学会発表Characteristics of bovine fibroblast cells after freeze drying
In general, mammalian somatic cells are preserved in the ultra low temperature freezer or the liquid nitrogen. However, the maintenance and transportation of frozen cells are costly, and there are problems with a safety aspect on liquid nitrogen. Freeze drying, which removes moisture through sublimation, has been used for a long-term storage of food, drug, yeast. To our knowledge, little work has been devoted to freeze dry for mammalian somatic cells. The objective of this study was to assess the physical properties on freeze-dried (FD) products of bovine fibroblast cells, and evaluate the characteristics. Bovine fibroblast cells were obtained from ear skin tissue of a Japanese Brown Cattle-Kochi. The cells were suspended in a freeze-drying solution, and lyophilized for 11.5 h. The eutectic point of freeze-drying solution was about -28℃, therefore, we compared the freezing method between rapid freezing and slow freezing (-0.3℃/min) at -30℃. The DNA damage of the FD cells was evaluated with comet assay. The FD cells were also observed by Transmission Electron Microscopy (TEM). The moisture content and glass transition temperature (Tg) were measured by Karl Fischer titration and Differential Scanning Calorimetry, respectively. The rate of DNA-damaged cells was significantly lower in slow freezing (14%; 68/500) than in rapid freezing (24%; 122/500) (P<0.05). After freeze-drying, the plasma membrane was of all cells was completely damaged, however, the nuclear membrane was intact. In the FD products, the moisture content was 27% and Tg was -28℃. These results suggest that bovine FD fibroblast cells would be useful as a donor cell for nuclear transfer, because they preserve their nuclear structure. However, FD conditions should be improved for a long-term storage at room temperature.