Proline-conditioning and chemically-programmed ice nucleation protects spheroids during cryopreservation.

Spheroids mimic 3-D tissue niches better than standard cell cultures. Cryopreserving spheroids, however, remains challenging as conventional cryoprotectants do not mitigate all damage mechanisms. Here chemically-programmed extracellular ice nucleation is used to prevent supercooling, alongside proline pre-conditioning, which are found to synergystically improve post-thaw recovery of spheroids. This validates the need to identify compounds and materials to address both biochemical and biophysical damage pathways beyond standard cryoprotectants.