Abstract
Hepatocyte transplantation is dependent on the availability of good quality human hepatocytes isolated from donor liver tissue. Hepatocytes obtained from livers rejected for transplantation on the grounds of steatosis are often of low viability and not suitable for clinical use. The aim of this study was to evaluate the effects of the antioxidant N-acetylcysteine (NAC) on the function of hepatocytes isolated from steatotic donor livers. Human hepatocytes were isolated from 10 severely steatotic (>60%) donor livers rejected for transplantation. The left lateral segment of the donor liver was dissected into two equal size pieces and randomized to NAC or control. NAC (5 mM) was added to the first perfusion buffer of the standard collagenase digestion technique. Cells from tissues perfused with NAC had a significantly higher mean viability (81.1 ± 1.7% vs. 66.0 ± 4.7%; p = 0.003) and cell attachment (1.08 ± 0.26 vs. 0.67 ± 0.18 OD units; p = 0.012). Addition of NAC during isolation of human hepatocytes from steatotic donor liver tissue significantly improved the outcome of cell isolation. Further studies are needed to investigate the mechanism(s) of this effect. Incorporation of NAC in the hepatocyte isolation protocol could increase the availability of hepatocytes for transplantation.
Introduction
Hepatocyte transplantation is emerging as alternative treatment for patients with liver-based metabolic disease and acute liver failure. With cell transplantation, major surgery is avoided and there is the possibility of using hepatocytes isolated from livers that have been rejected for organ transplantation. Eight children with inherited metabolic liver disease have undergone cell transplantation at King's College Hospital (3,12,24). As with all types of transplantation, shortage of donor organs is a major problem. There is a limited and decreasing supply of normal donor liver tissue for the isolation of highquality hepatocytes suitable for transplantation.
Hepatic steatosis has become a common problem in Western populations with the increased incidence of obesity. It affects about 25% of liver transplant donors and most steatotic donor livers are rejected for organ transplantation (15,22). This is thus an important potential source of tissue, but hepatocytes isolated from these livers are usually of low cell viability and poor function (1). Steatotic hepatocytes do not tolerate cryopreservation well, leading to a greater cell loss and impaired metabolic function compared to cells from nonsteatotic livers (26). With steatotic livers there is increased formation of damaging reactive oxygen species (ROS) on reperfusion after ischemia (21). As a result, steatotic livers are more vulnerable to damage during isolation of hepatocytes, which leads to cells with poor function.
Agents are needed to protect the liver from oxidative stress in order to improve the outcome of cell isolation from steatotic livers, so that hepatocytes can be used for transplantation. N-Acetylcysteine (NAC) has been widely studied as an antioxidant that mainly acts as a precursor for reduced glutathione protecting the liver against oxidant damage (28).
This study has investigated whether addition of NAC to the perfusion media during isolation of hepatocytes can improve the viability of the cells obtained from steatotic donor livers rejected for transplantation.
Materials and Methods
Ten donor livers (7 male, 3 female) rejected for transplantation on the grounds of being severely steatotic (>60%) on histology were used. The mean (±SEM) donor age was 56 ± 3.3 years. One liver (donor 8) was retrieved from a non-heart-beating donor (Table. 1). The mean cold ischemia time of the livers was 19.6 ± 1.7 h. Ethical approval for the study was obtained from King's College Hospital Local Research Ethics Committee.
Characteristics of Donor Liver Tissue and Viability of Isolated Hepatocytes
All processed tissues showed >60% macrosteatosis. Tissue No. 8 was from a non-heart-beating donor. CIT, cold ischemia time; NAC, N-acetylcysteine.
Human Hepatocyte Isolation and Addition of NAC to the Perfusion Media
Hepatocytes were isolated using a standard collagenase perfusion technique (18) with some modifications. Briefly, the left lateral segment (segments II and III—-Couinaud classification) of the donor liver was dissected and maintained in University of Wisconsin (UW) solution on ice until the perfusion was commenced. The tissue was further dissected into two pieces of approximately equal weight (100 ± 10 g). For each specimen, two IV cannulae (16 gauge) were sutured into two separate hepatic vessels of sufficient size on the cut surface of the tissue. In order to prevent efflux of the perfusate, further hepatic vasculature and bile duct branches were tied with 3/0 or 4/0 PDS sutures.
For each experiment the two pieces of tissue from the donor liver were randomized to perfusion with NAC (Sigma-Aldrich, Dorset, UK) or without NAC as control. The specimens were perfused with three buffer solutions maintained at 37°C: a) Hank's balanced salt solution (HBSS; Lonza Wokingham Ltd., Berkshire, UK) containing 0.5 mM EGTA (Sigma-Aldrich) and 5 mM NAC or without NAC (control) then b) plain HBSS (Lonza Wokingham Ltd.), followed by c) Eagle's minimum essential medium (EMEM; Lonza Wokingham Ltd.) containing 0.5 g/L collagenase P (Roche Diagnostics Ltd., East Sussex, UK). The digested tissue was minced in ice-cold EMEM, and the hepatocytes were pelleted by a low speed centrifugation (50 × g; 4°C; 5 min) then washed with ice-cold EMEM. Centrifugation/ wash steps were repeated three times, and the final hepatocyte pellets (control and NAC) were assessed for cell number and viability using a standard trypan blue exclusion technique and an improved Neubauer haemocytometer. Freshly isolated hepatocytes were seeded onto collagen type I-coated 96- and 6-well plates (5 × 104 and 4 × 106 cells/well, respectively) and maintained for 24 h in Williams' medium E [supplemented with heat-inactivated fetal calf serum (10%; v/v), 10 mM HEPES, 2 mM l-glutamine, penicillin (100 U/ml), and streptomycin (0.1 mg/ml); all purchased from Lonza Wokingham Ltd.] in a humidified incubator (37°C; 5% CO2). The following assays were then carried out.
Cell Attachment: Sulforhodamine B Assay
Twenty-four hours post-cell seeding, cell attachment of NAC-treated hepatocytes was compared to control, using the sulforhodamine B (SRB) assay (20). Culture medium was removed from each well of the 96-well plate and cells fixed using 10% trichloroacetic acid. Plates were incubated for 60 min at 4°C. Wells were rinsed five times with tap water and fixed cells were stained with 0.4% SRB (Sigma-Aldrich) solution for at least 60 min at room temperature. Wells were rinsed five times with 1% acetic acid to remove unbound dye, and left to air dry. The cell membrane bound SRB dye was then solubilized in unbuffered TRIZMA-base (Sigma-Aldrich) solution on a plate shaker for 1 h at room temperature. Plates were read at OD 564 nm using a Dynex MRX microplate reader (Dynex Technologies Ltd, Worthing, West Sussex, UK).
Mitochondrial Dehydrogenase Activity: MTT Assay
The mitochondrial dehydrogenase activity of the plated hepatocytes was assessed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay according to Mitry et al. (19). Briefly, the culture medium was removed and wells washed with fresh medium; 200 μl of medium containing MTT (0.5 mg/ml) was added to each well, and the plates were incubated at 37°C for 4 h. The supernatants were removed and the formazan produced was dissolved in acidified isopropanol (120 μl/well) on a plate shaker for 20 min at room temperature. Plates were read at OD 630 nm using the microplate reader.
Protein Synthesis: [14C]Leucine Incorporation Assay
At the time of hepatocyte plating, [14C]leucine (Amersham International, Buckinghamshire, UK) was added to the culture medium at 0.2 μCi/well and incubated for 24 h (10). Cells were harvested on glass fiber membranes using a Packard FilterMate (Packard Instruments, Berkshire, UK). The filters were left to dry for at least 1 h in an oven (60°C), then counted using a Packard Matrix 9600 β-counter (Packard Instruments, Berkshire, UK). The results were expressed as counts per minute per well (cpm/well).
Protein Content Determination
Hepatocyte cultures were gently washed with phosphate-buffered saline (PBS) and cells were lysed using lysis buffer solution [PBS containing aprotinin (30 μl/ml), NP-40 (1%), sodium deoxycholate (0.5%), sodium dodecyl sulphate (0.1%), phenylmethylsulfonyl fluoride (10 μl), and sodium orthovanadate (10 μl/ml); all purchased from Sigma-Aldrich]. The total protein content in the total cell lysate and supernatant samples (control and NAC) was estimated according to Schacterle and Pollack (25).
Urea Synthesis Using Quantitative Colorimetric Assay
Urea production by hepatocytes in each well of the control and NAC cultures was quantified using the QuantiChrom™ Urea Assay Kit (BioAssay Systems, CA, USA) according to the manufacturer's protocol. The results were expressed as mg/dl/mg protein.
Inducible Nitric Oxide Synthase (iNOS) Using ELISA
The iNOS content in the total cell lysate samples of the control and NAC cultures were measured using the Quantikine® Human iNOS Immunoassay (R&D Systems Europe Ltd., Oxfordshire, UK) according to the manufacturer's protocol. The results were expressed as U/mg protein.
Statistical Analysis
All data were expressed as mean ± SEM. Statistical significance was determined using the paired t-test (two-tailed) for comparisons between the control and NAC groups. A value of p < 0.05 was considered statistically significant.
Results
Cells isolated from liver tissues perfused with NAC had significantly higher mean cell viability than cells from control steatotic tissue (81.1 ± 1.7% vs. 66.0 ± 4.7%, p = 0.003) (Fig. 1a). There was also a significantly increased mean viable cell yield (number of viable cells/g of tissue) from NAC perfused tissue than from control tissue (2.59 ± 0.61 × 106 vs. 1.10 ± 0.40 × 106 cells/g, p = 0.015). There were no statistically significant correlations between cell viability and donor age or cold ischemia time. Cell attachment (SRB assay) was significantly higher for cells obtained after NAC perfusion, with a 61% increase in value compared with control (Fig. 1b): mean OD reading 1.08 ± 0.26 for NAC hepatocytes versus 0.67 ± 0.18 for control (p = 0.012).
Effects of N-acetylcysteine on human hepatocytes isolated from steatotic donor livers. (a) Cell viability; (b) cell attachment: SRB assay; (c) mitochondrial dehydrogenase activity: MTT assay. Horizontal lines are mean values. **p < 0.01, *p < 0.05.
Mitochondrial dehydrogenase activity (MTT assay) was 54% greater in hepatocytes from tissue perfused with NAC (Fig. 1c), the mean OD reading being 0.20 ± 0.02 for NAC cells compared with 0.13 ± 0.01 for control (p = 0.0001). A 22% increase in protein synthesis ([14C]leucine incorporation assay) was observed in hepatocytes from tissues perfused with NAC (Fig. 2); the mean count was 983 ± 187 cpm/well with NAC compared with 808 ± 149 cpm/well (p = 0.043) for control. There was no significant effect on urea synthesis in hepatocytes from tissues perfused with NAC. The mean urea synthesis was 7.95 ± 1.01 mg/dl/mg protein for tissues perfused with NAC compared with 7.62 ±1.18 mg/dl/mg protein for control.
Effects of N-acetylcysteine on protein synthesis as determined by [14C]leucine incorporation into hepatocytes isolated from steatotic donor livers. Results are shown as mean ± SEM. *p < 0.05.
Hepatocytes from tissues perfused with NAC showed a 25% decrease of iNOS content. This was not statistically significant as only five paired samples were available for this assay. The mean iNOS content was 13.20 ± 2.13 U/mg protein for cells from tissues treated with NAC compared with 17.70 ± 2.18 U/mg protein for control.
Discussion
This study has shown that perfusion of liver tissue with NAC significantly improves the viability and cell attachment of human hepatocytes isolated from severely steatotic donor liver tissue rejected for organ transplantation. Most of the steatotic livers used in this study had long cold ischemia times (>12 h) and came from donors of age >50 years, which are risk factors for poor graft function and patient survival in liver transplantation (2). However, hepatocytes that met the criteria for clinical transplantation (9) were obtained in all cases after NAC perfusion. When the results of this study are also compared with those of 26 steatotic liver tissues used to isolate hepatocytes in the last 5 years at our institution (unpublished), the mean cell viability (81% vs. 56%) and cell yield (2.59 × 106 vs. 1.28 × 106 cells/g) show a significant improvement with addition of NAC.
Although a significant increase in MTT activity was detected in hepatocytes isolated in the presence of NAC, this is likely to be due to the increase in the number of attached cells present in the MTT assay, rather than an increase in the activity of the cells per se. In keeping with this, if the MTT results are expressed as a ratio of MTT/SRB values, the increase observed is no longer statistically significant. This also applies to the protein synthesis results, where a small increase was observed.
The clinical use of NAC is well established for the management of paracetamol overdose and its hepatoprotective role is widely accepted when administered as an antidote. It was important to choose an antioxidant that could readily be translated to clinical use and would be safe to use during cell isolation. NAC has been shown to have protective effects against liver ischemia/reperfusion injury in perfused rat livers (4) and in pigs (6). Of particular relevance to the present study, administration of NAC to rabbits with hepatic steatosis during the late phase of reperfusion improved the hepatic microcirculation and decreased the extent of ischemic/reperfusion injury as shown by a reduced ALT and decreased production of reactive oxygen and nitrogen species (7). There have been a number of clinical studies where NAC has been investigated as a hepatoprotective agent during liver transplantation. This experience was recently reviewed (17), and although NAC given either to the liver donors or transplant recipients tended to give improvements in postoperative measures of liver function, such as serum AST and prothrombin time, there were no clear effects on the outcome of transplantation.
In steatosis of the liver, intracellular fat accumulation and resultant cell swelling impairs the microcirculation of the liver (5,10). This is responsible for the increased ischemia/reperfusion injury in these livers with production of ROS, proinflammatory cytokines, and tumor necrosis factor-α (TNF-α). The donor livers used in this study had been rejected for transplantation after retrieval and had been preserved and stored in cold UW solution. They were then rewarmed when perfused with buffers to isolate hepatocytes, giving both cold ischemia and warm reperfusion. The impaired microcirculation in steatotic liver may have been improved by the effects of NAC as good tissue perfusion is needed in order to obtain efficient digestion with collagenase to release viable hepatocytes.
NAC rapidly enters cells and is deacetylated to l-cysteine, a precursor of glutathione. NAC is a source of sulfhydryl groups in cells and a scavenger of free radicals as it interacts with ROS such as OH• and H2O2 (27). Other studies have found that NAC increases the level of reduced glutathione in human hepatocytes, which may have been an important mechanism for the effects observed in the present study. NAC increased reduced glutathione in Mg-deficient cultures of rat and human hepatocytes (16), and recently NAC prevented cell death induced by galactosamine by reducing mitochondrial ROS generation and improving the oxidized to reduced glutathione ratio in cultured hepatocytes (8). Mitochondrial dysfunction has been shown to be an important factor in nonalcoholic fatty liver disease (NAFLD) and is a source of oxidative stress (23). The hepatoprotective mechanism(s) of NAC is likely to be due to its antioxidant effects, preventing cell damage due to excess ROS.
Events related to nitric oxide signaling are considered to be important in steatosis (14) and also increased iNOS is involved in liver ischemia/reperfusion injury (11). Our preliminary measurements of iNOS in human hepatocytes isolated from steatotic livers indicated that NAC reduced iNOS in parallel with the improved cell viability and function. This consistent with a previous study, which showed that NAC inhibited cytokine-mediated induction of iNOS expression in human HepG2 cells (13), and may suggest NAC is having an anti-inflammatory effect that is beneficial to hepatocyte function. Further experiments are needed to determine the mechanisms involved in the effects of NAC on steatotic hepatocytes.
In conclusion, this study has shown a beneficial effect of NAC perfusion during the isolation of hepatocytes from steatotic donor liver tissue. As a result, NAC is now included in the protocol for tissue processing to prepare hepatocytes from steatotic livers for cell transplantation.
Footnotes
Acknowledgment
This project was supported by the Department of Health, and King's College Hospital Charity, UK.