The Cryopreservation of a Tissue Engineered Dermal Replacement by Programmed Freezing

Long-term storage 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% CO₂ atmosphere for 14 days, and dermal slices (10times3times1.5 mm) were used in the experiment. Two groups were defined - fresh control and experimental group. The freezing was carried out in a computer-controlled programmable freezer at 0.5 degree C/min, 1degree C /min and 2degree C /min in 1.4M, 2.1M and 2.8M DMSO from 4degree C to -60degree C and then plunged into the liquid nitrogen tank immediately. After 24 h of cryopreservation, the samples were removed from liquid nitrogen and placed at room temperature in air for 0.5-1 min, prior to submergence in a water bath at 37degree 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 -6degree 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 cryoprotectant medium, which was kept at 4degree C , seal the bag and hold for 15 min at 4degree C prior to freezing. The viability of the dermal replacement treated by this protocol was maintained 80% of the fresh control viability. And the micrograph confirmed that after 48 h inc- - ubation following thawing, the dermal fibroblast can regain its natural shape and functions. No fracture was observed