Chemically Induced Extracellular Ice Nucleation Reduces Intracellular Ice Formation Enabling 2D and 3D Cellular Cryopreservation

3D cell assemblies such as spheroids reproduce the in vivo state more accurately than traditional 2D cell monolayers and are emerging as tools to reduce or replace animal testing. Current cryopreservation methods are not optimized for complex cell models, hence they are not easily banked and not as widely used as 2D models. Here we use soluble ice nucleating polysaccharides to nucleate extracellular ice and dramatically improve spheroid cryopreservation outcomes. This protects the cells beyond using DMSO alone, and with the major advantage that the nucleators function extracellularly and hence do not need to permeate the 3D cell models. Critical comparison of suspension, 2D and 3D cryopreservation outcomes demonstrated that warm-temperature ice nucleation reduces the formation of (fatal) intracellular ice, and in the case of 2/3D models this reduces propagation of ice between adjacent cells. This demonstrates that extracellular chemical nucleators could revolutionize the banking and deployment of advanced cell models.

Automated summary

3D cell assemblies, such as spheroids, are more accurate in replicating in vivo conditions compared to traditional 2D cell monolayers, making them valuable tools for reducing or replacing animal testing. However, current cryopreservation methods are not well-suited for complex cell models, limiting their usage. By utilizing soluble ice nucleating polysaccharides, researchers have significantly improved spheroid cryopreservation outcomes, providing better cell protection compared to using DMSO alone. The extracellular function of these nucleators reduces the formation of fatal intracellular ice and prevents ice propagation between adjacent cells, potentially revolutionizing the banking and deployment of advanced cell models.