Gerardo Catapano

Mass transfer limitations to the performance of membrane bioartificial liver support devices.

A number of membrane bioartificial devices have been proposed for liver support. However, their design does not yet ensure the successful treatment of acute liver insufficiency. In this paper, the Author reviews the limitations of the mass transport phenomena to the performance of a membrane bioartificial liver support device. First of all the requirements that an optimal membrane bioartificial liver support device has to meet for the therapy to be effective are presented. On these grounds, the issues that are still to be addressed to optimize the performance of such devices are discussed: particular attention is devoted to the mass transport phenomena in each region of the membrane bioartificial device. Finally, the main transport features of the membrane bioartificial liver support devices proposed so far are illustrated and examined.

The effect of oxygen transport resistances on the viability and functions of isolated rat hepatocytes

The treatment of fulminant hepatic failure with a bioartificial liver support device relies on the possibility of replacing the detoxification and synthetic functions of the injured liver for as long as needed for patient recovery. In spite of progress in cell culture techniques, the effective use of isolated hepatocytes in liver support devices is currently hampered by a lack of information on the metabolic factors limiting long term hepatocyte culture. In this paper, we report our investigation on the effects of oxygen transport resistances on the viability and functions of isolated rat hepatocytes cultured on collagen coated Petri dishes. Detoxification and synthetic functions of the hepatocytes were studied with respect to ammonia and phenolsulphonphthalein elimination and urea synthesis. Lower resistances to oxygen transport favored hepatocyte survival. The isolated hepatocytes synthesized urea at rates that decreased as the resistance to oxygen transport increased. The rate at which urea was synthesized also decreased during the culture. Neither PSP, nor ammonia elimination rate was greatly affected by increasing oxygen transport resistances and remained rather constant up to a week of culture.

The effect of surface roughness of microporous membranes on the kinetics of oxygen consumption and ammonia elimination by adherent hepatocytes

In membrane hybrid liver support devices (HLSDs) using isolated hepatocytes where oxygen is transported only by diffusion to the cells, about 15-40% of the cell mass is likely to be in direct contact with the semipermeable membranes used as immunoselective barriers: quantitative effects of membrane surface properties on the kinetics of hepatocyte metabolic reactions may also affect HLSD performance. In this paper, we report our investigation of the effects of surface morphology of two microporous commercial membranes on the kinetics of oxygen consumption and ammonia elimination by primary hepatocytes in adhesion culture. Isolated rat hepatocytes were cultured on polypropylene microporous membranes with different surface roughness and pore size in a continuous-flow bioreactor whose fluid dynamics was optimized for the kinetic characterization of liver cell metabolic reactions. Collagen-coated membranes were used as the reference substratum. Hepatocyte adhesion was not significantly affected by membrane surface morphology. The rates of the investigated reactions increased with ammonia concentration according to saturation kinetics: the values of kinetic parameters Vmax and K(M) increased as cells were cultured on the membrane with the greatest membrane surface roughness and pore size. For the reaction of oxygen consumption, Vmax increased from 0.066 to 0.1 pmol h(-1) per cell as surface roughness increased from 70 to 370 nm. For the kinetics of ammonia elimination. K(M) increased from 0.23 to 0.32 mM and Vmax increased from 1.49 to 1.79 pmol h(-1) per cell with membrane surface roughness increasing from 70 to 370 nm. Cells cultured on collagen-coated membranes consistently yielded the highest reaction rates. The Vmax values of 0.18 and 2.84 pmol h(-1) per cell for oxygen consumption and ammonia elimination, respectively, suggest that cell functions are also affected by the chemical nature of the substratum.

Polymeric membranes for hybrid liver support devices: the effect of membrane surface wettability on hepatocyte viability and functions.

Extracorporeal therapies based on membrane hybrid liver support devices using primary hepatocytes are an interesting approach to the treatment of acute hepatic failure. In such devices, semipermeable polymeric membranes are effectively used as immunoselective barriers between a patients blood and the xenocytes in order to prevent the immune rejection of the graft. The membranes may act also as the substratum for cell adhesion, thus favouring the viability and functions of anchorage-dependent cells such as the hepatocytes. Membrane cytocompatibility is expected to depend on the surface properties of the polymer, such as its morphology and its physico-chemical properties. In this paper, we report our investigation on the effect of the surface wettability of membranes on hepatocyte viability and functions. Polypropylene microporous membranes were modified to increase their surface wettability and were used as substrata for rat hepatocyte adhesion culture. Isolated hepatocytes were also cultured on collagen as a reference substratum. Hepatocyte viability generally improved as the cells were cultured on more wettable membranes. In agreement with the viability data, the increasing wettability of the membrane surface also improved some metabolic functions.

Morphology and metabolism of hepatocytes cultured in Petri dishes on films and in non-woven fabrics of hyaluronic acid esters

Polymers of hyaluronic acid (Hyal) esters exhibit good tissue compatibility and are available in various geometrical configurations. These properties can be exploited for the design of innovative bioartificial liver support devices (BALSDs) using primary hepatocytes. In this paper, we report a preliminary investigation of the polymer feasibility of the ethyl and the benzyl Hyal ester in the form of films and non-woven fabrics for the in vitro culture of primary rat hepatocytes. Cell function was evaluated daily in Petri dishes with respect to the rate of ammonia elimination (AER) and urea synthesis (USR). Cells cultured in non-woven fabrics of the ethyl ester of Hyal (HYAFF7nw) exhibited an initial AER about 32% lower and synthesised urea 33% faster than that of cells on collagen films. After a week in culture, cells on collagen films retained only a minor fraction of their initial rates. Cells cultured in non-woven fabrics of HYAFF7nw retained about 62 and 44% of their initial AER and USR, respectively, and exhibited an AER approximately equal to and a USR 3.6 times greater than those of cells adherent to collagen. These results suggest that non-woven fabrics of HYAFF7nw are promising substrata for hepatocyte culture in BALSDs.

Physical-chemical and biological characterization of silk fibroin-coated porous membranes for medical applications.

Membranes in artificial organs and scaffolds for tissue engineering are often coated with biomimetic molecules (e.g., collagen) to improve their biocompatibility and promote primary cell adhesion and differentiation. However, animal proteins are expensive and may be contaminated with prions. Silk fibroin (SF) made by Bombyx Mori silk worms, used as a scaffold or grafted to other polymers, reportedly promotes the adhesion and growth of many human cell types. This paper describes how commercial porous membranes were physically coated with SF, and their physical-chemical properties were characterized by SEM, AFM, tensile stress analysis and dynamic contact angle measurements. The effect of the SF coating on membrane biocompatibility and resistance to bacterial colonization is also examined. The proposed technique yields SF coats of different thickness that strengthen the membranes and make their surface remarkably more wettable. The SF coat is not cytotoxic, and promotes the adhesion and proliferation of an immortalized fibroblast cell line. Similarly to collagen, SF-coated membranes also exhibit a much better resistance to the adhesion of S. epidermidis bacteria than uncoated membranes. These preliminary results suggest that SF is a feasible alternative to collagen as a biomimetic coating for 3D scaffolds for tissue engineering or bioartificial (as well as artificial) prosthesis.

Bioreactor Design and Scale-Up

Design and selection of cell culture bioreactors are affected by cell-specific demands, engineering aspects, as well as economic and regulatory considerations. Mainly, special demands such as gentle agitation and aeration without cell damage, a well controlled environment, low levels of toxic metabolites, high cell and product concentrations, optimized medium utilization, surface for adherent cells, and scalability have to be considered. This chapter comprises engineering aspects of bioreactor systems (design, operation, scale-up) developed or adapted for cultivation of mammalian cells, such as bioreactors for suspension culture (stirred-tank reactors, bubble columns, and air-lift reactors), fixed bed and fluidized bed reactors, hollow fiber and membrane reactors, and, finally, disposable bioreactors. Aspects relevant for selection of bioreactors are discussed. Finally, an example is given of how to grow mammalian suspension cells from cryopreserved vials to laboratory and pilot scale.

Mass and momentum transport in extra-luminal flow (ELF) membrane devices for blood oxygenation

Abstract In this paper, we report on the characterisation of transport in membrane modules for blood oxygenation where blood is circulated outside hollow fibre membranes arranged in double layer cross-laid mats at an angle with respect to the main direction of blood flow. The effect of design and operating variables on module performance was investigated with respect to oxygen transfer into water, as gaseous oxygen and water are circulated counter-currently, respectively inside the membrane lumen and through the membrane assembly. Increasing water flow rates and membrane angles enhanced oxygen transfer across the membrane and resulted in robust operation but also in high pressure drops. Module pressure drop and oxygen transfer rate were correlated to module geometry, fibre packing density, water flow rate and membrane angle with respect to the main direction of the liquid flow in non-dimensional equations that can be used by membrane module manufacturers for the design of optimal ELF blood oxygenators. The results suggest that an optimum membrane angle exists, beyond which module operation is not convenient in terms of energy.

Transport Advances in Disposable Bioreactors for Liver Tissue Engineering

Acute liver failure (ALF) is a devastating diagnosis with an overall survival of approximately 60%. Liver transplantation is the therapy of choice for ALF patients but is limited by the scarce availability of donor organs. The prognosis of ALF patients may improve if essential liver functions are restored during liver failure by means of auxiliary methods because liver tissue has the capability to regenerate and heal. Bioartificial liver (BAL) approaches use liver tissue or cells to provide ALF patients with liver-specific metabolism and synthesis products necessary to relieve some of the symptoms and to promote liver tissue regeneration. The most promising BAL treatments are based on the culture of tissue engineered (TE) liver constructs, with mature liver cells or cells that may differentiate into hepatocytes to perform liver-specific functions, in disposable continuous-flow bioreactors. In fact, adult hepatocytes perform all essential liver functions. Clinical evaluations of the proposed BALs show that they are safe but have not clearly proven the efficacy of treatment as compared to standard supportive treatments. Ambiguous clinical results, the time loss of cellular activity during treatment, and the presence of a necrotic core in the cell compartment of many bioreactors suggest that improvement of transport of nutrients, and metabolic wastes and products to or from the cells in the bioreactor is critical for the development of therapeutically effective BALs. In this chapter, advanced strategies that have been proposed over to improve mass transport in the bioreactors at the core of a BAL for the treatment of ALF patients are reviewed.

Characterization of the distribution of matter in hybird liver support devices where cells are cultured in a 3-D membrane network or on flat substrata

Bioreactors for liver assist tested on small animal models are generally scaled-up to treat humans by increasing their size to host a given liver cell mass. In this process, liver cell function in different culture devices is often established based on the metabolite concentration difference between the bioreactor inlet and outlet irrespective of how matter distributes in the bioreactor. In this paper, we report our investigation aimed at establishing whether bioreactor design and operating conditions influence the distribution of matter in two bioreactors proposed for liver assist. We investigated a clinical-scale bioreactor where liver cells are cultured around a three-dimensional network of hollow fiber membranes and a laboratory-scale bioreactor with cells adherent on collagen-coated flat substrata. The distribution of matter was characterized under different operating modes and conditions in terms of the bioreactor residence time distribution evaluated by means of tracer experiments and modeled as a cascade of N stirred tanks with the same volume. Under conditions recommended by the manufacturers, matter distributed uniformly in the clinical-scale bioreactor as a result of the intense backmixing (N=1) whereas axial mixing was negligible in the laboratory-scale bioreactor (N=8). Switching from recycle to single-pass operation definitely reduced axial mixing in the clinical-scale bioreactor (N=2). Increasing feed flow rate significantly enhanced axial mixing in the laboratory-scale bioreactor (N=4). The effects of design, operating mode and conditions on matter distribution in bioreactors for liver cell culture suggest that characterization of the distribution of matter is a necessary step in the scale-up of bioreactors for liver assist and when function of liver cells cultured in different bioreactors is evaluated and compared.