Hepatocyte isolation

In vitro cytotoxicity assays using isolated cells have been applied intermittently to cyanobacterial toxicity testing over several years." Cells investigated for suitability in cyanobacterial toxin assays include primary liver cells (hepatocytes) isolated from rodents and fish, established permanent mammalian cell lines, including hepatocytes, fibroblasts and cancerous cells, and erythrocytes. Earlier work suggested that extracts from toxic cyanobacteria disrupted cells of established lines and erythrocytes," but studies with purified microcystins revealed no alterations in structure or ion transport in fibroblasts or erythrocytes,... 

Biological characterization of the nanoparticles was carried out by monitoring in vitro interactions with hepatocytes isolated from rat liver [12], Haemagglutination inhibition test of erythrocytes with ricine agglutinin (RCA120) was carried out [13]. The fluorescence of the hepatocytes incubated with the FlTC-labeled nanoparticles was determined by means of a FACS Star Becto-Dickinson instmment. 

Oral exposure of rats to a single dose of 100 mg/kg carbon tetrachloride did not result in unscheduled DNA synthesis in hepatocytes isolated from the treated animals (Mirsalis and Butterworth 1980). The effect of other dose levels or repeated exposures was not investigated. In a similar experiment, Craddock and Henderson (1978) found that oral exposure of rats to carbon tetrachloride caused an increase in DNA synthesis associated with tissue regeneration, but no increase in unscheduled DNA synthesis. Furthermore, chromosome aberrations, micronuclei, or sister chromatid exchanges were not induced within 4-72 hours in hepatocytes take from rats treated with the relatively high dose of 1,600 mg/kg (Sawada et al. 1991). 

Mirsalis JC, Butterworth BE. 1980. Detection of unscheduled DNA synthesis in hepatocytes isolated from rats treated with genotoxic agents an in vivo-in vitro assay for potential carcinogens and mutagens. Carcinogenesis 1 621-625. 

Hepatocytes isolated from male Wistar rats (180-250 g) were treated with 0.2 mM mono(2-ethylhexyl) phthalate or 1 mM 2-ethylhexanol for 48 h (Gray et al., 1982). Both di(2-ethylhexyl) phthalate metabolites increased carnitine acetyltransferase activity about nine-fold. In studies with hepatocytes from male Sprague-Dawley rats (180-220 g), treatment with 0.2 mM mono(2-ethylhexyl) phthalate and 1.0 mM 2-ethylhexanol for 48 h resulted in induction of carnitine acetyltransferase activity about 15-fold and six-fold, respectively (Gray et al., 1983). Mono(2-ethylhexyl) phthalate was also shown to induce cyanide-insensitive palmitoyl-CoA oxidation and, by ultra-structural examination, to increase numbers of peroxisomes. Hepatocytes were isolated from Wistar-derived rats (180-220 g) and treated for 72 h with 0-0.5 mM mono(2-ethylhexyl) phthalate and some mono(2-ethylhexyl) phthalate metabolites (Mitchell etal., 1985). Treatment with mono(2-ethylhexyl) phthalate and metabolites VI and IX (see Figure 1) resulted in a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation. In addition, 0-0.5 mM mono(2-ethylhexyl) phthalate and 0-1.0 mM metabolite VI produced concentration-dependent increases in lauric acid hydroxylation. Treatment with metabolites I and V resulted in only small effects on the enzymatic markers of peroxisome proliferation. In another study with hepatocytes from Wistar-derived rats (180-220 g), metabolite VI was shown by subjective ultrastructural examination to cause proliferation of peroxisomes (Elcombe Mitchell, 1986). 

Species comparisons of hepatic peroxisomal proliferation have also included studies of human and non-human primate primary hepatocyte cultures. Hepatocytes isolated from Wistar-derived rats (180-220 g), male Alderley Park guinea-pigs (400-500 g), male marmosets (350-500 g) and three human liver samples (renal transplant donors) were treated with 0-0.5 mM mono(2-ethylhexyl) phthalate for 72 h (Elcombe Mitchell, 1986). While there was a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation in rat hepatocytes, no induction was observed in guinea-pig or human hepatocytes and only small non-concentration-dependent effects were observed in marmoset hepatocytes. Metabolite VI induced cyanide-insensitive palmitoyl-CoA oxidation and lauric acid hydroxylation in cultured... 

In hepatocytes isolated from rats and mice, treatment of primary cultures with metabolites of di(2-ethylhexyl) adipate increased peroxisomal palmitoyl-coenzyme A oxidation activity. The same treatment of primary cultures of hepatocytes from guinea-pigs and marmosets failed to cause any similar increase in activity. 

Bom et al. (2000) demonstrated that ort/20-hydroxyphenylacetaldehyde (4 mmol/L) was much more cytotoxic than coumarin (4 mmol/L) to Chinese hamster ovary cells Kj, a cell line that does not contain cytochromes P450. When both of these compounds were investigated in metabolically active hepatocytes isolated from male Sprague-Dawley rats, ort/20-hydroxyphenylacetaldehyde (0.8 mmol/L) caused a greater cytotoxic response compared with coumarin (0.8 mmol/L). 3-Hydroxycoumarin (0.8 mmol/L), not a product of coumarin epoxidation, did not cause a change in cell viability or an increase in lactate dehydrogenase activity. 

Hepatocytes isolated from rats injected with C. parvum also had increased NO cCMP levels while cCMP levels were approximately 30% higher in these animals. These data suggested that inducible NO synthesis in HC may act as... 

Tyson CA, Mitoma C, Kalivoda J. 1980. Evaluation of hepatocytes isolated by a nonperfusion technique in a prescreen for cytotoxicity. J Toxicol Environ Health 6 197-205. 

Sweeny, D.J. and Weiner, M., Metabolism of acetaminophen in hepatocytes isolated from mice and rats of various ages, Drug Metab. Dispos., 13, 377, 1985. 

Isolation of viable hepatocytes was first demonstrated by Howard et al. and rapidly increased in popularity with the further development of a high-yield preparative technique by Berry and Friend (6,7). Compared with liver slices, isolated hepatocytes are easier to manipulate and show a superior range of activities (8). For a detailed description of rat and human hepatocyte isolation techniques, the reader is referred to other reviews (8,9). 

The best procedure for hepatocyte isolation is the 2-step collagenase perfusion technique. Freshly isolated human hepatocytes are now also available from commercial vendors. However, the quality of the liver may be affected by the transportation period. One needs to ensure that the hepatocytes are near confluent and that no significant attachment occurs during the culturing period. 

Freshly isolated hepatocytes are universally accepted as the gold standard for enzyme induction assays. However, each experiment requires the availability of a liver for hepatocyte isolation. Fresh hepatocyte availability has been recently enhanced due to several commercial vendors effort to procure livers and provide isolated hepatocytes as a product. Studies with fresh hepatocytes cannot be planned, and sometimes may be delayed due to the lack of livers. To overcome this inconvenience of the use of freshly-isolated human hepatocytes, the following approaches for enzyme induction have been developed ... 

A recent study has shown that membranes made of a modified polyetheretherketone (PEEK-WC) are interesting materials for biomedical applications [23,24]. The cytocompatibility of PEEK-WC membranes was evaluated by culturing hepatocytes isolated from rat liver (Figure 43.6). The properties of PEEK-WC membranes were compared to polyurethane membranes prepared using the same technique, and commercial membranes (made of Nylon, polyethersulphone, and polyester). The results have shown that PEEK-WC membranes promoted hepatocyte adhesion most effectively and metabolic activities of cells cultured on these membranes improved significantly. 

Ferre, P Satabin, R, Dccaus, ).-R, Escriva, F., and Girard,. (1983). Development and regulation of ketogenesis in hepatocytes isolated from newborn rats. Biochern. J. 214, 937-942. 

In the past, the use of human hepatocytes was severely limited by their availability, as studies would be performed only if human livers were available for hepatocyte isolation. Further, hepatocyte isolation from human livers is not a technology available to most drug metabolism laboratories. This limitation has... 

Li AP. Human hepatocytes Isolation, cryopreservation and applications in drug development. Chem Biol Interact. 2007 168 16-29. 

Galactosamine is one of the hepatotoxicants known to have age-dependent hepatotoxicity (carbon tetrachloride-chlordecone combination, chloroform, and thioacetamide are other examples). It has been reported that neonatal (5 days old) and old rats (24 months old) are less susceptible to galactosamine-induced liver damage as compared to adult rats (5 months old) due to increased liver tissue repair. It is also known that primary hepatocytes isolated from old rats exhibited higher basal levels of UTP, UDP, and UMP in the liver, which may play a role in the... 

Knobeloch D, Ehnert S, Schyschka L, Buchler P, Schoenberg M, Kleeff J, Thasler WE, Nussler NC, Godoy P, Hengstler J, Nussler AK (2012) Human hepatocytes isolation, culture, and quality procedures. Methods Mol Biol 806 99-120... 

Riley, W.W. and J.G. Eales. Characterization of L-thyroxine transport into hepatocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss). Gen. Comp. Endocrinol. 90 31-42, 1993. 

A number of commercially available sources of human liver cytochrome enzymes exist (see above). There are deficiencies or drawbacks associated with many of them. Most sources of primary hepatocytes provide cells which are notoriously difficult to culture for any significant length of time in the laboratory without a loss of cytochrome phenotype. Cryopreservation techniques to prolong the storage lifetime of particular hepatocytes isolates also routi-... 

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