SUMMARY: Using a modified collagenase digestion procedure, we were successful in the isolation of viable hepatocytes from human liver surgical wastes, unused transplantable livers, and diseased livers from transplant recipients. The modified procedures for isolation involved collagenase perfusion of a sample of limited size (<50 g) with only one cut surface, perfusion via only one blood vessel, the use of an appropriate amount of collagenase, as well as manual "massaging" of the liver sample during critical stages of the collagenase perfusion. Using this modified procedures, we had close to 100% success rate in the isolation of viable hepatocytes from the human liver samples.

The cellular mechanisms associated with the replicative response of hepatocytes to growth factor simulation is incompletely understood. Murine hepatocyte DNA synthesis is altered by cyclic AMP, suggesting that protein kinase A is involved in the cellular mechanisms associated with liver growth. The purpose of this study was to evaluate the role of protein kinase A in human hepatocyte DNA synthesis. human hepatocytes were isolated and maintained in primary culture on rat tail collagen. DNA synthesis was evaluated by determining [3H] thymidine incorporation. Human hepatocytes between 24 and 96 hr following harvest increased DNA synthesis in response to epidermal growth factor but not in response to glucagon, a stimulant of adenyl cyclase, or dibutyryl cyclic AMP. Mitogen-stimulated DNA synthesis was decreased by dibutyryl cyclic AMP. Cyclic AMP isomers that block or stimulate the effect of cyclic AMP on protein kinase A did not significantly alter resting or mitogens-stimulated human hepatocyte DNA synthesis. The results suggest that increased protein kinase A activity does not produce human hepatocyte replicative DNA synthesis.

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