title: Freezing-induced uptake of trehalose into mammalian cells facilitates cryopreservation.
abstract: The aim of this study was to investigate if membrane-impermeable molecules are taken up by fibroblasts when exposing the cells to membrane phase transitions and/or freezing-induced osmotic forces. The membrane-impermeable fluorescent dye lucifer yellow (LY) was used to visualize and quantify uptake during endocytosis, and after freezing-thawing. In addition, trehalose uptake after freezing and thawing was studied. Fourier transform infrared spectroscopic studies showed that fibroblasts display a minor non-cooperative phase transition during cooling at suprazero temperatures, whereas cells display strong highly cooperative fluid-to-gel membrane phase transitions during freezing, both in the absence and presence of protectants. Cells do not show uptake of LY upon passing the suprazero membrane phase transition at 30-10°C, whereas after freezing and thawing cells show intracellular LY equally distributed within the cell. Both, LY and trehalose are taken up by fibroblasts after freezing and thawing with loading efficiencies approaching 50%. When using 250 mM extracellular trehalose during cryopreservation, intracellular concentrations greater than 100 mM were determined after thawing. A plot of cryosurvival versus the cooling rate showed a narrow inverted-'U'-shaped curve with an optimal cooling rate of 40°C min(-1). Diluting cells cryopreserved with trehalose in isotonic cell culture medium resulted in a loss of cell viability, which was attributed to intracellular trehalose causing an osmotic imbalance. Taken together, mammalian cells can be loaded with membrane-impermeable compounds, including the protective agent trehalose, by subjecting the cells to freezing-induced osmotic stress.
authors.affiliation: Institute of Multiphase Processes, Leibniz Universität Hannover, Germany.
authors.forename: Miao
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Clinic for Horses, Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Germany.
authors.forename: Harriëtte
authors.initials: H
authors.lastname: Oldenhof
authors.affiliation: Clinic for Horses, Unit for Reproductive Medicine, University of Veterinary Medicine Hannover, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Sieme
authors.affiliation: Institute of Multiphase Processes, Leibniz Universität Hannover, Germany. Electronic address: wolkers@imp.uni-hannover.de.
authors.forename: Willem F
authors.initials: WF
authors.lastname: Wolkers
mesh_terms: D016475:3T3 Cells; D000818:Animals; D015925:Cryopreservation; D005615:Freezing; D051379:Mice; D008856:Microscopy, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D014199:Trehalose / Q000378:metabolism*
chemical_list: D014199:Trehalose
keywords: Cryopreservation; Dehydration; Freezing; Membrane permeability; Membrane phase behavior; Trehalose loading