Hybrid Artificial Liver Research Articles

Cryopreservation of Porcine Hepatocyte Cultures

Cryopreservation of freshly isolated hepatocytes is regarded the standard procedure for long term storage of liver cells. However, a dramatic loss in cell number, viability, and differentiated cell function is usually inevitable, and, upon thawing, hepatocytes do not attach well in culture. Freezing of hepatocyte cultures has already been shown to reduce these hazardous effects, probably due to the beneficial impact of a repair phase that allows the cells to restore their membranes during the first days of culture prior to and after freezing. We have compared the performance of porcine hepatocytes from slaughterhouse organs that were frozen at day 3 of immobilizing gel culture and stored at −80°C for increasing time intervals up to 30 days (three groups;n= 3 each). Control groups consisted of batch identical hepatocyte cultures, either frozen at a fixed cooling rate (−1°C/min;n= 3), subjected to rapid freezing (−10°C/min;n= 3) or left without any freezing (n= 4). Upon thawing, light and electron microscopic studies revealed a cell survival rate of 70.3 ± 10.5% (mean ± SD;n= 9) in hepatocyte cultures after adjusted rate-controlled freezing. In these groups, the length of storage time did not influence results. Functional parameters, like albumin secretion values and ethoxycoumarin deethylase activity (cytochrome P450-IA1) were comparable to non–frozen cultures after a recovery time of 48 h post-thaw. Fixed cooling rates at −1°C and −10°C, respectively, showed a greater reduced viability (35.4 ± 7.4%; 9.87 ± 2.8%) and a severely impaired post-thaw functional performance of cultures. Cryopreservation of hepatocyte cultures could provide a practical means of establishing a bank of hepatocyte cultures. This could be extremely useful for the development of a hybrid artificial liver device, and it might support repeated investigations on batch-controlled hepatocyte cultures, especially with respect to human cells.

Isolation of Porcine Hepatocytes from Slaughterhouse Organs

Recent developments in the field of hybrid artificial liver research have increased the demand for an unlimited supply of primary hepatocytes. At present, liver cells are mainly isolated from anesthetized pigs, since slaughterhouse organs have been regarded as a cell-source of minor quality. A modified enzymatic isolation technique for the successful harvesting of viable porcine liver cells from slaughterhouse organs is introduced. Digestion of the left medial liver lobe (n = 114) resulted in 1.2 +/- 0.35 x 10E7 viable hepatocytes per gram tissue and an overall yield of 2.23 +/- 0.48 x 10E9 cells per isolation (viability: 94 +/- 2.4%). Morphological integrity of hepatocytes in long-term immobilization culture systems was assessed by electron microscopic follow-up. Stable DNA-contents (52 +/- 2 micrograms) and low alanine-amino-transferase (ALAT) release were measured after early culture adaptation. Urea production under NH4Cl, Albumin secretion, total bile acid synthesis (3.5 pmol/hr/micrograms DNA) and 7-ethoxicoumarin o-deethylase (ECOD) activity demonstrated functional activity and maintenance of Type IA1 cytochrome P450 (CYP450) dependent metabolism in cultured hepatocytes for at least 10 days. Compared to ex vivo isolation results in the literature, we could not see any disadvantages in the use of liver cells from slaughterhouse organs, but would like to point out four advantages. It saves animal lives, labor, costs, and time. Research groups especially with no animal housing and/or surgical facilities should evaluate the presented technique for their own needs to make use of this unlimited cell source.

Porcine Hepatocytes From Slaughterhouse Organs

Most recent strategies for the development of hybrid artificial liver devices focus on the use of parenchymal liver cells (hepatocytes). For clinical application of these devices, a sufficient cell supply is mandatory. Because human liver tissue is rarely available, isolated porcine hepatocytes from laboratory animals have been suggested for use in bioartificial livers. The authors introduce a modified isolation protocol to yield large scale numbers of viable porcine hepatocytes from slaughterhouse organs. Perfusion and enzymatic digestion of the left medial liver lobe (n = 74) resulted in 1.0 +/- 0.3 x 10(7) viable hepatocytes per gram of tissue, and an overall yield of 1.92 +/- 0.5 x 10(9) viable cells per isolation (viability: 93 +/- 2%). Collagen gel immobilization maintained morphologic integrity and functional activity of hepatocyte cultures over long-term periods. Cell morphology, as assessed by light microscopic evaluation, was maintained for 2 weeks. Stable DNA content (51 +/- 5 micrograms) and low values of alanine aminotransferase release (8 microU/hr/micrograms DNA) indicated structural stability of cultures after a short period of post isolational adaptation. Albumin secretion (4.5 micrograms/hr/micrograms DNA) and persistent Cytochrome P450 IA1 dependent deethylation of 7-ethoxycoumarin (4.5 nmol/hr/micrograms DNA) indicated long-term metabolic activity of cultured hepatocytes. Hepatocytes from livers of slaughtered pigs represent an unlimited resource of viable material for cell culture, and their usefulness as functional units of bioartificial liver support devices should be tested.

A Novel Bioreactor Design for In Vitro Reconstruction of In Vivo Liver Characteristics

We have constructed a bioreactor aimed at imitating the three-dimensional micro- and macroenvironment of the liver. In vivo hepatocytes are arranged in plates of cell monolayers and are specifically attached with both sinusoidal surfaces to the space of Disse which contains extracellular matrix. Nonparenchymal cells are located on the other side of the space of Disse toward the sinusoid. For supporting monolayer hepatocytes with bipolar attachment to the extracellular matrix, we used a double gel culture technique that sandwiches hepatocytes between two layers of collagen. In double gel cultures, albumin production increases during an adaptive period to the in vitro environment. In contrast to conventional single gel hepatocytes, double gel hepatocytes maintain expression of sinusoidal microvilli and a polyhedric cell shape in culture as seen by transmission electron microscopy. Albumin production in the bioreactor was stable. The organotypical bioreactor concept is an example of organ mimicry and may provide the basis for the organ-otypical development of a full-sized hybrid artificial liver.

High Cell‐Density Culture System of Hepatocytes Entrapped in a Three‐Dimensional Hollow Fiber Module with Collagen Gel

A compact three-dimensional (3D) module is needed for hepatocyte culture in order to develop an effective hybrid artificial liver system that can retain hepatocellular structure and differentiated functions. We treated the 3D module with collagen gel to entrap rat hepatocytes. This method yielded a high hepatocellular density (2 x 10(7) cells/ml) over a period of 14 days and maintained the secretion of albumin and ureogenesis at the same level as the control monolayer method. The ammonia removal remained at 43% of the Day 0 value over 8 days of perfusion. Our data show that this approach may be useful for liver support therapy in an extracorporeal circuit.

Influence of Warm Ischemia on Isolation and Primary Culture of Hepatocytes from Rat Liver for a Hybrid Artificial Liver

To assess the possibility of using hepatocytes from ischemic liver, as a bioreactor of a hybrid artificial liver, we investigated the influence of warm ischemia on the isolation and culture of hepatocytes in rats. Warm ischemia was induced by clamping the liver hilus and the animals were divided into 3 groups according to the duration of ischemia: group A (no ischemia), group B (10 minutes) and group C (20 minutes). Hepatocytes were isolated by the collagenase perfusion method and cultured for 5 days. The yield and viability of the isolated hepatocytes were lower in group C. Rate of attachment was decreased as the duration of ischemia increased. There was no significant difference observed in functions in culture. Sufficient hepatocytes, as a bioreactor, can be isolated and cultured from warm ischemic liver within 10 minutes. Though the number of available hepatocytes were diminished, hepatocytes procured from longer warm ischemic liver could be utilized as a bioreactor.

Is the biological artificial liver clinically applicable? A historic review of biological artificial liver support systems.

Hemoperfusion, hemodiafiltration, plasma exchange, and extracorporeal liver perfusion have already been adopted to treat patients with acute and chronic hepatic failure. However, the survival rate of patients with acute hepatic failure remains at approximately 30% and has not improved as expected. Current advances in biotechnology have opened the way for the development of a biological artificial liver, which is called the hybrid artificial liver because it consists of both biological and artificial materials. Isolated hepatocytes have been investigated for use in various types of hybrid artificial liver. In addition, the role of biomatrices, microcarriers, and the microencapsulation technique has been studied with respect to long-term maintenance of hepatocellular function and development of high-density culture systems for hepatocytes. Before clinical application of hybrid artificial liver support systems becomes possible, many problems have to be resolved, including large-scale preparation and long-term preservation of biomaterials, high-density and stable immobilization of biomaterials on artificial materials, control of immunological hazards, biocompatibility, safe transportation and sterilization of biomaterials, and the high cost. We review the history of biological artificial livers and discuss their future role.

Long‐Term continuous culture of hepatocytes in a packed‐bed reactor utilizing porous resin

As part of our attempt to develop a hybrid artificial liver support system using cultured hepatocytes, we investigated the long-term metabolic function of hepatocytes incubated in a packed-bed type reactor using reticulated polyvinyl formal (PVF) resin as a supporting material. Long-term (up to 1 week) perfusion culture experiments using the packed-bed reactor (20 mm i.d.) loaded with 500 PVF resin cubes (mean pore size 250 mum, 2 x 2 x 2 mm), together with conventional monolayer culture experiments as controls, were performed in serum-free or serum-containing medium. Ammonium metabolism and urea synthesis activities were evaluated quantitatively based on reaction kinetic analyses. Initial rates of ammonium metabolism and urea-N synthesis, as well as GPT enzyme activities, were adopted as indexes of the metabolic performance of the reactor and activities of the cultured hepatocytes.When serum-free medium was used in the perfusion cultures, ammonium metabolic and urea-N synthetic rates showed significant decay with elapse of the culture period, being less than 10% of those measured on day 1. This loss of activity was more prominent in the perfusion culture than in the monolayer cultures using this medium. In contrast, when serum-containing medium was used, approximately 50% of these activities obtained on day 1 were maintained even at the end of the cultures both in the perfusion and monolayer culture experiments.We concluded that the packed-bed reactor using PVF resin enabled high-density culture of hepatocytes, and showed a satisfactory ability to maintain the metabolic function of immobilized hepatocytes for relatively long periods of up to 1 week. This type of reactor is thus considered to represent a breakthrough in overcoming the difficulties involved in the development of a hybridtype artificial liver support system. (c) 1994 John Wiley & Sons, Inc.

Use of hepatocyte cultures for liver support bioreactors.

Hybrid artificial liver systems are being developed as extracorporeal temporary liver support therapy. Here, an overview is given with emphasis on hepatocyte culture models for bioreactors, in vitro studies, animal studies and the clinical application of hybrid liver support systems. In vitro studies show long term external metabolic functions of primary isolated hepatocytes in bioreactors. These systems are capable of supporting essential liver functions. Animal experiments show the possibility of upscaling the bioreactors for clinical treatment. Since there is no reliable animal model for investigations on the treatment of acute liver failure, the promising results of these studies have limited relevance. The small number of clinical studies are not sufficient to give statements about a clinical improvement of therapy of acute liver failure. Although important progress has been made in the development of the systems, multiple different hepatocyte culture models and bioreactor constructions are discussed in the literature, indicating competition in this field of medical research.

Design of hepatocyte-specific extracellular matrices for hybrid artificial liver.

For the development of an artificial liver which can assist multiple metabolic functions of liver, the effective cultivation of hepatocytes on substrate as extracellular matrix analogs was examined. Adult rat hepatocytes attached on an asialoglycoprotein model polymer, poly-N-p-vinylbenzyl-D-lactonamide (PLVA), formed anchored multilayer aggregates which had a stable three-dimensional structure when epidermal growth factor (EGF) and insulin were added to the culture medium. The formation of multilayer aggregates was also dependent on the specific cellular attachment mediated by asialoglycoprotein receptor of hepatocyte surface. Cells in the aggregates expressed higher level of albumin secretion and lower proliferative ability than those in monolayer cultures on collagen. It seemed likely that the cells in multilayer aggregates experienced stable differentiated states resembling in vivo status through the forming multilayer aggregates. The culture system described here has useful potentiality for the development of hepatic module systems for a hybrid artificial liver.

Immunologic Considerations in the Use of Cultured Porcine Hepatocytes as a Hybrid Artificial Liver

Cultured porcine hepatocytes with reasonable metabolic functions are a promising bioreactor for a hybrid artificial liver in the treatment of liver failure. A cytotoxic human serum (t-serum) against cultured porcine hepatocytes was accidentally discovered during preliminary experiments, however, which prompted the authors to study the mechanism of the cytotoxicity and the frequency of occurrence of the cytotoxic sera. Among 103 individual sera examined, seven (6.8%) sera showed cytotoxicity to cultured porcine hepatocytes. T-serum heated at 56 degrees C for 30 min completely lost its cytotoxic activity, but the inactivated cytotoxicity was restored by the addition of rabbit complement to the inactivated t-serum. IgM-depleted t-serum produced by 2-mercaptoethanol treatment abolished the cytotoxicity to porcine hepatocytes. The cytotoxic reactions were therefore thought to be mediated by IgM capable of complement activation. Although IgM binding to porcine hepatocytes was also seen in nontoxic sera, the IgM did not activate complement. This implies differences in hepatocyte surface antigens recognized by IgM from t- and nontoxic sera. Before clinical application of a hybrid artificial liver using cultured porcine hepatocytes, detection of cytotoxic human IgM against porcine hepatocytes is necessary, and the means of eliminating the cytotoxic IgM from the hybrid artificial liver system, or of inactivating complement in the system, will be desirable in cases positive for cytotoxic IgM.

Isolation and culture of human hepatocytes from resected liver tissue as a bioreactor for a hybrid artificial liver.

To use cultured human hepatocytes as a hybrid artificial liver, effective methods for isolating and culturing the hepatocytes from resected surgical specimens were investigated. Two different procedures for isolating hepatocytes, perfusion and agitation with a collagenase solution (Method 1) and perfusion with a mixed solution of collagenase and dispase (Method 2), were examined. The yield of isolated hepatocytes obtained by Method 2 (13.31 x 10(6) cells/g of liver) was significantly higher than that by Method 1 (0.94 x 10(6)). The warm ischemia time (0-90 min) of the liver fragments obtained did not disturb the viability and yield of the isolated hepatocytes. The gluconeogenesis and urea synthesis of the cultured human hepatocytes were well preserved for 10 days. These results show that for prolonged human hepatocyte culture (10 days), isolation from resected human liver tissues by a combination of the proteolytic enzymes collagenase and dispase was effective and warm ischemia was tolerated for up to 90 min, which indicates the possibility of using cultured human hepatocytes as a hybrid artificial liver.

Immunologic Considerations in the Use of Cultured Porcine Hepatocytes as a Hybrid Artificial Liver

Immunologic Considerations in the Use of Cultured Porcine Hepatocytes as a Hybrid Artificial Liver

Present and Future of a Hybrid Artificial Liver Support System

Present and Future of a Hybrid Artificial Liver Support System

Tissue reconstruction in primary cultured rat hepatocytes on asialoglycoprotein model polymer.

Adult rat hepatocytes attached on an asialoglycoprotein model polymer, poly-N-p-vinylbenzyl-D-lactonamide (PVLA), formed anchored multilayer aggregates that had stable three-dimensional structure when epidermal growth factor (EGF) and insulin were added to the culture medium. The formation of multilayer aggregates depended on the concentrations of EGF and insulin added. Furthermore, the formation was synergistically accelerated by the presence of both hormones. Cells in the aggregates expressed a higher level of albumin secretion and lower proliferative ability than those in monolayer cultures on collagen. It seemed likely that the cells in multilayer aggregates experienced stable differentiated states resembling those in vivo through the formation of multilayer aggregates. The culture system described here has potential use for the study of the process of liver regeneration and the development of hepatic module systems such as a bioreactor and a hybrid artificial liver.

Hepatocyte culture systems for artificial liver support: implications for critical care medicine (bioartificial liver support).

The primary purpose of this review article is to familiarize critical care practitioners with newly developing techniques of hybrid artificial liver support. Implantable and extracorporeal hepatocyte culture systems are emphasized based on their current experimental and clinical status. Data used to prepare this document were obtained from the authors' personal files, as well as the computerized MEDLINE database. Medical headings used include: liver, artificial organs, cell culture, growth hormones, extracellular matrix, and transplantation. Only articles published in English have been cited. All studies are discussed in which hepatocyte culture systems have been used to support human patients with liver failure. All studies reported the patient's condition before therapy, duration of therapy, and outcome after therapy in order to be included in this review. Since the number of clinical trials is small at this time, animal studies were used to demonstrate application of other systems in the treatment of experimentally induced liver failure. Similar selection criteria were used to select animal studies for review. All initially identified human studies met these selection criteria. Independent extraction by multiple observers. Liver failure, resulting from infection, drugs, or as a part of the multiple organ failure syndrome, remains a major cause of morbidity, mortality, and resource allocation. Current therapy is limited to supportive care, along with liver transplantation. Because of these therapeutic limitations, hybrid artificial liver systems have been proposed for temporary and long-term hepatic support. Several animal studies and a small number of preliminary human studies indicate that hepatocyte culture systems are capable of supporting nearly all essential hepatic functions and may supply biologically active substances that promote regeneration and repair of the damaged liver being supported. Hybrid systems may be constructed from materials that serve as immunoprotective barriers against host defenses. During the past decade, important progress has been made with hybrid artificial liver support systems. Cell culture technology has progressed sufficiently so that an artificial liver, composed of metabolically active hepatocytes, may be a potential reality in the foreseeable future. Both implantable and extracorporeal artificial liver support systems have been developed to provide metabolic support during acute liver failure, or to serve as a bridge to solid organ transplantation. Implantable hepatocyte systems, however, require a prolonged period for intraperitoneal engraftment and vascularization, not typically available to patients with acute liver failure. For this reason, extracorporeal hybrid designs offer the greatest hope for on-line treatment of acute liver failure. Such systems are entering the final stages of animal testing.

Does a porcine hepatocyte hybrid artificial liver prolong the survival time of anhepatic rabbits?

To examine cultured porcine hepatocytes as a bioreactor of a hybrid artificial liver (HAL) in rabbits, a small version of a multiplated HAL was manufactured. In vitro and ex vivo studies were performed to evaluate the small HAL. The HAL consisted of 50 glass plates (10 x 5 x 0.04 cm each) on which porcine hepatocytes were cultured in a monolayer at confluent cell density. After 2 days of standard cultivation, the glass plates with hepatocytes were placed in the module and were used for studies. The module contains about 5 grams of hepatocytes (1/10 of 2 kg.bw rabbit liver). After undergoing perfusion culture for 5 days, the HAL showed satisfactory hepatic function. Glucose and urea were synthesized at the rate of 4.06 +/- 0.7 mg/module/hr and 0.62 +/- 0.09 mg/module/hr, respectively, and loaded ammonia was metabolized at the rate of 1.29 +/- 0.26 mg/module/hr. Ex vivo extracorporeal plasma perfusion studies revealed that the module with cultured porcine hepatocytes, which were heterogeneous to rabbits, showed a tendency to prolong the survival time of anhepatic rabbits. These results indicate that the HAL system, using multiplated porcine hepatocyte monolayers, is a promising artificial liver support system for clinical cases.

A packed-bed reactor utilizing porous resin enables high density culture of hepatocytes.

To enable high density culture of hepatocytes for use as a hybrid artificial liver support system or a bioreactor system, a packed-bed reactor using collagen-coated reticulated polyvinyl formal (PVF) resin was applied to a primary culture of hepatocytes. Cubic PVF resins (2 x 2 x 2 mm, mean pore size: 100, 250 or 500 microns) were used as supporting substrates to immobilize hepatocytes. Two hundred and fifty cubes were packed in a cylindrical column, and 2.6-11.3 x 10(7) hepatocytes were seeded in the column by irrigating with 3 ml of the medium containing hepatocytes. Perfusion culture experiments using this packed-bed reactor, as well as monolayer cultures using conventional collagen-coated petri dishes as control experiments, were performed. Sufficient amounts of hepatocytes were found to be immobilized in the reticulated structure of the PVF resins. The highest density of immobilized hepatocytes attained with PVF resin was 1.2 x 10(7) cells/cm3 PVF, which showed levels of ammonium removal and urea-N secretion comparable to those in the monolayer culture. It is concluded that the packed-bed reactor system utilizing PVF resin is a promising process for developing a bioreactor or a bioartificial organ using hepatocytes.

Characterization of Immobilized Hepatocytes as Liver Support

Hepatocytes isolated from rat liver were immobilized within Ca-alginate. Immobilized hepatocytes could remove ammonia and other toxic substances causing hepatic coma, such as indole, phenol, bilirubin, and short chain fatty acids. Although free hepatocytes lost those activities within 2 days, immobilized hepatocytes maintained those activities for more than 7 days. Immobilized hepatocytes induced tyrosine aminotransferase (TAT) in the presence of dexamethasone and dibutyryl-cAMP and retained the ability to induce TAT for more than 7 days. Biologically active form of coagulation factor II, prothrombin could be synthesized and secreted into medium by immobilized hepatocytes. Moreover, immobilized hepatocytes produced glucose from lactate, alanine, fructose, and galactose. Like adult rat liver, growth-related function and liver-specific function in immobilized hepatocytes were reciprocally controlled by cell density. There are both alpha-, and beta-adrenergic receptors in membrane of liver cells, and the adrenergic action of epinephrine is alpha-predominant in adult rat liver. Monolayer-cultured hepatocytes can not maintain alpha-adrenergic response. However, immobilized hepatocytes maintained alpha-adrenergic response as shown in vivo. Those characteristics of immobilized and three-dimensionally cultured hepatocytes are regarded almost the same as liver cells in vivo. Furthermore, therapeutic effect of immobilized hepatocytes on the hepatic failure were confirmed in the experiment using hepatocytes damaged with D-galactosamine. Therefore, it is suggested that immobilized hepatocytes could be applied to a hybrid artificial liver support.

Performance of a new hybrid artificial liver support system using hepatocytes entrapped within a hydrogel.

To develop a hybrid artificial liver support system (ALSS), the authors constructed a rotating-disk type ALSS using hepatocytes entrapped within a calcium alginate hydrogel. This module was designed in imitation of the gas-liquid contactor that uses the same principle. Forty disks with film-shaped hydrogel were mounted to a horizontal rotating axis and were kept in contact with blood. The concentration of hepatocytes in the gel varied from 1.9 X 10(9) to 1.7 X 10(10) cells/L gel. Entrapping hepatocytes within hydrogel kept the hepatocytes viable, and results of in vitro experiments showed ammonium metabolism and urea synthesis. The results of ex vivo perfusion experiments using cats with acute hepatic insufficiency indicated that this module had the ability to replace liver function in vivo.