconstrained by absolute cell number. Thawed cells were cultured in 25 cm2 cell culture flasks to confluence and examined daily by phase contrast imaging. With regard to cell recovery immediately after thawing, DMSO was the most suitable CPA combined with K+TiP in vitrification (99 ±0.5%) and with DMEM in slow freezing (92 ±1.6%). The most viable cells in proliferation after three days of culture were obtained in cells vitrificated by using GLY with K+TiP (308 ±34%) and PG with DMEM in slow freezing (280 ±27%).
authors.affiliation: Technical University of Munich, Department of Maxillofacial Surgery, Langerstr. 3, 81675 Munich, Germany.
authors.forename: Achim
authors.initials: A
authors.lastname: von Bomhard
authors.affiliation: University Medical Center Ulm, Department of Otorhinolaryngology, Frauensteige 12, 89075 Ulm, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Elsässer
authors.affiliation: Technical University of Munich, Department of Maxillofacial Surgery, Langerstr. 3, 81675 Munich, Germany.
authors.forename: Lucas Maximilian
authors.initials: LM
authors.lastname: Ritschl
authors.affiliation: University Medical Center Ulm, Department of Otorhinolaryngology, Frauensteige 12, 89075 Ulm, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Schwarz
authors.affiliation: University Medical Center Ulm, Department of Otorhinolaryngology, Frauensteige 12, 89075 Ulm, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Rotter
mesh_terms: D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects; D005615:Freezing; D051379:Mice; D058989:Vitrification / Q000187:drug effects
chemical_list: D003451:Cryoprotective Agents