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.

Torsional fracture of viscoelastic liquid bridges

Short liquid bridges are stable under the action of surface tension. In applications like electronic packaging, food engineering, and additive manufacturing, this poses challenges to the clean and fast dispensing of viscoelastic fluids. Here, we investigate how viscoelastic liquid bridges can be destabilized by torsion. By combining high-speed imaging and numerical simulation, we show that concave surfaces of liquid bridges can localize shear, in turn localizing normal stresses and making the surface more concave. Such positive feedback creates an indent, which propagates toward the center and leads to breakup of the liquid bridge. The indent formation mechanism closely resembles edge fracture, an often undesired viscoelastic flow instability characterized by the sudden indentation of the fluid’s free surface when the fluid is subjected to shear. By applying torsion, even short, capillary stable liquid bridges can be broken in the order of 1 s. This may lead to the development of dispensing protocols that reduce substrate contamination by the satellite droplets and long capillary tails formed by capillary retraction, which is the current mainstream industrial method for destabilizing viscoelastic liquid bridges.