Glycerol improves the viability of a cryopreserved choanoflagellate

The colonial choanoflagellate Salpingoeca rosetta is a tractable model system for studying the origins of multicellularity, but long-term storage strategies for this species have not been tested. In this study, we probed each stage of cryopreservation (freeze-down, long-term storage, recovery) to identify the optimal protocol for recovery of S. rosetta and co-cultured bacterial cells. Dimethyl sulfoxide (Me2SO; commonly referred to as DMSO), the current cryoprotective agent (CPA) standard, proved to be worse than glycerol at comparable concentrations. Samples treated with either CPA at 5% showed the poorest recovery. Our results identified 15% glycerol as the most effective CPA for both S. rosetta and Echinicola pacifica . We also determined that ultra-low temperature freezers can be sufficient for short-term storage. We propose 15% glycerol and liquid phase nitrogen as the standard cryopreservation protocol for S. rosetta cultures and as a starting point for testing long-term storage strategies for other choanoflagellates and heterotrophic protists.