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Research ArticleArticle

Effect of Human Liver Source on the Functionality of Isolated Hepatocytes and Liver Slices

Peter Olinga, Marjolijn Merema, Ingrid H. Hof, Koert P. de Jong, Maarten J. H. Slooff, Dirk K. F. Meijer and Geny M. M. Groothuis
Drug Metabolism and Disposition January 1998, 26 (1) 5-11;

Abstract

In vitro experiments using human liver tissue to study drug metabolism and transport are usually performed and interpreted without real consideration of the differences in procurement of the tissue, if it is obtained from different sources. Therefore, in this study the functionality of isolated hepatocytes and liver slices prepared either from healthy human liver tissue obtained from patients undergoing partial hepatectomy [livers from partial hepatectomy (PH-livers)] or from donor tissue remaining after reduced-size or split-liver transplantation [livers from transplantation (Tx-livers)] was compared. From each liver sample, both liver slices and hepatocytes were prepared and compared with respect to viability and drug disposition. The viability of hepatocytes was assessed by trypan blue exclusion, ATP content, and energy charge and that of liver slices by potassium retention. In both preparations phase I metabolism was studied using lidocaine and testosterone as substrates, whereas phase I and II metabolism was assessed with 7-ethoxycoumarin. The membrane transport capability of the hepatocytes was investigated by measuring the uptake of taurocholic acid. The hepatocytes from PH-livers and Tx-livers showed similar viabilities and functional capacities. Metabolism in cells and slices from Tx-livers was found to be quantitatively comparable. However, liver slices from PH-livers showed a significantly lower metabolic capacity, compared with cells from the same tissue. This may indicate that only some of the hepatocytes in the liver slices from PH-livers participate in the metabolism of the compounds studied and that a selection of healthy cells takes place during isolation of the hepatocytes. Our results imply that hepatocytes isolated from Tx-livers and PH-livers can be used in the same study without consideration of the procurement of the tissue. However, the procurement of the tissue may significantly influence the functions of liver slices; the liver slices prepared from PH-livers showed significantly lower metabolic function, compared with slices prepared from Tx-livers.

The liver is the main organ responsible for the clearance of various endogenous and exogenous compounds. Clearance of substrates is a result of hepatic uptake, metabolism, and biliary and/or sinusoidal excretion. The mechanisms involved in these processes have been investigated using differentin vitro techniques. In decreasing order of tissue organization, the following techniques can be mentioned: isolated perfused livers, liver slices, isolated hepatocytes, isolated plasma membrane vesicles, microsomes, and isolated carrier proteins. The slice technique, introduced by Otto Warburg (1923), was commonly used inin vitro liver research (Krebs, 1933) until isolated hepatocyte and isolated perfused liver preparations were introduced and optimized (Berry and Friend, 1969; Meijer et al., 1981). Isolated hepatocyte and isolated perfused liver preparations have been used in many research fields, e.g. drug transport, metabolism, and toxicology (Berry et al., 1992; Meijer and Nijssen, 1991). Since the introduction of the Krumdieck slicer (Krumdieck et al., 1980) and a new incubation system for slices (Smith et al., 1985), tissue slices are increasingly being used in studies of drug metabolism and toxicity (Parrish et al., 1995).

Most of the in vitro drug research in liver is performed with liver preparations from animals. The results of such experiments are frequently discussed in relation to anticipated metabolic profiles in humans, but these extrapolations are often inappropriate because of large interspecies differences in drug metabolism. The use of human liver material for in vitro research would, in principle, make such scaling less hazardous. Moreover, in vitro data on the toxicity and metabolism of certain drugs in different species, including humans, would enable a more rational choice of appropriate animal model for toxicity or metabolism studies with the particular agents. This could also lead to a reduction in the number of animals used in drug research.

Until now, human liver microsomes have often been used to predict the metabolic profiles of compounds in humans (Rodrigues, 1994); however, microsome studies reflect only phase I and glucuronidation reactions. Introduction of the technique of isolation of human hepatocytes made it possible to investigate integrated phase I and II metabolism in human liver in more detail (Ballet et al., 1984; Groothuiset al., 1995; Strom et al., 1982; Vons et al., 1990; Bojar et al., 1976; Guguen-Guillouzoet al., 1982; Iqbal et al., 1991; Sandkeret al., 1994a). Human hepatocytes have also been used to predict the clearance of drugs in humans in vivo (Sandkeret al., 1994b). It should be noted, however, that the use of hepatocytes has inevitable drawbacks, because hepatocytes lose their polarity during isolation and primary cells in suspensions can be used only up to 4 hr without losing viability (Dickson and Pogson, 1977). Furthermore, the metabolic and transport capacities of hepatocytes in long-term culture decrease considerably after 24 hr of culture (Paine, 1991; Kwekkeboom et al., 1989). This decrease can be retarded only by using relatively complicated cell culture conditions, such as co-culturing with other epithelial cells (Guillouzo et al., 1990). In contrast, in liver slices the complex architecture of the liver is retained, and rat liver slices can be cultured for up to 72 hr without losing their metabolic capacity (Fisher et al., 1995).

Human liver tissue is only sparsely available for research purposes, and there are only a limited number of sources for healthy tissue,i.e. discarded transplantation livers, surgical waste material remaining after reduced-size or split-liver transplantations, and material from partial hepatectomy specimens. Results from in vitro experiments with human liver tissue so obtained are usually interpreted without real consideration of the differences among the various tissue sources. As far as we know, it is basically unknown whether the procurement of the human liver influences the metabolic and transport functions of the particular in vitro preparations.Vons et al. (1990) found significant differences in the ketogenesis of hepatocytes isolated from surgical biopsies and cold-stored donor livers. Those authors found impairment of glucagon stimulation in hepatocytes isolated from cold-stored donor livers, compared with hepatocytes from surgical biopsies (Vons et al., 1990).

In our laboratory, a comparison was made of the transport capacities and viabilities of human hepatocytes from discarded livers and livers accepted for transplantation (Groothuis et al., 1995). We found that hepatocytes could be isolated successfully from both sources, with comparable viabilities and transport capacities for taurocholic acid.

In the present study, we investigated two in vitropreparations (isolated hepatocytes and liver slices) from human liver tissue obtained from two different sources, i.e. healthy liver tissue from patients undergoing partial hepatectomies because of metastases of colorectal carcinoma (PH-livers1) and donor tissue remaining after reduced-size or split-liver transplantations (Tx-livers). There are essential differences in procurement for these two liver sources. Donor tissue is commonly perfused in situwith cold UW solution, which causes no warm ischemia, and subsequent cold preservation in UW solution may extend to 39 hr. This is in contrast to material from PH-liver specimens. The hepatectomy technique causes warm ischemia of the liver tissue during surgery (varying from 5 to 115 min), and the subsequent cold preservation in UW solution is limited to only 10–30 min. Both slices and freshly isolated hepatocytes were prepared from PH-livers and Tx-livers and compared with respect to viability and drug disposition. The viability parameters investigated for isolated hepatocytes were trypan blue exclusion and the overall energy state, as determined by the ATP content and the energy charge (Atkinson, 1968). The human liver slice viability was assessed by potassium retention. Transport function of the isolated hepatocytes was studied by the uptake of the bile acid taurocholic acid. Phase I metabolism was investigated using two model compounds, lidocaine and testosterone. Lidocaine is metabolized by cytochrome P450-mediated N-deethylation to MEGX (Bargetziet al., 1989). Testosterone is used as an indicator of metabolism for different isoenzymes of cytochrome P450 (Nedelcheva and Gut, 1994). Phase I and II metabolism was studied with the compound 7-EC, which is O-deethylated by several isoforms of cytochrome P450 to 7-HC, followed by glucuronidation and sulfation of 7-HC (Nedelcheva and Gut, 1994; Wishnies et al., 1991).

Materials and Methods

Materials.

The following compounds were obtained from the indicated sources: collagenase P from Boehringer Mannheim (Mannheim, Germany); BSA from Organon Teknika (Boxtel, The Netherlands); lidocaine from Centrachemie (Etten-Leur, The Netherlands); 7-EC, testosterone, and sodium taurocholate from Fluka (Buchs, Switzerland); 7-HC from Sigma Chemical Co. (St. Louis, MO); Percoll from Pharmacia AB (Uppsala, Sweden); [3H(G)]taurocholic acid (specific activity, 3.5 Ci/mmol) from Du Pont-NEN Research Products (Boston, MA); and UW solution from Du Pont Critical Care (Waukegab, IL). MEGX was a kind gift of ASTRA (Södertälje, Sweden), and 7-HC glucuronide and sulfate conjugates were kind gifts from P. Mutch, Glaxo Wellcome Research and Development (Herts, UK). The 24-well tissue-culture plates were obtained from Costar (Cambridge, MA). All other chemicals were of analytical grade and were obtained from commercial sources.

Liver Tissue.

Human liver tissue was obtained from livers procured from multi-organ donors or from patients undergoing partial hepatectomies because of metastases of colorectal carcinoma. Consent from the legal authorities and from the families concerned was obtained for the explantation of organs for transplantation purposes. The donor livers were reduced for reduced-size or split-liver transplantations. The donor livers were perfused in situ with cold UW solution before explantation. The livers were stored in cold UW solution on ice until reduction of the livers. Reduction or splitting of the donor organs was performed while the organs were immersed in UW solution with ice slush. The liver tissue remaining after bipartition was again stored in cold UW solution until the start of the isolation/slicing procedure. Total cold preservation time from in situ perfusion in the donor until slicing varied from 6 to 39 hr and was mainly the result of the time necessary for transportation and reduction of the liver. The research protocols were approved by the Medical Ethical Committee of our institution. In the case of PH-livers, consent from the patients was obtained for the use of liver tissue for research purposes. The research protocols were approved by the Medical Ethical Committee of our institution. The technique of partial hepatectomy was performed as described earlier (Brouwers et al., 1997). After partial hepatectomy, a wedge from the resected liver lobe was cut at a distance of at least 5 cm from the metastasis. Warm ischemia time for PH-livers, defined as the time after clamping of the branches of the hepatic artery and portal vein to the portion to be resected until perfusion with cold UW solution, varied from 5 to 115 min. Directly after excision of the piece of tissue, the biopsy wedge was perfused with cold UW solution (through cannulas in the portal venules) and was transported to the laboratory, where the isolation and/or slicing procedure was started within 30 min. Liver tissue from each individual was used to prepare both hepatocytes and liver slices.

Preparation of Human Hepatocytes.

Human hepatocytes were isolated using a modification of the method described earlier by Groothuis et al. (1995); the biopsy wedge was cannulated with two to four cannulas, as described (Groothuiset al., 1995). The cannulas were filled with ice-cold modified HBSS without Ca2+ [112 mM NaCl, 5.4 mM KCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 mM glucose, 25 mM NaHCO3, pH 7.42]. The biopsy wedge was placed in a cabinet at 37°C and was perfused (single pass) for 10 min with modified HBSS without Ca2+[containing 5 mM [ethylene bis(oxyethylenenitrilo)]tetraacetic acid and saturated with 95% O2/5% CO2 (carbogen)]. The flow rate was adjusted to a flow of 30 ml/cannula/min. Thereafter, the liver tissue was perfused (single pass) for 2 min with modified HBSS with 5 mM Ca2+ (saturated with carbogen). This buffer was supplemented with 0.05% (w/v) collagenase, and 250 ml of this collagenase buffer solution was perfused in recirculating mode for 30 min. After the recirculating period, the wedge was placed in ice-cold modified KHB [118 mM NaCl, 5.0 mM KCl, 1.1 mM MgSO4, 2.5 mM CaCl2, 1.2 mM KH2PO4, 25 mM NaHCO3, 10 mM glucose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, saturated with carbogen, pH 7.42] (Sandker et al., 1992) supplemented with 1% (w/v) BSA. The liver capsule was cut, and liver tissue was dissociated with a pair of forceps and then filtered through 250-, 100-, and 50-μm nylon filters. The cell suspensions were pooled, centrifuged at 50g for 4 min, and washed three times with ice-cold KHB. Nonviable cells were removed by Percoll density centrifugation (Groothuis et al., 1995), and viability was assessed by trypan blue exclusion (final concentration, 0.2%).

Preparation of Human Liver Slices.

Cores (diameter, 8 mm) were made from the pieces of liver tissue, as previously described (Olinga et al., 1993), and were stored in ice-cold UW solution until slicing. The slicing was performed with a Krumdieck slicer. Ice-cold KHB supplemented with glucose to a final concentration of 25 mM was used as slice buffer. Human liver slices (thickness, 200–300 μm; wet weight, 10–14 mg) were prepared with standard settings (cycle speed, 40; interrupted mode). After slicing, the human liver slices were stored in UW solution until the start of the experiment.

Incubation of Hepatocytes. Viability Parameters.

The viability of the hepatocytes was assessed by trypan blue exclusion, ATP content, and energy charge. ATP in the cells was determined directly after isolation and after 30 min of incubation under carbogen gassing at 37°C in a shaking water bath, to restore ATP content. The hepatocytes (1 ml of 1–2 × 106 cells in KHB plus 1%, w/v, BSA) were centrifuged at 10,000g for 30 sec. The supernatant was discarded, and the pellet was resuspended in 0.5 ml of 0.5 M perchloric acid. After 10 min on ice, the acid extract was centrifuged at 10,000g for 3 min. An aliquot of the supernatant was neutralized with 0.5 M K2CO3 to pH 7.0. The neutralized extract was centrifuged at 10,000g for 3 min to precipitate the insoluble KClO4. An aliquot of the supernatant was diluted with 250 mM Tris-HCl buffer, pH 7.0, frozen in liquid N2, and stored at −80°C until analysis by HPLC.2 With this HPLC method, all adenosine nucleotides can be separated from each other and from adenosine; therefore, the method allows calculation of the energy charge of the hepatocytes with the equation (ATP + ½ADP)/(ATP + ADP + AMP) (Atkinson, 1968).

Functional Parameters.

The uptake experiments with 21 μM taurocholic acid in isolated human hepatocytes were performed as described by Sandker et al.(1994b). The rate constant for uptake (kin) and excretion (kout) were calculated as described earlier (Sandker et al., 1994b).

For the metabolism experiments, 1.5 × 106hepatocytes/ml in KHB supplemented with 1% BSA were preincubated for 30 min under carbogen gassing at 37°C, in a shaking water bath. After preincubation, lidocaine, 7-EC, or testosterone was added to the cell suspension, to a final concentration of 5 mM, 500 μM, or 250 μM, respectively. Samples of 1 ml were taken at regular intervals for 30 or 60 min for the testosterone experiments.

For the lidocaine cell samples, the reaction was stopped by adding HClO4 in a final concentration of 6%. MEGX, the main metabolite of lidocaine, was analyzed by HPLC (Olinga et al., 1993).

For the 7-EC cell samples, the reaction was stopped by freezing the samples in liquid N2, and the samples were stored at −80°C until analysis. Immediately after thawing, the 7-EC samples were precipitated with perchloric acid (final concentration, 6%); after 10 min on ice, the acid extract was centrifuged at 10,000g for 3 min. An aliquot of the supernatant was neutralized with 0.5 M K2CO3 to pH 7.0. The neutralized extract was centrifuged at 10,000g for 3 min to precipitate the insoluble KClO4. An aliquot of the supernatant was diluted with 250 mM Tris-HCl buffer, pH 7.0, and analyzed by HPLC. The metabolites of 7-EC, i.e. 7-HC, 7-HC sulfate, and 7-HC glucuronide, were determined by HPLC (Walsh et al., 1995).

Testosterone metabolism was stopped by freezing the samples in liquid N2, and the samples were stored at −80°C until analysis. After thawing, the samples were homogenized and analyzed as described by van’t Klooster et al. (1993).

Incubation of Liver Slices. Viability Parameters.

Slices were incubated in 1.4 ml of KHB supplemented to 25 mM glucose, on stainless steel grids in 24-well tissue culture plates at 37°C, with separate magnetic stirrers and carbogen supplies in each well (Olinga et al., 1993). The potassium content was determined, after 1 hr of incubation, as described by Fisher et al.(1991).

Functional Parameters.

Lidocaine, 7-EC, or testosterone was added to a final concentration of 5 mM, 500 μM, or 250 μM, respectively. After different time periods, the reaction was terminated by separating the slices from the medium, weighing the slices, freezing (in liquid N2) slices and medium separately for the lidocaine and 7-EC samples and slices and medium together for the testosterone samples, and storing the samples at −80°C until analysis.

The lidocaine medium samples were treated as described for hepatocytes, but the MEGX content in the liver slices was not determined. Previously, it was shown that only a negligible amount is retained in the slices (Olinga et al., 1993). The 7-EC medium samples were treated as described for hepatocytes; the amount of metabolites in the slices was <5% (data not shown), as was also found by Worboyset al. (1995). The testosterone samples, containing medium and slice material, were sonicated and handled as specified for hepatocytes. The amount of metabolites formed by liver slices is expressed as nanomoles or micrograms per 1 × 106 cells, assuming that 1 g of human liver contains about 100 × 106 hepatocytes (Olinga et al., 1993).

Statistics.

Results were compared using two-tailed, unpaired, Student’st test or Wilcoxon signed rank test. Linear regression analysis was used to determine the possible significance of correlation. A p value of <0.05 was considered significant.

Results

The yield of viable hepatocytes, isolated from the total group (N = 57) of livers, varied widely (from 0 to 27 × 106 human hepatocytes/g liver). The viability of the hepatocytes, as assessed by trypan blue exclusion, was 93 ± 5%. In table 1, the yield of viable hepatocytes from the two groups of human livers (i.e.Tx-livers and PH-livers) is depicted. The age of the donors ranged from 2 to 63 years, and the average age was 26 years (median, 26 years). A negative linear correlation (r2 = 0.27,p < 0.001) between the age of the donor and the yield of viable cells was found. The age of the patients undergoing partial hepatectomy ranged from 41 to 76 years, with an average age of 53 years (median, 50 years). Therefore, in table 1 the data obtained for Tx-livers from donors above the age of 40 years are given separately, to match the age differences existing for the Tx-livers and PH-livers. However, no significant differences were found in the yields of viable cells from Tx-livers and PH-livers.

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Table 1

Yield of viable hepatocytes prepared from Tx-livers, from PH-livers, and from age-matched (>40 years) Tx-livers

The Tx-livers were stored in UW solution at 0°C for 6 to 39 hr (average, 17.8 hr). There was no (linear or exponential) correlation found between the storage time in UW solution and the yield of viable cells (data not shown). For patients undergoing partial hepatectomy, resection of the segment with the metastasis caused a period of warm ischemia ranging from 5 to 115 min, with a median of 79 min. A significant negative linear correlation (r2= 0.36, p = 0.037) was found between the warm ischemia time and the yield of viable cells (data not shown).

Hepatocytes isolated from PH-livers had a high energy state directly after isolation but lost 24% (not significant, Wilcoxon signed rank test) of their ATP content during 30 min of incubation (table2). The cells from Tx-livers tended to have a higher ATP content and energy charge after 30 min of incubation at 37°C, compared with the values determined directly after isolation.

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Table 2

ATP content and energy charge in human hepatocytes determined directly after isolation (0 min) and after 30 min of incubation at 37°C

The viability of the liver slices was assessed by measuring the potassium concentration in the slices after 1 hr of incubation at 37°C (table 3). The potassium concentration in the liver slices from Tx-livers was significantly higher than that in the slices from PH-livers.

View this table:
Table 3

Potassium concentration in liver slices after 1 hr of incubation at 37°C

The uptake and efflux of taurocholic acid were measured in the hepatocytes from the two liver sources. The rate constants for uptake and excretion of 21 μM taurocholate were calculated, and the data are depicted in table 4. It was found that the rate of uptake of taurocholic acid in human hepatocytes was consistently lower for cells obtained from livers of young children than for cells from livers of donors older than 15 years of age.3 Therefore, data obtained from livers of donors younger than 15 years were omitted from the data in table 4. No significant difference could be found in the rate constants for uptake and excretion of taurocholic acid for isolated hepatocytes from the two different sources.

View this table:
Table 4

Fractional rate constants for uptake (k in) and excretion (k out) of 21 μM taurocholic acid in human hepatocytes

Phase I metabolism was investigated by lidocaine deethylation, and the results are depicted in table 5. Hepatocytes isolated from the two tissue sources showed no significant differences in the amounts of MEGX formed. This is in contrast to the rate of metabolism of lidocaine in the liver slices prepared from the two sources; the slices from PH-livers showed a significantly lower rate of MEGX formation, compared with slices from Tx-livers. Hepatocytes and liver slices from Tx-livers had the same metabolic capacities for lidocaine biotransformation (expressed per 1 × 106 cells), whereas liver slices from PH-livers showed a significantly diminished rate of metabolism of lidocaine, compared with isolated hepatocytes from the same livers.

View this table:
Table 5

Lidocaine metabolism in hepatocytes and liver slices

Fig. 1 shows the interindividual differences in lidocaine biotransformation and the metabolic rate of MEGX formation in liver slices vs. hepatocytes prepared from the same human livers. A significant correlation was found between the amounts of MEGX formed in cells and slices from the same human livers from the two sources.

Figure 1
Figure 1

MEGX formation (nmol/106hepatocytes) in liver slices vs. hepatocytes, for each individual human liver.

○, Tx-livers; •, PH-livers. ——, Linear correlation for all livers (N = 20, r2 = 0.24,p < 0.05); – · –, Tx-livers (N = 12, r2 = 0.30); – – –, PH-livers (N = 8,r2 = 0.04).  

The rate of testosterone conversion to 6β-hydroxytestosterone showed large interindividual differences (table6). No significant differences between PH- and Tx-liver preparations were found. Phase I and II metabolism, as investigated by 7-EC metabolism, also showed considerable interindividual differences. Table 7shows the amounts of 7-EC metabolites formed in isolated cells, indicating that glucuronidation of the 7-HC product is the main phase II metabolic route. No differences were seen in the total amount of 7-HC (7-HC + 7-HC glucuronide + 7-HC sulfate), which represents total phase I metabolism of 7-EC, formed by hepatocytes from the two sources. However, the sulfation of 7-HC in the hepatocytes from PH-livers was significantly decreased, compared with the cells of Tx-livers.

View this table:
Table 6

Testosterone metabolism in hepatocytes and liver slices

View this table:
Table 7

Metabolism of 7-EC to 7-HC, 7-HC glucuronide, and 7-HC sulfate in hepatocytes and slices

Liver slices prepared from PH-livers showed significantly less total metabolism of 7-EC, compared with slices from Tx-livers. When the metabolism of 7-EC was compared between liver slices and hepatocytes from Tx-livers, no significant difference was found (table 7). However, slices prepared from PH-livers showed a significant impairment of both phase I and phase II metabolism of 7-EC, compared with hepatocytes isolated from the same source.

Discussion

To compare hepatocytes and slices from Tx- and PH-livers, cell yield, viability, and drug metabolism functions were assessed. The yield of viable cells isolated from human livers varied widely, as was found previously (Ballet et al., 1984; Groothuis et al., 1995; Iqbal et al., 1991; Dorko et al., 1994; Rogiers, 1993). A significant negative correlation with donor age and yield was found, as reported previously (Groothuis et al., 1995; Dorko et al., 1994). There was no significant difference between the yield of hepatocytes from livers from donors older than 40 years of age and the yield from liver tissue from PH-livers. In the Tx-liver group, the cold ischemia period did not influence the yield of viable hepatocytes, confirming the data of others (Groothuis et al., 1995; Dorko et al., 1994). Warm ischemia time (5–115 min) for the PH-livers did influence the yield of viable hepatocytes, in contrast to the findings ofTakahashi et al. (1993). The warm ischemia in their study did not exceed 90 min. In our study, when PH-livers with a warm ischemia time of >90 min were excluded from the analyses, there was no significant correlation between cell yield and warm ischemia time. The present results indicate that functional and viable hepatocytes can be isolated after a considerable time of warm ischemia of the tissue but warm ischemia of >90 min negatively influences the yield of viable cells.

The cells from PH-livers contained a greater amount of ATP immediately after isolation; however, during incubation of the hepatocytes, these cells showed no increase but, rather, a small decrease in ATP content. This is in contrast to the cells from Tx-livers, where ATP content and energy charge rose during incubation at 37°C. The significantly greater amount of ATP immediately after isolation in hepatocytes isolated from PH-livers may be a result of the fact that isolation of the hepatocytes was performed within 1–3 hr after resection. Only a relatively small amount of ATP may be degraded during storage of PH-livers, whereas during the relatively long-term cold storage of Tx-livers considerable ATP loss occurs (Guyomard et al., 1990). After 30 min of preincubation, the energy charge and ATP content in hepatocytes from the two sources were comparable. Additional experiments must be performed with longer incubation periods to investigate whether the ATP content and energy charge reach a plateau after 30 min of incubation.

The lower potassium contents in liver slices from PH-livers point to decreased viability, compared with liver slices from Tx-livers. Taurocholic acid uptake and excretion were not different in human hepatocytes isolated from the two sources, indicating equivalent transport capacities in hepatocytes obtained from the two sources, as was earlier reported by us for discarded and accepted donor livers (Groothuis et al., 1995).

The metabolism of lidocaine in in vitro liver preparations showed considerable interindividual differences (Olinga et al., 1995). This is similar to data obtained in in vivostudies (Oellerich et al., 1987). Hepatocytes isolated from Tx-livers showed lidocaine metabolism comparable to that of hepatocytes isolated from PH-livers. Moreover, for the individual Tx-livers the amount of MEGX formed per 1 × 106 cells was almost identical in liver slices and in hepatocytes. This suggests that virtually all hepatocytes in the liver slices are involved in the biotransformation of lidocaine. This observation is in agreement with results from a previous study, with fewer livers (Olinga et al., 1995). However, using PH-livers, MEGX formation in liver slices (expressed per 1 × 106 cells) was clearly lower, compared with the isolated hepatocytes from the same livers. This can be explained by assuming that not all hepatocytes in the slice are fully able to perform normal biotransformation of lidocaine or, in other words, a loss of functionality of some of the hepatocytes may play a role, which is in agreement with the relatively low potassium content. Apparently, during isolation only the viable hepatocytes are selectively isolated. The alternative explanation,i.e. that all cells in the slice exhibit decreased metabolic activity, is less probable, because after isolation the viable hepatocytes exhibit a higher metabolic rate than do the slices. The same characteristic differences between Tx-liver and PH-liver preparations were found for phase I and II metabolism of 7-EC. The data on 7-EC metabolism seem to indicate again that almost all hepatocytes in Tx-livers are involved in the biotransformation of 7-EC and at least some of the hepatocytes in the liver slices from PH-livers may have deteriorated. The data on the metabolism of testosterone to 6β-hydroxytestosterone are insufficient to allow conclusions regarding this point but at least do not contradict the hypothesis that not all hepatocytes in the slices from PH-livers perform normal biotransformation. The combined biotransformation results for hepatocytes and slices can be explained by assuming that apparently during the partial hepatectomy surgery some of the hepatocytes suffer from the warm ischemia and in the isolation of the hepatocytes some sort of selection occurs, in which the most viable cells of the tissue survive the entire procedure. This is shown by the negative correlation between warm ischemia time and the yield of viable hepatocytes. In addition, this aspect is obviously reflected in the data, because the metabolic rate in the isolated hepatocytes is expressed per number of functional cells finally present, whereas in the slices this parameter is expressed per number of total (functional + nonfunctional) hepatocytes present in the slice.

The main route of phase II 7-EC metabolism was glucuronidation of 7-HC. Sulfation activity was low in almost all human livers, as also shown by others (Wishnies et al., 1991; Barr et al., 1991). The almost complete absence of 7-HC sulfate formation in thein vitro preparations of PH-livers was remarkable. It could be hypothesized that sulfation is very vulnerable to warm ischemia damage, possibly because of the loss of the necessary cosubstrates.

Human liver slices and isolated hepatocytes prepared from Tx-liver tissue showed almost identical metabolic capacities. This in contrast to the data obtained with rat livers by Ekins et al. (1995). Those authors compared the metabolism of 7-EC, testosterone, and 1-chloro-2,4-dinitrobenzene in rat hepatocytes and liver slices and found a lower metabolic rate in slices, compared with hepatocytes (ranging from a 2.2- to 29-fold difference). Recently, we found a 3–4-fold lower rate of formation of MEGX from lidocaine in rat liver slices, compared with isolated hepatocytes.4 This in contrast to in vitro preparations of livers of cynomolgus monkeys, where no difference was found in this metabolic process between hepatocytes and liver slices (Olinga et al., in press, 1998). The difference between the lower metabolic rate in rat liver slices, compared with rat hepatocytes, and the similar metabolic rates in slices and hepatocytes prepared from human and monkey liver is not likely to be the result of low viability of rat liver slices but may be the result of limited diffusion of the substrates into the rat liver slices. This difference between rat, human, and monkey liver may be caused by differences in procurement of the livers. All human and monkey livers in our studies were perfused with UW solution. In contrast, in the rat liver experiments the livers were not perfused before slicing, and an aggregation of blood cells in the sinusoids may inhibit diffusion of substrates into the slices. More experiments are currently underway to investigate this possibility.

In conclusion, hepatocytes isolated from PH-livers and Tx-livers showed similar viabilities and functional capacities. Comparable metabolism in cells and slices from Tx-livers was found. However, liver slices from PH-liver specimens showed lower viability and metabolic capacity, compared with cells from the same tissue. The data indicate that only some of the hepatocytes in the liver slices prepared from PH-livers are involved in the metabolism of the compounds studied. This can be explained by assuming an intrinsic decrease in the metabolic capacity of some of the cells, related to changes in enzymatic activity and/or the absence of adequate amounts of cosubstrates. Evidently this is not the result of the slice technique itself, because slices from Tx-livers exhibited normal metabolic rates. Rather, the warm ischemia time may contribute to the effects shown. These results suggest that, in the hepatocyte isolation procedure, a selection of hepatocytes occurs and only the most healthy cells survive isolation.

It can be concluded that hepatocytes from Tx-livers and PH-livers can be used in the same study, without consideration of the procurement of the tissue. This is in contrast to human liver slices; the tissue procurement of the tissue influences the functions of the liver slices.

Acknowledgments

We thank Dr. R. de Kanter and Dr. H. Koster from Solvay Duphar BV for analysis of the testosterone samples.

Footnotes

  • Send reprint requests to: P. Olinga, Department of Pharmacokinetics and Drug Delivery, University Centre for Pharmacy, Ant. Deusinglaan 1, 9713 AV Groningen, The Netherlands.

  • This study was supported by grants from the Alternatives to Animal Experiments Platform, Organon International BV, and Solvay Duphar BV. The experiments were performed in cooperation with the Human Liver Group, Groningen.

  • ↵2 Olinga P, Merema MT, Hof IH, Visser J, Meijer DKF and Groothuis GMM. Fast analysis of ATP, ADP, AMP and adenosine with gradient ion-pair reversed-phase liquid chromatography as viability test for hepatocytes. Submitted for publication.

  • ↵3 Olinga P, Merema MT, Sandker GW, Slooff MJH, Meijer DKF and Groothuis GMM. Uptake of taurocholic acid in human hepatocytes isolated from livers of donors of different ages. Submitted for publication.

  • ↵4 Olinga P, unpublished data.

  • Abbreviations used are::
    PH-liver
    liver from partial hepatectomy
    Tx-liver
    liver from transplantation
    UW solution
    University of Wisconsin organ preservation solution
    7-EC
    7-ethoxycoumarin
    7-HC
    7-hydroxycoumarin
    MEGX
    monoethylglycinexylidide
    KHB
    Krebs-Henseleit buffer
    HBSS
    Hanks’ balanced salt solution
    BSA
    bovine serum albumin
    • Received January 31, 1997.
    • Accepted September 23, 1997.

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Effect of Human Liver Source on the Functionality of Isolated Hepatocytes and Liver Slices

Peter Olinga, Marjolijn Merema, Ingrid H. Hof, Koert P. de Jong, Maarten J. H. Slooff, Dirk K. F. Meijer and Geny M. M. Groothuis
Drug Metabolism and Disposition January 1, 1998, 26 (1) 5-11;
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