Alginate microencapsulated human hepatocytes for the treatment of acute liver failure in children.

BACKGROUND & AIMS: Liver transplantation (LT) is the most effective treatment for patients with acute liver failure (ALF), but is limited by surgical risks and the need for life-long immunosuppression. Transplantation of microencapsulated human hepatocytes in alginate is an attractive option over whole liver replacement. The safety and efficacy of hepatocyte microbead transplantation have been shown in animal models. We report our experience of this therapy in children with ALF treated on a named-patient basis. METHODS: Clinical grade human hepatocyte microbeads (HMBs) and empty microbeads were tested in immunocompetent healthy rats. Subsequently, 8 children with ALF, who were awaiting a suitable allograft for LT, received intraperitoneal transplantation of HMBs. We monitored complications of the procedure, assessing the host immune response and residual function of the retrieved HMBs, either after spontaneous native liver regeneration or at the time of LT. RESULTS: Intraperitoneal transplantation of HMBs in healthy rats was safe and preserved synthetic and detoxification functions, without the need for immunosuppression. Subsequently, 8 children with ALF received HMBs (4 neonatal haemochromatosis, 2 viral infections and 2 children with unknown cause at time of infusion) at a median age of 14.5 days, range 1 day to 6 years. The procedure was well tolerated without complications. Of the 8 children, 4 avoided LT while 3 were successfully bridged to LT following the intervention. HMBs retrieved after infusions (at the time of LT) were structurally intact, free of host cell adherence and contained viable hepatocytes with preserved functions. CONCLUSION: The results demonstrate the feasibility and safety of an HMB infusion in children with ALF. LAY SUMMARY: Acute liver failure in children is a rare but devastating condition. Liver transplantation is the most effective treatment, but it has several important limitations. Liver cell (hepatocyte) transplantation is an attractive option, as many patients only require short-term liver support while their own liver recovers. Human hepatocytes encapsulated in alginate beads can perform the functions of the liver while alginate coating protects the cells from immune attack. Herein, we demonstrated that transplantation of these beads was safe and feasible in children with acute liver failure.

Effect of Dimethyl Sulfoxide and Melatonin on the Isolation of Human Primary Hepatocytes.

The availability of fully functional human hepatocytes is critical for progress in human hepatocyte transplantation and the development of bioartificial livers and in vitro liver systems. However, the cell isolation process impairs the hepatocyte status and determines the number of viable cells that can be obtained. This study aimed to evaluate the effects of using dimethyl sulfoxide (DMSO) and melatonin in the human hepatocyte isolation protocol. Human hepatocytes were isolated from liver pieces resected from 10 patients undergoing partial hepatectomy. Each piece was dissected into 2 equally sized pieces and randomized, in 5 of 10 isolations, to perfusion with 1% DMSO-containing perfusion buffer or buffer also containing 5 mM melatonin using the 2-step collagenase perfusion technique (experiment 1), and in the other 5 isolations to standard perfusion or perfusion including 1% DMSO (experiment 2). Tissues perfused with DMSO yielded 70.6% more viable hepatocytes per gram of tissue (p = 0.076), with a 26.1% greater albumin production (p < 0.05) than those perfused with control buffer. Melatonin did not significantly affect (p > 0.05) any of the studied parameters, but cell viability, dehydrogenase activity, albumin production, urea secretion, and 7-ethoxycoumarin O-deethylase activity were slightly higher in cells isolated with melatonin-containing perfusion buffer compared to those isolated with DMSO. In conclusion, addition of 1% DMSO to the hepatocyte isolation protocol could improve the availability and functionality of hepatocytes for transplantation, but further studies are needed to clarify the mechanisms involved.

BMPER protein is a negative regulator of hepcidin and is up-regulated in hypotransferrinemic mice.

The BMP/SMAD4 pathway has major effects on liver hepcidin levels. Bone morphogenetic protein-binding endothelial cell precursor-derived regulator (Bmper), a known regulator of BMP signaling, was found to be overexpressed at the mRNA and protein levels in liver of genetically hypotransferrinemic mice (Trf(hpx/hpx)). Soluble BMPER peptide inhibited BMP2- and BMP6-dependent hepcidin promoter activity in both HepG2 and HuH7 cells. These effects correlated with reduced cellular levels of pSMAD1/5/8. Addition of BMPER peptide to primary human hepatocytes abolished the BMP2-dependent increase in hepcidin mRNA, whereas injection of Bmper peptide into mice resulted in reduced liver hepcidin and increased serum iron levels. Thus Bmper may play an important role in suppressing hepcidin production in hypotransferrinemic mice.

Character and temporal evolution of apoptosis in acetaminophen-induced acute liver failure*.

OBJECTIVE: To evaluate the role of hepatocellular and extrahepatic apoptosis during the evolution of acetaminophen-induced acute liver failure. DESIGN AND SETTING: A prospective observational study in two tertiary liver transplant units. PATIENTS: Eighty-eight patients with acetaminophen-induced acute liver failure were recruited. Control groups included patients with nonacetaminophen-induced acute liver failure (n = 13), nonhepatic multiple organ failure (n = 28), chronic liver disease (n = 19), and healthy controls (n = 11). MEASUREMENTS: Total and caspase-cleaved cytokeratin-18 (M65 and M30) measured at admission and sequentially on days 3, 7, and 10 following admission. Levels were also determined from hepatic vein, portal vein, and systemic arterial blood in seven patients undergoing transplantation. Protein arrays of liver homogenates from patients with acetaminophen-induced acute liver failure were assessed for apoptosis-associated proteins, and histological assessment of liver tissue was performed. MAIN RESULTS: Admission M30 levels were significantly elevated in acetaminophen-induced acute liver failure and non-acetaminophen induced acute liver failure patients compared with multiple organ failure, chronic liver disease, and healthy controls. Admission M30 levels correlated with outcome with area under receiver operating characteristic of 0.755 (0.639-0.885, p < 0.001). Peak levels in patients with acute liver failure were seen at admission then fell significantly but did not normalize over 10 days. A negative gradient of M30 from the portal to hepatic vein was demonstrated in patients with acetaminophen-induced acute liver failure (p = 0.042) at the time of liver transplant. Analysis of protein array data demonstrated lower apoptosis-associated protein and higher catalase concentrations in acetaminophen-induced acute liver failure compared with controls (p < 0.05). Explant histological analysis revealed evidence of cellular proliferation with an absence of histological evidence of apoptosis. CONCLUSIONS: Hepatocellular apoptosis occurs in the early phases of human acetaminophen-induced acute liver failure, peaking on day 1 of hospital admission, and correlates strongly with poor outcome. Hepatic regenerative/tissue repair responses prevail during the later stages of acute liver failure where elevated levels of M30 are likely to reflect epithelial cell death in extrahepatic organs.

Ammonia and the neutrophil in the pathogenesis of hepatic encephalopathy in cirrhosis.

Hepatic encephalopathy (HE) constitutes a neuropsychiatric syndrome which remains a major clinical problem in patients with cirrhosis. In the severest form of HE, cirrhotic patients may develop varying degrees of confusion and coma. Ammonia has been regarded as the key precipitating factor in HE, and astrocytes have been the most commonly affected cells neuropathologically. Although the evidence base supporting a pivotal role of ammonia is robust, in everyday clinical practice a consistent correlation between the concentration of ammonia in the blood and the manifest symptoms of HE is not observed. More recently the synergistic role of inflammation and infection in modulating the cerebral effects of ammonia has been shown to be important. Furthermore, it has been recognized that infection impairs brain function both in the presence and absence of liver disease. Thus it could be postulated that in the presence of ammonia, the brain is sensitized to a systemic inflammatory stimulus and is able to elicit an inflammatory response involving both proinflammatory and neurotransmitter pathways. Ammonia is not only directly toxic to astrocytes but induces neutrophil dysfunction with the release of reactive oxygen species, which contribute to oxidative stress and systemic inflammation. This may further exacerbate the cerebral effects of ammonia and potentially reduce the capacity of the neutrophil to fight microbial attack, thus inducing a vicious circle. This evidence supports the neutrophil in addition to ammonia as being culpable in the pathogenesis of HE, making the neutrophil a target for future anti-inflammatory therapeutic strategies in addition to ammonia lowering therapies.

Transferrin receptor 2 is crucial for iron sensing in human hepatocytes.

Hepcidin expression in vivo is regulated in proportion to iron status (i.e., increased by iron loading and decreased in iron deficiency). However, in vitro studies with hepatoma cell lines often show an inverse relationship between iron status and hepcidin expression. Here, we investigated possible molecular mechanisms responsible for the differences in iron sensing between hepatoma cell lines and human primary hepatocytes. RNA was collected from primary human hepatocytes, and HepG2 and HuH7 hepatoma cells were treated with either transferrin-bound and non-transferrin-bound iron. Expression of hepcidin, transferrin receptor 2, HFE, and hemojuvelin were quantified by real-time PCR. Hepcidin expression was increased in primary human hepatocytes following 24-h exposure to holoferric transferrin. In contrast, hepcidin mRNA levels in hepatoma cells were decreased by transferrin. Hepcidin expression was positively correlated with transferrin receptor 2 mRNA levels in primary human hepatocytes. Compared with primary hepatocytes, transferrin receptor 2 expression was significantly lower in hepatoma cell lines; furthermore, there was no correlation between transferrin receptor 2 and hepcidin mRNA levels in either HepG2 or HuH7 cells. Taken together our data suggest that transferrin receptor 2 is a likely candidate to explain the differences in iron sensing between hepatoma cell lines and primary human hepatocytes.

Optimization of the cryopreservation and thawing protocol for human hepatocytes for use in cell transplantation.

Cryopreservation of human hepatocytes is important for their use in hepatocyte transplantation. On thawing, cryopreserved hepatocytes often have reduced viability and metabolic function in comparison with fresh cells. The aim of this study was to modify the different steps in the standard cryopreservation procedure in an attempt to improve the overall outcome. Human hepatocytes with a viability of 69% +/- SD 16% were isolated from donor livers with a collagenase perfusion technique. Different cell densities, concentrations, rates, and methods of addition of dimethyl sulfoxide were tested for the freezing solution. Modified controlled-rate freezer programs were tested to obtain a linear decrease in the temperature. Once they were frozen, the storage time and thawing method for hepatocytes were investigated. The effects on thawed cell viability and attachment, lactate dehydrogenase release, cytochrome P450 1A1/2 activity, and albumin synthesis were determined. The results were used to produce an improved cryopreservation protocol suitable for good manufacturing practice conditions. With a cell density of 10(7) cells/mL in University of Wisconsin solution containing 300 mM glucose, 10% (vol/vol) dimethyl sulfoxide was added dropwise over 5 minutes, and was immediately frozen. Thawing was done rapidly at 37 degrees C, and dilution was performed with Eagle's minimum essential medium containing 300 mM glucose and 4% human serum albumin. Hepatocytes could be stored at -140 degrees C without significant further loss of function for up to 3 years. With this protocol, hepatocytes had a viability of 52% +/- 9%, an attachment efficiency of 48% +/- 8%, and lactate dehydrogenase leakage of 17% +/- 4%. This protocol is currently in use to cryopreserve hepatocytes for use in cell transplantation at our center.

An optimised method for cryopreservation of human hepatocytes.

Successful cryopreservation of hepatocytes is essential for their use in hepatocyte transplantation. Cryopreservation allows hepatocytes to be available for emergency treatment of acute liver failure and also for planned treatment of liver-based metabolic disorders. In addition, cryopreservation of human hepatocytes can facilitate their use in metabolism and toxicity studies. Cryopreservation can adversely affect the viability and function, especially reduce the attachment efficiency, of hepatocytes on thawing.The cryopreservation process can be divided into steps so that improvements can be made on the 'standard' protocols that are followed in some laboratories. These steps are as follows: pre-incubation of cells; freezing solution, cryoprotectants and cytoprotectants; freezing process; storage; thawing; post-thawing culture. This chapter presents an optimised protocol for cryopreservation of human hepatocytes as developed at King's College Hospital.

In vitro effects of sera from children with acute liver failure on metabolic and synthetic activity of cryopreserved human hepatocytes.

OBJECTIVE: The aim of the study was to investigate the in vitro effects of sera from children with acute liver failure (ALF) who had received N-acetylcysteine (NAC) on the metabolic and synthetic activity of cryopreserved human hepatocytes. MATERIALS AND METHODS: Cryopreserved human hepatocytes were plated on collagen-coated culture plates and incubated in cell culture medium containing pooled sera at 20% (v/v) obtained from children with ALF (ALF) who received treatment with NAC (ALF + NAC), no treatment with NAC and from normal controls (normal sera [NS]). The effects of the sera on cell metabolic functions were assessed using methylthiazolyldiphenyltetrazolium bromide, [14C]-leucine incorporation, and cytochrome P-450 (CYP1A1/2) activity assays. RESULTS: The overall hepatocyte metabolic activity was lower with ALF sera than with NS and ALF + NAC sera. [14C]-leucine incorporation was higher with both ALF sera (ALF and ALF + NAC) than with NS sera. There was a slightly higher activity of cytochrome P450 (CYP1A1/2 activity) in cultures treated with ALF and ALF + NAC than with normal sera treated hepatocyte cultures. CONCLUSIONS: Sera from children with ALF who received NAC did not impair the overall cell metabolic activity of cryopreserved human hepatocyte in vitro, which is encouraging for the use of hepatocytes transplantation in these patients.

Improving engraftment of hepatocyte transplantation using alpha-1 antitrypsin as an immune modulator.

For patients with non-cirrhotic liver-based metabolic disorders, hepatocyte transplantation can be an effective treatment. However, long-term function of transplanted hepatocytes following infusion has not been achieved due to insufficient numbers of hepatocytes reaching the liver cell plates caused by activation of the instant blood-mediated inflammatory reaction (IBMIR). Our aim was to determine if the natural immune modulator, alpha-1 antitrypsin (AAT), could improve engraftment of transplanted hepatocytes and investigate its mechanism of action. A tubing loop model was used to analyse activation of the IBMIR when human hepatocytes were in contact with ABO-matched blood and 4 mg/ml AAT. Platelet and white cell counts, complement and cytokine expression were analysed. To determine if AAT could improve short-term engraftment, female rats underwent tail vein injection of AAT (120 mg/kg) or water (control) prior to the intrasplenic transplantation of 2 × 107 male hepatocytes. At 48 h and 1 week, livers were collected for analysis. In our loop model, human hepatocytes elicited a significant drop in platelet count with thrombus formation compared to controls. Loops containing AAT and hepatocytes showed no platelet consumption and no thrombus formation. Further, AAT treatment resulted in reduced IL-1ß, IL-6 and IFN-γ and increased IL-1RA compared to untreated loops. In vivo, AAT significantly improved engraftment of rat hepatocytes compared to untreated at 48 h. AAT infusion may inhibit the IBMIR, thus improving short-term engraftment of donor hepatocytes and potentially improve the outcomes for patients with liver-based metabolic disease. KEY MESSAGES: • Alpha-1 antitrypsin (AAT) acts as an immune modulator to improve the efficacy of hepatocyte transplantation. • Treatment with AAT decreased thrombus formation and pro-inflammatory cytokine expression in a tubing loop model. • AAT significantly improved engraftment of donor hepatocytes within the first 48 h post transplantation.

Liver after hepatocyte transplantation for liver-based metabolic disorders in children.

There are limited data regarding donor hepatocyte engraftment into recipient liver after human hepatocyte transplantation (HHTx). We reviewed the explant livers of seven children with metabolic disorders [ornithine-transcarbamylase deficiency (one), coagulation factor VII deficiency (three), Crigler-Najjar syndrome (one), progressive familial intrahepatic cholestasis type 2 (PFIC-2) deficiency (two)] who received allograft hepatocytes by intraportal infusion with improvement in phenotype, although all later underwent liver transplantation (LT). Immunohistochemistry for bile salt export protein (BSEP) in the PFIC-2 patients and genetic typing following laser capture microdissection (LCM) of liver cells in the others were used to identify donor hepatocytes in recipient explant livers. Explant livers usually showed a preserved lobular architecture. In one patient, hepatocytes were identified inside portal vein thrombi. No donor hepatocytes in liver cell plates were identified immunohistochemically or by genetic typing. HHTx was generally followed by partial recovery of metabolic function; the procedure was well tolerated; any increase in portal vein pressure was transient. Hepatocytes were identified in portal vein thrombi, even months after portal vein infusion. Further studies are needed to monitor donor hepatocytes in vivo, to quantify better the efficacy of the procedure and to find ways of improving engraftment and function.

Cryopreservation-induced nonattachment of human hepatocytes: role of adhesion molecules.

Good quality cryopreserved human hepatocytes are becoming an important source for clinical hepatocyte transplantation. However, the process of cryopreservation leads to both structural and functional impairment of hepatocytes. The aim of this study was to investigate the mechanisms of cryopreservation-induced nonattachment in human hepatocytes. Hepatocytes were cryopreserved after isolation from unused donor liver tissue. Cell attachment to collagen-coated plates was measured. A cDNA gene array system for 96 cell adhesion-related molecules was used to determine mRNA expression in fresh and cryopreserved hepatocytes. Two cell adhesion molecule proteins were investigated further: beta1-integrin, a cell-matrix adhesion molecule, and E-cadherin, a cell-cell adhesion molecule. Attachment efficiency was significantly decreased after cryopreservation of human hepatocytes. Twenty-two genes were downregulated after cryopreservation including integrins, cadherins, catenins, and matrix metalloproteinases (MMPs). Beta1-Integrin gene and protein expression were significantly decreased in cultured cryopreserved hepatocytes compared to fresh hepatocytes. There was a significant correlation between loss of beta1-integrin and attachment in cryopreserved cells. Degradation of E-cadherin was increased in cryopreserved hepatocytes. The process of cryopreservation leads to downregulation of cell adhesion molecules at the gene and the cellular level. New cryopreservation protocols are needed to prevent these effects on cell attachment.

Serum hepatocyte growth factor and vascular endothelial growth factor in children with acute liver failure.

OBJECTIVES: Hepatocyte growth factor (HGF), a potent mitogen, and vascular endothelial growth factor (VEGF), a potent angiogenic factor, may play roles in hepatocyte regeneration in patients with acute liver failure (ALF). The aim of this study was to investigate serum levels of HGF and VEGF in children with ALF. PATIENTS AND METHODS: Serum samples were collected on admission from 25 children with ALF (median age, 11.1 y; range, 1.3-17.1 y; 11 male, 14 female) and 12 normal children (9.1 y; range, 5.4-15.4 y; 6 male, 6 female). Aetiology of ALF was 13 non-A to E hepatitis, 3 viral, 3 toxic, and 6 other. HGF and VEGF in sera were assayed by enzyme-linked immunosorbent assay. RESULTS: Median HGF levels in patients (10,157 pg/mL; range, 3412-73,420 pg/mL) were significantly higher than in controls (855 pg/mL, 510-1253 pg/mL; P < 0.001). Median VEGF levels in patients (164 pg/mL, 0-1588 pg/mL) were not significantly different from those in controls (214 pg/mL, 11-527 pg/mL). There was no relationship of HGF or VEGF levels to the aetiology of liver failure. There was a positive correlation between serum HGF and international normalized ratio (r = 0.73, P < 0.001), but not with levels of serum aspartate aminotransferase, bilirubin, or VEGF. There was no correlation between VEGF levels and international normalized ratio, aspartate aminotransferase or bilirubin. CONCLUSIONS: Serum levels of HGF but not VEGF are increased in children with acute liver failure.

Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation.

Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimized protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300 mM) glucose and CryoStor CS10 showed better postthawing cell viability, attachment, and hepatocyte functions than with histidine-tryptophan-ketoglutarate/10% DMSO/5% glucose and Bambanker. The 2 freezing solutions that gave better results were studied with human and rat hepatocytes microbeads. Similar effects on cryopreserved microbead morphology (external and ultrastructural), viability, and hepatocyte-functions post thawing were observed over 7 d in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone [ZVAD]), an antioxidant (desferoxamine [DFO]), and a buffering and mechanical protectant (human serum albumin [HSA]) on RMBs. ZVAD (60 µM) had a beneficial effect on cell viability that was greater than with DFO (1 mM), HSA (2%), and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes and a lower degree of cell apoptosis were observed with all 3 cytoprotectants, with ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the 3 cytoprotectant groups than with fresh microbeads. In conclusion, developing an optimized cryopreservation protocol by adding cytoprotectants such as ZVAD could improve the outcome of cryopreserved hepatocyte microbeads for future clinical use.

The effects of immunosuppressive agents on the function of human hepatocytes in vitro.

Calcineurin inhibitors (tacrolimus) and steroids continue to be an important component of hepatocyte transplantation protocols, despite reports of hepatotoxicity and inhibitory effects of steroids on cell proliferation. The aim of the study was to investigate whether isolated human hepatocytes were more vulnerable to the toxicity of these agents and also to investigate their effects on hepatocyte VEGF secretion, a vascular permeability factor suggested to be involved in the cell engraftment process. Human hepatocytes were isolated from donor livers/segments rejected or unused for orthotopic liver transplantation using a collagenase perfusion technique. Hepatocytes were plated for cell function tests and to determine VEGF production. Tacrolimus (0-50 ng/ml) and methylprednisolone (0-500 ng/ml) were added to the culture media and cells incubated for 24 h. Cell metabolic activity was assessed using the MTT assay, cell number using the SRB assay, and cell attachment from hepatocyte total protein content and protein synthesis using [14C]leucine incorporation. VEGF in culture supernatants was measured by ELISA. Tacrolimus and methylprednisolone had no statistically significant inhibitory effects on metabolic activity or protein synthesis compared to controls at all concentrations of the agents tested when added after plating. There were also no significant effects on cell attachment when tacrolimus or methylprednisolone was added at the time of cell plating. There were no differences in the responses obtained when either fresh or cryopreserved hepatocytes were used. The amount of VEGF secreted by untreated hepatocytes was highly variable (0-1400 pg/10(6) cells/24 h). VEGF levels in the culture supernatant from hepatocytes isolated from < or = 20-year-old donors (687 +/- 59 pg/10(6) cells/24 h) was significantly greater than from older donors (61 +/- 7 pg/10(6) cells/24 h; p = 0.003). Tacrolimus and methylprednisolone did not significantly affect VEGF secretion by hepatocytes. Tacrolimus and methylprednisolone did not have detrimental effects on the metabolic function of human hepatocytes, cell attachment, or VEGF secretion after cell isolation.

Analysis of the effects of cryopreservation on rat hepatocytes using SELDI-TOF mass spectrometry.

Successful cryopreservation of hepatocytes is essential to the future of hepatocyte transplantation as a treatment for liver disease, and also for the improved in vitro use of hepatocytes for research. However, hepatocyte function is adversely affected by even the best cryopreservation protocols. To investigate possible mechanisms for these changes, total cell lysates were prepared from fresh and cryopreserved rat hepatocytes and the proteome profiles compared using SELDI-TOF-MS ProteinChip technology. In addition, in vitro functional assays (viability, attachment efficiency, and lactate dehydrogenase leakage) were performed on the corresponding fresh and cryopreserved hepatocytes. Sixty-one peptides were identified as being significantly changed after cryopreservation. Thirty-seven peaks were significantly increased and 24 were significantly decreased after cryopreservation. The peak intensity of a number of these peptides was found to correlate with the in vitro function of the hepatocytes. Seven peptides correlated with in vitro function after cryopreservation and 10 peptides correlated with both fresh and cryopreserved function. The peptides significantly decreased after cryopreservation could include cytosolic enzymes or cofactors, which leaked out of the cells due to cryopreservation-induced membrane damage. The peptides significantly increased after cryopreservation could be retained products of cleavage of larger intracellular polypeptides and proteins or the result of aggregation of peptides caused by physical changes in the cell due to the cryopreservation process. Proteome profiling using SELDI-TOF-MS could be a useful tool to assess the effects of isolation and cryopreservation of hepatocytes, particularly if the findings are extended to human hepatocytes.

Hepatocyte transplantation for liver-based metabolic disorders.

Hepatocyte transplantation is being investigated as an alternative to orthotopic liver transplantation in patients with liver-based metabolic disorders. The progress made in this field to date is reviewed. Protocols have been developed using collagenase perfusion to isolate human hepatocytes from unused donor liver tissue. Hepatocytes with a high viability can often be obtained and can be cryopreserved for later use, though with loss of function on thawing. For clinical use, hepatocytes must be prepared in clean GMP conditions with cells meeting criteria of function and lack of microbial contamination before patient use. Hepatocytes are infused intraportally into the patient's liver, where a proportion of cells will engraft and replace the deficient metabolic function without the need for major surgery. Twenty patients have now received hepatocyte transplantation, including eight children at King's College Hospital. There was a range of aetiologies of liver disease: familial hypercholesterolaemia, Crigler-Najjar syndrome type 1, urea cycle defects, infantile Refsum disease, glycogen storage disease type Ia, inherited factor VII deficiency and progressive familial intrahepatic cholestasis type 2. Clinical improvement and partial correction of the metabolic abnormality was observed in most cases. Considerable progress has been made in developing the technique, but hepatocyte transplantation is limited by the available supply of liver tissue. Hepatocytes derived from stem cells could provide alternative sources of cells in the future.

The Effect of Intraoperative N-Acetylcysteine on Hepatocellular Injury During Laparoscopic Bariatric Surgery. A Randomised Controlled Trial.

BACKGROUND: The combination of pneumoperitoneum and intraoperative retraction of the left lobe of the liver leads to hepatocellular injury during laparoscopic gastric surgery. Fatty livers are more susceptible to ischaemic insults. This trial investigated whether the antioxidant N-acetylcysteine (NAC) reduced liver injury during laparoscopic sleeve gastrectomy (LSG). METHODS: Patients undergoing LSG were randomised (single blinded) to receive intraoperative NAC infusion or standard anaesthetic treatment. Blood samples were taken before and after surgery (days 0 to 4). Primary endpoints included serum aminotransferases. Secondary measures were C-reactive protein, weight cell count (WCC), cytokines (interleukin 6 and 10) and cytokeratin-18 as markers of apoptosis. Intraoperative liver biopsy samples were assessed using a locally developed injury score. RESULTS: Twenty patients (14 females, mean age 44.5 (SEM ± 2.9) years, mean BMI 60.8 (SEM ± 2.4) kg/m(2)) were recruited (NAC n = 10, control n = 10). The trial was stopped early after a planned interim analysis. Baseline liver function was similar. The peak rise in liver enzymes was on day 1, but levels were not significantly different between the groups. Rates of complications and length of stay were not significantly different. Secondary outcome measures, including white cell count (WCC), cytokines and cytokeratin (CK)-18 fragments, were not different between groups. Liver injury scores did not differ significantly. CONCLUSIONS: NAC did not reduce intraoperative liver injury in this small number of patients. The heterogenous nature of the study population, with differences in co-morbidities, body mass index and intraabdominal anatomy, leads to a varied post-operative inflammatory response. Significant hepatocyte injury occurs through both necrosis and apoptosis.

Changes in Bile Acid Profile After Laparoscopic Sleeve Gastrectomy are Associated with Improvements in Metabolic Profile and Fatty Liver Disease.

BACKGROUND: Bile acids (BA) modulate lipid and glucose metabolism in a feedback loop through production of fibroblast growth factor (FGF) 19 in the terminal ileum. Changes in BA after bariatric surgery may lead to improvements in the metabolic syndrome, including fatty liver disease. This study investigated the relationship between BA and metabolic and inflammatory profiles after laparoscopic sleeve gastrectomy (LSG). METHODS: Patients undergoing LSG had fasting blood samples taken pre-operatively and 6 months post-surgery. Liver injury was measured using cytokeratin (CK) 18 fragments. BA were measured using liquid chromatography tandem-mass spectrometry. FGF-19 was measured using enzyme-linked immunosorbent assay. RESULTS: The study included 18 patients (12 females), with mean age 46.3 years (SEM ± 2.9) and BMI 60.1 kg/m(2) (±2.6). After 6 months, patients lost 39.8 kg (±3.1; p < 0.001). Fourteen patients (78 %) had steatosis. FGF-19 increased from median 128.1 (IQR 89.4-210.1) to 177.1 (121.8-288.9, p = 0.045) at 6 months. Although total BA did not change, primary glycine- and taurine-conjugated BA, cholic acid decreased, and secondary BA, glycine-conjugated urodeoxycholic acid increased over the study period. These changes are associated with reduction in insulin resistance, pro-inflammatory cytokines and CK-18 levels. CONCLUSIONS: The profile of individual BA is altered after LSG. These changes occur in the presence of reductions in inflammatory cytokines and markers of liver injury. This study supports evidence from recent animal models that LSG may have an effect on fatty liver through changes in BA metabolism.

The effects of cryopreservation on human hepatocytes obtained from different sources of liver tissue.

Successful cryopreservation of human hepatocytes is important to establish hepatocyte banks for clinical use or in vitro research. The availability of donor tissue from unused liver segments/lobes and non-heart-beating donors (NHBD) has provided newer sources of hepatocytes. The quality of hepatocytes at the time of cryopreservation is important as cells isolated from liver tissue of borderline quality may not withstand the stresses associated with cryopreservation and subsequent thawing. Human hepatocytes were cryopreserved after isolation from mainly donor tissues (n = 40). In vitro assessment of the viability and function of the fresh and thawed cryopreserved hepatocytes was performed. Viability, attachment efficiency, enzyme activity, and albumin production of hepatocytes were all significantly decreased, and LDH leakage significantly increased, on thawing after cryopreservation. The viability of cryopreserved hepatocytes isolated from tissue rejected for orthotopic liver transplantation (36 +/- 15%) was significantly lower than those isolated from tissue where part was used for liver transplantation (47 +/- 14%, p = 0.002), but there were no significant differences in functional parameters. The viability of cryopreserved hepatocytes isolated from NHBD tissue (29 +/- 9%, p = 0.001) and from steatotic donor tissue (35 +/- 11%, p = 0.019) was significantly lower than those isolated from normal donor tissue (49 +/- 14%). There was no difference in functional parameters, except for albumin production of hepatocytes from NHBD tissue (2.9 +/- 1.0 microg/h/mg protein) being significantly lower than those from normal donor tissue (4.8 +/- 2.8 microg/h/mg protein, p = 0.03). The viability and attachment efficiency of cryopreserved hepatocytes isolated from liver tissue from resections for tumors was significantly higher, and the LDH leakage significantly lower, than those isolated from all donor tissue. Hepatocytes isolated from NHBD and steatotic tissue were more vulnerable to the effects of cryopreservation. Further research is required to improve hepatocyte isolation and cryopreservation protocols for different types of liver tissue.