Cold-Storage of Synthetic Human Epidermis in HypoThermosol

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Abstract
There is a growing need for engineered tissues in a wide variety of medical applications and as alternatives to animal tissues for in vitro toxicology testing. While techniques for the preparation of a variety of synthetic tissue constructs have been devised, little attention has been focused upon the optimum conditions necessary for storage and shipping of these tissue devices. This study investigates the effects of hypothermic storage on synthetic human epidermis (EpiDerm, MatTek Corp., Ashland, MA) and specifically examines the quality of storage in keratinocyte growth medium (KGM), a standard skin culture medium, compared with storage in HypoThermosol, a new hypothermic preservation solution. EpiDerm samples were immersed in HypoThermosol for 1 to 13 days at 4°C and were assayed using the noninvasive, viability indicator dye, Alamar Blue (AB). Samples immersed for 1 to 9 days in HypoThermosol retained their viability subsequent to warming to 37° C and for at least 7 days thereafter in culture. During this time samples previously stored in HypoThermosol continued to generate a stratum corneum and their ultrastructural characteristics were similar to EpiDerm that were not exposed to hypothermic solutions. This profile, however, was not apparent in EpiDerm maintained for 1 to 13 days in KGM and subsequently warmed. These samples appeared viable immediately upon warming in most cases, but viability was not retained thereafter. EpiDerm maintained in KGM and allowed to recover at 37°C appeared necrotic and failed to continue to differentiate. The conclusions of this study are the following: (1) HypoThermosol protects the viability of EpiDerm during cold-storage, (2) HypoThermosol preserves EpiDerm's ability to differentiate subsequent to warming, and (3) the inferior preservation of samples stored in KGM was most apparent 24 h after warming.