Abstract: Objective: The purpose of this study was to explore the influences of different cooling methods and cryoprotectants on the viability of periodontal ligament cells in vitro. Methods: A total of 25 fresh teeth (the first premolar or second premolar) were collected and randomly divided into five groups. Four experimental groups were cooled to -196 ℃ by programmed freezer or rapid freezer in cryoprotectants with or without trehalose for 1 week. In the control group, the fresh teeth were not given any treatment. The PDL attached to the middle third of roots was shaved from the root surface using scalpel knife. Filtered fluid was collected after digestion using trypsin for 5 mins and repeated for 5 times. After centrifugation, the supernatant was discarded. After resuspending the cells with phosphate buffered saline (PBS), the living PDLCs were stained by Trypan blue and counted under high magnification. The cell survival rates of different groups were calculated and statistically analysed. Results: There were no statistically significant difference in survival rates of PDLCs between all groups. But in the group cryopreserved by programmed freezer with trehalose, the survival rates was the highest compared to that of the others. Conclusions: The cryopreservation do not cause obvious damage to the periodontal ligament cells. In our opinion, this technique could be applied in teeth preservation. On the other hand, using the programmed freezer with trehalose in cryoprotectants have minimal effect on the viability of periodontal ligament cells.