Development and validation of a bioartificial liver device with fluidized bed bioreactors hosting alginate-encapsulated hepatocyte spheroids.

Acute and acute-on-chronic liver failure are associated to high mortality when transplantation is not possible. The lack of donors has resulted in an important demand for liver support devices. This paper describes the design and validation of a new bioartificial liver (BAL) device including fluidized bed bioreactors hosting alginate-encapsulated hepatocytes spheroids. To ensure the efficacy of the BAL and the safety of the patients, a complex extracorporeal circulation was designed to be compatible with a commercial medical device, the Prismaflex(®) monitor, already used in intensive care units. Preclinical studies on large animal show that the treatment was well tolerated in terms of hemodynamics considerations. A method using non adhesive coating in petri dish led to the production of large amount of viable spheroids in vitro that were further encapsulated to follow up bioartificial liver activity during four days.

Impact of alginate type and bead diameter on mass transfers and the metabolic activities of encapsulated C3A cells in bioartificial liver applications.

Liver-assist devices have been developed in the last few decades to support patients with liver failure on the road to recovery or transplantation. Fluidised bed bio-artificial livers--where liver cells are encapsulated within alginate beads--appear to be a valuable alternative to hollow fibre devices for improving mass transfers and enhancing treatment efficacy. This approach nevertheless deserves optimization in terms of bead production. The aim of this study was to investigate the impact of alginate type and of two bead diameters (1000 µm and 600 µm) on mass transfers within beads and on the biological functions of encapsulated C3A cells. After assessing the effect of the encapsulation process on bead quality, we investigated cell viability and metabolic activities (ammonia, albumin, alpha-fetoprotein synthesis and glucose consumption). They were successfully maintained over 48 h within fluidised bed bioreactors, independently of alginate type and bead diameter. Mass transfers were not significantly influenced by the latter parameters. Finally, suggestions are made for improving the entrapment process as a means of enhancing the treatment efficiency of the fluidised bed bioartificial liver.

Artificial and bioartificial liver devices: present and future.

Liver failure is associated with high morbidity and mortality without transplantation. There are two types of device for temporary support: artificial and bioartificial livers. Artificial livers essentially use non-living components to remove the toxins accumulated during liver failure. Bioartificial livers have bioreactors containing hepatocytes to provide both biotransformation and synthetic liver functions. We review here the operating principles, chemical effects, clinical effects and complications of both types, with specific attention paid to bioartificial systems. Several artificial support systems have FDA marketing authorisation or are CE labelled, but the improvement they provide in terms of patient clinical outcome has not yet been fully demonstrated. At present, different bioartifical systems are being investigated clinically on the basis of their promises and capacity to provide and replace most liver functions. However, important issues such as cost, cell availability, maintenance of cell viability and functionality throughout treatment, and regulatory issues, as well as difficult challenges, including implementing cell-housing devices at the patient's bedside on an emergency basis, have delayed their appearance in intensive care units and on the market. Bioreactors are, nevertheless, when combined with artificial components, a pragmatic approach for future treatment of liver failure.

Three-dimensional growth of human hepatoma C3A cells within alginate beads for fluidized bioartificial liver.

OBJECTIVES: Alginate beads allow cultivation of cells in a 3-dimensional environment. The aim of our study was to assess the influence of a 3-dimensional culture in alginate microbeads, on hepatic cell metabolism. METHODS: We used 2 types of alginate: low viscosity (LV) and medium viscosity (MV). The hepatic cell line C3A was encapsulated in alginate beads. Cells were cultured for 2 weeks. Using scanning electron microscopy, the morphology of 3D structures and the surfaces of cells were analyzed. Fluidized bed bioartificial liver experiments were performed 24 hours, 7, and 14 days after bead formation. RESULTS: Two different cell growth types in alginate beads were observed: channel-like structures and spherical aggregates characteristic of LV and MV alginate, respectively. A significant increase in albumin synthesis was observed in long-term culture. Formation of characteristic hepatic cell microvilli on cell surfaces was observed under scanning electron microscopy for both types of alginate. Prolonged static cultivation of C3A cells within the alginate beads in both types of alginates caused significant increases in albumin production in the fluidized bioreactor. CONCLUSIONS: Cultivation of the hepatic C3A cells within the alginate microbeads significantly improved bioreactor effectiveness in albumin production. The presence of extensions of cell membranes on the surface of hepatoma cells in 3-dimensional culture within the alginate beads indicated formation of microvilli-like structures characteristic of normal hepatocytes.

The place of adsorption and biochromatography in extracorporeal liver support systems.

Artificial and bioartificial liver devices aim at replacing some or all liver functions in the cases of end stage or fulminant disorders. Among all of its function, liver plays a key role in detoxification of substances that are hydrosoluble or bound to albumin. In this paper, the authors first reviewed the requirements for temporary liver support, then the adsorption-based systems that can be found on the market and finally propose new applications of biochromatography using perfusion-based bioartificial systems.

Culture of C3A cells in alginate beads for fluidized bed bioartificial liver.

Extracorporeal bioartificial liver has been designed to sustain the detoxification and synthetic function of the failed liver in patients suffering from acute liver failure until the time of liver allotransplantation or regeneration of their own. A fluidized bed, bioartificial liver improves the mass transfer velocity between the medium and the hepatocytes. Detoxification functions of the liver could be replaced by completely artificial systems, but the synthetic functions of hepatocytes may be obtained only by metabolically active cells. The aim of our study was to investigate the influence of C3A cell culture in alginate beads on synthetic function in a fluidized bed, bioartificial liver. Cells in alginate beads were prepared using an electrostatic droplet generator of our own design using low-viscosity alginate. Beads were cultured for 24 hours then 7 days in static conditions and then 24 hours of fluidization in the bioreactor to assess albumin production. We observed significantly increased albumin production by C3A cells entrapped in alginate beads during static culture. Fluidization increased albumin production compared with static culture. Fluidization performed after 7 days of static culture resulted in a significant increase in albumin synthesis. In conclusion, static culture of alginate beads hosting hepatic cells facilitates restoration of cell function.

Arterio-venous fistula: two cases realistic numerical blood flow simulations.

Patients with ESRD "End-stage renal disease" require hemodialysis. The most desirable form of vascular access is the arterio-venous fistula (AVF). It is placed surgically joining an artery and a vein together. We propose an investigation protocol to understand the correlation between hemodynamics in this vessel and the associated clinical complications. Realistic 3D reconstruction of the fistula is the result of segmenting CT angiography images for two cases. Then the geometrical model is preprocessed by hybrid meshing and CFD results are confronted with medical image data such as calcifications.

Investigations into the relationship between hemodynamics and vascular alterations in an established arteriovenous fistula.

Arteriovenous fistula are specific vessels created by a vascular operation in order to provide sufficient blood access for extracorporeal circulation in hemodialysis. They are subject to numerous pathologies that may be caused by hemodynamic effects. To better understand these effects, a specific patient's arteriovenous fistula was reconstructed from computed tomography angiography. Computational fluid dynamics software made it possible to solve fluid mechanics equations under physiological conditions. An accurate map of unsteady velocity profiles and wall shear stress was drawn up. The computed velocity profiles were successfully confronted with Echo Doppler investigation. Selected regions with or without calcification, the end stage of wall alteration, were examined in terms of the mechanical constraints generated by blood flow. In contrast with other authors, we did not observe any association between calcification and areas of oscillating shear stress. Nevertheless, a statistical analysis of the whole vessel envelop and specific sites of calcification suggested a potential association between calcification and high temporal wall shear stress gradients.

Mechanical properties of alginate beads hosting hepatocytes in a fluidized bed bioreactor.

Fluidized bed bioartificial liver has been proposed as a temporary support to bridge patients suffering from acute liver failure to transplantation. In such a bioreactor, alginate beads hosting hepatocytes are in continuous motion during at least six hours. After having shown in vitro the functionality of such a device, the present study aims at analyzing the potential mechanical alterations of the beads in the bioreactor, perfused by different surrounding media. Compression experiments are performed and coupled for analysis with Hertz theory. They provide qualitative and quantitative data. The average value of the shear modulus, calculated for the different cases studied varied from 2.4 to 10.4 kPa, and could therefore be considered as a quantitative measure of the beads mechanical properties. From the compression experiments and the estimated values of the shear modulus, we could now evaluate the effect of different operating conditions (fluidization, presence of cells, surrounding medium) on the mechanical behavior of alginate beads. On the one hand, the motion during six hours in the bioreactor does not alter the beads significantly. On the other hand, the presence of different substances in the fluid phase might change their mechanical strength. These results can be considered as new encouragements to use such a device as a bioartificial organ.

Mass transfers in a fluidized bed bioreactor using alginate beads for a future bioartificial liver.

Fluidized bed bioreactor with alginate beads may be an alternative to hollow fiber cartridge to host hepatocytes for bioartificial liver purposes. After the bioreactor design and the characterization of fluid mechanics, the present study was aimed at analyzing bi-directional mass transfers of calibrated species between external fluid and empty beads. Static (batch) and dynamic (fluidized bed bioreactor) experimental conditions were analyzed. A simple modelling approach permitted the definition of mass transfer coefficients. The motion of beads within the bioreactor clearly enhanced mass transfer kinetics, but did not alter the amount exchanged. The shear enhanced diffusion coefficient for VitB12 was 20 times higher in the fluidized bed bioreactor than under batch conditions, proving the efficiency of such a device.

Blood flow patterns in an anatomically realistic coronary vessel: influence of three different reconstruction methods.

Many clinical studies suggest that local blood flow patterns are involved in the location and development of atherosclerosis. In coronary diseases, this assumption should be corroborated by quantitative information on local hemodynamic parameters such as pressure, velocity or wall shear stress. Nowadays, computational fluid dynamics (CFD) algorithms coupled to realistic 3-D reconstructions of such vessels make these data accessible. Nevertheless, they should be carefully analysed to avoid misinterpretations when the physiological parameters are not all considered. As an example, we propose here to compare the flow patterns calculated in a coronary vessel reconstructed by three different methods. In the three cases, the vessel trajectory respected the physiology. In the simplest reconstruction, the coronary was modelled by a tube of constant diameter while in the most complex one, the cross-sections corresponded to the reality. We showed that local pressures, wall shear rates and velocity profiles were severely affected by the geometrical modifications. In the constant cross-section vessel, the flow resembled to that of Poiseuille in a straight tube. On the contrary, velocity and shear rate exhibited sudden local variations in the more realistic vessels. As an example, velocity could be multiplied by 5 as compared to Poiseuille's flow and area of very low wall shear rates appeared. The results obtained with the most complex model clearly outlined that, in addition to a proper description of the vessel trajectory, the section area changes should be carefully taken into account, confirming assumptions already highlighted before the rise of commercially available and efficient CFD softwares.

Protein adsorption on histidyl-aminohexyl-Sepharose 4B. II. Application to the negative one-step affinity purification of human beta2-microglobulin and immunoglobulin G.

The adsorption of two human proteins, beta2-microglobulin and Immunoglobulin G, from uremic patient's blood ultrafiltrate and plasma, respectively, was investigated on the histidyl-aminohexyl-Sepharose 4B adsorbent. Both target proteins could be adsorbed on the gel through a low affinity for immobilized histidine ligand. However, a fine adjustment of the operating conditions (ionic strength, buffer, pH) prevented their adsorption and thus allowed their "negative affinity" purification (purity estimated by silver nitrate SDS-PAGE) by the removal of the contaminating proteins. This simple and efficient method provides purification under gentle chromatographic conditions and a further characterization of both molecules.

In vitro removal of human IgG autoantibodies by affinity filtration using immobilized L-histidine onto PEVA hollow fiber membranes.

Histidine was immobilized onto PEVA membrane to obtain an affinity support for human IgG removal from serum with a view to clinical apheresis for the treatment of autoimmune diseases. These membranes were able to remove in vitro several autoantibodies from the serum of SLE patients.

Design of a fluidized bed bioartificial liver.

The aim of this study was to design a bioreactor for extracorporeal liver supply containing alginate beads in a fluidized bed regimen. The objective was to achieve a satisfactory mixing into the bioreactor to promote the potential exchanges and mass transfers. First, we checked whether both present phases (solid: alginate beads; liquid: saline solution at 20 degrees C) might allow for this fluidization. Then the optimal design was defined as a function of the required operating conditions, bead volume, and perfusion flow rate; the bioreactor cross section and height especially needed to be adjusted. The efficient fluidization, under optimized conditions, was proven through the follow-up of the head losses generated by the fluidized bed. Criteria for scaling up were also determined.

Functionalized hollow fiber membrane cartridge for adsorption of Anticofactor/Antiphospholipid antibodies: a potential tool for treatment.

It is of considerable interest to ascertain whether a hollow fiber cartridge containing histidine immobilized on polyethylenevinyl alcohol membrane (His-PEVA) is able to retain specific autoantibodies involved in antiphospholipid syndrome. To this end diluted patient pathogenic plasma containing high levels of anti-beta2-glycoprotein I (anti-beta2GPI) and antiprothrombin antibodies was processed through the functionalized cartridge. The adsorbed material was then eluted under mild conditions and analyzed; an enrichment of the eluted fractions in total IgG and more specifically in IgG2 subclass was observed, compared with the injected sample. Enzyme-linked immunosorbent assay tests showed a higher specific binding of antiprothrombin and anti-beta2GPI in these fractions. This was in accordance with the concomitant higher anticoagulant activity measured on the same fractions. All in vitro results clearly demonstrated the ability of the His-PEVA cartridge to preferentially adsorb these autoantibodies. Hence the functionalized cartridge represents a potential tool for the treatment of antiphospholipid syndrome by selective extracorporeal removal of IgG.

A new concept of bioartificial liver based on a fluidized bed bioreactor.

Many bioartificial livers have been developed, but most of them suffer from difficulty when being scaled up and from poor efficiency of mass transfer between the plasma and the immobilized hepatocytes. We present a new concept of bioartificial liver based on the fluidized bed motion of hepatocytes entrapped in alginate beads. The bioreactor is designed to offer stable behavior. The maximum fluid perfusion velocity is determined to avoid any bead release from the bioreactor. The fluidized bed height depends on the amount of beads and the velocity employed. Under the optimized operating conditions, the mass transfer between perfusion fluid and beads is very efficient; only 10 min are necessary to reach concentration equilibrium. Hence, this fluidized bed bioartificial liver appears to be a promising tool for a liver support system in the treatment of acute liver failure.

The dependency of calcium set point on basal plasma calcium in dialysis patients: a better explanation for the discrepancies regarding its link with PTH secretion than methodological differences.

BACKGROUND: The increase of calcium (Ca) set point in uremic hyperparathyroid patients and its decrease with calcitriol therapy are controversial. Besides methodological differences regarding the experimental protocol for obtaining the sigmoidal curve, mainly differences in definitions of maximal PTH (peak or steady value) and of calcium set point itself have been proposed for the discrepant conclusions. However, two other explanations are possible: the various aluminum load of the patients and the dependency of Ca set point upon the basal plasma ionized calcium (PCa). PATIENTS AND METHODS: Therefore the Ca set point was measured in 2 groups of patients on maintenance dialysis never exposed to aluminum, one of 7 patients with normosecretion of PTH (NPT) and the other of 8 patients with hyperparathyroidism (HPT) before and after 3 intravenous administration of 4 microg of alfacalcidol in a week. The sigmoidal curve was established during a zero Ca dialysis, without Ca replacement for the first 90 minutes and with intravenous infusion of 41 mmoles of Ca during the 150 last minutes. The curvilinear decrease of PCa induced a peak of PTH followed by a decrease while PCa was still decreasing up to the 90th minute. Therefore PTHmax was taken both at the peak and at its lower value observed at the 90th minute (steady PTHmax). Experimental determinations of the Ca set point were made using both definitions of Brown and Felsenfeld and both PTHmax values. In basal conditions, while using any of the values given by the same calculation methodology, Ca set point was not different in NPT and HPT patients. After alfacalcidol, no change in plasma PTH nor in Ca set point was observed in HPT patients. In contrast, in NPT patients alfacalcidol induced a significant decrease of plasma PTH concentrations in association with an increase in basal PCa and in Ca set point, whatever the definitions of the latter and of PTHmax. Calcitriol induced changes in Ca set point and basal PCa were correlated. CONCLUSIONS: 1) In normocalcemic dialysis patients never exposed to aluminium hyperparathyroidism is not explained by an increased Ca set point 2) Calcitriol suppressive effect on PTH secretion is neither explained by a decrease in Ca set point. 3) Ca set point as measured in vivo does not reflect an intrinsic characteristic of the parathyroid glands since it varies with basal PCa. Better than methodological differences, this dependency may explain the discrepant conclusions between the various clinical investigations.

Endotoxin removal with poly(ethyleneimine)-immobilized adsorbers: Sepharose 4B versus flat sheet and hollow fibre membranes.

Poly(ethyleneimine) was immobilized on poly(vinyl alcohol)-coated nylon flat sheet membranes, poly(vinyl alcohol) and poly(ethylenevinyl alcohol) hollow fibre membranes as well as Sepharose 4B. The resulting poly(ethyleneimine)-immobilized adsorbers were used for removal of E. coli derived endotoxin from buffers and bovine serum albumin solutions. The efficiency of poly(ethyleneimine) proved to be constant over a wide pH range, including phosphate buffered saline. The performance depended upon the matrix type employed: endotoxin clearance factors varied from 100 to 120,000 in protein-free solutions and 40 to 33,000 in solutions of bovine serum albumin using 6000 EU/ml as feed concentration. The best adsorber was the flat sheet membrane-immobilized poly(ethyleneimine), followed by the hollow fibre-immobilized poly(ethyleneimine) and poly(ethyleneimine)-Sepharose. The factors influencing endotoxin clearance were the mass transport (convective systems were superior to the diffusive system), the chemical composition and the surface structure of the underlying matrix.