Autophagy as a life support marker of isolated hepatocytes

AIM: The work aimed to reveal structural signs of autophagy in the cytoplasm of isolated hepatocytes in the dynamics of their cultivation. MATERIALS AND METHODS: The cultivated hepatocyte culture cell cycle was studied by flow cytofluorometry. The cells were cultured for 1, 24, and 48 hours. Morphometric analysis was performed using of the computer program Image J. The diameters of the nuclei and cytoplasm of hepatocytes, the volumes of nuclei and cytoplasm, and the nuclear-cytoplasmic ratio were determined. The concentration of intracellular organelles and autophagy was evaluated with magnification by 30000 times. RESULTS: The cell cycle arrest in the G0/G1 stage after 24 hours of hepatocyte cultivation and the preservation of their viability by hour 48 of the experiment without increase in the percentage of cells in the apoptosis stage were revealed. The decrease in the absolute count of cells was registered, as well as an increase in the nuclear-cytoplasmic ratio indicating a decrease in the proportion of hepatocyte cytoplasm in the course of cultivation. After 24 hours of cultivation, autophagosomes with fragments of cytoplasm, glycogen rosettes, and autolysosomes with partially degraded material were revealed in the cell cytoplasm. By hour 48 of the study, a significant decrease in the volume density of glycogen and mitochondria was noted, as well as an increase in basal autophagy in hepatocytes, with a prevalence of glycophagy and mitophagy. CONCLUSIONS: Autophagy maintains cellular homeostasis of isolated hepatocytes under standard culture conditions, as evidenced by a decrease in the volume density of glycogen and mitochondria, and an increase in basal autophagy in the hepatocyte cytoplasm. The findings indicate the contribution of autophagy to the survival of the primary culture of hepatocytes and can be used as an indicator of the adequacy of culturing conditions.