Optimization of rat hepatocyte culture in citrated human plasma

Background. Maintenance of liver-specific functions in hepatocyte cultures during plasma exposure is critical for the clinical application of bioartif icial liver assist systems. Sodium citrate is a common anticoagulant but ha s been shown to be cytotoxic to hepatocytes. We have tested the effect of v arious supplements on the viability and function of adult primary rat hepat ocytes exposed to citrated plasma. Materials and methods. Freshly isolated rat hepatocytes were cultured in th e collagen gel sandwich configuration in culture medium for 6 days followed by exposure to citrated human plasma with various supplements for 1 week. Controls were left in culture medium throughout. Viability and synthetic fu nctions were evaluated. Results. Hepatocytes exposed to unsupplemented citrated plasma lost signifi cant viability and function within the first 2 days. Cells cultured in plas ma supplemented with a fivefold concentrate of standard hepatocyte culture medium maintained urea (1.2-2.1 mu mol/day/10(6) cells) and albumin (51-62 mug/day/10(6) cells) synthesis rates equal to or higher than those of contr ols. Among the various components of the concentrated medium supplement, ca lcium chloride (1.8 mM), magnesium sulfate (0.8 mM), amino acids (fourfold Basal Medium Eagle amino acids including 4 mM glutamine), and glucagon (14 ng/ml) were found to be essential in maintaining urea synthesis. Maintenanc e of a high albumin synthesis rate also required the addition of hydrocorti sone (7.5 mug/ml) and insulin (0.5 U/ml). Conclusions. Appropriate metabolic and hormonal supplementation of citrated human plasma prevents its cytotoxic effects and may be used in conjunction with in vivo use of bioartificial liver assist systems. (C) 2000 Academic Press.