Effects of two-step freezing on the ultra-structural components of 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 possible targets of cryopreservation-induced damage. Immuno-fluorescence was used to assess the status of the actin filaments (F-actin), focal adhesions (vinculin) and gap junctions (connexin-43) of murine osteoblasts attached to hydroxyapatite (HA) discs and plastic coverslips for a two-step freezing process. The freezing process de-polymerized and distorted the actin filaments of dead cells, while those of live cells experienced little change. Vinculin and connexin-43 structures were rarely seen in dead cells, while a portion of vinculin (8.14+/-2.27 percent) and connexin-43 (21.7+/-4.7 percent) structures remained in live cells. These results suggest that focal adhesions and gap junctions may support cell robustness during cryopreservation. The present study contributes to our knowledge of the damage mechanisms associated with attached cells during a freezing process.
Liu
Baolin
B
Institute of Cryobiology, Shanghai University of Science and Technology, Shanghai, China. blliuk@163.com
McGrath
John J
JJ
D000199:Actins / Q000187:drug effects / Q000648:ultrastructure; D000818:Animals; D002460:Cell Line; D018031:Connexin 43 / Q000187:drug effects / Q000648:ultrastructure; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000648:ultrastructure*; D022001:Focal Adhesions / Q000187:drug effects / Q000648:ultrastructure*; D017629:Gap Junctions / Q000187:drug effects / Q000648:ultrastructure*; D051379:Mice; D009930:Organic Chemicals / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000648:ultrastructure*; D016596:Vinculin / Q000187:drug effects / Q000648:ultrastructure
D000199:Actins; D018031:Connexin 43; D003451:Cryoprotective Agents; D009930:Organic Chemicals; C420642:alpha minimal essential medium; D016596:Vinculin