Induced Extracellular Ice Nucleation Protects Cocultured Spheroid Interior and Exterior during Cryopreservation

Spheroids and other 3D cellular models more accurately recapitulate physiological responses when compared to 2D models and represent potential alternatives to animal testing. The cryopreservation of spheroids remains challenging, limiting their wider use. Standard DMSO-only cryopreservation results in supercooling to low subzero temperatures, reducing viability, shedding surface cells, and perforating spheroid interiors. Here, cocultured spheroids with differentially labeled outer cell layers allow spatial evaluation of the protective effect of macromolecular ice nucleators by microscopy and histology. Extracellular nucleation is shown to reduce damage to both interior and exterior regions of the spheroids, which will support the development of "off-the-shelf" 3D models.