Abstract
It is crucial for the cryopreserved dermal equivalent to recover both metabolic activity and protein expression as well as its intact structure. In this study, dermal fibroblasts were cultured on a three-dimensional scaffold to form a dermal equivalent, and the effects of culture time and cryopreservation protocols on the metabolic activity, structural and functional properties of the equivalent were evaluated. Two groups were defined, a fresh control group and an experimental group. Viabilities of the fresh control and the cryopreserved dermal slices were determined using the modified MTT assay. Histology staining and scanning electron microscopy of the samples were performed. The cooling was carried out in a computer-controlled programmable cooler. For the dermal equivalent cultured for 14 days, a cryopreservation protocol of 1°/min from 4° to -60°C (seeding at -7°C), then plunged into the liquid nitrogen Dewar immediately proved optimal, provided the dermal slice was transferred into the cryopreservation bag containing 1 mL of 1.4M DMSO and held for 15 min at 4°C prior to freezing. The tissue-engineered dermal replacement can maintain a relatively high metabolic activity (81% of the fresh control viability) and retain proper functional and structural characteristics.
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