Bioartificial Liver Assist Device Research Articles

Hepatocyte growth on polycapronolactone and 2-hydroxyethylmethacrylate nanofiber sheets enhanced by bone marrow-derived mesenchymal stromal cells.

The development of hepatocyte-based Bioartificial Liver Assist Devices, intended for the therapy of chronic and fulminant liver failure, is one of the important tasks in the area of tissue engineering. New advances in the development of semipermeable non-woven nanofiber biomaterials and the co-cultivation of bone marrow mesenchymal stromal cells (BMSC) and hepatocytes could be utilized in order to maintain hepatocyte cultures in these devices. We have compared rat hepatocyte growth on nanofiber biomaterials from different polymers, 2-hydroxyethylmethacrylate (HEMA) and ethoxyethylmethacrylate (EOEMA) copolymers, polyurethane (PUR), chitosan and polycapronolactone (PCL) spun from different solvent mixtures. In all cases the adhesion of hepatocytes to nanofibers was significantly better/stronger than to unstructured polymer surfaces; coating the nanofibers with collagen did not increase cell adhesion. We found the best hepatocyte adhesion on HEMA/EOEMA copolymer nanofibers and PCL nanofibers spun from a mixture of ethylacetate and dimethyl sulphoxide. Using a migration assay, we observed the migration of BMSC towards hepatocytes; hepatocytes cocultivated with BMSC excreted lower amounts of stress enzymes. The results demonstrate that nonwoven nanofiber layers, particularly those containing BMSC, are a suitable biocompatible support for functional hepatocyte cultures and that they can be used in a laboratory bioreactor or potentially in clinical setting.

Computational fluid modeling and performance analysis of a bidirectional rotating perfusion culture system

A myriad of bioreactor configurations have been investigated as extracorporeal medical support systems for temporary replacement of vital organ functions. In recent years, studies have demonstrated that the rotating bioreactors have the potential to be utilized as bioartificial liver assist devices (BLADs) owing to their advantage of ease of scalability of cell-culture volume. However, the fluid movement in the rotating chamber will expose the suspended cells to unwanted flow structures with abnormally high shear conditions that may result in poor cell stability and in turn lower the efficacy of the bioreactor system. In this study, we compared the hydrodynamic performance of our modified rotating bioreactor design with that of an existing rotating bioreactor design. Computational fluid dynamic analysis coupled with experimental results were employed in the optimization process for the development of the modified bioreactor design. Our simulation results showed that the modified bioreactor had lower fluid induced shear stresses and more uniform flow conditions within its rotating chamber than the conventional design. Experimental results revealed that the cells within the modified bioreactor also exhibited better cell-carrier attachment, higher metabolic activity, and cell viability compared to those in the conventional design. In conclusion, this study was able to provide important insights into the flow physics within the rotating bioreactors, and help enhanced the hydrodynamic performance of an existing rotating bioreactor for BLAD applications.

Electrospun Chitosan Nanofibers for Hepatocyte Culture

In this study, we developed a method to obtain high surface area nanofiber meshes composed of chitosan of a number of molecular weights. This method required decreasing the viscosity and surface tension of the chitosan solution as well as optimization of the electrospinning parameters such as applied voltage and environmental humidity. These chitosan nanofiber meshes were developed as a culture substrate for hepatocytes. The fibers exhibited a uniform diameter distribution (average diameter: 112 nm) and FTIR results indicate that the chemical structure of chitosan is stable during the electrospinning process. The attachment, morphology and activity of hepatocytes cultured on the chitosan nanofiber meshes were tested. The results showed that the chitosan nanofibers are biocompatible with hepatocytes and that these chitosan nanofiber meshes could be useful tissue culture substrates for various applications, including bioartificial liver-assist devices and tissue engineering for liver regeneration.

Hemoglobin Regulates the Metabolic, Synthetic, Detoxification, and Biotransformation Functions of Hepatoma Cells Cultured in a Hollow Fiber Bioreactor

Hepatic hollow fiber (HF) bioreactors constitute one type of extracorporeal bioartificial liver assist device (BLAD). Ideally, cultured hepatocytes in a BLAD should closely mimic the in vivo oxygenation environment of the liver sinusoid to yield a device with optimal performance. However, most BLADs, including hepatic HF bioreactors, suffer from O2 limited transport toward cultured hepatocytes, which reduces their performance. We hypothesize that supplementation of hemoglobin-based O2 carriers into the circulating cell culture medium of hepatic HF bioreactors is a feasible and effective strategy to improve bioreactor oxygenation and performance. We examined the effect of bovine hemoglobin (BvHb) supplementation (15g/L) in the circulating cell culture medium of hepatic HF bioreactors on hepatocyte proliferation, metabolism, and varied liver functions, including biosynthesis, detoxification, and biotransformation. It was observed that BvHb supplementation supported the maintenance of a higher cell mass in the extracapillary space, improved hepatocyte metabolic efficiency (i.e., hepatocytes consumed much less glucose), improved hepatocyte capacity for drug metabolism, and conserved both albumin synthesis and ammonia detoxification functions compared to controls (no BvHb supplementation) under the same experimental conditions.

Fluidized-bed bioartificial liver assist devices (BLADs) based on microencapsulated primary porcine hepatocytes have risk of porcine endogenous retroviruses transmission.

Bioartificial liver assist devices (BLADs) are expected to bridge liver failure patients to liver transplantation, but porcine endogenous retroviruses (PERVs) still pose a potential risk in pig-to-human xenotransplantation and thereby limit the use of bioartificial liver therapy. In our lab, fluidized-bed BLADs based on microencapsulated primary porcine hepatocytes have been successfully used to treat liver failure pigs. We detected the risk of PERVs transmission of microencapsulated primary porcine hepatocytes-the key component of fluidized-bed BLADs, to evaluate the biosafety of this device for further clinical applications. Microencapsulated primary porcine hepatocytes (cell diameter=300μm) were cultured in Dulbecco's modified Eagles medium (DMEM). Microencapsulated cell culture supernatants were collected at 6, 12, 24 and 72h. HEK-293 were cocultured with these supernatants, and the cocultured cells were harvested every 7days. RT-PCR was used to detect PERVs transmission. RT-qPCR was used to get the number of virus copies. PK-15 was used as the positive control whereas HepG2 was used as the negative control. PERV was detected in all supernatants, and the viral load of the supernatants increased with time. Moreover, cocultured 293cells were positive for PERV-specific sequences. The kind of fluidized-bed BLADs based on microencapsulated primary porcine hepatocytes have risk of PERVs transmission. Further extensive pre-clinical study focused on biosafety is warranted.

Mixtures of hemoglobin‐based oxygen carriers and perfluorocarbons exhibit a synergistic effect in oxygenating hepatic hollow fiber bioreactors

Hepatic hollow fiber (HF) bioreactors are being developed for use as bioartificial liver assist devices (BLADs). In general, BLADs suffer from O(2) limited transport, which reduces their performance. This modeling study seeks to investigate if O(2) carrying solutions consisting of mixtures of hemoglobin-based oxygen carriers (HBOCs) and perfluorocarbons (PFCs) can enhance O(2) transport to hepatocytes cultured in the extra capillary space (ECS) of HF bioreactors. We simulated supplementing the circulating cell culture media stream of the HF bioreactor with a mixture containing these two types of oxygen carriers (HBOCs and PFCs). A mathematical model was developed based on the dimensions and physical characteristics of a commercial HF bioreactor. The resulting set of partial differential equations, which describes fluid transport; as well as, mass transport of dissolved O(2) in the pseudo-homogeneous PFC/water phase and oxygenated HBOC, was solved to yield the O(2) concentration field in the three HF domains (lumen, membrane and ECS). Our results show that mixtures of HBOC and PFC display a synergistic effect in oxygenating the ECS. Therefore, the presence of both HBOC and PFC in the circulating cell culture media dramatically improves transport of O(2) to cultured hepatocytes. Moreover, the in vivo O(2) spectrum in a liver sinusoid can be recapitulated by supplementing the HF bioreactor with a mixture of HBOCs and PFCs at an inlet pO(2) of 80 mmHg. Therefore, we expect that PFC-based oxygen carriers will be more efficient at transporting O(2) at higher O(2) levels (e.g., at an inlet pO(2) of 760 mmHg, which corresponds to pure O(2) in equilibrium with aqueous cell culture media at 1 atm).

Perfluorocarbon facilitated O2 transport in a hepatic hollow fiber bioreactor

A mathematical model describing O(2) transport in a hepatic hollow fiber (HF) bioreactor supplemented with perfluorocarbons (PFCs) in the circulating cell culture media was developed to explore the potential of PFCs in properly oxygenating a bioartificial liver assist device (BLAD). The 2-dimensional model is based on the geometry of a commercial HF bioreactor operated under steady-state conditions. The O(2) transport model considers fluid motion of a homogeneous mixture of cell culture media and PFCs, and mass transport of dissolved O(2) in a single HF. Each HF consists of three distinct regions: (1) the lumen (conducts the homogeneous mixture of cell culture media and PFCs), (2) the membrane (physically separates the lumen from the extracapillary space (ECS), and (3) the ECS (hepatic cells reside in this compartment). In a single HF, dissolved O(2) is predominantly transported in the lumen via convection in the axial direction and via diffusion in the radial direction through the membrane and ECS. The resulting transport equations are solved using the finite element method. The calculated O(2) transfer flux showed that supplementation of the cell culture media with PFCs can significantly enhance O(2) transport to the ECS of the HF when compared with a control with no PFC supplementation. Moreover, the O(2) distribution and subsequent analysis of ECS zonation demonstrate that limited in vivo-like O(2) gradients can be recapitulated with proper selection of the operational settings of the HF bioreactor. Taken together, this model can also be used to optimize the operating conditions for future BLAD development that aim to fully recapitulate the liver's varied functions.

In Vitro Large-Scale Cultivation and Evaluation of Microencapsulated Immortalized Human Hepatocytes (HepLL) in Roller Bottles

Microencapsulated hepatocytes have been proposed as promising bioactive agents for packed-bed or fluidized-bed bioartificial liver assist devices (BLaDs) and for hepatocyte transplantation because of the potential advantages they offer of high mass transport rate and an optimal microenvironment for hepatocyte culture. We developed a large-scale and high-production alginate-chitosan (AC) microcapsule roller bottle culture system for the encapsulation of hepLL immortalized human hepatocytes. In this study, the efficacy of upscaling encapsulated hepLL cells production with roller bottle cultivation was evaluated in vitro. Microencapsulated hepLL cells were grown at high yield in large-scale roller bottles, with free cells cultured in roller bottle spinners serving as controls. The mechanical stability and the permeability of the AC microcapsules were investigated, and the growth, metabolism and functions of the encapsulated hepLL cells were evaluated as compared to free cells. The microcapsules withstood well the shear stress induced by high agitation rates. The microcapsules were permeable to albumin, but prevented the release of immunoglobulins. Culture in roller bottles of immortalized human hepatocytes immobilized in the AC microcapsules improved cell growth, albumin synthesis, ammonia elimination and lidocaine clearance as compared with free cells cultured in roller bottles. Encapsulated hepLL cells may be cultured on a large scale in roller bottles. This makes them possible candidates for use in cell-based liver assist therapies.

The effect of nanofibrous galactosylated chitosan scaffolds on the formation of rat primary hepatocyte aggregates and the maintenance of liver function

Liver tissue engineering requires a perfect extracellular matrix (ECM) for primary hepatocytes culture to maintain high level of liver-specific functions and desirable mechanical stability. The aim of this study was to develop a novel natural nanofibrous scaffold with surface-galactose ligands to enhance the bioactivity and mechanical stability of primary hepatocytes in culture. The nanofibrous scaffold was fabricated by electrospinning a natural material, galactosylated chitosan (GC), into nanofibers with an average diameter of ∼160 nm. The GC nanofibrous scaffolds displayed slow degradation and suitable mechanical properties as an ECM for hepatocytes according to the evaluation of disintegration and Young's modulus testing. The results of morphology characterization, double-staining fluorescence assay and function detection showed that hepatocytes cultured on GC nanofibrous scaffold formed stably immobilized 3D flat aggregates and exhibited superior cell bioactivity with higher levels of liver-specific function maintenance in terms of albumin secretion, urea synthesis and cytochrome P-450 enzyme than 3D spheroid aggregates formed on GC films. These spheroid aggregates could be detached easily during culture period from the flat GC films. We suggest such GC-based nanofibrous scaffolds could be useful for various applications such as bioartificial liver-assist devices and tissue engineering for liver regeneration as primary hepatocytes culture substrates.

Convection and Hemoglobin-Based Oxygen Carrier Enhanced Oxygen Transport in a Hepatic Hollow Fiber Bioreactor

Hepatic hollow fiber bioreactors are a promising class of bioartificial liver assist device (BLAD). The development of this type of device is currently hindered by limited oxygen transport to cultured hepatocytes, due to low solubility of oxygen in aqueous media. In order to increase the oxygen spectrum to cultured hepatocytes housed within a hollow fiber bioreactor, several different engineering strategies were explored in this study. These included: supplementing the circulating media stream of the hollow fiber bioreactor with a hemoglobin-based oxygen carrier (bovine red blood cells) with defined oxygen binding and release kinetics and operating the bioreactor with media flow through the hollow fiber membrane into the extracapillary space (ECS). We hypothesize that these two strategies can be used to improve hepatocyte oxygenation and possibly attain an in vivo-like pO(2) spectrum, similar to that observed in vivo in the liver sinusoid. This work is significant, since provision of an in vivo-like pO(2) spectrum should create a fully functional BLAD that could potentially bridge thousands of liver failure patients towards native liver regeneration of damaged tissue or, if necessary, orthotopic liver transplantation.

Acute liver failure: liver support therapies

We summarize the therapeutic approach to patients with acute liver failure with the main focus on bioartificial and artificial liver support. We also describe specific and general therapeutic approaches based upon recent advances in the understanding of the pathophysiology of acute liver failure. Bioartificial liver support systems use hepatocytes in an extracorporeal device connected to the patient's circulation. Artificial liver support is intended to remove protein-bound toxins and water-soluble toxins without providing synthetic function. Both systems improve clinical and biochemical parameters and can be applied safely to patients. Although bioartificial liver-assist devices have not been shown to improve the survival of patients with acute liver failure, further development is underway. Artificial liver support systems have been shown to alter several pathophysiological mechanisms involved in the development of acute liver failure but survival data are still limited. Mortality in patients with acute liver failure is still unacceptably high. The most effective treatment, liver transplantation, is a limited resource and so other therapeutic options to bridge patients to recovery or stabilization have to be considered. Better understanding of the pathophysiology of acute liver failure and device development is necessary to achieve the elusive goal of effective extracorporeal liver assist.

Enhanced Oxygen Delivery to Primary Hepatocytes within a Hollow Fiber Bioreactor Facilitated via Hemoglobin-Based Oxygen Carriers

The production of a fully functional bioartificial liver assist device (BLAD) would greatly enhance available treatment options for patients suffering from acute liver failure. Currently, inadequate oxygen provision to hepatocytes seeded within hollow fiber bioreactors hampers development of a viable hollow fiber-based BLAD. Experimentally, oxygen provision to primary rat hepatocytes cultured within hollow fiber bioreactors was measured, it was observed that supplementation with an oxygen carrier (bovine red blood cells at approximately 2% human hematocrit) did not significantly improve oxygenation compared to the absence of an oxygen carrier. Therefore, an oxygen transport model of an individual hollow fiber within the bioreactor was developed and simulated (up to approximately 10% human hematocrit) to more fully examine the effect of oxygen carrier supplementation on oxygenation within the bioreactor. The modeling analysis, supported via the experimental results, was utilized to predict optimal bioreactor operating conditions for the delivery of in vivo-like oxygen gradients to cultured hepatocytes in clinically relevant settings.

Targeted Oxygen Delivery within Hepatic Hollow Fiber Bioreactors via Supplementation of Hemoglobin‐Based Oxygen Carriers

Hepatic hollow fiber bioreactors are considered a promising class of bioartificial liver assist device (BLAD). Unfortunately, limited oxygen (O(2)) transport to hepatocytes within this device hinders further development. Hepatocytes in vivo (in the liver sinusoid) experience a wide range of oxygen tensions (pO(2) = 25-70 mmHg), which is important for development of proper differentiated function (zonation). Previously, we observed that bovine red blood cell (bRBC) supplementation of the circulating media stream enhanced oxygenation of cultured C3A hepatoma cells compared to a culture with no O(2) carrier (Gordon, J.; Palmer, A. F. Artif. Cells, BloodSubstitutes, Biotechnol. 2006, 33 (3), 297-306). Despite this success, the cells were not exposed to the desired in vivo O(2) spectrum (Sullivan, J.; Gordon, J.; Palmer, A. Biotechnol. Bioeng. 2006, 93 (2) 306-317). We hypothesize that altering the kinetics of O(2) binding/release to/from hemoglobin-based O(2) carriers (HBOCs) could potentially target O(2) delivery to cell cultures. High P(50) (low O(2) affinity) HBOCs preferentially targeted O(2) delivery at high inlet pO(2) values. Conversely, low P(50) (high O(2) affinity) HBOCs targeted O(2) delivery at low inlet pO(2) values. Additionally, inlet pO(2), flow rate, and HBOC concentration were varied to find optimal bioreactor operating conditions. Our results demonstrate that HBOCs can enhance O(2) delivery to cultured hepatocytes, while exposing them to in vivo-like O(2) tensions, which is critical to create a fully functional BLAD.

Simulation of oxygen carrier mediated oxygen transport to C3A hepatoma cells housed within a hollow fiber bioreactor

A priori knowledge of the dissolved oxygen (O2) concentration profile within a hepatic hollow fiber (HF) bioreactor is important in developing an effective bioartificial liver assist device (BLAD). O2 provision is limiting within HF bioreactors and we hypothesize that supplementing a hepatic HF bioreactor's circulating media with bovine red blood cells (bRBCs), which function as an O2 carrier, will improve oxygenation. The dissolved O2 concentration profile within a single HF (lumen, membrane, and representative extra capillary space (ECS)) was modeled with the finite element method, and compared to experimentally measured data obtained on an actual HF bioreactor with the same dimensions housing C3A hepatoma cells. Our results (experimental and modeling) indicate bRBC supplementation of the circulating media leads to an increase in O2 consumed by C3A cells. Under certain experimental conditions (pO2,IN) = 95 mmHg, Q = 8.30 mL/min), the addition of bRBCs at 5% of the average in vivo human red blood cell concentration (% hRBC) results in approximately 50% increase in the O2 consumption rate (OCR). By simply adjusting the operating conditions (pO2,IN) = 25 mmHg, Q = 1.77 mL/min) and increasing bRBC concentration to 25% hRBC the OCR increase is approximately 10-fold. However, the improved O2 concentration profile experienced by the C3A cells could not duplicate the full range of in vivo O2 tensions (25-70 mmHg) typically experienced within the liver sinusoid with this particular HF bioreactor. Nonetheless, we demonstrate that the O2 transport model accurately predicts O2 consumption within a HF bioreactor, thus setting up the modeling framework for improving the design of future hepatic HF bioreactors.

Molecular Adsorbent Recirculating System (MARS)

Introduction: Molecular adsorbent recirculating system (MARS) for albumin liver dialysis has been used as a bridge to liver transplantation in patients with fulminant hepatic failure (FHF). This review examines the available data on its clinical use, its technical aspects and present gaps in knowledge. Methods: Peer-reviewed journals and monographs on the subject were covered. Results: FHF is associated with elevation in various substances including bilirubin, ammonia, lactate, free fatty acids and aromatic amino acids. Some of these toxic metabolites, such as ammonia and bilirubin, are believed to be central to the clinical manifestations of hepatic encephalopathy and acute renal failure. MARS ameliorates both biochemical and clinical manifestations of FHF by removing both water-soluble and protein-bound toxins. Among the benefits of MARS is the attenuation of severe cerebral oedema and raised intracranial pressure found in FHF, possibly through reduction in high concentrations of these toxins. Although MARS has been shown to be useful in FHF, its clinical efficacy in subfulminant hepatic failure and less severe forms of acute liver failure (ALF) remains uncertain. The current literature also suggests that it may be beneficial to treat cases of acute-on-chronic liver failure (AoCLF). Deranged systemic chemistries can be similarly ameliorated, but the impact of MARS on the natural history of AoCLF remains uncertain. The difficulty lies in being able to accurately quantify residual liver function and variability in the course of acute intercurrent events. The broader question is whether MARS can favourably change the natural history of ALF and FHF. For this, large multi-centre, randomised controlled trials are needed. Furthermore, it is also uncertain how hepatic excretory-assist devices, such as MARS, compare with bio-artificial liver-assist devices which have both synthetic and excretory hepatic functions in ALF treatment in intensive care unit patients. Nevertheless, MARS has proven to be a valuable homeostatic tool that may be useful in restoring the biochemical and clinical status quo in much the same way that continuous veno-venous haemofiltration and mechanical ventilation provide temporary artificial organ support while these organs are in distress. This is the evolving concept of multi-organ support therapy. Other major unresolved issues with MARS include the timing of initiation of albumin liver dialysis, the clinical and/or biochemical parameters to base this decision on, the intensity of MARS therapy (continuous versus intermittent) and the saturation capacity of the system for different metabolites in intermittent MARS. Conclusions: MARS is an effective and, thus far, safe homeostatic tool in treating FHF. More studies are needed to delineate its role as a homeostatic tool in less severe forms of ALF, including that which occurs in multi-organ failure and in AoCLF. Other studies need to focus on the optimal timing of initiation of and intensity of MARS albumin liver dialysis. The larger issue is to compare MARS with bio-assist liver devices in treating the whole spectrum of ALF.

Microcapsules with improved mechanical stability for hepatocyte culture

Packed-bed or fluidized-bed bioreactor filled with microencapsulated hepatocytes has been proposed as one of the promising designs for bioartificial liver assist device (BLAD) because of potential advantages of high mass transport rate and optimal microenvironment for hepatocyte culture. Recently, we have developed a microcapsule system for the encapsulation of hepatocytes. The microcapsules consist of an inner core of modified collagen and an outer shell of terpolymer of methyl methacrylate, methacrylate and hydroxyethyl methacrylate. Cells encapsulated in these microcapsules exhibit enhanced cellular functions. Improving the mechanical stability of the microcapsules to withstand the shear stress induced by high perfusion rate would be crucial to the success of BLAD applications. In this study, we investigated the effects of terpolymer molecular weight ( M w) on the mechanical property of these microcapsules and the differentiated functions of encapsulated hepatocytes. Six terpolymers with different M w were synthesized using radical polymerization in solution by adjusting the reaction temperature and the initiator concentration. All the terpolymers formed microcapsules with the methylated collagen. While the terpolymer M w had little effect on the capsule membrane thickness and permeability of serum albumin, the mechanical property of the microcapsules was significantly improved by the higher M w of the terpolymer. Differentiated functions of the hepatocytes cultured in the microcapsules, including urea synthesis, albumin synthesis and cytochrome P450 metabolic activity, were not significantly affected by the terpolymer M w.

Advances in bioartificial liver assist devices.

Rapid advances in development of bioartificial liver assist devices (BLADs) are exciting clinical interest in the application of BLAD technology for support of patients with acute liver failure. Four devices (Circe Biomedical HepatAssist, Vitagen ELAD, Gerlach BELS, and Excorp Medical BLSS) that rely on hepatocytes cultured in hollow-fiber membrane technology are currently in various stages of clinical evaluation. Several alternative approaches for culture and perfusion of hepatocytes have been evaluated in preclinical, large animal models of liver failure, or at a laboratory scale. Engineering design issues with respect to xenotransplantation, BLAD perfusion, hepatocyte functionality and culture maintenance, and ultimate distribution of a BLAD to a clinical site are delineated.

New approaches to supporting the failing liver.

With the continued, growing disparity between the numbers of organ donations and patients waiting for liver transplantation, various efforts have been made to optimize the allocation of organs, as well as to devise means to support the failing liver. Over the years, the development of bioartificial liver-assist devices has aimed at replacing the three main functions of hepatocytes, which are synthetic, metabolic, and excretory. The application of porcine hepatocytes in humans to carry out biotransformation, as well as other metabolic functions and refinement of the membrane separator, have yielded some promising results in supporting patients with acute liver failure. Further advances will need to be made before these bioartificial devices can be considered for routine application in clinical settings.