Bruce Amiot

Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function

The culture of primary hepatocytes as spheroids creates an efficient three‐dimensional tissue construct for hepatic studies in vitro. Spheroids possess structural polarity and functional bile canaliculi with normal differentiated function. Thus, hepatocyte spheroids have been proposed as the cell source in a variety of diagnostic, discovery, and therapeutic applications, such as a bioartificial liver. Using a novel rocking technique to induce spheroid formation, kinetics of spheroid formation, cell‐cell adhesion, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. Evidence was provided that the formation of spheroids occurred faster and with fewer nonadherent hepatocytes in rocked suspension culture compared to a traditional rotational system. Hepatocyte spheroids in rocked culture showed stable expression of more than 80% of 242 liver‐related genes including those of albumin synthesis, urea cycle, phase I and II metabolic enzymes, and clotting factors. Biochemical activity of rocked spheroid hepatocytes was superior to monolayer culture of hepatocytes on tissue culture plastic and collagen. Conclusion: Spheroid formation by rocker technique was more rapid and more efficient than by rotational technique. Rocker‐formed spheroids appear suitable for application in a bioartificial liver or as an in vitro liver tissue construct. (HEPATOLOGY 2009.)

Rapid, Large-Scale Formation of Porcine Hepatocyte Spheroids in a Novel Spheroid Reservoir Bioartificial Liver

We have developed a novel bioreactor based on the observation that isolated porcine hepatocytes rapidly and spontaneously aggregate into spheroids under oscillation conditions. The purpose of this study was to characterize the influence of oscillation frequency (0.125 Hz, 0.25 Hz), cell density (1‐10 × 106 cells/mL), and storage condition (fresh, cryopreserved) of porcine hepatocytes on the kinetics of spheroid formation. The viability and metabolic performance of spheroid hepatocytes was also compared to monolayer culture. We observed that both fresh and cryopreserved porcine hepatocytes began formation of spheroids spontaneously at the onset of oscillation culture. Spheroid size was directly related to cell density and time in culture, though inversely related to oscillatory frequency. Spheroid formation by fresh porcine hepatocytes was associated with decreased cell death (lactate dehydrogenase release, 1.3 ± 1.0 vs. 3.1 ± 0.7 U/mL, P < 0.05) and increased metabolic performance (albumin production, 14.7 ± 3.3 vs. 4.6 ± 1.4 fg/c/h, P < 0.0001; ureagenesis from ammonia, 267 ± 63 vs. 92 ± 13 μmol/L/h, P < 0.001) compared with monolayer culture. In conclusion, based on the favorable properties of rapid spheroid formation, increased hepatocellular function, and ease of scale‐up, the spheroid reservoir bioreactor warrants further investigation as a bioartificial liver for support of liver failure. (Liver Transpl 2005;11:901–910.)

Cell therapies for liver diseases

Cell therapies, which include bioartificial liver support and hepatocyte transplantation, have emerged as potential treatments for a variety of liver diseases. Acute liver failure, acute‐on‐chronic liver failure, and inherited metabolic liver diseases are examples of liver diseases that have been successfully treated with cell therapies at centers around the world. Cell therapies also have the potential to be widely applied to other liver diseases, including noninherited liver diseases and liver cancer, and to improve the success of liver transplantation. Here we briefly summarize current concepts of cell therapy for liver diseases. Liver Transpl 18:9–21, 2012.

A technique for porcine hepatocyte harvest and description of differentiated metabolic functions in static culture.

Current bioartificial liver devices are based on the use of a large mass of hepatocytes exhibiting differentiated metabolic function. The pig has become a source of interest for the acquisition of such cells-however, harvesting a large mass of highly viable cells has met with difficulty. This study describes a technique for harvesting large quantities of hepatocytes at viabilities greater than 90% and also describes several features documenting differentiated function. Pigs, 6 to 10 kg body weight, underwent in situ two-step whole liver perfusion (ethylene glycol tetraacetic acid and collagenase) and ex vivo cell harvest. Harvests yielded an average of 19.5 billion cells with an average viability of 94.6%. Hepatocytes were then entrapped in type I collagen (3 x 10(5) cells/well) and cultured in serum-free media for 5 days. Pig hepatocytes produced stable amounts of albumin and maintained cytochrome P-450 and glucuronidation activity over 5 days, as shown by the metabolism of lidocaine and 4-methylumbelliferone. These data indicate that pig hepatocytes can be harvested with high yields and can retain viability and differentiated function over at least 5 days of culture, and therefore should prove to be an excellent source of hepatocytes for bioartificial liver devices.

Hepatocyte-like cells differentiated from human induced pluripotent stem cells: Relevance to cellular therapies

UNLABELLED Maturation of induced pluripotent stem cells (hiPSCs) to hepatocyte-like cells (HLCs) has been proposed to address the shortage of human hepatocytes for therapeutic applications. The purpose of this study was to evaluate hiPSCs, HLCs and hepatocytes, all of human origin, in terms of performance metrics of relevance to cell therapies. hiPSCs were differentiated to HLCs in vitro using an established four-stage approach. We observed that hiPSCs had low oxygen consumption and possessed small, immature mitochondria located around the nucleus. With maturation to HLCs, mitochondria showed characteristic changes in morphology, ultrastructure, and gene expression. These changes in mitochondria included elongated morphology, swollen cristae, dense matrices, cytoplasmic migration, increased expression of mitochondrial DNA transcription and replication-related genes, and increased oxygen consumption. Following differentiation, HLCs expressed characteristic hepatocyte proteins including albumin and hepatocyte nuclear factor 4-alpha, and intrinsic functions including cytochrome P450 metabolism. But HLCs also expressed high levels of alpha fetoprotein, suggesting a persistent immature phenotype or inability to turn off early stage genes. Furthermore, the levels of albumin production, urea production, cytochrome P450 activity, and mitochondrial function of HLCs were significantly lower than primary human hepatocytes. CONCLUSION - hiPSCs offer an unlimited source of human HLCs. However, reduced functionality of HLCs compared to primary human hepatocytes limits their usefulness in clinical practice. Novel techniques are needed to complete differentiation of hiPSCs to mature hepatocytes.

Pivotal preclinical trial of the spheroid reservoir bioartificial liver

BACKGROUND & AIMS The neuroprotective effect of the spheroid reservoir bioartificial liver (SRBAL) was evaluated in a porcine model of drug-overdose acute liver failure (ALF). METHODS Healthy pigs were randomized into three groups (standard therapy (ST) alone, ST+No-cell device, ST+SRBAL device) before placement of an implantable intracranial pressure (ICP) monitor and a tunneled central venous catheter. One week later, pigs received bolus infusion of the hepatotoxin D-galactosamine and were followed for up to 90h. RESULTS At 48h, all animals had developed encephalopathy and biochemical changes confirming ALF; extracorporeal treatment was initiated and pigs were observed up to 90h after drug infusion. Pigs treated with the SRBAL, loaded with porcine hepatocyte spheroids, had improved survival (83%, n=6) compared to ST alone (0%, n=6, p=0.003) and No-cell device therapy (17%, n=6, p=0.02). Ammonia detoxification, peak levels of serum ammonia and peak ICP, and pig survival were influenced by hepatocyte cell dose, membrane pore size and duration of SRBAL treatment. Hepatocyte spheroids remained highly functional with no decline in mean oxygen consumption from initiation to completion of treatment. CONCLUSIONS The SRBAL improved survival in an allogeneic model of drug-overdose ALF. Survival correlated with ammonia detoxification and ICP lowering indicating that hepatocyte spheroids prevented the cerebral manifestations of ALF (brain swelling, herniation, death). Further investigation of SRBAL therapy in a clinical setting is warranted.

Fumarylacetoacetate hydrolase deficient pigs are a novel large animal model of metabolic liver disease

Hereditary tyrosinemia type I (HT1) is caused by deficiency in fumarylacetoacetate hydrolase (FAH), an enzyme that catalyzes the last step of tyrosine metabolism. The most severe form of the disease presents acutely during infancy, and is characterized by severe liver involvement, most commonly resulting in death if untreated. Generation of FAH(+/-) pigs was previously accomplished by adeno-associated virus-mediated gene knockout in fibroblasts and somatic cell nuclear transfer. Subsequently, these animals were outbred and crossed to produce the first FAH(-/-) pigs. FAH-deficiency produced a lethal defect in utero that was corrected by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3 cyclohexanedione (NTBC) throughout pregnancy. Animals on NTBC were phenotypically normal at birth; however, the animals were euthanized approximately four weeks after withdrawal of NTBC due to clinical decline and physical examination findings of severe liver injury and encephalopathy consistent with acute liver failure. Biochemical and histological analyses, characterized by diffuse and severe hepatocellular damage, confirmed the diagnosis of severe liver injury. FAH(-/-) pigs provide the first genetically engineered large animal model of a metabolic liver disorder. Future applications of FAH(-/-) pigs include discovery research as a large animal model of HT1 and spontaneous acute liver failure, and preclinical testing of the efficacy of liver cell therapies, including transplantation of hepatocytes, liver stem cells, and pluripotent stem cell-derived hepatocytes.

Serum-Free Medium and Mesenchymal Stromal Cells Enhance Functionality and Stabilize Integrity of Rat Hepatocyte Spheroids

Long-term culture of hepatocyte spheroids with high ammonia clearance is valuable for therapeutic applications, especially the bioartificial liver. However, the optimal conditions are not well studied. We hypothesized that liver urea cycle enzymes can be induced by high protein diet and maintain on a higher expression level in rat hepatocyte spheroids by serum-free medium (SFM) culture and coculture with mesenchymal stromal cells (MSCs). Rats were feed normal protein diet (NPD) or high protein diet (HPD) for 7 days before liver digestion and isolation of hepatocytes. Hepatocyte spheroids were formed and maintained in a rocked suspension culture with or without MSCs in SFM or 10% serum-containing medium (SCM). Spheroid viability, kinetics of spheroid formation, hepatic functions, gene expression, and biochemical activities of rat hepatocyte spheroids were tested over 14 days of culture. We observed that urea cycle enzymes of hepatocyte spheroids can be induced by high protein diet. SFM and MSCs enhanced ammonia clearance and ureagenesis and stabilized integrity of hepatocyte spheroids compared to control conditions over 14 days. Hepatocytes from high protein diet-fed rats formed spheroids and maintained a high level of ammonia detoxification for over 14 days in a novel SFM. Hepatic functionality and spheroid integrity were further stabilized by coculture of hepatocytes with MSCs in the spheroid microenvironment. These findings have direct application to development of the spheroid reservoir bioartificial liver.

Cytotoxic immune response to a xenogeneic bioartificial liver.

Prior studies have suggested the possibility of immune-mediated death of xenogeneic hepatocytes in a bioartificial liver (BAL) during hemoperfusion. This study was designed to elucidate how immunity may cause death of xenogeneic hepatocytes in the BAL. Healthy dogs were treated with a BAL containing hollow fiber membranes with large pores (200 nm) or small pores (400 kDa). The immune response of recipient dogs to BAL therapy was monitored over 3 h of treatment. We observed significantly greater loss of viability of hepatocytes in the 200 nm group compared with the 400 kDa group (p < 0.001). Low viability after treatment with the large pore membrane was associated with positive staining for dog IgG, dog IgM, and dog complement on dead hepatocytes. Significant levels of dog antibody were detected in samples of BAL medium from the 200 nm group. These canine antibodies were cytotoxic to porcine hepatocytes. In contrast, medium from the 400 kDa group contained only trace levels of dog IgG and were noncytotoxic. We conclude that antibody-mediated cytotoxicity contributed to the death of hepatocytes during treatment with a xenogeneic BAL. Immune-mediated death of hepatocytes was reduced by increasing selectivity of the BAL membrane.

Membrane pore size impacts performance of a xenogeneic bioartificial liver

Background. We have developed a novel bioartificial liver (BAL) composed of porcine hepatocyte spheroids in a reservoir design. A semipermeable membrane is used to protect the spheroids from immune-mediated damage. This study was designed to assess the influence of membrane pore size on performance of the spheroid reservoir BAL. Methods. Eight healthy dogs were studied during primary and secondary exposures to the spheroid reservoir BAL using membranes with small (10 nm) or large (200 nm) pores. BAL performance was assessed by multiple functional assays. Spheroids were examined microscopically before and after all BAL treatments. Titers of xenoreactive antibody were monitored until elective death of animals on day 42. Results. Viability and functional performance of spheroids were significantly greater after all BAL treatments that used membranes with 10-nm versus 200-nm pores. Reduced performance in the 200 nm group was associated with 7.7-fold and 78.0-fold rise in xenoreactive antibody titers after first and second treatments, respectively. Dogs in the 10 nm group remained hemodynamically stable during all BAL treatments, whereas those in the 200 nm group experienced acute hypotension (P <0.001) during second BAL exposures. Microscopic examination of spheroids after BAL treatments indicated that deposition of canine proteins, including complement, was associated with reductions in both viability and functional performance of the BAL. Conclusions. The elicited immune response of healthy dogs to a xenogeneic BAL was blocked and BAL performance significantly improved by reducing the permeability of the BAL membrane.