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An important challenge in neuronal tissue engineering is to create innovative tools capable of promoting cellular response in terms of neuronal differentiation and neurite orientation that may be used as investigational platforms for studying neurobiological events and neurodegenerative disorders. A novel membrane bioreactor was created to provide a 3D well-controlled microenvironment for neuronal outgrowth. The bioreactor consisted of poly-L-lactic acid highly aligned microtube array (PLLA-MTA) membranes assembled in parallel within a chamber that establish an intraluminal and an extraluminal compartment whose communication occurs through the pores of the MTA membrane walls. The bioreactor configuration provided a wide surface area for cell adhesion in a small volume, and offered a peculiar arrangement that directed neuronal orientation. The combination of an appropriate membrane porosity, pore interconnectivity and very thin walls ensured optimal indirect perfusion to cell compartment, and enhanced the mass transfer of metabolites and catabolites protecting neurons from shear stress. The PLLA-MTA membrane bioreactor promoted the growth and differentiation of SH-SY5Y cells toward a neuronal phenotype, and guided neurite alignment giving rise to a 3D neuronal tissue-like construct. It provides an innovative platform to study neurobiological phenomena in vitro and by guiding neuronal orientation for repair and/or regeneration.

Biocatalytic membrane reactors have been widely used in different industries including food, fine chemicals, biological, biomedical, pharmaceuticals, environmental treatment and so on. This article gives an overview of the different immobilized enzymatic processes and their advantages over the conventional chemical catalysts. The application of a membrane bioreactor (MBR) reduces the energy consumption, and system size, in line with process intensification. The performances of MBR are considerably influenced by substrate concentration, immobilized matrix material, types of immobilization and the type of reactor. Advantages of a membrane associated bioreactor over a free-enzyme biochemical reaction, and a packed bed reactor are, large surface area of immobilization matrix, reuse of enzymes, better product recovery along with heterogeneous reactions, and continuous operation of the reactor. The present research work highlights immobilization techniques, reactor setup, enzyme stability under immobilized conditions, the hydrodynamics of MBR, and its application, particularly, in the field of sugar, starch, drinks, milk, pharmaceutical industries and energy generation.

Peripheral blood lymphocytes isolated from healthy human donors' buffy coat were cultured in membrane bio-reactors (MBR) designed in two different configurations: a conventional hollow-fiber (HF) bundle of modified polyetheretherketone (PEEK-WC) arranged in parallel, and a cross-assembled PEEK-WC and polyethersulfone (PES) HF membranes having different structural properties. Both bioreactors were experimentally compared in terms of metabolic activity of cultured cells, monitored over 8 days with respect to glucose uptake rate (GUR) and lactate production rate (LPR), and mathematically modelled by Computational Fluid Dynamics (CFD) method in order to investigate the impact of geometrical configuration and transport properties of biomaterials. The almost uniform trend of GUR from day 2 to day 7 (average of 0.0497 ± 0.0076 ng/h cell) and the low LPR (that decreased from an initial value of 2.92 ± 0.0055 pg/h cell to practically zero at day 8) provided evidence for superior performance of crossed-HFMBR in reproducing an optimal in vitro physiological environment with quite uniform concentration distribution of species in the extracellular space of the bioreactor and able to maintain lymphocyte viability and functions. The crossed HFMBR also resulted in an enhanced production of interleukin IL-2 over 8 days (average of 0.995 ± 0.25 pg/h/Mcell) and IL-10 in the first 3 days (average of 6.46 ± 0.28 pg/h/Mcell) which were up to one order of magnitude higher with respect to values measured in the parallel configuration., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

An important challenge in liver tissue engineering is the development of bioartificial systems that are able to favour the liver reconstruction and to modulate liver cell behaviour. A crossed hollow fiber membrane bioreactor was developed to support the long-term maintenance and differentiation of human hepatocytes. The bioreactor consists of two types of hollow fiber (HF) membranes with different molecular weight cut-off (MWCO) and physico-chemical properties cross-assembled in alternating manner: modified polyetheretherketone (PEEK-WC) and polyethersulfone (PES), used for the medium inflow and outflow, respectively. The combination of these two fiber set produces an extracapillary network for the adhesion of cells and a high mass exchange through the cross-flow of culture medium. The transport of liver specific products such as albumin and urea together with the transport of drug such as diazepam was modelled and compared with the experimental metabolic data. The theoretical metabolite concentration differed 7.5% for albumin and 5% for urea with respect to experimental data. The optimised perfusion conditions of the bioreactor allowed the maintenance of liver functions in terms of urea synthesis, albumin secretion and diazepam biotransformation up to 18 days of culture. In particular the good performance of the bioreactor was confirmed by the high rate of urea synthesis (28.7 microg/h 10(6) cells) and diazepam biotransformation. In the bioreactor human hepatocytes expressed at high levels the individual cytochrome P450 isoenzymes involved in the diazepam metabolism. The results demonstrated that crossed HF membrane bioreactor is able to support the maintenance of primary human hepatocytes preserving their liver specific functions for all investigated period. This device may be a potential tool in the liver tissue engineering for drug metabolism/toxicity testing and study of disease pathogenesis alternatively to animal experimentation.

This paper reports on human hepatocytes cultured in a galactosylated membrane bioreactor in order to explore the modulation of the effects of a pro-inflammatory cytokine, Interleukin-6 (IL-6) on the liver cells at molecular level. In particular the role of IL-6 on gene expression and production of a glycoprotein, fetuin-A produced by hepatocytes, was investigated by culturing hepatocytes in the membrane bioreactor, both in the absence and presence of IL-6 (300 pg/ml). IL-6 modulated the fetuin-A gene expression, synthesis and release by primary human hepatocytes cultured in the bioreactor. A 75% IL-6-induced reduction of fetuin-A concentration in the medium was associated with a 60% increase of C-reactive protein in the same samples. Real-time-PCR demonstrated an 8-fold IL-6-induced reduction of fetuin-A gene expression. These results demonstrate that the hepatocyte galactosylated membrane bioreactor is a valuable tool to study IL-6 effects and gave evidence, for the first time, that IL-6 down-regulates the gene expression and synthesis of fetuin-A by primary human hepatocytes. The human hepatocyte bioreactor behaves like the in vivo liver, reproducing the same hepatic acute-phase response that occurs during the inflammatory process.

In this study we developed a PEEK-WC hollow fiber (HF) membrane bioreactor for the maintenance of human peripheral lymphocytes as model system for the in vitro investigation of disease pathogenesis, chemical effects and individual drug sensitivity. Peripheral lymphocytes isolated from donor's human buffy coat were cultured in the shell compartment of the PEEK-WC-HF bioreactor and stimulated with PHA 5microg/mL for the first 48h of culture to enhance cytokine production and cell proliferation. Thereafter, cells were cultured in the presence of Hypericum perforatum (St. John's wort) in order to induce cytochrome P450s enzymes, CYP2E, involved in the biotransformation of endogenous molecules and exogenous compounds. The metabolic activity of cells with respect to glucose consumption and oxygen uptake was maintained for all the culture time without the addition of mitogen. Two cytokines IL-2 and IL-10, which are specific pattern of lymphocytes T helper 1 and T helper 2, respectively, were produced in the bioreactor up to 14 days of culture. Lymphocytes were also able to biotransform acetaminophen through the formation of the main metabolite paracetamidofenil-beta-glucuronide, which is the product of glucuronidation reaction, as a result of the Hypericum perforatum administration that induced the catalytic activity of the CYP2E1. These results demonstrated the usefulness of the bioreactor as the support system that reproduces physiological parameters such as a constant perfusion of medium, nutrients and oxygen maintaining the in vitro integrity of lymphocyte viability and functions.

The viability and liver specific functions of human hepatocytes in a multibore fiber bioreactor are reported. Human hepatocytes were cultured in the intraluminal compartment of the bioreactor. Human hepatocytes on the membranes maintained their round shape and showed focal adhesions as sites of interaction with the membrane surface. Cells in the bioreactor expressed liver specific functions, including synthetic and detoxification activity up to 14 d of culture. The results demonstrate that human hepatocytes cultured in the multibore fiber bioreactor are able to sustain the same in vivo liver functions in vitro.

Progresses in polymeric membrane preparation and in the understanding and control of their transport properties make possible the design of novel membranes to be used for cell culture (e.g., hepatocytes, lymphocytes, pancreatic islets) in biohybrid systems such as therapeutic device or as in vitro model systems for studying the effects of various drugs and chemicals on cell metabolism. Special attention is paid to the design of the membrane with defined microstructure and physicochemical properties as well as to the importance of transport and physicochemical properties of the membrane in contact with the cells. The development of new biomaterials and bioreactors able to activate a specific response of the cells and to maintain cell differentiation for a long time is one of the most pertinent issues in the field of tissue engineering and regenerative medicine. Polymeric membranes are attractive for their selectivity and biostability characteristics in the use of biohybrid systems for cell culture. Semipermeable membranes act as a support for the adhesion of anchorage-dependent cells and allow the specific transport of metabolites and nutrients to cells and the removal of catabolites and specific products. Moreover, new membrane systems that have been recently realized as the membrane contactors might also potentially contribute to regenerative medicine and tissue engineering.

An oxygen-permeable membrane bioreactor utilizing human hepatocytes has been tested in this study. In the bioreactor, human hepatocytes were cultured between flat-sheet gas-permeable polymeric membranes, which ensure the diffusion of O(2) and CO(2) providing a support for cell anchorage and growth and permit the online observation of the cells with an inverse microscope. This bioreactor allows a direct oxygenation of cells adhered on membranes and of the medium overlaying cells simulating in vivo sinusoidal organization. Human hepatocytes were cultured in the presence of some therapeutic molecules to assess the temporal liver-specific functions of the cells. Interleukin 6 (IL-6), which is a multifactorial proinflammatory cytokine involved in a variety of host defences and pathological processes, and diclofenac, an arylacetic non-steroidal anti-inflammatory drug, were used as therapeutic molecules. The aim of this study was to evaluate the in vitro performance of the small oxygen-permeable membrane bioreactor in the long-term maintenance and differentiation of human hepatocytes under in-vivo-like conditions. The fluid dynamics of the bioreactor were characterized before using it for human cell culture. The functional response to a step challenge in the medium of IL-6 (120 pg/ml), diclofenac (80 microm) and IL-6 and diclofenac together was investigated. The ability of hepatocytes to perform liver-specific functions in terms of urea and albumin synthesis, as well as secretion of total proteins, was maintained for 32 days. Also, the diclofenac biotransformation functions were sustained as the formation of the metabolites 4'-OH-diclofenac and 5-OH-diclofenac lactam demonstrated. This study attested the feasibility of the membrane bioreactor as an in vitro simple model system that allows human hepatocytes to be maintained in a differentiated state similar to that in vivo.

The enantiocatalytic performance of immobilized lipase in an emulsion membrane reactor using stable emulsion prepared by membrane emulsification technology was studied. The production of optical pure (S)-naproxen from racemic naproxen methyl ester was used as a model reaction system. The O/W emulsion, containing the substrate in the organic phase, was fed to the enzyme membrane reactor from shell-to-lumen. The enzyme was immobilized in the sponge layer (shell side) of capillary polyamide membrane with 50 kDa cut-off. The aqueous phase was able to permeate through the membrane while the microemulsion was retained by the thin selective layer. Therefore, the substrate was kept in the enzyme-loaded membrane while the water-soluble product was continuously removed from the reaction site. The results show that lipase maintained stable activity during the entire operation time (more than 250 h), showing an enantiomeric excess (96 +/- 2%) comparable to the free enzyme (98 +/- 1%) and much higher compared to similar lipase-loaded membrane reactors used in two-separate phase systems (90%). The results demonstrate that immobilized enzymes can achieve high stability as well as high catalytic activity and enantioselectivity., (Copyright 2003 Wiley Periodicals, Inc.)

In the experimental work reported here the optical resolution of racemic naproxen methyl ester with crude lipase immobilized in a membrane reactor was studied. The multiphasic enantiocatalytic enzyme membrane reactor consisted of an organic phase that dissolved the naproxen methyl ester, a lipase-loaded membrane, and an aqueous phase that extracted the reaction product. Lipase preferentially converted the (S)-naproxen methyl ester to (S)-naproxen acid that was simultaneously separated by a membrane. The effect of the immobilization site and membrane type on the performance of the enzyme membrane reactor was studied. Capillary polyamide membrane with 10 kDa nominal molecular weight cutoff (NMWCO) and polysulfone membrane with 30 kDa NMWCO were applied, with lipase loaded in the sponge layer or on a membrane thin layer. With various immobilization sites and membrane types, the enzyme membrane reactors showed different productivity and enantioselectivity resulting from the varying amounts of immobilized enzyme and varying microenvironment of the hydrolysis reaction. Higher amounts of immobilized enzyme led to increased productivity and generally higher enantioslectivity of the membrane reactor. It seems that the location of organic/aqueous interface on membrane, which plays an important role in a multiphasic enzyme membrane reactor, was influenced by the immobilization site and membrane type and this affected the productivity and enantioselectivity. As much as 90% enantioexcess was obtained with a polyamide membrane, and lower values with a polysulfone membrane. A sponge layer of polyamide membrane is the preferred immobilization site for its higher productivity than a thin membrane layer, and for its higher enantioselectivity than a polysulfone membrane. In comparison with the unstable hydrolysis activity of free lipase in stirred tank reactor, a stable lipase activity can be obtained with the lipase-immobilized membrane reactor, irrespective of whether a polyamide or polysulfone membrane is used.

Membranes and biotechnological tools can be used for improving traditional production systems to maintain the sustainable growth of society. Typical examples include: new and improved foodstuffs, in which the desired nutrients are not lost during thermal treatment; novel pharmaceutical products with well-defined enantiomeric compositions; and the treatment of waste-water, wherein pollution by traditional processes is a problem.

Abstract: Enzymatic membrane bioreactors (MBR) have been studied for very different applications since many years. Submerged MBR has also been successfully used for treatment of wastewater. In the existing submerged configuration, the membrane works as the separation unit operation while the bioconversion is carried out by microorganisms suspended in the tank reactor. In the present work, a novel approach that combines the concept of biocatalytic membranes and submerged modules is proposed for the treatment of biomass. Lipase enzyme from Candida rugosa has been immobilized in polyethersulphone hollow fiber (PES HF) membrane in order to develop a two separate phase biocatalytic submerged membrane reactor in which the membrane works as both catalytic and separation unit. Furthermore, the submerged biocatalytic membrane reactor is intended for production of valuable components from waste biomass, and different physical, chemical and fluid dynamics has been optimized. Response surface methodology (RSM) has been used to model the operating parameters and Box-Behnken method has been applied to maximize the fatty acids production and optimization. At TMP 80 ± 2 kPa, pH 7.40 ± 0.1, temperature 35 ± 0.5 °C with an axial velocity of 0.07 ± 0.01 ms−1 and organic stirring 89.01 rad s−1 the system showed the global examined value within the experimental scope. The proof of principle using fried cooked oils has been performed in later period. [Copyright &y& Elsevier]

Abstract: In the present work a biocatalytic membrane reactor operating in water phase while extracting the water unstable reaction product in organic phase by water-in-oil droplet formation via membrane emulsification has been optimized and tested. The performance of the system as a function of pH, temperature, residence time, and immobilized enzyme amount has been investigated with identification of optimal parameter values for best system performance. In addition to optimal reaction conditions practical aspects such as energy and chemical consumptions have been taken into account. Therefore experiments to test the process efficiency at temperature and pH different from the ideal ones have been performed. The stability of the catalytic process has been evaluated on a lab scale showing no activity decay for more than three days of continuous operation with a constant production of oleuropein aglycon of about 9g/m2 h. The suitability of the system for the conversion of oleuropein present in olive mill waste water (OMWW) has also been proved. [Copyright &y& Elsevier]

Abstract: Water gas shift reaction for hydrogen production was studied in a catalytic membrane reactor using a supported silica membrane at 220–290°C temperature and 2–6bar pressure ranges. A CO conversion higher than the thermodynamic equilibrium of a traditional reactor was obtained. The best result, 95% CO conversion, was achieved at 4bar and 280°C. The membrane was also characterized in terms of permeance and selectivity by means of permeation tests carried out before and after reaction. In addition, permeance and separation factor were also measured during the reaction. Permeance of all species (H2: 9.7–29; CO: 0.3–1.1; CO2: 0.4–1.5nmol/m2 sPa), selectivity (H2/CO, H2/CO2 and H2/N2) ranging from 15 to 40 and separation factors (H2/CO=20–45), showed no dependence on the related permeation driving force. Differences between selectivity and separation factor were registered. Furthermore, no inhibition effects of other gases on the hydrogen flux were observed. The membrane was prepared by the soaking roller procedure depositing a silica layer on a stainless steel support with an intermediate γ-alumina layer. The membrane reactor allowing selective hydrogen permeation presents a good performance exceeding also the equilibrium conversion of a traditional reactor. [Copyright &y& Elsevier]

Abstract: Aim of this work is the theoretical analysis of the potential of integrated membrane systems to recover hydrogen at a very high purity level (with CO content lower than 10ppm), suitable for fuel cell applications. Both polymeric and palladium (Pd) separators as well as a Pd-based membrane reactor have been investigated in order to identify the appropriate operation conditions for each unit taking advantage of the synergic effects of their combination in the design of the whole integrated system. A feed stream containing hydrogen, carbon monoxide and carbon dioxide has been considered. By means of a screening of different materials it has been possible to identify a membrane able to achieve high hydrogen purity and recovery. In order to improve the single membrane stage performance, a two stage system has been analysed taking into account both compression and membrane surface requirements. At fixed stage cut values, higher purity levels are reached in a two membrane stage arrangement with an increase of membrane surface and compression requirements. High hydrogen purities can also be achieved by operating at high feed pressure values. The presence of a membrane reactor to further enhance the hydrogen amount by converting the carbon monoxide has also been investigated. As a low driving force is available (e.g. 5atm), the combination that seems to be the most convenient assumes that the syngas mixture is first fed to the two-stage polymeric membrane unit, then the permeate stream is further treated in a palladium separator (Pd-Sep) while the retentate streams are processed in two palladium membrane reactors (Pd-MRs). On the contrary, at 10atm a single polymeric stage followed by a Pd-Sep and a Pd-MR represents the most adequate solution because a comparable membrane surface is combined to lower compression power and H2 losses. [Copyright &y& Elsevier]

Abstract: The concepts of process intensification and renewable sources use are becoming more popular in process design as a consequence of the shortage of raw materials and of the impact of modern society on the environment. Membrane reactors can combine reaction and separation steps in a single unit, improving the efficiency of operations. Mass and heat transport in a non-isothermal membrane reactor for an exothermic reaction as a function of working temperature, pressure, sweep flow rate, molar feed ratio and feed flow rate of the reagents was investigated by computer simulation. The analysis of the inherent safety aspects of the membrane reactor at different working conditions was developed using the HAZOP technique. [Copyright &y& Elsevier]

In hydrogen production, the syngas streams produced by reformers and/or coal gasification plants contain a large amount of H 2 and CO in need of upgrading. To this purpose, reactors using Pd-based membranes have been widely studied as they allow separation and recovery of a pure hydrogen stream. However, the high cost of Pd-membranes is one of the main limitations for scaling up technology. Therefore, many researchers are now pursuing the possibility of using supported membranes with as thin as possible Pd-alloy layers. In this work, the upgrading of a syngas stream is experimentally investigated in a water gas shift membrane reactor operated in a high temperature range with an ultra-thin supported membrane (3.6 micron-thick). The membrane permeance was measured before and after catalyst packing and also after reaction for 2100 h of operation in total. Membrane reactor performance was evaluated as a function of operating conditions such as temperature, pressure, gas hourly space velocity, feed molar ratio, and sweep gas. A CO conversion significantly higher than the thermodynamics upper limit of a traditional reactor was achieved, even at high gas hourly space velocities and a 25% less reaction volume than that of a traditional reactor was enough to achieve a 90% equilibrium conversion. [ABSTRACT FROM AUTHOR]

Abstract: Pure hydrogen production is of fundamental interest for its use as feed of proton exchange membrane fuel cells. In this work an innovative configuration for a membrane reactor (MR) was proposed: the membrane is located only in the second part of the catalytic bed. This configuration is of particular importance in all the reactions characterized by low kinetics such as, e.g., water gas shift (WGS). Permeation of hydrogen, in the desired direction, takes place when its partial pressure on the reaction side exceeds that on the permeate side. In an MR where the membrane starts at the reactor entrance there is a part of membrane area giving back permeation to the reaction side. The length used in a bad way depends on the kinetics: for fast kinetics reactions (e.g., methane steam reforming) it is very short but is significantly higher for reactions characterized by low kinetics (e.g., WGS). The innovative configuration proposed allows a good exploitation of the whole membrane area for the permeation. This innovative Pd-alloy (60μm thick) MR was used to analyse the WGS reaction at 280–320°C, up to 600kPa, GHSV ranging from 2000h−1 to 4500h−1. A significant reduction of the reaction volume required to achieve the same CO conversion was shown by the novel configuration of the MR with respect to the “typical” one used up to now. [Copyright &y& Elsevier]

Abstract: A new combined method is reported to localize the sites of enzyme immobilization and to determine its catalytic activity on a polymeric capillary membrane reactor. The useful new method resulted from the merging of the classic in situ enzyme activity assay and western blot technique whose both results are easily detectable either at low than at high magnification in light microscopy. β-Glucosidase from olive fruit was selected as enzyme model because of its suitable relevance in the industrial processing of foods, in biotechnology and in pharmaceuticals and for its activity against the synthetic substrate 5-brome-4-chloro-3-indolyl-β-d-glucopyranosyde which develops an insoluble dyed product. The enzyme was physically immobilized within 30kDa cut-off capillary polysulphone membranes and results obtained by means of a polyclonal antibody against β-glucosidase and the synthetic substrate clearly showed a coherent localization of the immobilization enzyme sites and its activity. [Copyright &y& Elsevier]

Abstract: The methanol steam reforming (MSR) reaction to produce hydrogen was studied from an experimental point of view in membrane reactors (MRs). In order to investigate the behaviour of methanol conversion, the influence of the membrane characteristics, the sweep gas flow rate, as well as the influence of the operating temperature and the different flux configurations were analysed. Experimental results, in terms of methanol conversion as well as hydrogen production and gases selectivity in MRs and a traditional reactor (TR), are proposed. A set of values of key parameters to obtain the maximum hydrogen production is given. [Copyright &y& Elsevier]

Abstract: For an efficient membrane bioreactor design, transport phenomena determining the overall mass flux of metabolites, catabolites, cell regulatory factors, and immune-related soluble factors, need to be clarified both experimentally and theoretically. In this work, experiments and calculations aimed at discerning the simultaneous influence of both diffusive and convective mechanisms to the transport of metabolites. In particular, the transmembrane mass flux of glucose, bovine serum albumin (BSA), APO-transferrin, immunoglobulin G, and ammonia was experimentally measured, under pressure and concentration gradients, through high-flux microporous hydrophilic poly-ether-sulphone (PES-HFMs) and poly-sulphone hollow fiber membranes (PS-HFMs). These data were analyzed by means of a model based on the mechanism of capillary pore diffusion, assuming that solute spherical molecules pass through an array of solvent-filled cylindrical pores with a diffusive permeation corrected for friction and steric hindrances. Additionally, resistances to the mass transfer were taken into account. Convective permeation data were discussed in terms of morphological properties of the polymeric membranes, molecular Stokes radius, and solute-membrane interactions according to information given by contact angle measurements. The observed steady-state hydraulic permeance of PS-HFMs was 0.972L/m2 hmbar, about 15.6-fold lower than that measured for PES-HFMs (15.2L/m2 h); in general, PS-HFMs provided a significant hindrance to the transport of target species. Diffusion coefficients of metabolites were found to be similar to the corresponding values in water through PES-HFMs, but significantly reduced through PS-HFMs (, , ). [Copyright &y& Elsevier]