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In cryobiology, the freezing of cells and tissues requires further detailed study to clarify the mechanisms of freezing injury and protection by cryoprotectants, and to extend the application of cryopreservation. Generally, the freezing and thawing behavior of cells depends on the properties of the cells. The freezing of neurons forming neuronal network has largely been neglected, despite the fact that freezing these cells benefits the study of the cryopreservation of cells in medicine/poison screening. The freezing of these cells is also attractive for studying cell morphology because of their characteristic long, thread-like neurites extending from the cell body. In the present study, the freezing of adherent neuron-like cells (differentiated PC12 cells) with neurites, which are necessary to form neuronal network, in physiological saline were investigated to understand their basic characteristics on the freezing and thawing. First, the microscopic freezing behavior of the cells with different cooling rates was observed. Next, the post-thaw morphological changes of the cells including cytoskeleton were investigated and the post-thaw cell viability was evaluated from the dye exclusion using propidium iodide. The relationship among these characteristics was also discussed.

Freezing and Thawing Characteristics and Post-Thaw Viability of Differentiated PC12 Cells Adhered to Substrate