Vitrification Solutions for the Cryopreservation of Tissue-Engineered Bone

Abstract

Vitrification solutions were explored for the cryopreservation of tissue-engineered bone constructs using differential scanning calorimetry, visual inspection, toxicity and viability assays. Two vitrification "cocktails" (VS55 and VEG; concentration range of 80–100%)—40% (vol/vol) DMSO and 40% (vol/vol) glycerol solutions—were studied with and without an ice blocker (IB). Results show that 95% VEG + IB solution is the best option to preserve the OB:HA scaffold-cell complex from the standpoint of critical cooling rate (5°C/min), critical warming rate (35°C/min), minimal scaffold damage, toxicity, and preliminary cryopreservation results (92.0% ± 1.4% for suspended cells, 43.0% ± 2.7% for attached cells). Ice blocker was found to reduce the critical warming rates of VS55 and VEG solutions at higher concentrations but worsen devitrification of lower concentration solutions. The fact that the critical cooling and warming rates are larger for larger systems as judged by visual inspection indicates that visual inspection is a reasonable practical guide to determine the critical cooling and warming rates for larger volume solutions, especially with embedded HA samples. Fracture of vitrified solutions is detrimental to cells, but such fracture should be avoidable by slow and uniform cooling combined with a minimum storage temperature just below the glass transition temperature rather than lower temperatures.