Improvements in the infectivity of cryopreserved third-stage larvae of Angiostrongyluscantonensis using a programmable freezer

Abstract

Although there have been some advances in the cryopreservation of Angiostrongyluscantonensis, the degrees of viability and infectivity of the cryopreserved developmental stages have not been high. A two-step freezing protocol using a programmable freezer was determined to be effective in improving the infectivity of the cryopreserved third-stage larvae of this parasite. After washing steps and suspension in 10% (v/v) dimethylsulfoxide and equilibrium at room temperature the larvae were placed into the freezer. The temperature was lowered first at 0.8 °C/min from room temperature to −40 °C and then at 10 °C/min to −70 °C. The samples were plunged into liquid nitrogen. After storage in liquid nitrogen for 7–15 days the larvae were thawed rapidly in 37 °C water and 27.6% were found to show vigorous “S-shape” movement without significant changes in appearance. These larvae (50/rodent) could develop to the fifth stage in mice (42.6%) and establish patent infection in rats (40.4%). Moreover, there was no significant difference in the recovery rates of cryopreserved worms and their unfrozen counterparts. These findings indicate that steady precooling conditions may decrease damage with regard to the infectivity of cryptopreserved third-stage larvae of A. cantonensis.