The cryopreservation of a tissue engineered dermal replacement by programmed freezing

Long-term storag of engineered bio-artificial tissues is required to ensure the off-the-shelf availability to clinicians due to their long production cycle. Cryopreservation is likely the choice for long-term preservation. This study investigated the effects of dimethyl sulfoxide (DMSO) concentrations, cooling rates, cryoprotectant medium treatment methods and seeding on the cell viability of a tissue-engineered dermal substitute. The dermal fibroblast was cultured on a polyglycolic acid (PGA) scaffolding at 37degree C and a 5% CO2atmosphere for 14 days, and dermal slices (10×3×1.5mm) were used in the experimental group. The freezing was carried out in a computer-controlled programmable freezer at 0.5degree C/min, 1degree C/min and 2degree C/min in1.4M, 2.1M and 2.8M DMSO from 4degree C to -60degree C and then plunged into the liquid nitrogen tank immediately. After 24th of cryopreservation, the samples wereped in removed from liquid nitrogen and placed at room temperature in air for 0.5-1min, prior to submergence in a water bath at 37 degree C. viability of fresh control and cryopreserved dermal slices were determined with our modification of the MTT (3-[4,5-Dimethythiazol-2-yl] -2,5-diphenyltetrazolium bromide) assay. The effects of two cryoprotectant medium treatment methods and seeding (at -7degree C)on cell viability were also assessed. A cryopreservation protocol of 1degree C/min in 1.4M DMSO from 4degree C to -60degree C, and seeding at -7degree C proved optimal for this tissue-engineered dermal replacement, provided the dermal slice was transferred into the cryopreservation bag containing 1mL 1.4M DMSO bag and hold for 15min at 4degree C prior to freezing.