Liver-assist Devices Research Articles

Flow cytometric characterization of isolated porcine hepatocyte suspensions for liver support.

The high yield hepatocyte isolation necessary for hybrid liver assist devices (LAD) unavoidably increases contamination by nonparenchymal cells and depresses hepatocyte viability and functions. We have developed a flow cytometric procedure that improves quality control of the isolations. Cells present in these preparations were labeled by immunofluorescent antibody staining against cytokeratin 8, 18 as well as vimentin to identify hepatocytes, fibroblasts, and endothelial cells. Antibody staining against albumin and carbamoylphosphate synthetase allowed assessment of levels of albumin and carbamoylphosphate synthetase based on the hepatocyte relative fluorescence intensity. Hepatocyte P450 enzyme activity was measured by its ability to convert 5,6-methoxycarbonylfluorescein, a nonfluorescent substrate, to an intracellular fluorescent product. Flow cytometric methods of cell type identification and cell function assessment are fast and accurate and can be applied to commercial cell production. They may also provide an avenue for the enrichment of otherwise heterogeneous hepatocyte suspensions with cells presenting the specific functions desired for an hybrid liver assist devices.

Effect of extracellular matrix topology on cell structure, function, and physiological responsiveness: hepatocytes cultured in a sandwich configuration.

Extracellular matrix (ECM) geometry is an important modulator of cell polarity and function. For example, 3-dimensional matrices often more effectively induce differentiated cell function than traditional 2-dimensional substrates. The effect of ECM topology can be investigated in a controlled fashion using a technique whereby cells cultured on a single surface are overlaid with a second layer of ECM, thereby creating a "sandwich" configuration. Confluent monolayers of epithelial or endothelial cells overlaid in this fashion often reorganize into structures that are reminiscent of their native tissue. In the case of hepatocytes, the overlay causes a dramatic reorganization of the cytoskeleton, adoption of in vivo-like morphology and polarity, and expression of a wide array of liver-specific functions. In this short review, we use the sandwiched hepatocyte culture system to illustrate the effect of ECM geometry on cellular function. Pertinent studies are summarized in the context of defining the parallels, strengths, and limitations of this culture system as an in vitro model to study the physiology and morphogenesis of liver tissue. We also explore some of its potential uses as a model to study liver pharmacology and toxicology, and for the development of liver preservation techniques and liver-assist devices.

Isolation and culture of porcine hepatocytes for artificial liver support

The primary requirement of cells in a liver support system is the preservation of the in vivo metabolic functions that prevent or decrease the progress of hepatic encephalopathy (HE) by providing interim support to liver failure patients. While rodent hepatocytes offer a model for liver assist device (LAD) research, their limited number per animal prohibits direct scale up to human devices. Healthy human liver cells are seldom available in adequate numbers to support clinical LAD use; consequently, a large animal source of liver cells is needed. The study presented here explored the potential of porcine hepatocytes to proliferate and maintain metabolic function in vitro. Porcine hepatocytes were isolated from approximately 12 kg swine by a modification of Seglen's method. Hepatocytes cultured up to 10 days were shown to metabolize ammonia and maintain both Phase I and II detoxification functions. In addition, the cultures showed proliferative activity both as an increase in total protein content and by thymidine incorporation. Immunocytochemical staining identified cell proliferation through Day 4 to be primarily hepatocytes while Days 6 and 10 showed nonparenchymal cells to be increasing. The detoxification functions measured showed peak activity on Day 4 and gradually declined through Day 10. The ability of porcine hepatocytes to proliferate and maintain a diversity of hepatic functions in culture strongly suggests their potential for use as the biological component of artificial LADs.

Isolation and culture of porcine hepatocytes for artificial liver support.

The primary requirement of cells in a liver support system is the preservation of the in vivo metabolic functions that prevent or decrease the progress of hepatic encephalopathy (HE) by providing interim support to liver failure patients. While rodent hepatocytes offer a model for liver assist device (LAD) research, their limited number per animal prohibits direct scale up to human devices. Healthy human liver cells are seldom available in adequate numbers to support clinical LAD use; consequently, a large animal source of liver cells is needed. The study presented here explored the potential of porcine hepatocytes to proliferate and maintain metabolic function in vitro. Porcine hepatocytes were isolated from approximately 12 kg swine by a modification of Seglen's method. Hepatocytes cultured up to 10 days were shown to metabolize ammonia and maintain both Phase I and II detoxification functions. In addition, the cultures showed proliferative activity both as an increase in total protein content and by thymidine incorporation. Immunocytochemical staining identified cell proliferation through Day 4 to be primarily hepatocytes while Days 6 and 10 showed nonparenchymal cells to be increasing. The detoxification functions measured showed peak activity on Day 4 and gradually declined through Day 10. The ability of porcine hepatocytes to proliferate and maintain a diversity of hepatic functions in culture strongly suggests their potential for use as the biological component of artificial LADs.

In vivo evaluation of a hollow fiber liver assist device

Orthotopic liver transplantation is the only effective form of therapy currently available for patients with fulminant hepatic failure (FHF). The use of an extracorporeal (EC) liver assist device (LAD) may result in improved presurgical clinical management. Alternatively, patients treated with LADs could avoid the transplantation procedure if they are able to regenerate a critical mass of hepatocytes that will sustain functional viability. In this study, the efficacy of a prototype hollow fiber LAD seeded with rabbit hepatocytes was assessed in vivo by the use of two different animal models: (1) normal rabbits injected with diazepam or lidocaine, and (2) a galactosamine (Gal)-intoxicated rabbit model of FHF. The EC LAD clearly decreased the blood levels of the two drugs and significantly generated diazepam and lidocaine metabolites indicating the maintenance of active P450 forms in the cellular component of the devices. A 6-hour EC treatment significantly increased the survival time and delayed the onset of hepatic encephalopathy (HE) in the Gal-intoxicated rabbits. Histological evaluations of postmortem livers showed greater hepatocyte regenerative activity in the animals treated with hepatocyte-seeded LADs than in the two control groups, e.g., rabbits not treated or treated with unseeded devices. These findings support the concept that a microporous hollow fiber LAD seeded with rabbit hepatocytes is able to sustain drug detoxification in vivo as well as to modify the course of FHF in a well-characterized animal model.

In vivo evaluation of a hollow fiber liver assist device

Orthotopic liver transplantation is the only effective form of therapy currently available for patients with fulminant hepatic failure (FHF). The use of an extracorporeal (EC) liver assist device (LAD) may result in improved presurgical clinical management. Alternatively, patients treated with LADs could avoid the transplantation procedure if they are able to regenerate a critical mass of hepatocytes that will sustain functional viability. In this study, the efficacy of a prototype hollow fiber LAD seeded with rabbit hepatocytes was assessed in vivo by the use of two different animal models: (1) normal rabbits injected with diazepam or lidocaine, and (2) a galactosamine (Gal)-intoxicated rabbit model of FHF. The EC LAD clearly decreased the blood levels of the two drugs and significantly generated diazepam and lidocaine metabolites indicating the maintenance of active P450 forms in the cellular component of the devices. A 6-hour EC treatment significantly increased the survival time and delayed the onset of hepatic encephalopathy (HE) in the Gal-intoxicated rabbits. Histological evaluations of postmortem livers showed greater hepatocyte regenerative activity in the animals treated with hepatocyte-seeded LADs than in the two control groups, e.g., rabbits not treated or treated with unseeded devices. These findings support the concept that a microporous hollow fiber LAD seeded with rabbit hepatocytes is able to sustain drug detoxification in vivo as well as to modify the course of FHF in a well-characterized animal model.

Acute liver failure

Acute liver failure is a multiorgan syndrome with dramatic clinical features and, often, a fatal outcome. It is characterized by the onset of coma and coagulopathy within 6 months, and usually in <6 weeks, from onset of illness. Viral hepatitis, drug-related liver injury, and the alcohol-acetaminophen syndrome are the most common etiologies. Altered mental status accompanied by jaundice is a hallmark of acute liver failure. A unique feature is the evolution of increased intracranial pressure due to cerebral edema. The resulting cerebral ischemia and brainstem herniation account for approximately 50% of deaths in patients with acute liver failure. Mannitol therapy may successfully treat most patients with high intracerebral pressure. Most patients demonstrate features of the multiple organ failure syndrome, including a shock-like state, renal failure, and occasionally respiratory distress syndrome. Close monitoring of volume status is necessary, since administration of large quantities of fluid may be required. Infection is also common; most pathogens are gram-positive, and fungal infections are also seen. Because an optimum therapy for acute liver failure does not yet exist, liver transplantation should be considered early, before advanced levels of coma develop. Alternative, experimental treatment modalities include heterotopic liver grafting, administration of hepatocyte growth factor, use of an extracorporeal liver-assist device, and liver cell transplantation, but none of these has attained widespread use.

Use of the Hepatix Extracoproreal Liver Assist Device in the treatment of fulminant hepatic failure

Use of the Hepatix Extracoproreal Liver Assist Device in the treatment of fulminant hepatic failure

Assessment of an Extracorporeal Liver Assist Device in Anhepatic Dogs

We have used an anhepatic dog model to demonstrate the efficacy of a bioartificial liver assist device. Six dogs underwent total hepatectomy. Three received only medical care (controls) while the remainder were connected to an extracorporeal liver assist device (ELAD). The control dogs failed to regain consciousness after anesthesia although all lived 4-5 h postoperatively. Plasma ammonia concentration increased by an average of 250 mumol/L between the end of surgery and the demise of the animals. The treated dogs lived 3-12.5 h, and 2 of them required repeated doses of thiamylal sodium to maintain sedation. Plasma ammonia concentration was unchanged after connection to the ELAD except in the longest survivor, whose ammonia began to rise after 8 h on the ELAD. The short survival in the other 2 treated dogs was the result of uncontrolled intraabdominal bleeding. This device is capable of replacing the metabolic function of the liver, and might provide hepatic support in patients awaiting transplantation or in fulminant hepatic failure.

Long-term cultures of adult mammalian hepatocytes in hollow fibers as the cellular component of extracorporeal (hybrid) liver assist devices.

A discussion of the treatment of liver insufficiency with extracorporeal (hybrid) liver assist devices (LADs) should address a definition of the types of liver failure susceptible to being treated by these devices as well as the modalities of in vivo and in vitro testing. Relevant to the first subject is the subject of pathogenesis of hepatic coma, which should be the target for the design of these LADs. Although this modality of therapy is new, it can be predicted that these devices will demand minimal safety conditions, i.e., the seeding with cells that are not tumorigenic or carrying viral particles. Among other topics to be considered in the development of LADs is the proper choice of hollow fiber to be used and the testing on proper animal models of hepatic failure. It is our philosophy that the long-term culture of adult mammalian hepatocytes in hollow fibers is the basis for appropriate designs of this type of temporary liver support.

Mammalian hepatocytes as a foundation for treatment in human liver failure.

Technological advances in the separation and culture of mammalian hepatocytes have facilitated the use of these cells as the foundation for either hepatocyte transplantation or hepatocyte-seeded hollow fiber liver assist devices (LAD). To fully appreciate the practical applications of these tissue engineering solutions, it is necessary to understand the types of human liver failure as well as the corresponding animal models. The most immediate application of this type of technology is the treatment of hepatic encephalopathy (HE), an acute and highly fatal complication of fulminant hepatic failure. Although the pathogenesis of HE is unknown, failure of the detoxification function of the liver is accepted as playing an important role in this disorder. Consequently, the assaying and preservation of P450 activity in the grafted cells or in the LAD must be among the main targets of this research. This review explores the problems in hepatocyte transplantation and culture that deserve special consideration and emphasizes the conditions contributing to the in vitro maintenance of phenotypic expression of these cells.

Cells Cultured on Artificial Capillaries and Their Use as a Liver Assist Device

ABSTRACTMammalian cells in culture can be maintained and, in some cases, will proliferate to form tissue‐like masses on the exterior surfaces of artificial capillaries made of semi‐permeable membrane when nutrient medium is circulated within the capillaries. Published experience with the system in a variety of applications is reviewed with particular attention to the use of cells of liver origin to provide hepatic metabolic functions when the blood of a host animal replaces the nutrient medium and a symbiotic relationship is established between host and device through the semipermeable capillary walls. The rationale for liver assist in acute fulminant hepatic failure (FHF) and animal models for FHF are reviewed. Exploratory studies of bil‐irubin metabolism with congenitally jaundiced host rats are described which provide a quantitative measure of the capillary culture liver assist device's functional capacity and suggest that substantial increases in the metabolic capacity of the device are needed. Potential means to increase capacity are considered, especially the use of con vective mass transfer to supplement diffusion by inducing ultrafiltration through the capillary walls, and the application of relevant theoretical methods for the analysis of convective transfer in the capillary culture chamber is proposed.