Abstract: Objective: To explore the influences of different cooling methods and cryopreservation with different cryoprotectants on biomechanical performances of in vitro dental tissues. Methods: A total of 50 fresh teeth (the first premolar or second premolar) were collected and divided into five groups. The tissues of four experimental groups were cooled to -196 ℃ by programmed cooling method and rapid cooling method in cryoprotectant with or without trehalose, for 1 week cryostorage. And the fresh teeth of the control group were not given any treatment. Five teeth from each group were made into solid mass dental tissues, which were measured for compression strength and modulus of elasticity; another 5 teeth, in which Ⅴ-type dental cavities were made in the maxillofacial region and filled with resin, were given dye penetration test with 2% methylene blue solution after 500 temperature cycles. Micro leaking at the edge of dental filling materials in each group was measured and compared. Results: There were no significant differences between groups with different cooling methods and different cryoprotectants in dental compression strength, modulus of elasticity, and micro leaking at the edge of dental filling materials. Conclusions: There are no signicant differences in biomechanical properties of dental tissue and micro leaking at the edge of filling materials in cryopreservation for teeth preservation.