Cryopreservation of Kudoa septempunctata sporoplasm using commercial freezing media

Cryopreservation methods for Kudoa septempunctata have not been established. This prevents an effective study of K. septempunctata, which cannot be artificially cultivated in the laboratory. In this study, we attempted to establish a cryopreservation method for K. septempunctata sporoplasm using Cellbanker® 1, a commercial preservation medium for mammalian cells. Spores were purified from the meat of Paralichthys olivaceus (olive flounder). These purified spores were suspended in Cellbanker® 1 and were stored at -80 °C for up to 16 months. Although the spores stored at -80 °C for 16 months were damaged, the sporoplasms maintained its amoeba-like indeterminate morphology, and their motility was well preserved. The viability of sporoplasms was variable among vials but was not below 70 %. In addition, the sporoplasms stored at -80 °C for 16 months could decrease the transepithelial electrical resistance of Caco-2 cells. These results indicate that this cryopreservation method using Cellbanker® 1 could preserve the viability and pathogenesis of K. septempunctata sporoplasm.