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.