Hepatocytes cell viability

Isolated hepatocytes incubated with ionic iron rapidly undergo lipid peroxidation. Some studies have not shown a consequent decrease in viability (as indicated by uptake of trypan blue or release of enzymes). This is probably a result of short incubation times, as changes in viability lag behind increases in lipid peroxidation, and may not occur for more than 2 h after lipid peroxidation begins (Bacon and Britton, 1990). Recent studies have shown strong correlations between increased lipid peroxidation [production of thiobarbituric acid (TBA) reactants] and loss of cell viability (trypan blue staining) (Bacon and Britton, 1989). The significance of the lag between lipid peroxidation and decreases in cell viability is as yet uncertain. 

Treatment with iron chelators and a-tocopherol protect against lipid p>eroxidation and hepatocellular injury in iron-overloaded rats (Sharma etal., 1990). When hepatocytes are isolated from rats, which have been pretreated with a-tocopherol, there is a significant reduction in iron-induced lipid peroxidation and improvement in cell viability in vitro (Poli et al., 1985). Similar effects were seen when hepatocytes were incubated with iron chelators (Bacon and Britton, 1990). Treatment of moderately, but not heavily, iron-loaded rats with desferrioxamine in vivo inhibits the pro-oxidant activity of hepatic ultrafiltrates (Britton et al., 1990b). 

FIGURE 10.2 Effects of alkylphenols on cell viability compared to the amounts of p-QMs formed in isolated rat hepatocytes. Source From Ref. 28, with permission from the American Chemical Society. 

To be useful to both, clinicians and the pharmaceutical industry, a bioartificial liver will need to maintain a large number of hepatocytes at high cell densities and in a fully differentiated state for prolonged periods of time. Development of such a system has been impeded by three principal problems a) a requirement for large numbers of cells (>25 10 ) b) loss of liver-specific functions in cultured cells (primary and immortalized) c) nutrient and waste product gradients in high density cultures leading to lowered cell viability and impaired function. 

The cultivation of hepatocytes in a stationary suspension culture is actually ineffective. The hepatocytes lose their differentiation within hours. An improvement was the attachment culture. Thereby, the cells are cultivated either in self- or microcarrier-induced multicellular aggregates or on membranes. When standard monolayer culture was adequate to maintain the cell viability for 1 to 2 weeks the differentiation was lost after a few days. Different modifications as described beneath allowed the maintenance of differentiation for 2 to 3 weeks. 

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. 

Tissue slices and whole organ culture maintain many spatial aspects of the intact tissue and, like whole cells, maintain the linkage between Phase I and Phase II enzymes (Sipes et al., 1987). Precision-cut tissue slices are more easily and reproducibly prepared than primary hepatocytes. However, viability limitations often restrict studies to a few hours duration. Histological examination of the material after exposure to a xenobiotic is possible with tissue slices. 

Rat Hepatocytes Rat hepatocytes are freshly isolated by perfusion and are diluted with Krebs-bicarbonate buffer (pH 7.4) to 1 x 10 cells/mL. Cell viability is analyzed by the trypan blue exclusion method. 

Apart from the concentrafion of sericin, its extraction method has been shown to influence cell viability, also. If compared to heat, acid, or alkaline extraction methods, urea-extracted silk sericin is linked to the lowest cell viability of L929 mouse fibroblast (Aramwit et al., 2010). Sericin can also be used to replace bovine semm in cell freezing media. It cryopreserves cells as effectively as standard freeze medium containing foetal bovine serum. This effect has been demonstrated for various cell lines including P3U1 myeloma cell line, Chinese hamster ovary CHO cells, human dermal fibroblasts, human epidermal keratinocytes, the rat phaeochromocytoma cell line PC 12 and Sf9 insect cells (Sasaki et al., 2005), human primary hepatocytes (Miyamoto et al., 2010), rat pancreatic... 

Table 1. CELL VIABILITY AND HMGCoA REDUCTASE ACTIVITY OF HEPATOCYTES AFTER INCUBATION ...

Figure 2. Suppression of the cytotoxicity induced by Trp-P-1 in P-NF-treated rat hepatocytes. Hepatocytes were isolated from fi-naphthoflavone (P-NF) and com oil (Controlftreated rats and cultured for 2 or 20 h. The cells were treated with Trp-P-1 (30 pM) for 2, 4, and 6 h and cell viability was determined by MTT test. Data are presented as means S.D. (n=10).

There are several reports using animal cells to test the MEIC chemicals (Shrivastava et al., 1992 Rommert et al, 1994 Rouget et al., 1993). Shrivastava et al., used freshly isolated rat hepatocyte cells and two transformed cell lines. Comparable results were obtained with the three cell types indicating that rat hepatocytes are no more resistant to toxic chemicals than are other cells. They determined viability of die cells with 1) morphological studies 2) lactic dehydrogenase activity in the hepatocytes 3) the ability of the cells to take up the dye tryptan blue. They got much the same results with die diree teeh-niques. Rommert et al, simply determined the effect of the toxins on the abihty of the cells to replicate. They determined cell numbers with a Coulter Counter after 48 hours. Rouget et al., followed the effect of the toxins by determining the ability of cells to reduee the tetrazolium dye MTT and the ability of cells to take up the neutral red dye. The authors maintained that the two methods provided comparable results. 

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