A mixture of innate cryoprotectants is key for cryopreservation of a drosophilid fly larva.

Background: Organisms evolved biochemical strategies to cope with environmental stressors. For instance, insects that naturally tolerate internal freezing produce complex mixtures of multiple cryoprotectants (CPs). Better knowledge on composition of these mixtures, and on mechanisms of how the individual CPs interact, could inspire development of laboratory CP formulations optimized for cryopreservation of cells and other biological material. Results: Here we identify and quantify (using high resolution mass spectrometry) a range of putative CPs in larval tissues of a subarctic fly, Chymomyza costata, that survives long-term cryopreservation in liquid nitrogen. The CPs (proline, trehalose, glutamine, asparagine, glycine betaine, glycerophosphoethanolamine, glycerophosphocholine, and sarcosine) accumulate in hemolymph in a ratio of 313:108:55:26:6:4:3:0.5 mmol.L-1. Using calorimetry, we show that the artificial mixtures, mimicking the concentrations of major CPs' in hemolymph of freeze-tolerant larvae, suppress the melting point of water and significantly reduce the ice fraction. We demonstrate in a bioassay that mixtures of CPs administered through the diet act synergistically rather than additively to enable cryopreservation of otherwise freeze-sensitive larvae. Using MALDI-MSI, we show that during slow extracellular freezing of whole larvae trehalose becomes concentrated in partially dehydrated hemolymph and stimulates transition to the amorphous glass phase. In contrast, proline moves to the boundary between extracellular ice and dehydrated hemolymph and tissues where it likely forms a layer of dense viscoelastic liquid. Conclusion: Our results suggest that different components of innate cryoprotective mixtures of freeze-tolerant insect act in synergy during extracellular freezing. We propose that transitions to amorphous glass (stimulated by trehalose) and viscoelastic liquids (having proline as major component) may protect macromolecules and cells from thermomechanical shocks associated with freezing and transfer into and out of liquid nitrogen.

Review of Single Bubble Motion Characteristics Rising in Viscoelastic Liquids

The emphasis of this review is to discuss three peculiar phenomena of bubbles rising in viscoelastic fluids, namely, the formation of a cusp, negative wake, and velocity jump discontinuity, and to highlight the possible future directions of the subject. The mechanism and influencing factors of these three peculiar phenomena have been discussed in detail in this review. The evolution of the bubble shape is mainly related to the viscoelasticity of the fluid. However, the mechanisms of the two-dimensional cusp, tip-streaming, “blade-edge” tip, “fish-bone” tip, and the phenomenon of the tail breaking into two different threads, in some special viscoelastic fluids, are not understood clearly. The origin of the negative wake behind the bubbles rising in a viscoelastic fluid can be attributed to the synergistic effect of the liquid-phase viscoelasticity, and the bubbles are large enough; thus, leading to a very long relaxation time taken by the viscoelastic stresses. For the phenomenon of bubble velocity jump discontinuity, viscoelasticity is the most critical factor, and the cusp of the bubbles and the surface modifications play only ancillary roles. It has also been observed that a negative wake does not cause velocity jump discontinuity.