Hepatocytes isolated/cultured

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... 

In vitro metabolism of pH]PbTx-3 was studi in isolated rat hepatocytes (25). Hepatocyte monolayers cultured in 6-well plates containing 1 ml modified Williams E medium were incubated with 0.1 fig radiolabeled toxin at 37 C for 24 hr. The... 

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. 

Liver Uptake Blood Parenchymal cells Isolated, cultured cryopreserved hepatocytes, sinusoidal membrane vesicles, transporter expressions system... 

To achieve longer incubation times, cultivation of freshly isolated hepatocytes in culture on monolayer (Maslansky 1982 Wang 2002), sandwich culture on variable matrixes (e.g. Maurel 1996 Kem 1997 Wang... 

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. 

Primary hepatocytes are not responsive to inducers immediately after isolation. Primary hepatocytes require culturing at 37 °C for approximately 1-2 days before they are responsive to inducers. This recovery period is also important for hepatocytes that have been subjected to culturing at temperature... 

II.K.4 CYP Inhibition Studies Using Isolated/Cultured Hepatocytes or Liver Slices 557... 

Interaction studies in isolated/cultured hepatocytes and liver slices have to be assessed critically since a couple of competing reactions occur e.g. uptake pathways or phase II metabolism of the NCE and/or marker substrate, which makes it extremely difficult to interpret the data mechanistically. Additionally, metabolic capacity of hepatocytes in culture change with time (e.g. decrease/increase of phase I and phase II enzyme activities and internalization of transporters). Usually, Michaelis-Menten kinetics does not apply directly. For reliable results, interactions studies have to be performed in hepatocytes from at least three different donors since pooled hepatocytes are not available yet. Similar problems are also discussed for interaction studies in liver slices (Ekins 1998). 

Our present knowledge concerning the extraordinarily complex process of liver regeneration stems almost exclusively from experiments on animals and isolated hepatocytes in culture. In principle, this exceedingly... 

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. 

Chromosome breakage and micronuclei were not induced in human lymphocytes in whole blood or isolated cultures following in vitro exposure to the single congener 3,3 4,4 -hexaCB (PCB 77) (Belpaeme et al. 1996). In another study, PCB 77, but not Aroclor 1254, induced DNA adducts in the Hep G2 human cell line and in primary fetal rat and quail hepatocytes (Dubois et al. 1995). 

Moreover, myopathic Syrian hamsters given a calcium-deficient diet exhibit fewer lesions in skeletal and cardiac muscle (17). Conversely, facilitation of calcium uptake with ionophores or membrane-active toxins such as lysophospholipids or macrolide antibiotics accelerate necrosis of isolated skeletal muscle (19) and rat hepatocytes in culture (10). 

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-... 

Richard B, Fabre G, De Sousa G, Fabre I, Rahmani R, Cano JP. Interspecies variability in mitoxantrone metabolism using primary cultures of hepatocytes isolated from rat, rabbit and humans. Biochem Pharmacol 1991 41 255-262. 

Strom SC, Jirtle RL, Jones RS, Novicki DL, Rosenberg MR, Novotny A, Irons G, McLain JR, and Michalopoulos G. Isolation, culture and transplantation of human hepatocytes. 1982 J Nat Cane Inst 68 771-78... 

CYP inhibition assays include ones that utilize liver microsomes, isolated/cultured hepatocytes, and human cDNA-expressed enzymes. Typically, LC-MS/MS quantification is used for the microsome and hepatocyte methods while fluorimetric assays are used for the human cDNA-expressed enzymes. Typically, a weak inhibitor is defined as having a fei>20 rmolN, whereas a potent inhibitor has a ki[Pg.882]

The synthesis and secretion of RBP has been observed and studied with isolated liver cells in culture in vitro. Detailed studies have been carried out with two differentiated rat hepatoma cell lines (Smith et al., 1978). The production of RBP by primary rat hepatocytes in culture in vitro has also been observed (unpublished studies from the author s laboratory). Finally, the specific in vitro synthesis of rat RBP using isolated messenger RNA from rat liver and the rabbit reticulocyte in vitro protein-synthesizing system has been demonstrated (Soprano et al., 1981). Taken together, these various studies clearly establish the liver, and specifically the hepatic parenchymal cell, as the locus of RBP synthesis. The molecular and cellular mechanisms that are involved in the processes of RBP synthesis and secretion by the liver and the factors that regulate these processes are discussed in detail below. While it is clear that the liver is the major site of RBP synthesis, there is no evidence as to whether or not the liver is the only site of RBP synthesis in the body. 

To study the effect of some inhibitors on cholesterol synthesis we have developed a model where freshly Isolated hepatocytes were used. Differently from cultured cells hepatocytes isolated and incubated under our conditions show a dephosphorylated phosphorylated ratio as that found in the rat liver. 

Several studies on BA synthesis by isolated rat hepatocytes have been reported in the past[12-14]. These studies indicated that hepatocytes isolated from cholestyramine fed rats maintained a much higher rate of BA synthesis than hepatocytes isolated from control rats. The BA formed were identified as taurine conjugates of cholic and muricholic acid. The viability of free hepatocytes rapidly deteriorated, therefore in order to conduct long term experiments cultivated hepatocytes seem preferable. In the present study we used cultured chick-embryo hepatocytes. 

Fig. 1. Decay of stimulated System A activity in cultured hepatocytes from glucagon-injected rats. Three hours prior to hepatocyte isolation, a normal rat was injected (ip) with glucagon, 2 mg/100 g body weight. Actinomycin (ACT, 4/iM) or cycloheximide (CHX, 1 mM) was added to a portion of the hepatocytes during the final cold wash of the cell isolation procedure as well as to the culture medium. The Na -dependent uptake of 50 iM AIB was measured for 1 minute at 37 °C. The data presented are the averages SD of three individual assays. From Handlogten and Kilberg (75) with permission.

In another study, primary mammalian hepatocytes isolated from a three-month-old female Lewis rat were cultured on either untreated pSi, fetal bovine serum-treated pSi or coUagen-coated pSi [91 ]. After a 24 h incubation period, measurements of cell adhesion showed the collagennitrogen metabolic pathways, was monitored in the cell culture for 14 days and shown to be comparable to values observed in the presence of polystyrene. Taken together, these data suggest that pSi does not exhibit any significant cytotoxic effects towards primary mammalian cell lines. 

Green et al. (1986) compared the metabolism of amphetamine in isolated hepatocyte suspensions from rat, dog, squirrel, monkey, and human livers. The metabolite profile of hepatocytes from each species corresponded to the profile of urinary metabolites identified previously. These results indicate that species-specific differences in the metabolic activation of compounds seen in vivo can be reproduced in vitro by the utilization of primary hepatocyte cultures. 

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