Abstract

Shortage of donor liver tissue for the isolation of hepatocytes requires the development of improved cryopreservation techniques for long-term storage of these cells for more efficient use. The aim of this study is to compare two cryoprotectants for preserving living cells in culture by vitrification. The viability of hepatocytes after cryopreservation was determined by the intracellular concentration of potassium (K⁺) and lactate dehydrogenase (LDH). Cryomicroscopy was used to check the state of vitrification and morphology of these cells subjected to the process of freezing and thawing. This experiment made it possible to observe an early vitrification with 40% of Ethylene glycol (EG) or 1,2-propanedio (1, 2-PD) to reach the final vitrified state with 60%. The treatment of these cells in culture with solutions containing EG or 1, 2-PD revealed a failure in functional properties of the plasma membrane. Thus, a decrease of intracellular concentration of LDH and K⁺ was noticeable. This decrease was more significant when it came with 60% EG or 1, 2 - PD. The use of a cryopreservation solution additionally containing NaCl, KCl, Ca²⁺ and sucrose and the treatment of hepatocytes in culture at +4°C greatly minimized the functional impairment of the plasma membrane. These results suggest that the osmo-diffusional mechanism is at the basis of membrane damage letting out LDH and K⁺ into the extracellular compartment. However, this mechanism can be controlled by a selection of treatment solutionparameters such as temperature, osmolarity and electrolytes concentration while taking the cryoprotectants coefficient of permeability into consideration.

Key words: Hepatocyte, cryopreservation, cryoprotectant, vitrified state, vitrification.