This was both in terms of the cell recovery at 24 h post-thaw, an

This was both in terms of the cell recovery at 24 h post-thaw, and minimising differences in doubling time from the non-frozen control. Freezing media consisting of 10% Me2SO and 90% FBS was chosen as the control cryopreservation

media. Media such as this has been widely used in previous studies [23], [36] and [37]. The 24 h cell recovery for the optimum PP-50 concentration (103 ± 4%) was found to be less than that for the Me2SO control (130 ± 14%), AZD0530 although this difference was not statistically significant. In part, this may be explained by proliferation of the SAOS-2 cells during the first 24 h post-thaw. Assuming the cell doubling times remained constant throughout the experiment, the number of viable cells capable of proliferating immediately post-thaw for the PP-50/trehalose and Me2SO protocols was estimated to be comparable (64 ± 5% and 70 ± 11%, respectively). This estimated cryosurvival was similar to that achieved for mesenchymal stem cells by Wang et al. [42]. Hence the cryosurvival of proliferative cells achieved using the PP-50/trehalose treatment may have been comparable to the Me2SO control. It should be noted that MTS assays were not performed on the cells immediately post-thaw, as the presence of early apoptotic cells can yield CT99021 chemical structure misleading results [24],

as could the presence of cells incapable of substrate attachment. The cryosurvival immediately post-thaw was tested further for these protocols, using a flow cytometry based Annexin V/PI assay. The proportion of viable cells for the PP-50/trehalose and

Me2SO protocols were found to be comparable to those calculated above (80 ± 3% and 60 ± 2%, respectively). This could indicate that there is not a significant sub-population of cells for either protocol that appears viable, but is non-proliferative during subsequent culture. As discussed previously, Me2SO is currently the cryoprotectant of choice for most cell culture and therapeutic applications. Although FAD there is scope for improving the number of cells that survive the freezing process, the two most concerning problems associated with the use of Me2SO are loss of cell functionality, and toxicity to patients. Therefore, of the outcome measures tested, the comparison of the cell doubling times to the non-frozen control was thought to be the more important. It was found that the rate of proliferation was abnormally high for the cells cryopreserved using Me2SO compared to non-frozen SAOS-2 cells (Fig. 5). Indeed the cell doubling times were found to be significantly different from the non-frozen control by 41 ± 4%. In contrast, the doubling time for the cells cryopreserved using the optimum PP-50/trehalose protocol did not significantly affect the doubling time (Fig. 6). These data suggest that the normal processes of the cells were affected less when cryopreserved using PP-50/trehalose than Me2SO, while maintaining high cell recovery.

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