It can be debated that functional assays can produce misleading results when performed on cellular systems as these cells can become metabolically more this website active after cooling stresses, as demonstrated by the work of Jomha et al. [49]. These results were the first positive demonstration of liquidus-tracking application in tissues; however, it suffered from one significant limitation: actual human cartilage with up to 5 mm thickness would require significantly longer times for equilibration

of the CPA than cartilage discs with 0.7 mm thickness. The equilibration time would be even longer when the cartilage-on-bone grafts are to be cryopreserved using this method as cartilage on bone has only half of the surface available for CPA diffusion compared to a cartilage disc. Using one type of CPA during stepwise cooling for vitrification may be possible in thinner tissues as shown by Pegg but would result in excessive CPA toxicity in larger tissues because of the prolonged exposure to very high concentrations of the CPA during the final Doxorubicin mouse stages. This is complicated by the fact that for most CPAs, cytotoxicity increases nonlinearly with concentration [26]. Therefore, to decrease concentration-dependent CPA cytotoxicity during cooling steps, another approach is to use combinations of CPAs each at a lower final concentration so that individually the CPAs are

less toxic to the cells but the overall final concentration is sufficient to vitrify [20], [31] and [61]. The idea of cryoprotectant toxicity neutralization using certain amides as structural analogues of cryoprotectants Clostridium perfringens alpha toxin was discussed previously by Fahy [28]. Recently,

Jomha et al. showed positive interactions between commonly used CPAs [6] and [53]. This indicates that a lowered cumulative CPA toxicity occurs in multiple-CPA solutions compared to single-CPA solutions of similar total concentration [53]. The same group showed that multiple-CPA solutions can be beneficial by increasing the glass stability above that of an equivalent molar single-CPA solution [108]. These conclusions were indirectly supported by Brockbank et al. (2010) [17] who recorded good chondrocyte recovery after vitrification of pig articular cartilage using different combinations of Me2SO, formamide and propylene glycol (VS55 and VS83 both loaded at 4 °C followed by cooling to −135 °C at various cooling rates). Cartilage thickness remained an issue as the results showed increasingly lower recovery with thicker cartilage. Again in this study the cartilage had been removed from its bone base prior to vitrification. To address three main obstacles to cartilage cryopreservation, including CPA permeation to prevent ice formation within the matrix, CPA toxicity and CPA vitrifiability, vitrification with multi-CPA solutions using stepwise cooling is perhaps the most viable approach.