Liver 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,... 

These studies represent the first report of the metabolism of brevetoxins by mammalian systems. PbTx-3 was rapidly cleared from the bloodstream and distributed to the liver, muscle, and gastrointestinal tract. Studies with isolated perfused livers and isolated hepatocytes conflrmed the liver as a site of metabolism and biliary excretion as an important route of toxin elimination. [ H]PbTx-3 was metabolized to several compounds exhibiting increased polarity, one of which appeared to be an epoxide derivative. Whether this compound corresponds to PbTx-6 (the 27,28 epoxide of PbTx-2), to the corresponding epoxide of PbTx-3, or to another structure is unknown. The structures of these metabolites are currently under investigation. 

Data from both in vivo and in vitro systems showed PbTx-3 to have an intermediate extraction ratio, indicating in vivo clearance of PbTx-3 was equally dependent upon liver blood flow and the activity of toxin-metabolizing enzymes. Studies on the effects of varying flow rates and metabolism on the total body clearance of PbTx-3 are planned. Finally, comparison of in vivo metabolism data to those derived from in vitro metabolism in isolated perfused livers and isolated hepatocytes suggested that in vitro systems accurately reflect in vivo metabolic processes and can be used to predict the toxicokinetic parameters of PbTx-3. 

Fish may be more resistant to lead than mammals. For example, isolated liver hepatocytes of channel catfish were about 40 times more resistant to lead than rat liver hepatocytes as judged by ALAD inhibition (Conner and Fowler 1994). 

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. 

Freshly isolated hepatocytes can be obtained from animal or human liver. Hepatocytes taken from these sources, however, are very vulnerable to contamination and technically difficult to preserve in a fresh state. Although it is not generally possible until now to proliferate human hepatocytes in vitro, the advantages of homology and human-specific functions demand that resources continue to be directed towards the elucidation of the conditions necessary for maintaining viable human hepatocyte cultures. 

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... 

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 ... 

The induction of g-glutamyltranspeptidase (GTP) foci, which are putative preneoplastic lesions, in isolated rat liver hepatocytes correlates well with carcinogenicity. 1,1-Dichloroethane failed to induce GTP foci in liver hepatocytes obtained from rats and mice treated with 1,1-dichloroethane for 7 days followed by promotion with phenobarbital (Herren-Freund and Pereira 1986). This suggests that... 

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. 

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... 

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... 

DJ Carlile, N Hakooz, JB Houston. Kinetics of drug metabolism in rat liver slices. IV Comparison of ethoxycoumarin clearance by liver slices, isolated hepatocytes, and hepatic microsomes from rats pretreated with known modifiers of cytochrome P-450 activity. Drug Metab Dispos 24 526—532, 1999. 

S Bezek, M Kukan, Z Kallay, T Tmovec, M Stefek, LB Piotrovskiy. Disposition of ethimizol, a xanthine-related inotropic drug, in perfused rat liver and isolated hepatocytes. Drug Metab Dispos 18 88, 1990. 

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-... 

Biotransformation of Sulfamethazine and B-Nortestosterone by Pig Hepatocytes. Incubation of pig hepatocytes, isolated from livers of a number of different animals, with the antibacterial agent sulfamethazine resulted in the formation of a single... 

Hepatoma cell lines, such as human HepG2 hepatoma cells and McArdle 7777 rat hepatoma cells, are frequently used for studying VLDL secretion. In addition, primary hepatocytes isolated from livers of rats, mice, or hamsters are commonly used. Each model has advantages and limitations. Hepatoma cells are convenient laboratory models since they can be... 

Wang, H., M.E. Figueiredo-Pereira, and M.A. Correia (1999). CYP 3A degradation in isolated rat liver hepatocytes 26S proteasome inhibitors as prohes. Arch. Biochem. Biophys. 365, 45-53. 

Administration of killed Corynebacterium parvunt to rodents induces massive circulating NOx production as well as liver necrosis (Billiar et al., 1990 Geller etal., 1993), and also decreases hepatic CYP levels and attenuates CYP-dependent hepatotoxicant injury (Raiford and Thigpen, 1994). Table II shows that there is a substantial loss of total hepatic microsomal heme and total CYP heme in vivo under these inflammatory conditions. As demonstrated previously (Billiar et al., 1990), hepatocytes isolated from C. parvum-tKattd animals continue to produce -NO for at least 24 hr after... 

Nefazodone Human hepatocytes, isolated rat liver mitochondria Dykens et ai. (2008)... 

Gallucci RM, Simeonova PP, Toriumi W, Luster MI (2000) TNF-alpha regulates transforming growth factor-alpha expression in regenerating murine liver and isolated hepatocytes. J Immunol 164 872-878... 

Blackmore PF, Mojsov S, Exton JH, Habener JF. Absence of insnlinotropic glncagon-like peptide-I (7-37) receptors on isolated rat liver hepatocytes. FEES Lett 1991 283 7-10. 

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