Conversely, the CFU-F number was shown to increase after thawing (data not shown). The fresh SVF cells were successively challenged by a freezing and thawing cycle. N = 15 samples were used in the freezing/thawing procedure as described above. SVF samples ranging from 6.66 × 105 to 3.94 × 106 total cells were taken in consideration. Table 2 shows the results of these experiments. Cell samples were kept RGFP966 manufacturer frozen for periods ranging from 14 to 193 days. Viability of SVF cells was

measured by FACS analysis and gave an average value of 89.6% ranging from 81% to 98%. The total ASCs content of each fresh sample ranged from 237,938 to 1,092,925 with an average value of 587,753 cells. After thawing, cells were counted for ASCs number and viability. We could demonstrate the viability results over 15 samples, ranging from BIRB 796 in vivo 71.7% to 98.3% and average recovery rates of 79.82% of living ASCs after the freeze/thaw procedure. Alive cells after a freezing/thawing cycle are important because the freezing process prolongs cells’ life and makes them available for future therapies based on expanded ASCs. To check whether the thawed cells can grow and differentiate again after the freezing/thawing cycle, we cultivated and differentiated 3 samples of thawed SVF-cells

in 0.1% human serum supplemented medium. The results are showed in Fig. 3. Three different samples were plated at 3000 cells/cm2 and cultured for 20 days. Cells showed a classical growth pattern with an early

lag-phase in the first 7 days and a subsequent exponential growth. After 20 days in culture, cells reached a concentration many of 42,550 cells/cm2 i.e. a 13× expansion of the initial seeded number. The same cells were induced to differentiate into adipocytes, osteocytes and chondrocytes and representative results are shown in Fig. 4. Cells were clearly inducible to the specified phenotypes. Oil Red staining evidenced adipoinduction by red deposits in vacuoles (Fig. 4, panel A: induced and D: control), whereas Alizarin-S staining was used for osteoinduction and showed the formation of red calcium deposits as a marker of osteogenic differentiation (Fig. 4, panel B: induced and E: control). Sections of chondro-induced samples stained with Alcian Blue showed a strong blue signal (Fig. 4, panel C, induced and F, control). We found all tested samples to be inducible for the differentiation of adipocytes, osteocytes and chondrocytes. Tissue engineering keeps promise for the restoration of the soft tissue esthetic function and for the treatment of known diseases that have currently no therapy option [22]. In this regard, the storage of ASCs is still for long time the initial step for future cell therapies using ASCs for regenerative purposes.