Slow-cooling protocols for microcapsule cryopreservation.

Abstract

The relatively large size (300-400 microm) and fragile semi-permeable membrane of microcapsules makes them particularly prone to cryodamage. This study investigated slow-cooling protocols for the cryopreservation of microcapsules. Instead of a programmable freezing-machine, slow cooling was carried out directly within a -80 degrees C refrigerator. A range of increasing cryoprotectant (DMSO and EG) concentrations with slow cooling was investigated. The results showed that 2.8 M (20% v/v) DMSO and 2.7 M (15% v/v) EG were optimal for microcapsule cryopreservation, resulting in approximately 55-60% of the microcapsules remaining intact, with a relatively high post-thaw cell viability of 80-85%. Post-thaw cell viability and microcapsule integrity were consistently higher at equivalent molarities of DMSO compared to EG. Hence, all subsequent studies utilized only DMSO. Post-thaw cell viability upon slow cooling with 2.8 M (20% v/v) DMSO was significantly improved in the presence of 0.25 M sucrose (> 95%), but there was no enhancement in microcapsule integrity. Neither post-thaw cell viability nor microcapsule integrity was improved with multi-step exposure and removal of sucrose, compared to a single-step protocol. There was also no improvement in either post-thaw cell viability or microcapsule integrity in the presence of 20% (w/v) Ficoll. Hence, the optimal condition for microcapsule cryopreservation by slow-cooling is with 2.8 M (20% v/v) DMSO and 0.25 M sucrose.