A refinement to the liquidus-tracking method for vitreous preservation of articular cartilage

The recent liquidus-tracking method developed by Pegg et al. (2006a), as an alternative pathway to vitrification, achieved reasonable survival of post-thawing chondrocytes in situ. One of the main drawbacks of this method is the long duration of the cryoprotectant addition/removal process. This study was conducted to investigate the possibility of reducing the time by rationalizing the final dimethyl sulfoxide (Me2SO) concentration loaded in tissue before being plunged into liquid nitrogen. Using the differential scanning calorimetric technique, the critical cooling and warming rates for solutions of Me2SO in CPTes2 (a potassium-rich medium, modified slightly from Taylor's original formulation by Pegg et al.) were obtained. The critical cooling and warming rates for 47.5 percent (w/w) solution are < 2.5 degree C per min and < 10 degree C per min, respectively, which could be readily realized for 4 ml solution samples held in polypropylene cryovials as demonstrated by experiments. For articular cartilage, 47.5 percent (w/w) may be recommended as the final concentration of Me2SO loaded in the tissue, which will lead to a time cut of about one-third compared with the original protocol of Pegg et al. (2006a).