Effects of Freezing on the Cytoskeleton, Focal Adhesions and Gap-Junctionsin Murine Osteoblast Cultures

Understanding the ultra-structural response of cells to the cryopreservation process is important for designing cryopreservation strategies for cells and tissues. Cell-cell interaction and cell-scaffold interactions alter cryopreservation response and, in turn, the cellular structures involved in adhesion and intercellular contact are targets of cryopreservation-induced damage. Immuno-fluorescence was used to assess the status of the actin filaments, vinculin (focal adhesion) and connexin-43 (gap junction) of murine osteoblasts attached to hydroxyapatite (HA) discs glass coverslips for a two-step freezing process. The freezing process de-polymerized and distorted the F-actin of dead cells, while that of live cells has little change. Vinculin and connexin-43 structures were rarely seen in dead cells, a portion of vinculin and connexin-43 remained in live cells. These results suggest that focal adhesions and gap junctions may support the cells robustness during cryopreservation. The present study helps investigate the damage mechanism of attached cells during freezing process