Biotechnological challenges of bioartificial kidney engineering

With the world-wide increase of patients with renal failure, the development of functional renal replacement therapies have gained significant interest and novel technologies are rapidly evolving. Currently used renal replacement therapies insufficiently remove accumulating waste products, resulting in the uremic syndrome. A more preferred treatment option is kidney transplantation, but the shortage of donor organs and the increasing number of patients waiting for a transplant warrant the development of novel technologies. The bioartificial kidney (BAK) is such promising biotechnological approach to replace essential renal functions together with the active secretion of waste products. The development of the BAK requires a multidisciplinary approach and evolves at the intersection of regenerative medicine and renal replacement therapy. Here we provide a concise review embracing a compact historical overview of bioartificial kidney development and highlighting the current state-of-the-art, including implementation of living-membranes and the relevance of extracellular matrices. We focus further on the choice of relevant renal epithelial cell lines versus the use of stem cells and co-cultures that need to be implemented in a suitable device. Moreover, the future of the BAK in regenerative nephrology is discussed.     

Enhancement of permeability in endothelial cells for the development of an antithrombogenic bioartificial hemofilter

For the development of an antithrombogenic bioartificial hemofilter, in which the inner surface of hollow fibers is lined by endothelial cells, it is essential to increase the permeability of the cells in order to achieve a sufficient ultrafiltrate. We tried to increase it by using an actin microfilament polymerization inhibitor, cytochalasin B (CyB). Fifty microg/mL CyB was added for 2 h to the culture medium of confluent rat glomerular endothelial cells (RGEC) and human umbilical vein endothelial cells (HUVEC). Under the 130 mmHg hydrostatic pressure, the CyB-treated group produced significantly more ultrafiltration than the non-treated control group and this increase was maintained for at least 7 days. Horseradish peroxidase (HRP) permeability acutely and reversibly increased in the CyB-treated group compared with the non-treated control group. Scanning electron microscopy revealed a larger average diameter and increased number of fenestrae on the CyB-treated endothelial cells, compared with the non-treated cells. This phenomenon also lasted for at least 7 days. The platelet adherence test showed that CyB did not deteriorate the antithrombogenic property of endothelial cells. These results indicate that CyB is potentially applicable for the enhancement of endothelial cell permeability in an antithrombogenic bioartificial hemofilter.     

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.     

Isolated hepatocytes in a bioartificial liver: A single group view and experience

Despite recent advances in medical supportive therapy, patients with severe fulminant hepatic failure (FHF) have mortality rate approaching 90%. Investigators have attempted to improve survival by using various extracorporeal liver support systems loaded with sorbents and liver tissue preparations. None of them succeeded in gaining clinical acceptance and orthotopic liver transplantation (OLT) remains a primary therapeutic option for patients with FHF. In this study, authors discuss the systems which utilize isolated hepatocytes. Most of these devices were tested in vitro and in animals with chemically and surgically induced liver failure. In some studies, signficant levels of detoxification and liver functions were achieved. The authors describe their own hepatocyte-based artificial liver (BAL). It is based on plasma perfusion through a hollow-fiber module seeded with matrix-anchored porcine hepatocytes. The BAL was used 14 times to treat 9 patients with acute liver failure. On 10 occasions, a charcoal column was included in the plasma circuit. Each treatment lasted 7 +/- 1 h. All procedures were tolerated well and 8 patients (including 6 patients with FHF) underwent OLT. Five patients with increased intracranial pressure (ICP) and evidence of decerebration had normalization of ICP and enjoyed full neurologic recovery after OLT. Laboratory data showed evidence for bilirubin conjugation, decrease in blood ammonia, maintenance of low lactic acid levels, and increase in the ration between the branched chain and aromatic amino acids. No allergic reactions to xenogeneic hepatocytes were observed. The authors conclude that BAL treatment with porcine hepatocytes appears to be safe and can help maintain patients alive and neurologically intact until a liver becomes available for transplantation. (c) 1994 John Wiley & Sons, Inc.     

Mini-review: Islet transplantation to create a bioartificial pancreas

Donor scarcity precludes the use of pancreatic transplantation to treat type I diabetes. Xenogeneic islet transplantation offers the possibility of overcoming this problem; however, it entails the use of immunoisolation devices to prevent immune rejection of the transplanted islets. These devices consist of a semipermeable membrane, which surrounds the islets and isolates them from the host's immune system, while allowing the passage of insulin and essential nutrients, including glucose. Problems associated with proposed device designs include diffusion limitations, biocompatibility, device retrieval in the event of failure, and mechanical integrity. Microencapsulation appears to be the most promising system of immunoisolation, however, the design of a device suitable for human clinical use remains a challenge. (c) 1994 John Wiley & Sons, Inc.     

Hepatocyte culture in bioartificial livers with different membrane characteristics

Rat hepatocytes were cultured in three polysulfone, hollow-fiber cartridges, characterized by two membrane variables: pore size and inner diameter (ID). Hepatocytes entrapped in a micro-filtration (MF) cartridge with the membrane pore size 0.1 microm had twice the production of urea and 4-fold the amount of albumin in comparison to the control cartridge, a ultra-filtration (UF) cartridge with a molecular weight cut-off (MWCO) of 100 kDa. Hepatocytes entrapped in a UF cartridge with ID of 0.5 mm secreted twice the amount of urea and 10-fold the amount of albumin compared with the control UF cartridge.     

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.     

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.     

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.     

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     

A novel model of solute transport in a hollow-fiber bioartificial pancreas based on a finite element method

Extravascular bioartificial pancreas based on hollow fiber seems to be a promising treatment of diabetes mellitus. However, solutes mass-transport limitations in such a device could explain its lack of success. To determine critical device parameters, we have developed a novel tridimensional model based on finite element method for glucose, insulin, and oxygen diffusion around an islet of Langerhans encapsulated in a hollow-fiber section. A glucose ramp stimulation was applied outside the fiber and diffused to the islet. Concomitantly, a stationary oxygen partial pressure was applied outside the fiber, and determined local oxygen partial pressure on the islet environment. An insulin secretion model stimulated by a glucose concentration ramp and corrected by the local oxygen partial pressure was also implemented. Insulin secretion by the islet was thus computed as a response to glucose signal. The model predictions notably showed that the fiber radius had to be small enough to favor a fast response for insulin secretion and to ensure a maximal oxygen partial pressure in the islet environment. Besides the effect of fiber radius, a better islet oxygenation could be achieved by adjustments on the islet density, i.e., on the fiber length dedicated to a single islet. These hints should allow the future proposal of an optimal design for an implantable bioartificial pancreas.     

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 study of oxygen transfer including cell necrosis for the design of a bioartificial pancreas

In an extravascular bioartificial pancreas (BAP), islet functions are probably limited by diffusive mass transfer and local consumption, leading to low oxygenation. A mathematical model based on finite elements and focusing on local oxygen transport in both the alginate core and the islets of Langerhans has been proposed to help design an efficient pancreas supply. It was possible to randomly localize islets in a hollow fiber at different densities, and the effects of hypoxia and necrosis were included in the mass transfer simulations. Thorough study of the numerical results first led to the analysis of several relevant parameters, such as necrosis factor and efficacy in terms of insulin secretion, as a way to optimize fiber geometry. The approach was then to calculate the number of islets that needed to be implanted in order to obtain a correct response in terms of insulin secretion. In most configurations, it was found to be much higher than that of ultimately functional islets, because of hypoxia and necrosis. Fiber length should thus be adjusted accordingly. Finally, we demonstrated that the compromise to be found between the reduction of the number of implanted islets and fiber length and diameter did not correspond to realistic hollow fiber systems. The alternative of using flat geometry was also envisaged with more optimistic feasibility assessments.     

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.