Microfabricated grooved substrates as platforms for bioartificial liver reactors

An extracorporeal bioartificial liver device has the potential to provide temporary hepatic support for patients with liver failure. Our goal was to optimize the flow environment for the cultured hepatocytes in a flat-plate bioreactor, specifically focusing on oxygen delivery using high medium flow rates while reducing the detrimental effects of the resulting shear stresses. We used photolithographic techniques to fabricate microgrooves onto the underlying glass substrate. The microgrooves, perpendicular to the axial flow direction, protected the hepatocytes from the shear stress induced by the flowing medium. Using finite element analysis, we found that the velocity gradient change near the cell surface (i.e., bottom of the grooves) was smaller than that near the top surface of the flow channel, indicating that the grooves would provide protection to the attached cells from the mechanical effects of the flowing medium. We also determined that the shear stress at the cell surface could be reduced by as much as 30 times (channel height of 100 microm) in the grooved-substrate (0.5 dyn/cm(2)) bioreactor compared to the flat-substrate (15 dyn/cm(2)) bioreactor for a medium flow rate of 4.0 mL/min. Albumin and urea synthesis rates of hepatocytes cocultured with 3T3-J2 fibroblasts remained stable over 5 days of perfusion in the grooved-substrate bioreactor, whereas in the flat-substrate bioreactor they decreased over the same time period. These studies indicate that under "high" flow conditions the microgrooved-substrate in the bioreactor can decrease the detrimental effects of shear stress on the hepatocytes while providing adequate oxygenation, thereby resulting in stable liver-specific function.     

Method for evaluating metabolic functions of drugs in bioartificial liver

Lidocaine and galactose loading tests were performed on a bioartificial liver (BAL), an extracorporeal medical device incorporating living hepatocytes in a cartridge without a transport barrier across the membranes. The concentration changes were analyzed using pharmacokinetic equations to evaluate the efficacy and limitation of the proposed method. Lidocaine and galactose were found to be suitable drugs for a quantitative evaluation of the BAL functions, as they did not interact with the plasma proteins or blood vessels, making their concentrations easy to determine. The drug concentration changes after drug loading were easily analyzed using pharmacokinetic equations, and the BAL functions quantitatively expressed by pharmacokinetic parameters, such as the clearance (CL) and galactose elimination capacity (GEC). In addition, these two drugs have already been used in clinical tests to evaluate human liver functions over long periods, and lidocaineCL values andGEC values reported for a normal human liver. Thus, a comparison of theCL andGEC values for theBAL and a natural liver revealed what proportion of normal liver functions could be replaced by the BAL.     

Characterization of the three-compartment gel-entrapment porcine hepatocyte bioartificial liver

A hybrid bioartificial liver device supporting a large mass of cells expressing differentiated hepatocyte metabolic capabilities is necessary for the successful treatment of fulminant hepatic failure. The three-compartment gel-entrapment porcine hepatocyte bioartificial liver was designed to provide "bridge" support to transplantation or until native liver recovery is achieved for patients with acute liver failure. The device is an automated mammalian cell culture system supporting 6-7 × 109 porcine hepatocytes entrapped in a collagen matrix and inoculated into the capillary lumen spaces of two 100 kDa molecular mass cut-off hollow fiber bioreactors. Gel contraction recreates a small lumen space within the hollow fiber which allows for the delivery of a nutrient medium. This configuration supported hepatocyte viability and differentiated phenotype as measured by albumin synthesis, ureagenesis, oxygen consumption, and vital dye staining during both cell culture and ex vivo application. The hollow fiber membrane was also shown to isolate the cells from xenogenic immunoglobulin attack. The gel-entrapment bioartificial liver maintained a large mass of functional hepatocytes by providing a three-dimensional cell culture matrix, by delivering basal nutrients through lumen media perfusion, and by preventing rejection of the xenocytes. These features make this device a favorable candidate for the treatment of clinical fulminant hepatic failure.     

Cell-based therapy of acute liver failure: The extracorporeal bioartificial liver

The need for an alternative ttreatment to orthotopic liver transplantation for acute liver failure is a major issue, and systems capable of temporalily providing liver functions are being actively tested. Liver assist devices based on detoxication by dialysis or hemoperfusion through various membranes or cartridges proved to be inefficient because of their lack of metabolic function. An extracorporeal hybrid bioartificial liver might be an appropriate treatment, since it can provide liver-specific functions, maintain the patient alive, and allow spontaneous recovery of the patient's own liver or act as a bridge toward liver transplantation. Many devices have been proposed, including flat culture substrates, hollow-fiber bioreactors, or microcarriers, using xenogenic hepatocytes or hepatoma cell lines. Various drawbacks of these devices led us to attempt to develop a reliable extracorporeal bioartificial liver based on alginate bead-entrapped hepatocytes. This system was used successfully for the correction of the Gunn rat genetic defect, which results in lack of bilirubin conjugation. The development of this system for clinical purposes requires large yields of functional hepatocytes. We have isolated normal porcine hepatocytes by collagenase perfusion of the liver. Cells were immobilized in membrane-coated alginate gel beads, which were subsequently inoculated into a bioreactor. Porcine hepatocytes expreessed liver-specific functions at high levels, particularly protein neosysnthesis and enzymatic activities involved in detoxication and biotransformation processes. In addition, hepatocytes entrapped in coated alginate beads were isolated from immunoglobulins. This system represents a promising tool for the design of anoartificial liver in human beings.Abbreviations ALF acute liver failure - EBAL extracorporeal bioartificial liver - OLT orthotopic liver transplantation     

Improving the next generation of bioartificial liver devices

Several extracorporeal bioartificial liver (BAL) devices are currently being evaluated as an alternative or adjunct therapy for liver disease. While these hybrid systems show promise, in order to become a clinical reality, BAL devices must clearly demonstrate efficacy in improving patient outcomes. Here, we present aspects of BAL devices that could benefit from fundamental advances in cell and developmental biology. In particular, we examine the development of human hepatocyte cell lines, strategies to stabilize the hepatocyte phenotype in vitro, and emphasize the importance of the cellular microenvironment in bioreactor design. Consideration of these key components of BAL systems will greatly improve next generation devices.     

Improved survival of porcine acute liver failure by a bioartificial liver device implanted with induced human functional hepatocytes

Acute liver failure (ALF) is a life-threatening illness. The extracorporeal cell-based bioartificial liver (BAL) system could bridge liver transplantation and facilitate liver regeneration for ALF patients by providing metabolic detoxification and synthetic functions. Previous BAL systems, based on hepatoma cells and non-human hepatocytes, achieved limited clinical advances, largely due to poor hepatic functions, cumbersome preparation or safety concerns of these cells. We previously generated human functional hepatocytes by lineage conversion (hiHeps). Here, by improving functional maturity of hiHeps and producing hiHeps at clinical scales (3 billion cells), we developed a hiHep-based BAL system (hiHep-BAL). In a porcine ALF model, hiHep-BAL treatment restored liver functions, corrected blood levels of ammonia and bilirubin, and prolonged survival. Importantly, human albumin and α-1-antitrypsin were detectable in hiHep-BAL-treated ALF pigs. Moreover, hiHep-BAL treatment led to attenuated liver damage, resolved inflammation and enhanced liver regeneration. Our findings indicate a promising clinical application of the hiHep-BAL system.     

Reversal of liver failure using a bioartificial liver device implanted with clinical-grade human-induced hepatocytes

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A theoretical method to improve and optimize the design of bioartificial livers

Bioartificial livers (BALs) are a potentially effective countermeasure against liver failure, particularly in cases of acute or fulminant liver failure. It is hoped these devices can sustain a patient's liver function until recovery or transplant. However, no large‐scale clinical trial has yet proven that BALs are particularly effective and evidently design issues remain to be addressed. One aspect of BAL design that must be considered is the mass transfer of adequate oxygen to the hepatocytes within the device. We present here a mathematical modeling approach to oxygen mass transport in a BAL. A mathematical model based upon Krogh cylinders is outlined to describe a diffusion‐limited hollow fiber bioreactor. In addition, operating constraints are defined on the system—cells should not experience hypoxia and the cell population should be of adequate size. By combining modeling results with these operating constraints and presenting the results graphically, “operating region” charts can be constructed for the hollow fiber BAL (HF‐BAL). The effects of varying various operating parameters on the BAL are then established. It is found that smaller radii and short, thin walled fibers are generally advantageous while cell populations in excess of 10 billion could be supported in the BAL with a plasma flow rate of 200 mL/min. For fibers of intermediate length and lumen radius, the minimum number of fibers required to produce a viable design ranges approximately from 7,000–10,000. In theory, this may be enough to support patients with failing livers. Biotechnol. Bioeng. 2010;106: 980–988. © 2010 Wiley Periodicals, Inc.     

Preclinical characterization of alginate‐poly‐L‐lysine encapsulated HepaRG for extracorporeal liver supply

We recently demonstrated that HepaRG cells encapsulated into 1.5% alginate beads are capable of self‐assembling into spheroids. They adequately differentiate into hepatocyte‐like cells, with hepatic features observed at Day 14 post‐encapsulation required for external bioartificial liver applications. Preliminary investigations performed within a bioreactor under shear stress conditions and using a culture medium mimicking acute liver failure (ALF) highlighted the need to reinforce beads with a polymer coating. We demonstrated in a first step that a poly‐l ‐lysine coating improved the mechanical stability, without altering the metabolic activities necessary for bioartificial liver applications (such as ammonia and lactate elimination). In a second step, we tested the optimized biomass in a newly designed perfused dynamic bioreactor, in the presence of the medium model for pathological plasma for 6 h. Performances of the biomass were enhanced as compared to the steady configuration, demonstrating its efficacy in decreasing the typical toxins of ALF. This type of bioreactor is easy to scale up as it relies on the number of micro‐encapsulated cells, and could provide an adequate hepatic biomass for liver supply. Its design allows it to be integrated into a hybrid artificial/bioartificial liver setup for further clinical studies regarding its impact on ALF animal models.     

Primary culture of rat hepatocytes entrapped in cylindrical collagen gels: An in vitro system with application to the bioartificial liver

A static culture model employing cylindrical collagen-hepatocyte gels is reported for large scale testing of conditions relevant to the three compartment hollow fiber bioartificial liver. High density hepatocyte cultivation was achieved by cell entrapment within the collagen-hepatocyte gel. Hepatocyte viability was assessed by vital staining, gel contraction, and insulin utilization. Measures of hepatocyte-specific function included albumin synthesis, ureagenesis, lidocaine biotransformation, and cholate conjugation. Although hepatocyte viability remained stable through the seven day incubation period, hepatocyte functions were not uniformly preserved. Albumin synthesis remained stable, while representative P-450 and conjugation activities decreased with time. This static culture system will facilitate the development of a hollow fiber bioartificial liver which utilizes cylindrical collagen-hepatocyte gels.Abbreviations FDA fluorescein diacetate - EB ethidium bromide - MEGX monoethylglycinexylidide     

Effect of low temperature preservation and cell density on metabolic function in a bioartificial liver

Difficulties associated with bioartificial liver (BAL) preservation limit not only the commercialization of BAL, but also its clinical trials. In this study, the possibility of cold preservation of BAL cartridges containing porcine hepatocytes was examined at 4 °C. In anin vitro perfusion culture system, BAL cartridges maintained cytochrome P450 metabolic function for at least 50 days. However, all BAL cartridges completely lost their ammonia eliminating ability when stored at 4 °C. We also studied the effect of cell density on the maintenance of BAL liver function in a highly differentiated and healthy state. As expected, BALs containing a larger number of hepatocytes demonstrated higher metabolic functions. When metabolic functions were compared per gram of hepatocytes, no large differences were observed between devices containing different densities of hepatocytes. Decreased cell density did not successfully prolong BAL function. The viability and function of isolated hepatocytes highly depend on the culture conditions, such as cell density, substrata, culture media, and additives to the culture media. Perfusion culture of BAL cartridges at 4°C gave a promosing result with respect to the maintenance of P450 activity. However, as indicated by the rapid loss of ammonia metabolic activity, many factors still remain to be optimized for preservation of BAL keeping high metabolic functions for a longer time.     

Radial flow hepatocyte bioreactor using stacked microfabricated grooved substrates

Bioartificial liver (BAL) devices with fully functioning hepatocytes have the potential to provide temporary hepatic support for patients with liver failure. The goal of this study was to optimize the flow environment for the cultured hepatocytes in a stacked substrate, radial flow bioreactor. Photolithographic techniques were used to microfabricate concentric grooves onto the underlying glass substrates. The microgrooves served to protect the seeded hepatocytes from the high shear stresses caused by the volumetric flow rates necessary for adequate convective oxygen delivery. Finite element analysis was used to analyze the shear stresses and oxygen concentrations in the bioreactor. By employing high volumetric flow rates, sufficient oxygen supply to the hepatocytes was possible without an integrated oxygen permeable membrane. To implement this concept, 18 microgrooved glass substrates, seeded with rat hepatocytes cocultured with 3T3-J2 fibroblasts, were stacked in the bioreactor, creating a channel height of 100 microm between each substrate. In this bioreactor configuration, liver-specific functions (i.e., albumin and urea synthesis rates) of the hepatocytes remained stable over 5 days of perfusion, and were significantly increased compared to those in the radial flow bioreactor with stacked substrates without microgrooves. This study suggests that this radial flow bioreactor with stacked microgrooved substrates is scalable and may have potential as a BAL device in the treatment of liver failure.     

Effects of oxygenation and flow on the viability and function of rat hepatocytes cocultured in a microchannel flat-plate bioreactor

The goal of this study was to investigate the viability and synthetic function of rat hepatocytes cocultured with 3T3-J2 fibroblasts in a small-scale microchannel flat-plate bioreactor with and without an internal membrane oxygenator under flow. Bioreactor channel heights ranged between 85 and 500 microm and medium flow rates ranged between 0.06 and 4.18 mL/min. The results showed that the bioreactor without the oxygenator resulted in significantly decreased viability and function of hepatocytes, whereas hepatocytes in the bioreactor with internal membrane oxygenator were able to maintain their viability and function. The shear stress calculations showed that, at lower wall shear stresses (0.01 to 0.33 dyn/cm(2)), hepatocyte functions, measured as albumin and urea synthesis rates, were as much as 2.6- and 1.9-fold greater, respectively, than those at higher wall shear stresses (5 to 21 dyn/cm(2)). Stable albumin and urea synthesis rates for 10 days of perfusion were also demonstrated in the bioreactor with internal membrane oxygenator. These results are relevant in the design of hepatocyte bioreactors and the eventual scaling-up to clinical devices.     

Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: A potential bioartificial liver

Summary Conventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell contact, and are continuously perfused with blood. We report here a novel bioreactor system for the culturing of primary hepatocytes with cuboidal cell shape, extensive cell-cell contact, and perfusing medium. The hepatocytes were inoculated into the bioreactor and allowed to recirculate at a rate optimal for them to collide and form aggregates. These newly-formed aggregates were subsequently entrapped in a packed bed of glass beads. The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen tension, pH, and medium perfusion rate. The hepatocytes were viable for up to the longest time point studied of 15 days in culture based on urea synthesis, albumin synthesis and cell morphology. Light microscopy studies of hepatocytes cultured for 15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver organ. Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes. We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial applications including toxicology, drug metabolism, and protein/peptide synthesis. The hepatocyte bioreactor concept may also be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with hepatic failure.     

Optimization of mass transfer for toxin removal and immunoprotection of hepatocytes in a bioartificial liver

This study was designed to determine optimal operating conditions of a bioartificial liver (BAL) based on mass transfer of representative hepatotoxins and mediators of immune damage. A microprocessor‐controlled BAL was used to study mass transfer between patient and cell compartments separated by a hollow fiber membrane. Membrane permeability (70, 150, or 400 kDa molecular weight cut‐off—MWCO), membrane convection (high: 50 mL/min; medium: 25 mL/min; low: 10 mL/min; diffusion: 0 mL/min), and albumin concentration in the cell compartment (0.5 or 5 g%) were considered for a total of 24 test conditions. Initially, the patient compartment contained pig plasma supplemented with ammonia (0.017 kDa), unconjugated bilirubin (0.585 kDa), conjugated bilirubin (0.760 kDa), TNF‐α (17 kDa), pig albumin (67 kDa), pig IgG (147 kDa), and pig IgM (900 kDa). Mass transfer of each substance was determined by its rate of appearance in the cell compartment. Membrane fouling was assessed by dextran polymer technique. Of the three tested variables (membrane pore size, convection, and albumin concentration), membrane permeability had the greatest impact on mass transfer (P < 0.001). Mass transfer of all toxins was greatest under high convection with a 400 kDa membrane. Transfer of IgG and IgM was insignificant under all conditions. Bilirubin transfer was increased under high albumin conditions (P = 0.055). Fouling of membranes ranged from 7% (400 kDa), 24% (150 kDa) to 62% (70 kDa) during a 2‐h test interval. In conclusion, optimal toxin removal was achieved under high convection with a 400‐kDa membrane, a condition which should provide adequate immunoprotection of hepatocytes in the BAL. Biotechnol. Bioeng. 2009; 104: 995–1003. © 2009 Wiley Periodicals, Inc.     

Salivary flow rate and risk of malnutrition – a study among dentate,community‐dwelling older people

doi: 10.1111/j.1741‐2358.2012.00679.x Salivary flow rate and risk of malnutrition – a study among dentate, community‐dwelling older people Objective: To analyse the relation between unstimulated and stimulated salivary secretion and the risk of malnutrition among home‐dwelling elderly people. Background: Saliva has an important role in eating. Despite this, there are only a few studies on the role of salivary secretion in the development of malnutrition among elderly people. Materials and methods: The study population consisted of 157 subjects aged 75 or older. This was a part of GeMS study carried out in Kuopio, in eastern Finland. The data used in this study were collected by means of interviews and geriatric and oral clinical examinations. The risk of malnutrition was measured using the Mini Nutritional Assessment Short‐Form. Logistic regression models were used to estimate odds ratios (OR) and their 95% Confidence Intervals (CI). Results: Subjects with a low unstimulated salivary flow rate (<0.1 ml/min) or stimulated salivary flow rate (<1.0 ml/min) had no statistically significant increase in risk of malnutrition, OR: 1.3, CI: 0.5–3.9, OR: 1.5, CI: 0.5–4.2, respectively, when compared with those with a normal unstimulated and stimulated salivary flow rate. Conclusion: Our results do not support the concept that low salivary secretion is an important risk factor for malnutrition among community‐dwelling elders.     

Perfusion of 3D encapsulated hepatocytes—A synergistic effect enhancing long‐term functionality in bioreactors

Long‐term primary cultures of hepatocytes are essential for bioartificial liver (BAL) devices and to reduce and replace animal tests in lead candidate optimization in drug discovery and toxicology tests. The aim of this work was to improve bioreactor cultures of hepatocyte spheroids by adding a more physiological perfusion feeding regime to these bioreactor systems. A continuous perfusion feeding was compared with 50% medium replacement (routinely used for in vitro tests) at the same dilution rate, 0.125 day⁻¹, for three operative weeks. Perfusion feeding led to a 10‐fold improvement in albumin synthesis in bioreactors containing non‐encapsulated hepatocyte spheroids; no significant improvement was observed in phase I drug metabolizing activity. When ultra high viscous alginate encapsulated spheroids were cultured in perfusion, urea synthesis, phase I drug metabolizing activity and oxygen consumption had a threefold improvement over the 50% medium replacement regime; albumin production was the same for both feeding regimes. The effective diffusion of albumin in the alginate capsules was 7.75.10⁻⁹ cm² s⁻¹ and no diffusion limitation for this protein was observed using these alginate capsules under our operational conditions. In conclusion, perfusion feeding coupled with alginate encapsulation of hepatocyte spheroids showed a synergistic effect with a threefold improvement in three independent liver‐specific functions of long‐term hepatocyte spheroid cultures. Biotechnol. Bioeng. 2011; 108:41–49. © 2010 Wiley Periodicals, Inc.