Culture and characterization of human hepatocytes obtained after graft reduction for liver transplantation: a reliable source of cells for a bioartificial liver

This article describes results obtained when human liver cells obtained from reduced grafts are cultured in a chemically defined medium. Remnants of livers after reduction for pediatric transplantation were processed by a multiple cannulation system through the existing vasculature, which allowed the homogeneous perfusion of collagenase. The graft weight ranged between 55 and 1000 g (median value: 145.6 g). The yield ranged between 0.13 x 10(6) and 38 x 10(6) cells/g of tissue (median value 14.73 x 10(6) cells/g), and the viability was 61.17 +/- 27.43%. The total number of cells ranged between 57.6 x 10(6) and 12 150 x 10(6) cells (median value: 740 x 10(6) cells). Cells were cultured for 30 days. Albumin synthesis was observed during the first 2 weeks, with a peak value at day 6 (27.85 +/- 1.77 micro g/mL). Urea production was detected during the first week (peak value at day 6: 17.12 +/- 2.11 mg/dL). Light microscopy showed the presence of cells in a monolayer. Biliary pigments were observed at day 20. By immunohistochemistry, positive cells for albumin, for hepatocyte marker, cytokeratin 19, CD 34, CD 68, and for alpha actin for smooth muscle, were observed. Our results showed that hepatocytes obtained from reduced liver grafts are easily cultured and are able to maintain viability and functionality in vitro. This alternative source of human cells maintained under controlled culture conditions may play an important role in the development of a bioartificial liver.     

Improved function of microencapsulated hepatocytes in a hybrid bioartificial liver support system

Conventional methods of microencapsulating isolated hepatocytes with Type I collagen matrix have provided metabolic liver support in experimental animal models of acute liver failure and congenital metabolic liver disease. We compared the biological function of transplanted microencapsulated hepatocytes cultured on standard Type I collagen (Vitrogen) and a commercially available liver basement-membrane-like extract from a mouse sarcoma (Matrigel). Isolated hepatocytes were microencapsulated with Matrigel and Vitrogen within an alginate-poly-L-lysine composite membrane. Isolated encapsulated hepatocytes (IEH) were transplanted intraperitoneally into homozygous Gunn rats that exhibit congenital hyperbilirubinemia. Control Gunn rats received empty or no microcapsules. Total serum bilirubin and conjugated bilirubin in bile were measured at weekly intervals for one month. Significant (p < 0.01) decreases in total serum bilirubin were observed in all IEH transplanted animals. No such decrease was seen in control animals. Gunn rats that received Matrigel had significantly (p < 0.05) lower serum bilirubin values and significantly (p < 0.05) higher conjugated bilirubin in bile than those that received Vitrogen. We conclude that hepatocytes microencapsulated with Matrigel functioned better than those with Vitrogen. This improved in vivo biological response underscores the importance of using the appropriate cell attachment substratum to enhance the function of a hybrid bioartificial liver support system based on transplanted hepatocytes.     

Cryopreservation of immobilized rat hepatocytes for the development of a bioartificial liver system

Liver transplantation, the only effective treatment for end-stage disease, is limited by donor availability. Cell transplantation offers the possibility to restore liver mass and function. Cryopreserved primary hepatocytes that can be thawed as needed might address this problem. Hepatocytes were harvested from a male Sprague Dawley rat, weighing approximately 250 g, using a 2-step in situ collagenase perfusion technique modified from the method described by Seglen. Hepatocytes were immobilized using a 100-mmol/L calcium with 1.5% alginate solution. Primary, immobilized hepatocytes transferred to various cryopreservation solutions containing 15% dimethyl sulfoxide were immediately placed into an isopropanol progressive freezing container at −80°C. We analyzed 4 cryopreservation solutions: Hormonally defined medium, histidine-tryptophan-ketoglutarate (HTK), fetal bovine serum (FBS), and HTK-modified cryopreservation solution (JH). After thawing, we measured viability, plating efficiency, urea synthesis, and albumin secretion to assess the effects of cryopreservation. Primary hepatocytes in HTK solution showed the better results in hepatocytes viability and urea synthesis after thawing. Cryopreserved immobilized hepatocytes in FBS maintained their viability and urea synthesis function. However, JH seemed to be the most effective medium for albumin secretion by both cyropreserved primary and immobilized hepatocytes.Our study suggested that HTK and JH cryopreservation solutions without FBS can be used to develop a bioartificial liver system.     

Apoptotic cell death and function of cryopreserved porcine hepatocytes in a bioartificial liver

We have previously shown that cryopreservation leads to increased apoptotic death of porcine hepatocytes intended for use in a bioartificial liver (BAL). This study was designed to determine if a broad-spectrum caspase inhibitor, IDN-1965, reduced apoptosis and increased function of cryopreserved porcine hepatocytes in static culture or in a BAL. Porcine hepatocytes were studied immediately after isolation and after 2 weeks of cryopreservation in liquid nitrogen using medium supplemented with 25 micromol/L IDN-1965 or vehicle. Both apoptotic and necrotic cells were observed in cultures of fresh and cryopreserved hepatocytes, but the percentage of apoptotic cells increased after cryopreservation. Cryopreservation in IDN-1965 improved hepatocyte viability and reduced apoptotic cell death determined by TUNEL assay. Cryopreservation of hepatocytes in IDN-1965 was also associated with reduced caspase 3-like activity, decreased release of cytochrome c from mitochondria, and a slower decline in mitochondrial membrane potential after thawing. These markers of apoptosis were lowest after cryopreservation when IDN-1965 was added to both the culture and cryopreservation medium. Functional markers of hepatocyte activity (albumin production, diazepam metabolism, urea production) were also increased after cryopreservation and culture of hepatocytes in medium supplemented with 25 micromol/L IDN-1965. Cryopreservation of porcine hepatocytes in the presence of caspase inhibitor IDN-1965 was associated with reduced apoptosis and improved function of porcine hepatocytes in both static culture and a perfused BAL. These data demonstrate that inhibition of apoptosis also preserves cell function.     

Development of a hybrid bioartificial liver.

OBJECTIVE: The authors developed an extracorporeal liver support system and tested its efficacy in experimental animals with liver failure. The first clinical use of this system to treat a patient with liver failure is reported. SUMMARY BACKGROUND DATA: Multiple attempts have been made, ranging from plasma exchange to use of charcoal columns, to develop liver support systems for treating patients with acute severe liver failure. None of these systems has achieved wide clinical use. There is a need for providing liver support as a "bridge" to transplantation and for treating patients with potentially reversible liver dysfunction. METHODS: A hybrid liver support system has been developed consisting of plasma perfusion through a charcoal column and a porous hollow fiber module inoculated with 5 x 10(9) matrix-attached hepatocytes. The system was tested in dogs with ischemic liver failure (n = 7) who underwent plasmapheresis; a control group (n = 6) underwent charcoal perfusion alone. A patient with liver failure was treated with this hybrid system. RESULTS: After 6 hours of hybrid liver support treatment, animals had significantly decreased serum ammonia and lactate levels, increased glucose level, normal prothrombin time, and increased systolic blood pressure compared with controls treated with charcoal perfusion alone. Use of the system to treat a patient was well tolerated with evidence of clinical improvement. CONCLUSIONS: Plasma perfusion through a system consisting of a charcoal column and matrix-attached porcine hepatocytes had significant beneficial effects in animals with liver failure and was well tolerated by a patient with liver failure.     

Cytoprotective influence of ZVAD-fmk and glycine on gel-entrapped rat hepatocytes in a bioartificial liver

BACKGROUND: This study was designed to determine if an anti-necrotic compound, glycine, and/or an anti-apoptotic agent, ZVAD-fmk, improved the viability and function of hepatocytes in a bioartificial liver. METHODS: Isolated rat hepatocytes were entrapped in collagen gel (1.0-10.0 x 10(6) cells/mL) and cultured in serum-free medium (1:10 ratio of gel:media) supplemented with glycine alone, ZVAD-fmk alone, or glycine and ZVAD-fmk. The cytoprotective effects of glycine and ZVAD-fmk on gel-entrapped rat hepatocytes (GERH) were determined after anoxic exposure (0-20 hours). Cell functionality (measured by urea production), cell viability (quantitated by vital staining with fluorescein diacetate:ethidium bromide [FDA:EB]), and the mechanism of cell death (verified by electron microscopy and DNA fragmentation studies) were determined for each condition. RESULTS: The viability of GERH declined gradually and then stabilized 12 hours after hepatocyte isolation. The rate of urea production by GERH was directly proportional to the number of viable hepatocytes. Apoptotic death predominated at low cell density, and necrotic cell death became significant at high cell density. Hepatocyte necrosis became more significant after exposure to longer periods of anoxia (4, 8, 12, and 20 hours). ZVAD-fmk provided dose-dependent cytoprotection to GERH with an optimum benefit at a concentration of 60 mumol/L. After anoxic exposure or under high cell density culture, glycine demonstrated a maximum benefit of inhibiting necrosis at a concentration of 3 mmol/L. The beneficial effects of glycine and ZVAD-fmk were additive. CONCLUSIONS: The metabolic activity of a hepatocyte bioartificial liver may benefit from the use of cytoprotective agents such as ZVAD-fmk and glycine.     

Microencapsulated parathyroid cells as a bioartificial parathyroid. In vivo studies

Parathyroid cells were isolated from healthy rats, encapsulated in alginate-polylysine membranes, and injected intraperitoneally into rats on which total parathyroidectomies had been performed. Three days posttransplant, serum calcium and PTH-M concentrations had increased to near-normal levels in the recipient animals. Similar results were observed in a separate group of parathyroidectomized rats 3 days after free parathyroid cells were implanted, but within 4 weeks serum calcium and PTH-M concentrations had decreased almost to pretransplant levels in these rats. In the rats with encapsulated cell transplants, by contrast, serum calcium and PTH-M levels were significantly higher, even after 8 weeks. No therapeutic effects were observed in rats injected with empty capsules or in the control group, which received no capsules or cells. These results indicate that transplants of microencapsulated parathyroid cells can temporarily reverse aparathyroidism in rats without the use of immunosuppressive drugs, and that further studies are warranted to investigate possible future clinical applications of this treatment.     

Liver stem cells: a potential source of hepatocytes for the treatment of human liver disease

Severe liver injury often leads to the proliferation of oval cells, which differentiate along hepatocytic and biliary lineages. Because oval cells proliferate only when hepatocyte replication is impaired, they are considered to be the progeny of facultative liver stem cells (FLSCs). Identification and isolation of FLSCs has been hampered by the lack of markers that delineate these bipotential progenitors. We hypothesized that transition ductal cells are FLSCs because they are located in a unique anatomical niche sharing tight junctions with a neighboring hepatocyte and another terminal ductular cell. Alternatively, it has been proposed recently that bone marrow-derived stem cells are FLSCs since these cells differentiate along the hepatic lineage following colonization of the liver. The intent of this review is to provide insight into the nature and origin of liver stem cells and to explore the possibility that stem cell technology may lead to the development of clinical modalities for the treatment of human liver disease.     

Cytotoxicity of Bile in Human Hep G2 Cells and in Primary Cultures of Rat Hepatocytes

There has been increasing interest in the development of a hepatocyte bioreactor for the treatment of acute hepatic failure; however, little is known about the effect of hepatocyte byproducts on the viability of the cells in the bioreactor environment. We investigated the effects of increasing concentrations of bile on the growth and viability of the human hepatoma cell line Hep G2 and on the cytochrome P-450 content and dependent mixed function oxidase (MFO) activities, reduced glutathione (GSH) content, and glutathione S-transferase (GST) activity of primary cultures of rat hepatocytes. Our purpose was to determine whether or not it would be necessary to pretreat the plasma from patients with acute liver failure to remove elevated bile concentrations which might be toxic to the hepatocytes in an artificial liver device. Bile was found to inhibit Hep G2 cell growth at concentrations as low as 0.1% and to decrease viability at concentrations above 0.5%. The cytochrome P-450 and GSH contents and the activities of the MFO system and of GST were decreased in the primary cultures of hepatocytes following 24 h treatment with concentrations of bile at and above 0.5%. The MFO activities associated with different cytochrome P-450 isoenzymes decreased to different extents in the presence of bile with the O-dealkylation of pentoxyresorufin being more labile than that of ethoxyresorufin. Our data indicate that elevated bile concentrations are cytotoxic to liver cells, and it may be necessary to pretreat patient plasma to decrease its bile content to protect the cells during the clinical operation of a hepatocyte bioreactor device.     

Enrichment of hepatocytes differentiated from mouse embryonic stem cells as a transplantable source

BACKGROUND: We previously reported that hepatocytes can be differentiated from embryonic stem (ES) cells by way of embryoid body (EB) formation and are transplantable into the mouse liver. However, the transplantation of EB-derived cells frequently resulted in teratoma formation in the recipient liver. In the present study, we eliminated the tumorigenic cells from EB outgrowths and examined the effects of enriched ES-cell-derived hepatocyte transplantation into an injured liver. METHODS: On day 15 in culture, the EBs were partially disaggregated and subcultured. Hepatocytes in the subcultured cells were examined by the expression of hepatocyte markers. Undifferentiated cells contaminating in the EB-derived cells were eliminated by Percoll discontinuous gradient centrifugation. Furthermore, undifferentiated cells, endothelial cells, and macrophages were eliminated by magnetic cell sorting using platelet/endothelial cell adhesion molecule (PECAM)-1 and Mac-1 antibodies. These enriched ES-cell-derived hepatocytes were then transplanted into the injured mouse liver. RESULTS: Percoll centrifugation and PECAM-1 antibodies eliminated the undifferentiated cells expressing Oct-3/4 from the EB-derived cells. ES-cell-derived hepatocytes showed expression of liver-related genes, synthesis of urea and glycogen, and structural characteristics during subculture. A transplantation study showed that the enriched ES-cell-derived hepatocytes integrated into the injured mouse liver and produced no teratomas. When the ES-cell-derived hepatocytes were transplanted into a CCl4-injured liver, the liver function was subsequently improved. CONCLUSIONS: Functional hepatocytes can be differentiated from mouse ES cells by way of EB formation. The elimination of undifferentiated cells from the EBs provides transplantable cells for liver failure without tumorigenicity.     

Comparison of porcine hepatocytes with human hepatoma (C3A) cells for use in a bioartificial liver support system

Cells from primary porcine hepatocytes (PPH) and the immortalized human hepatoma cell line C3A are both used in bioartificial liver support systems (BALSS). In this work the viability and metabolic capacity of PPH and C3A cells cultured in different media were compared. Also, because the cells come into direct or indirect contact with human blood components in BALSS, the effects of human complement on survival and functions of the cells was evaluated. For short-term culture, maintenance of PPH viability was essential for retention of P450IA1 activity (r = 0.882, p < 0.01) and effective ammonia clearance (r = −0.791, p < 0.01). When cell viability was below 60% P450IA1 activity could not be recorded and nitrogen elimination activity significantly diminished. In contrast to PPH, ammonia levels were markedly increased for C3A cells in all culture media tested (p < 0.01). Ammonia increase correlated with C3A viability (r = 0.896, p < 0.05). PPH metabolic function was superior to that of the C3A cell line when evaluated by P450IA1 activity, ammonia removal, and amino acid metabolism. When PPH were incubated in human plasma (HP) or human serum (HS) there was rapid and irreversible deterioration of viability occurring within 9 h. This toxic effect could be prevented by the inactivation of complement. When sodium citrate dissolved in dextrose was added to medium, there was considerable damage to both PPH and the C3A cell line. However, there was no demonstrable toxic effect when hepatic cells of either type were exposed to heparin. We conclude that PPH cultivated in complement-inactivated HP or HS are to be preferred to C3A for clinical application of BALSS, and that heparin should be preferred for anticoagulation in BALSS.     

Primary culture of the rat epididymal epithelial cells as a source of oestrogen

Studies were performed on the rat epithelial cells of the caput and cauda epididymidis cultured in a full medium enriched with foetal calf serum without or with exogenous testosterone. After 3 days of culture, the cells formed a monolayer. The cytoplasm of epididymal epithelial cells cultured with testosterone was rich in lipid droplets, glycogen and PAS-positive substances, while their content was decreased in the cytoplasm of cells cultured without testosterone. The activity of 3beta-hydroxysteroid dehydrogenase was observed both in the cytoplasm of cultured epididymal epithelial cells and epithelial cells of epididymal sections. Hormone assays showed very low levels of dehydroepiandrosterone, androstenedione and testosterone, and the absence of progesterone in the media of cells cultured without testosterone and higher testosterone concentrations when the cells were cultured with exogenous testosterone. However, the concentration of 17beta-oestradiol found in the medium of cells was high, and exceeded many-fold its levels in the control media. Lentaron (Formestan), steroidal inhibitor of cytochrome P450 aromatase added to the culture decreased the secretion of oestradiol. RT-PCR analysis yielded cDNA products of 333 bp in length when primers were chosen to amplify a highly conserved sequence in the 3' region of the cytochrome P450 aromatase gene. This study demonstrates the ability of epididymal epithelial cells in vitro to synthesize androgens and mRNA for cytochrome P450 aromatase in the cultured epididymal epithelial cells of the rat as well as the ability to aromatise the synthesized androgen to 17beta-oestradiol.     

Construction and performance of a minibioreactor suitable as experimental bioartificial liver

Abstract:  This work deals with the construction and performance of a hollow fiber-based minibioreactor (MBR). Due to its simple design and the utilization of standard materials, it could serve as a suitable tool to evaluate the behavior and performance of cold preserved or cultured hepatocytes in bioartificial liver devices. The system consists of 140 fiber capillaries through which goat blood is pumped at a flow of 9 mL/min. The cell compartment contains 90 × 10⁶ rat hepatocytes (volume 10 mL) and an internal oxygenator made of silicone tubing. To test the in vitro function of the system, 2-h perfusion experiments were performed, the evolution of hematocrit, plasma and extra-fiber fluid osmolality, and plasma urea and creatinine concentrations were evaluated. The detoxication efficiency of an ammonia overload was tested, showing that the system has enough capacity to remove ammonium. Also, the MBR oxygen transfer capacity to hepatocytes was tested, showing that the cells received an adequate oxygen supply.     

A bioartificial liver support system using primary hepatocytes: a preclinical study in a new porcine hepatectomy model

BACKGROUND: Bioartificial liver support systems providing a bridge to transplantation or even a definitive treatment of acute hepatic failure are a current focus of research. Different devices are used, although an impact on patient survival is doubtful for the time being. After developing a new flat membrane bioreactor, further preclinical studies have become necessary. Existing animal models of fulminant hepatic failure show common difficulties in defining a reproducible loss of functional liver tissue while considering systemic side effects. We now present a reproducible model with total hepatectomy in pigs, suitable to test the safety and efficacy of liver support systems. METHODS: Twelve pigs underwent total hepatectomy by using silicone tubes and a Y-adapter as vascular prosthesis; intracranial pressure was measured via a subdural probe. Anhepatic pigs were monitored under general anesthesia until death occurred. All were treated with a new flat membrane bioreactor (FMB) that contained porcine hepatocytes in 6 of the 12 cases. RESULTS: Our hepatectomy technique proved to be successful without requiring venovenous bypass circulation. Mean vascular clamping time was 9 minutes. The mean survival time was longer in animals treated with hepatocyte-equipped bioreactors than in untreated animals (24.8 +/- 4.3 vs 16.4 +/- 4.7 hours), which already showed increased intracranial pressure after 10 to 12 hours. Serum albumin levels indicated stable cell-specific functions of the FMB. CONCLUSIONS: The described technique of total hepatectomy is a well-reproducible animal model. The presented FMB maintained stable cell-specific functions and is a safe and efficient device.     

Mild hypothermic preservation for transport purposes of the AMC bioartificial liver charged with porcine hepatocytes

BACKGROUND: Preservation conditions play a crucial role during transport of a bioartificial liver (BAL) from the laboratory to the hospital. We assessed the possibility to preserve the AMC-BAL loaded with freshly isolated porcine hepatocytes at mild hypothermic temperatures. METHODS: Two laboratory-scale AMC-bioreactors were loaded with 1 billion freshly isolated porcine hepatocytes per experiment (n=6). Bioreactors in the control group were kept for three days at 37 degrees C. Bioreactors in the transport group were kept at 37 degrees C during day 1, at 15 degrees C during day 2, and again at 37 degrees C during day 3. In addition, long-term mild hypothermic preservation periods of 45 and 110 hr at 15 degrees C and 26 degrees C, respectively, were assessed. The effect of mild hypothermic preservation on hepatocytes inside the bioreactors was tested by determination of cell damage parameters, as well as metabolic and hepatocyte-specific functions. RESULTS: A 24-hour period of mild hypothermic preservation did not reduce any hepatocyte-specific function. LDH release was significantly higher only at day 2. Albumin production at day 2 and lidocaine elimination at day 3 were significantly higher with glucose consumption and lactate production being significantly lower at both test days. Long-term mild hypothermic preservation had a drastic negative effect on cellular viability and hepatocyte-specific function. CONCLUSIONS: Mild hypothermic preservation at temperatures as low as 15 degrees C and for a duration of 24 hr is a feasible method to preserve BAL systems loaded with freshly isolated porcine liver cells and will simplify the logistics of BAL transport from the laboratory to the hospital.