Silymarin mediated osmotic responses and damage in HepG2 cell suspensions and monolayers

Maintenance of cells within a volume range compatible with their functional integrity is a critical determinant of cell survival after cryopreservation, and quantifying this osmotically induced damage is a part of the rational design of improved cryopreservation protocols. The degree that cells tolerate osmotic stress significantly impacts applicable cryoprotocols, but there has been little research on the time dependence of this osmotic stress. Additionally, the flavonoid silymarin has been shown to be hepatoprotective. Therefore, here we test the hypotheses that osmotic damage is time-dependent and that flavonoid inclusion reduces osmotic damage. In our first experiment, cells were exposed to a series of anisosmotic solutions of graded hypo- and hypertonicity for 10-40 min, resulting in a conclusion that osmotically induced damage is time dependent. In the next experiment, adherent cells preincubated with silymarin at the concentration of 10^-4 mol/L and 10^-5 mol/L showed a significant increase in cell proliferation and metabolic activity after osmotic stress compared to untreated matched controls. For instance, when adherent cells preincubated with 10^-5 mol/L silymarin were tested, resistance to osmotic damage and a significant increase (15%) in membrane integrity was observed in hypo-osmotic media and a 22% increase in hyperosmotic conditions. Similarly, significant protection from osmotic damage was observed in suspended HepG2 cells in the presence of silymarin. Our study concludes that osmotic damage is time dependent, and the addition of silymarin leads to elevated resistance to osmotic stress and a potential increase in the cryosurvival of HepG2 cells.