Vitamin e and EDTA Improve the Efficacy of Hypothermosol-Implication of Apoptosis
Mathew; Hollister; Addona; Baust; Van Buskirk RG
In Vitro and Molecular Toxicology 12(3): 163-172
The emergence of engineered tissues and new cell strains has called for the need to explore solutions that can be used to store these cells and tissues in a state of near suspended animation without using cryopreservation. The ability of the hypothermic solutions ViaSpan (DuPont Merck Pharmaceutical Company, Wilmington, DE), HypoThermosol (Cryomedical Sciences, Rockville, MD), HypoThermosol supplemented with either ethylenediaminetetraacetic acid (EDTA) or Vitamin E and HypoThermosol supplemented with apoptosis protease inhibitors were tested for their abilities to cold-protect Madin Darby Canine Kidney (MDCK) cells at 4 degrees C. Alamar Blue, a nontoxic metabolic indicator was used to measure cell viability. The order of cold protection was HypoThermosol with Vitamin E and EDTA > HypoThermosol with Vitamin E > HypoThermosol with EDTA > HypoThermosol > ViaSpan > Dulbecco's Modified Eagle's Medium (DMEM). Membrane integrity tests supported the Alamar Blue data that EDTA and Vitamin E conferred a benefit to the cold-storage capabilities of HypoThermosol. MDCK cells that died subsequent to 1 to 6 days cold-storage detached from the substratum and their DNA was harvested after being placed at 37 degrees C. This DNA was compared to DNA retrieved from adherent cells in the same cultures that survived the cold-storage regime. Gel electrophoresis of cells dying due to 1 to 4 days of cold-storage showed a DNA ladder indicating that cells died through apoptosis, programmed cell death. Dead cells harvested at 5 to 6 days of cold storage, however, had randomly cleaved DNA indicative of necrotic cell death. HypoThermosol supplemented with apoptosis protease inhibitors was better able to cold-protect cells than the base HypoThermosol. These data suggest that the inhibition of apoptosis should be considered in the future cold-storage formulations.