Inhibition of oxidative stress and apoptosis enables extended maintenance of integrity and function of isolated hepatocytes in suspension

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Abstract

Aims:

Isolated hepatocytes in suspension may offer an alternative culture system for bioartificial liver devices. However, maintenance of isolated hepatocyte suspensions in conventional media leads to rapid loss of cell integrity. The aim of this study was to develop a modified medium to better maintain hepatocyte integrity.

Methods:

Isolated rat hepatocytes were prepared by collagenase digestion. Hepatocytes were purified in a Percoll gradient, suspended in bicarbonate buffered isotonic saline supplemented with d-α-tocopherol succinate and glucose and medium changed at 24 h (modified medium). The properties of cells treated this way were compared with those prepared by collagenase digestion and suspension in bicarbonate buffered isotonic saline (basic medium). Both media were maintained at 30°C for 48 h on an orbital shaker. Markers for oxidative stress, apoptosis and metabolic function were measured enzymatically. Cell morphology was assessed by electron microscopy.

Results:

When compared to collagenase-isolated hepatocytes maintained in basic medium, hepatocytes purified by Percoll (Amersham Biosciences, Castle Hill, Australia) and maintained in modified medium demonstrated significantly increased glutathione (GSH) and GSH: glutathione disulphide (GSSH) ratios, decreased lipid peroxidation product formation, decreased caspase-3 protease activity, reduced uptake of trypan blue and loss of lactate dehydrogenase (LDH) and increase preservation of cellular adenosine triphosphate concentration ([ATP]), urea synthesis, ammonia removal and glycogen content. Cell morphology was substantially preserved following 48 h of maintenance in the modified medium.

Conclusions:

The use of Percoll and modified medium reduces cell injury and apoptosis and greatly improves maintenance of cell function and morphology. The modifications reported here and the use of isolated hepatocyte suspensions in bioartificial liver devices are worthy of further investigation.

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