Extracellular Antifreeze Protein Significantly Enhances the Cryopreservation of Cell Monolayers

Abstract

The cryopreservation of cells underpins many areas of biotechnology, healthcare, and fundamental science by enabling the banking and distribution of cells. Cryoprotectants are essential to prevent cold-induced damage. Here, we demonstrate that extracellular localization of antifreeze proteins can significantly enhance post-thaw recovery of mammalian cell monolayers cryopreserved using dimethyl sulfoxide, whereas they show less benefit in suspension cryopreservation. A type III antifreeze protein (AFPIII) was used as the macromolecular ice recrystallization inhibitor and its intra/extracellular locations were controlled by using Pep-1, a cell-penetrating peptide. Flow cytometry and confocal microscopy confirmed successful delivery of AFPIII. The presence of extracellular AFPIII dramatically increased post-thaw recovery in a challenging 2-D cell monolayer system using just 0.8 mg·mL–1, from 25% to over 60%, whereas intracellularly delivered AFPIII showed less benefit. Interestingly, the antifreeze protein was less effective when used in suspension cryopreservation of the same cells, suggesting that the cryopreservation format is also crucial. These observations show that, in the discovery of macromolecular cryoprotectants, intracellular delivery of ice recrystallization inhibitors may not be a significant requirement under “slow freezing” conditions, which will help guide the design of new biomaterials, in particular, for cell storage.

Coaxial Alginate Hydrogels: From Self-Assembled 3D Cellular Constructs to Long-Term Storage

“…This could have a number of reasons: poor penetration of the CPAs into the capsules, increased thermal stresses at the intersection of the alginate membrane with a core material, unequal temperature distribution and spontaneous ice formation at suboptimal negative temperatures. Thus, the following approaches can be considered to minimize or avoid the damaging effects occurring during freezing and thawing: modelling of the heat transfer and thermomechanical stress [ 78 , 79 ], application of ice-binding proteins in the alginate membrane and core material [ 80 ], induced ice formation technologies [ 81 , 82 ], freezing containers with improved heat transfer (such as cryobags, [ 83 ], as well as the validation of the ice-free vitrification approach [ 48 ] and radiofrequency nanowarming [ 84 ].…”

Proline pre-conditioning of cell monolayers increases post-thaw recovery and viability by distinct mechanisms to other osmolytes

“…19,20 During thawing, ice recrystallization can damage the cells by both mechanical and osmotic stress, and controlling this process by using ice recrystallization inhibitors (IRIs) has been shown to result in enhanced post-thaw recovery. 21,22 Antifreeze (glyco)proteins (AF(G)Ps) are potent IRIs and can reduce damage to cell membranes, [23][24][25] however, AF(G)Ps are unsuitable for cryopreservation applications due to their potential toxicity/immunogenicity, their secondary effect of dynamic ice shaping (DIS), 21 and their low availability/ expense. Considering this, there is significant interest in the discovery of AF(G)P mimics.…”

Ice recrystallisation inhibiting polymer nano-objects via saline-tolerant polymerisation-induced self-assembly

“… 19 One particularly desirable property is ice recrystallisation inhibition (IRI); the prevention of ice crystals from growing (distinct from nucleation). IRI-active materials have been found to improve post-thaw recovery of cryopreserved cells, 17 , 20 , 21 proteins 22 and bacteria. 23 It is still not possible to rationally design new IRIs, and in particular generating IRI-active polymer colloids/nanomaterials is challenging.…”