The cryopreservation of a tissue engineered dermal replacement by programmed freezing

Area of Science:

• Regenerative Medicine
• Biomaterials Science
• Cryobiology

Background:

• Engineered bio-artificial tissues require long-term storage for clinical availability due to lengthy production cycles.
• Cryopreservation is a key technology for achieving long-term preservation of these tissues.

Purpose of the Study:

• To investigate the impact of various cryopreservation parameters on the cell viability of tissue-engineered dermal substitutes.
• To determine optimal conditions for cryopreservation to ensure the viability of dermal fibroblast cells within a polyglycolic acid scaffold.

Main Methods:

• Dermal fibroblast-seeded polyglycolic acid scaffolds were cryopreserved using varying dimethyl sulfoxide (DMSO) concentrations (1.4M, 2.1M, 2.8M) and cooling rates (0.5°C/min, 1°C/min, 2°C/min).
• Evaluated effects of cryoprotectant medium treatment methods and seeding at -7°C on cell viability using a modified MTT assay.
• Post-thaw viability assessed after controlled rewarming from liquid nitrogen storage.

Main Results:

• A cryopreservation protocol involving 1.4M DMSO and a cooling rate of 1°C/min from 4°C to -60°C yielded optimal cell viability.
• Pre-freezing seeding at -7°C significantly improved cell viability.
• Holding the dermal slice in 1mL of 1.4M DMSO at 4°C for 15 minutes prior to freezing was crucial for the optimal protocol.

Conclusions:

• The identified cryopreservation protocol (1°C/min, 1.4M DMSO, -7°C seeding, and pre-freezing incubation) is effective for preserving tissue-engineered dermal substitutes.
• This optimized method supports the goal of 'off-the-shelf' availability for clinical applications.