Assessment of hypothermie storage of normal human epidermal keratinocytes (NHEK) using alamar blue

Two key factors critical to the improvement of hypothermie solutions are the preservation of cellular viability and the maintenance of cell-specific function. We consider these two parameters distinct from each other, i.e., cells can be viable upon warming from cold storage but, nonetheless, lose their differentiated function as a consequence of hypothermie exposure. Normal human epidermal keratinocytes (NHEK) were used as a model cell culture system to (1) modify the cold-storage solution, HypoThermosol (HTS) as well as (2) to compare the cold-storage efficacy of HTS with that of Viaspan [University of Wisconsin (UW) Solution]. Alamar Blue (AB), a nontoxic metabolic indicator, was used to assess viability, but not the cell-specific function, of NHEK cold stored for a variety of times in keratinocyte growth medium (KGM), Viaspan, or HTS. After warming to 37°C, NHEK were assayed daily with AB. The data indicate that (1) magnesium, but not calcium, is critical to the NHEK cold-storage capability of HTS; (2) the differences in the ability of the cold-storage solutions to protect viability was often not fully manifested until 24 h subsequent to rewarming; and (3) the ability of the solutions to protect NHEK during cold storage was HTS>Viaspan>KGM. We suggest that AB be used in the future to optimize coldstorage solutions. Furthermore, HTS's ability to better protect viability of NHEK compared to Viaspan suggests that HTS might be a better cold-storage solution for human skin and other epidermal tissues.

Duke Scholars

Author Scott Matthew Robert Medicine, General Internal Medicine