Cold-Storage of Synthetic Human Epidermis in HypoThermosol

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 degrees 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 degrees 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 degrees 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.

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing

“…These features facilitate preservation of cell homeostasis, which is not achievable when using just culture medium as a preservation formulation. For example, normal human epidermal keratinocytes can be maintained for periods exceeding 1 week at 4°C in HTS and only 24 hours in normal growth medium at 4°C [21]. Currently, the incorporation of protective agents, including antioxidants, ion chelators, and membrane stabilizers, in preservation media has been reported to markedly improve preservation efficacy.…”

“…A variety of preservation solutions, such as ViaSpan (University of Wisconsin solution), Celsior, Custodiol histidine‐tryptophan‐ketoglutarate solution and, more recently, HypoThermosol (HTS), were developed taking these factors into consideration [15] and are commercially available today. These solutions showed to be effective for hypothermic storage of a large diversity of cells, tissues, and organs (including neonatal rat ventricular cardiomyocytes [16], renal cells [17], hepatocytes [18, 19], liver tissue [20], synthetic human epidermis tissue [21], kidney [22], pancreas [23], liver [20], and heart [24, 25]). Importantly, some of these formulations can be directly administered into patients, avoiding additional poststorage culture manipulations/costs and washing steps.…”

Cryoprotection by urea in a terrestrially hibernating frog

“…This assay, which yields a colorimetric change in proportion to cellular respiration, has been used to evaluate cell viability following hypothermic exposure (Acker and McGann, 2002;Cook et al, 1995). Organ samples were transferred 60·min after thawing to a 1.5-ml centrifuge tube containing 90·l alamarBlue diluted (1:10) with PBS, or PBS containing 80·mmol·l -1 urea, and incubated at 15°C with gentle orbital agitation for 120·min.…”

Cryopreservation and hypothermic storage of lacrimal gland: towards enabling delivery of regenerative medicine therapies for treatment of dry eye syndrome

“…Storage at 4 °C is only utilised over relatively short time periods: hours for organs prior to transplantation; or days to weeks for tissue‐engineered products prior to application (Cook et al , ). Maintaining a cold chain at 4 °C is considerably simpler than maintaining one at cryogenic temperatures without a requirement for specialised equipment, and so it is conceivable that a cold chain from clinic (where tissue could be harvested), to tissue bank or cleanroom (where tissue could be stored or reconstructed tissue produced), to theatre (where transplant could be performed) could be devised without much complication.…”