Cell, tissue, and organ preservation with insect-derived antifreeze peptides

Abstract

We describe our efforts to date using Dendroides- and, to a lesser extent, Cujucus-derived recombinant antifreeze proteins for short- and long-term mammalian cell, tissue, and organ storage strategies. Some single Dendroides antifreeze proteins (DAFPs), DAFP-6 and DAFP-2 concentrations in combination with dimethylsulfoxide increased post-cryopreservation cell viability. However, no significant improvement in viability was observed with either DAFP-1 or DAFP-4. Furthermore, more complex DAFP formulations resulted in loss of viability enhancement observed with DAFP-6 and 2. Experiments were also performed to see if the combination of DAFPs-1, -2, and -4 plus thaumatin-like protein had a benefit for hypothermic storage at -7 versus +4 °C. Thaumatin-like proteins are polypeptides of about 200 residues originally described in plants that are synthesized in response to fungal infection. Wang and Duman (Biochemistry 45:1278-1284, 2006) had previously described a thaumatin-like protein from larvae of the beetle Dendroides canadensis that enhances the activity of AFPs. Subzero storage of veins resulted in better contractile and relaxation properties than +4 °C storage. Further studies went on to compare Dendroides- and Cujucus-derived AFPs in combination with 0.5 M glycerol and 150 mM trehalose. DAFP-10 and CAFP-3 were very effective at maintaining venous contractile functions compared with control storage solutions with just glycerol and trehalose. We focused on DAFP-10 and demonstrated that this AFP in combination with glycerol and trehalose was effective for maintaining functions during hypothermic vein storage for up to 6 days in a dose-dependent manner. However, no benefit of AFPs were observed using arteries. Controls with just glycerol and trehalose maintained excellent arterial contractile functions for up to 12 days of cold storage. Porcine liver storage was also evaluated with DAFP-10 combined with glycerol and trehalose. Trends were observed to higher venous flow and lower hepatic artery flow after 24 h of storage, suggesting an organ level impact of the differences observed in vein versus artery physiology preservation observed in isolated blood vessels. No clear-cut benefits of DAFP-10 were observed compared with cold storage without DAFP-10, although both enzymes alanine aminotransferase and lactate dehydrogenase were similar to short-term positive controls stored for only 2 h. Based upon observations with fish AFPs and insect AFPs we believe it is likely that AFPs may have benefits for cell, tissue, and organ preservation at both frozen and hypothermic, nonfrozen temperatures. However, the published literature and our results to date are inconclusive, although suggestive of benefits. More research is needed on AFPs in combination with cryopreservation and nonfrozen hypothermic storage.