L. De Bartolo

A Novel Full‐Scale Flat Membrane Bioreactor Utilizing Porcine Hepatocytes: Cell Viability and Tissue‐Specific Functions

When designing an extracorporeal hybrid liver support device, special attention should be paid to providing the architectural basis for reconstructing a proper cellular microenvironment that ensures highest and prolonged functional activity of the liver cells. The common goal is to achieve high cell density culture and to design the bioreactor for full‐scale primary liver cell cultures under adequate mass transfer conditions. An important aim of this study was to evaluate the biochemical performance of a flat membrane bioreactor that permits high‐density hepatocyte culture and simultaneously to culture cells under sufficient oxygenation availability conditions comparable to the in vivo‐like microenvironment. In such a bioreactor pig liver cells were cultured within an extracellular matrix between oxygen‐permeable flat‐sheet membranes. In this investigation we used a novel scaled‐up prototype consisting of up to 20 modules in a parallel mode. Each module was seeded with 2 × 108 cells. Microscopic examination of the hepatocytes revealed morphological characteristics as found in vivo. Cell concentration increased in the first days of culture, as indicated by DNA measurements. The performance of the bioreactor was monitored for 18 days in terms of albumin synthesis, urea synthesis, ammonia elimination, and diazepam metabolism. The ability of the hepatocytes to synthesize albumin and urea increased during the first days of culture. Higher rates of albumin synthesis were obtained at day 9 and remained at a value of 1.41 pg/h/cell until day 18 of culture. The rate of urea synthesis increased from 23 ng/h/cell to 28 ng/h/cell and then remained constant. Cells eliminated ammonia at a rate of about 56 pg/h/cell, which was constant over the experimental period. Hepatocytes in the bioreactor metabolized diazepam and generated three different metabolites: nordiazepam, temazepam, and oxazepam. The production of such metabolites was sustained until 18 days of culture. These results demonstrated that the scale‐up of the bioreactor was assessed, and it could be demonstrated that the device design aimed at the reconstruction of the liver‐specific tissue architecture supported the expression of liver‐specific functions of primary pig liver cells.

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 influence of polymeric membrane surface free energy on cell metabolic functions

In membrane bioartificial organs using isolated cells, polymeric semipermeable membranes are used as immunoselective barriers, means for cell oxygenation and also as substrata for adhesion of anchorage-dependent cells. The selection of cytocompatible membranes that promote in vitro cell adhesion and function could be dependent on its membrane properties. In this study we investigated the physicochemical aspects of the interaction between the membrane and mammalian cells in order to provide guidelines to the selection of cytocompatible membranes. We evaluated the metabolic behavior of isolated liver cells cultured on various polymeric membranes such as the ones modified by protein adsorption. The physico-chemical properties of the membranes were characterized by contact angle measurements. The surface free energy of membranes and their different parameters acid (γ+), base (γ-) and Lifshitz-van der Waals (γLW) were calculated according to Good-van Osss model. The adsorption of protein modified markedly both contact angle and membrane surface tension. In particular, membrane surface free energy decreased drastically with increased water contact angle. For each investigated membrane we observed that liver specific functions of cells improve on hydrophilic membrane surfaces. For all investigated membranes the rate of ammonia elimination increased with increasing of membrane surface free energy.© 2001 Kluwer Academic Publishers

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.

Chapter 9 – Membrane bioreactors for biotechnology and medical applications

ABSTRACT In this chapter we will discuss the properties and applications of bioartificial hybrid systems used in biotechnological and medical applications. In the first part we will focused on membrane bioreactors using biocatalysts such as enzymes, microrganisms and cells for the production of biological molecules or for separation of pharmaceutical products. In the second part we will discuss on membrane bioreactors using isolated mammalian cells (i.e., liver cells, pancreatic cells) as bioartificial organs in temporary or continuous substitution of injured organ. The properties of membranes to be used in these devices will be reported including mass transport, morphological and physico-chemical properties that influence their performance. The discussion of the biotechnological part will consider the effect of immobilization on functional stability and activity of biocatalysts, including membrane material and morphology, physico-chemical properties of reaction environment. The successful examples of bioreactors running at large scale, of which the authors are aware, will be also presented. In the medical application part, the development of membrane bioartificial organs, e.g. bioartificial pancreas and bioartificial liver, as well as the properties of membranes for these systems will be discussed. Particular attention will be given to the recent achievements in cytocompatibility and biocompatibility of membranes in bioartificial organs.

Technique for the kinetic characterization of the metabolic reactions of hepatocytes in adhesion culture

In this paper, we report on the development of a technique for the kinetic characterization of the metabolic reactions of liver cells in adhesion culture. The technique is based on the use of a continuous‐flow bioreactor which is designed and operated in such a way as to ensure a uniform distribution of metabolite at the cell site: hence, the metabolite concentration at the surface of cells cultured in adhesion at the bottom of the bioreactor equals that in the stream leaving the bioreactor. Under steady conditions, the rate of a given cell reaction is directly estimated from the metabolite concentration difference in the streams entering and leaving the bioreactor and can be correctly related to the actual concentration at the cell surface. Such a technique was used for a preliminary investigation of the kinetics of ammonia elimination, urea synthesis, and phenolsulfonphthalein (PSP) elimination by primary rat hepatocytes cultured in adhesion on collagen, with respect to ammonia and PSP concentration, respectively. The rate at which the hepatocytes eliminated ammonia increased with increasing ammonia concentrations according to a Michaelis−Menten kinetics. The hepatocytes synthesized urea also in the absence of ammonia in the medium: as ammonia concentration increased, the cells synthesized urea at a rate that increased according to a saturation kinetics. In the concentration range investigated, the hepatocytes eliminated PSP at a rate that increased linearly with the actual PSP concentration in the medium. Such kinetic information can be coupled to the mechanism of metabolite transport in a hybrid liver support device to yield an effective device design for the treatment of acute liver failure.

IMPORTANCE OF THE KINETIC CHARACTERIZATION OF LIVER CELL METABOLIC REACTIONS TO THE DESIGN OF HYBRID LIVER SUPPORT DEVICES

Hybrid liver support devices (HLSDs) developed for the treatment of fulminant hepatic failure often perform well on a laboratory scale but rapidly lose their metabolic functions, or are not therapeutically effective, on a clinical scale. This suggests that the procedures adopted so far for the design of HLSDs are susceptible to improvement. In this paper, we discuss how essential a reliable and thorough kinetic characterization of the liver cell metabolic reactions is to the design of a clinically effective membrane HLSD. The features of the bioreactors used for the kinetic characterization of liver cell reactions are presented and discussed on the basis of the multifactorial nature of such reactions. The relevance of kinetics to the design of a membrane HLSD is also discussed with respect to the effect of the kinetics of oxygen consumption on the performance of the device.

The effect of catabolite concentration on the viability and functions of isolated rat hepatocytes

The treatment of patients with hepatic failure by means of hybrid liver support devices using primary xenogeneic hepatocytes is currently hindered by the rapid loss of cell metabolic functions. Similarly to what happens with other mammalian cells, accumulation of catabolites in the neighborhood of cultured hepatocytes might significantly affect their viability and functions. In this paper, we investigated the effects of high concentrations of catabolites, such as ammonia and lactic acid, on the viability and functions of rat hepatocytes cultured on collagen coated Petri dishes. The effects on hepatocyte functions were established with respect to their ability to synthesize urea and to eliminate ammonia. Indeed, high catabolite concentrations effected both hepatocyte viability and functions. The number of viable hepatocytes decreased with increasing ammonia concentrations in the culture medium. High ammonia concentrations had also both an inhibitory and a toxic effect on hepatocyte functions. In fact, the hepatocytes synthesized urea and eliminated ammonia at rates that decreased with increasing ammonia concentrations. Similarly, high lactic acid concentrations were toxic to the cells and also inhibited their synthetic functions.