Construction of Time-Lapse Scanning Electrochemical Microscopy with Temperature, Control and Its Application To Evaluate the Preservation Effects of Antifreeze Proteins on Living Cells

Antifreeze proteins (AFPs) can protect cells from hypothermic damage; however, their mechanism of action remains unclear. Scanning electrochemical microscopy (SECM) can evaluate the size and activities of cells, although long-term continuous monitoring has been unsuccessful. We constructed a novel, fully automated, timelapse SECM system and investigated the cell preservation effect of AFPs by analyzing single cellular topography at low temperatures. From the SECM measurements, mammalian cells (HepG2), treated in Euro-Collins (EC) solution at 4 degrees C, began to swell at 8 h and then immediately ruptured. In AFP-containing EC solution, the cellular size did not change until 16 h and then gradually increased and finally ruptured. In addition, the cellular height at rupture point significantly increased in the presence of AFF's. These results suggest that AFPs stabilize the cellular membrane and protect cells from hypothermic damage. This SECM system allowed us to observe the single cellular response to hypothermia by long-term automatic scanning and will be applicable for analysis to other cellular activities and topographies.