Functional Assessment of the Quality of Human Hepatocyte Preparations for Cell Transplantation

Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. Good quality freshly isolated or cryopreserved human hepatocytes are needed for clinical transplantation. However, isolation, cryopreservation, and thawing processes can seriously impair hepatocyte viability and functionality. The aim of the present study was to develop a fast and sensitive procedure to estimate the quality of hepatocyte preparations prior to clinical cell infusion. To this end, cell viability, attachment efficiency, and metabolic competence (urea synthesis and drug-metabolizing P450 activities) were selected as objective criteria. Viability of hepatocyte suspension was estimated by trypan blue staining. DNA content of attached cells 50 min after hepatocyte platting to fibronectin/collagen-coated dishes was quantified to estimate adherence capacity. Urea production was determined after incubating hepatocyte suspensions with 2 mM ClNH4 for 30 min. The cytochrome P450 function was assayed by a 30-min incubation of hepatocyte suspension with a cocktail mixture containing selective substrates for seven individual P450 activities (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4). The assay can be applied to both freshly isolated and cryopreserved hepatocyte suspensions, and the results are available within 1 h, which could help to make short-term decisions: 1) to assess the suitability for cell transplantation of a preparation of freshly isolated hepatocytes or a particular batch of thawed cells, or 2) to estimate the convenience of banking a particular cell preparation.

Protective effect of melatonin on paraquat-induced cytotoxicity in isolated rat hepatocytes

Paraquat (PQ) is a known herbicide that causes acute cell injury by undergoing redox cycling. In previous reports, it has been reported that melatonin reduces PQ-induced hepatic toxicity in vivo, but, at the moment, there is no evidence that this effect occurs in this organ in vitro. In the present study we examined the effect of melatonin on PQ-induced oxidative damage in the liver using a hepatocyte suspension as a biological model. Preincubation of hepatocytes with melatonin (0.5, 1 or 2 mM), 30 min prior to PQ (10 mM) addition, prevented in a dose-and time-dependent manner the loss of viability, the leakage of lactate dehydrogenase, depletion of intra-cellular glutathione and malondialdehyde accumulation induced by the herbicide. Melatonin at the highest dose assayed (2 mM) completely prevented cell damage caused by PQ. These effects of melatonin are similar to those described in studies carried out in vivo. These results confirm that melatonin confers protection against PQ-induced hepatic oxidative stress and show that freshly isolated hepatocyte suspension is an adequate in vitro system for evaluating the cytoprotective effects of melatonin on oxidative injury caused by xenobiotics.

Functional role of ecto-ATPase activity in goldfish hepatocytes

Extracellular [γ-32P]ATP added to a suspension of goldfish hepatocytes can be hydrolyzed to ADP plus γ-32Pidue to the presence of an ecto-ATPase located in the plasma membrane. Ecto-ATPase activity was a hyperbolic function of ATP concentration ([ATP]), with apparent maximal activity of 8.3 ± 0.4 nmol Pi ⋅ (106cells)−1 ⋅ min−1and substrate concentration at which a half-maximal hydrolysis rate is obtained of 667 ± 123 μM. Ecto-ATPase activity was inhibited 70% by suramin but was insensitive to inhibitors of transport ATPases. Addition of 5 μM [α-32P]ATP to the hepatocyte suspension induced the extracellular release of α-32Pi[8.2 pmol ⋅ (106cells)−1 ⋅ min−1] and adenosine, suggesting the presence of other ectonucleotidase(s). Exposure of cell suspensions to 5 μM [2,8-3H]ATP resulted in uptake of [2,8-3H]adenosine at 7.9 pmol ⋅ (106cells)−1 ⋅ min−1. Addition of low micromolar [ATP] strongly increased cytosolic free Ca2+([Formula: see text]). This effect could be partially mimicked by adenosine 5′- O-(3-thiotriphosphate), a nonhydrolyzable analog of ATP. The blockage of both glycolysis and oxidative phosphorylation led to a sixfold increase of[Formula: see text] and an 80% decrease of intracellular ATP, but ecto-ATPase activity was insensitive to these metabolic changes. Ecto-ATPase activity represents the first step leading to the complete hydrolysis of extracellular ATP, which allows 1) termination of the action of ATP on specific purinoceptors and 2) the resulting adenosine to be taken up by the cells.

Conversion of esterified fura-2 and indo-1 to Ca2+-sensitive forms by mitochondria

Rat liver mitochondria are shown to convert the acetoxymethyl ester forms of fura-2 and indo-1 into Ca2+-dependent forms of these indicators. The excitation spectrum of the Ca2+-dependent conversion product of fura-2 acetoxymethyl ester is shown to be similar to that of the pentacarboxylic acid form of fura-2. A systematic investigation of the ionic strength and pH dependences of the fluorescence of the pentacarboxylic acid forms of these indicators shows small changes within the ranges thought to obtain within the mitochondrial matrix after Ca2+ uptake. Intramitochondrial free Ca2+ levels are studied both before and after Ca2+ sequestration by mitochondria, and a rough estimate is made of the mitochondrial contribution to the Ca2+-dependent fura-2 fluorescence of a hepatocyte suspension.

Conformational change in plasma albumin due to interaction with isolated rat hepatocyte

The electron spin resonance spectroscopy of 4-isothiocyanato-tempo labeled to bovine serum albumin (BSA) and the absorption spectroscopy of eosin maleimide labeled to BSA in the isolated rat hepatocyte suspension indicate conformational change occurring in the albumin molecule during interaction with the hepatocellular membrane. The conformational change in the albumin molecule may possibly accelerate the dissociation of albumin-organic anion complexes at the surface of the liver cell. The conformational change in the albumin molecule may explain in part the mechanism of albumin-mediated hepatic transport.