C-1003: Cryopreservation of rat hepatocytes with wheat proteins: Role in oxidative stress protection

Hepatocytes are an essential model for the pharmaceutical industry, as they are used for drug toxicity testing. Their availability is dependent on the surgical isolation of fresh cells from animal liver, and cryoconservation allows for the storage of isolated cells for future use. However, the freeze/thaw step associated with this procedure leads to structural damage to the cells, which lowers post-thaw viability and metabolic functions. To circumvent such damage, cryoprotective agents such as dimethylsulfoxide (Me2SO) are used, but these are often toxic for cells. The development of alternative cryoprotectants that are less toxic would therefore be beneficial. We have previously shown that wheat protein extracts are efficient for cryopreservation of hepatocytes and other cell types. To identify cryoactive proteins in these extracts, fractions from chromatographic separation were analysed by tandem mass spectrometry. Data analysis allowed us to determine that enolase is a good candidate as cryoprotector, revealing a novel function for this glycolysis-associated protein. The cDNA was cloned and the protein was produced in a bacterial system. Viability tests confirmed that the recombinant enolase is more efficient than Me2SO for cryopreservation of rat hepatocytes and that it causes no cellular toxicity. As a first step to elucidate its mechanism of action in the protection of cells, we have determined that the protein decreases the oxidative stress resulting from cryopreservation. Levels of peroxides and nitric oxide after thawing of cells frozen with enolase are lower than those in cells frozen with Me2SO. These results show that this plant protein has tremendous potential as a cryoprotective agent for hepatocytes, and possibly other cell types. The use of non-toxic agents that preserve viability and metabolic functions of mammalian cells would represent a major breakthrough in the field. It would allow increasing the availability of functional cells used for toxicity testing required for medical and pharmaceutical advances. FRQNT équipe, Québec, Canada