Systematic optimization of Caenorhabditis elegans cryopreservation

Caenorhabditis elegans (C. elegans) is a non-parasitic roundworm widely utilized as a versatile model organism for studying fundamental biological processes. Despite the availability of multiple cryopreservation methods, variations in the selection of developmental stage, cryoprotectant composition, and storage conditions may sometimes cause inconsistencies and uncertainty among researchers. In this study, we followed a step-by-step approach to optimize the C. elegans cryopreservation process. Firstly, different larval stages (L1-gravid adult) of C. elegans were cryopreserved using a fixed concentration of trehalose-DMSO and glycerol, followed by the post-thaw survival rate assessment. We found that the starved L1 larvae has the highest survival rate among other developmental stages. Subsequently, the starved L1 larvae was cryopreserved using different concentrations (5%, 10%, and 15%) of DMSO and glycerol and found that 5% DMSO has the most effective cryoprotection. Finally, the impact of storage conditions on worm viability was evaluated by placing cryogenic vials in different storage boxes, including standard cardboard cryogenic boxes, Styrofoam boxes, and isopropanol-based freezing containers, and found that standard cardboard cryogenic boxes provided higher worm viability. The finalized protocol was then validated across multiple mutant and transgenic strains to confirm its reproducibility and broad suitability. Taken together, our results demonstrate that starved L1 larvae cryopreserved with 5% DMSO using a standard cardboard cryogenic box provided the highest survival rate (>90%) across wild-type, mutant, and transgenic strains. Here we propose a simple, reproducible, and strain-independent protocol for cryopreservation of C. elegans . The proposed protocol can be easily adopted in C. elegans research laboratories and stock centers globally.