Cell membrane permeability coefficients determined by single-step osmotic shift are not applicable for optimization of multi-step addition of cryoprotective agents: As revealed by HepG2 cells

HepG2 cells have a number of research applications and cryopreservation of these cells would improve supply and thus facilitate the study. Development of effective cryopreservation protocols relies on knowledges of the fundamental mass transport characteristics of HepG2 cell membrane. Currently, the permeability parameters estimated from single-step addition are routinely used to predict the osmotic responses of the cells in multistep protocols, as well as used for prediction of optimal cooling rates. However, the reasonability of this approach has not been rigorously studied. Here we measured the hydraulic conductivity (Lp) and the permeability coefficient (Ps) of HepG2 cells in the absence/presence of dimethyl sulfoxide (Me2SO) at various temperatures with single and multistep addition of Me2SO. We found that the permeability yielded via one-step addition of the Me2SO cannot exactly predict the volume change of the cells when the CPA was added in multiple steps.