In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.