Chitosan hollow fiber membranes

Chitosan hollow fibers were produced by wet spinning, taking advantage of the unique rheological properties of highly viscous chitosan solutions in acetic acid. The mechanical and separation properties of hollow fibers were tested. The mechanical properties were determined by measuring tensile force, tensile stress, elongation, and initial elasticity module. The separation properties were specified by determining retention coefficients of particular blood components and determining cut-off of the membrane by the analysis of dextran molecular weight distribution in the feed and permeate using a technique of gel chromatography (GPC)-Shimadzu gel chromatograph.     

Diffusive and hydraulic transfer of solutes through homogeneous membranes

Rates of hydraulic transport of water, solute permeabilities, and sieving coefficients of homogeneous kappa-carrageenan and bovine serum albumin membranes were measured. These values increased with the water content of membranes. The data show good agreement with the predictions based on the pore model.     

Protein adsorption in polysulfone hollow fiber bioreactors used for serum-free mammalian cell culture

The recovery of serum-free medium proteins from poly-sulfone hollow fiber bioreactors (HFBRs) was investigated. More than 99% of the initial transferrin was adsorbed to the hydrophobic hollow fibers within 2 h of HFBR operation. A methodology to minimize transferrin adsorption by pre-adsorption of bovine serum albumin (BSA) was developed. BSA adsorption on suspended cut fibers was virtually complete within 1 h. BSA-coated fibers adsorbed only 5% of the transferrin within 10 days, whereas uncoated cut fibers adsorbed more than 99% of the transferrin within 1 h. An improved HFBR startup procedure, using a BSA-coating step before inoculation, resulted in substantially higher transferrin recovery. Additional factors influenced extracapillary space (ECS) transferrin concentrations. Pronounced downstream polarization of transferrin was observed in the ECS. In addition, the 30-kDa nominal molecular weight cutoff ultrafiltration membranes rapidly leaked transferrin from the ECS to the lumen. (c) 1993 John Wiley & Sons, Inc.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

The effect of intracapillary media feed protocols on hollow fiber cell culture

Summary Two intracapillary (IC) media feed protocols termed media rich and media lean were examined in an effort to understand the effect of this variable on hollow fiber cell cultures. The media rich protocol emphasized a high volume IC media per day (5 liters) containing no serum and a normal amount of extracapillary (EC) media serum (10% v/v). Alternatively, the media lean protocol used up to 1.0 liter of IC media per day containing 5% v/v serum and increased EC media serum (20% v/v). Both protocols produced substantial amounts of antibody in 25 days using HFN7.1 hybridoma cells (ATCC CRL 1606), however the media rich protocol produced twice as much antibody as the media lean protocol. The metabolism of the cells was dramatically different as measured by glucose uptake rate (GUR) with media lean cells having a six-fold lower GUR. Our results indicate that the media rich protocol is useful for producing larger amounts of antibody in a short time frame. The media lean protocol may be considered when the production costs of antibody, particularly media and serum, is the overriding concern.     

Development of optimized transfectoma cell lines for production of chimeric antibodies in hollow fiber cell culture systems

Methods for the selection of transfectoma cells that express large quantities of mouse-human chimeric antibodies have been develped. SP2/0 mouse myeloma cells were transfected with pSV2-gpt and pSV2-neo based immunoglobulin expression vectors. Double transfectants were selected using the xanthine-guanine phosphoribosyl transferase (gpt)and the neomycin (neo) selection marker genes. ELISA-based screening of transfectoma clones resulted in the isolation of IgG-producing transfectomas. Introduction of the kappa light-chain 3'-enhancer into the light-chain expression vector significantly increased immunoglobulin expression, but only when the enhancer was located at its physiological site, 9 kb downstream of the kappa constant region exon. With some of the transfectomas, final yields of up to 80 mg/L of chimeric IgG were obtained in conventional flask cultures using serum-free growth medium. A pilot-scale AcuSyst Maximizer hollow fiber cell culture system was used for the production of gram amounts of chimeric IgG. Results obtained with different transfectoma clones in conventional culture were not fully predictive for yields in the hollow fiber system. In contrast, differences in productivity between individual clones in the laboratory-scale Tecnomouse cell culture unit were comparable with those in the Maximizer system. Up to 200 mg of chimeric IgG were produced per day in one Maximizer bioreactor. (c) 1995 John Wiley & Sons, Inc.     

A theoretical and experimental evaluation of a novel radial-flow hollow fiber reactor for mammalian cell culture

A hollow fiber perfusion reactor constructed from pairs of concentric fibers forming a thin annular space is analyzed theoretically in terms of mass transfer resistances, and is shown experimentally to support the growth of an anchorage-dependent cell line in high-density culture. Hollow fiber perfusion reactors described in the literature typically employ a perfusion pathlength much greater than the distance that could be supported by diffusion alone, and analyses of these reactors typically incorporate the assumption of uniform perfusion throughout the cell mass despite many reported observations of inhomogeneous cell growth in perfusion reactors. The mathematical model developed for the annular reactor predicts that the metabolism of oxygen, carbon substrates, and proteins by anchorage-dependent cells can be supported by the reactor even in the absence of perfusion. The implications of nonuniform cell growth in perfusion reactors in general is discussed in terms of nutrient distribution. In the second part of the paper, the growth and metabolism of the mouse adrenal tumor line Y-1 in flask culture and in the annular reactor are compared. The reactor is shown to be a promising means for culturing anchorage-dependent cells at high density.List of Symbols c mol/dm³ substrate concentration - D _mm²/s effective diffusivity of substrate in the membrane - D _tm²/s effective diffusivity of substrate in the cell region - L _pm²s/kg hydraulic permeability of fiber - Pe _m Peclet number for membrane transport, _wR₁/D _m - Pe _t Peclet number for transport through cell mass, v _wR₂/D _t - Q mol/m³s zero-order consumption rate of substrate per unit volume of cell mass - r m radial distance from centerline of fiber lumen - R ₁, R ₂ m inner and outer radii of inner annular fiber (Fig. 1) - R ₃, ₄ m inner and outer radii of outer annular fiber (Fig. 1) - v _wm/s fluid velocity through the fiber wall at R ₁ - fraction of shell side filled with cells - dimensionless radial distance, R ₃/R₁ - dimensionless radial distance, R ₂/R ₁ - cm² hydraulic conductivity - viscosity - ², Thiele modulus - dimensionless radial distance, R ₄/R ₁     

Theoretical analysis of the effect of convective flow on solute transport and insulin release in a hollow fiber bioartificial pancreas

The bioartificial pancreas, in which transplanted pancreatic tissue or isolated cells are cultured on a hollow fiber membrane, is an attractive approach to restore physiologic insulin delivery in the treatment of diabetes. Insulin response in prototype devices has been unacceptable due to the large mass transport limitations associated with the membrane and the surrounding shell region. Although available theoretical analyses provide some insight into the combined effects of transport and reaction in the bioartificial pancreas, they cannot quantitatively account for the effects of convective recirculation flow, complex intrinsic insulin secretory kinetics, and non-uniform distribution of pancreatic cells. We have developed a detailed model for glucose and insulin transport and insulin secretion in the hollow fiber bioartificial pancreas based on the solution of the mass and momentum conservation equations describing flow and transport in the lumen, matrix, and shell. Model predictions are in good agreement with literature data obtained in a hollow fiber device with minimal radial convective flow. Although no quantitative data are available for a device with significant radial convection, model simulations demonstrate that convective recirculation flow can dramatically improve insulin response, allowing the device to accurately capture the bi-phasic insulin secretion characteristic of the normal physiologic response. Results provide fundamental insights into the coupling between kinetics and transport in the hollow fiber system and a rational basis for the design of clinical devices.     

Carbon dioxide transport through membranes

Several membrane channels, like aquaporin-1 (AQP1) and the RhAG protein of the rhesus complex, were hypothesized to be of physiological relevance for CO(2) transport. However, the underlying assumption that the lipid matrix imposes a significant barrier to CO(2) diffusion was never confirmed experimentally. Here we have monitored transmembrane CO(2) flux (J(CO2)) by imposing a CO(2) concentration gradient across planar lipid bilayers and detecting the resulting small pH shift in the immediate membrane vicinity. An analytical model, which accounts for the presence of both carbonic anhydrase and buffer molecules, was fitted to the experimental pH profiles using inverse problems techniques. At pH 7.4, the model revealed that J(CO2) was entirely rate-limited by near-membrane unstirred layers (USL), which act as diffusional barriers in series with the membrane. Membrane tightening by sphingomyelin and cholesterol did not alter J(CO2) confirming that membrane resistance was comparatively small. In contrast, a pH-induced shift of the CO(2) hydration-dehydration equilibrium resulted in a relative membrane contribution of about 15% to the total resistance (pH 9.6). Under these conditions, a membrane CO(2) permeability (3.2 +/- 1.6 cm/s) was estimated. It indicates that cellular CO(2) uptake (pH 7.4) is always USL-limited, because the USL size always exceeds 1 mum. Consequently, facilitation of CO(2) transport by AQP1, RhAG, or any other protein is highly unlikely. The conclusion was confirmed by the observation that CO(2) permeability of epithelial cell monolayers was always the same whether AQP1 was overexpressed in both the apical and basolateral membranes or not.     

Enzymatic synthesis of glucuronides using lipophilic hollow fiber membranes

A lipophilic hollow fiber membrane preparation was used for the enzymatic glucuronidation of lipophilic aromatic compounds. A crude solubilized microsomal enzyme preparation was circulated on the external side of the lipophilic membrane while the phenol containing buffer solution was circulated through the internal side of the hollow fiber membrane. Phenols, which accumulate in and penetrate the lipophilic membrane, were converted by UDP-glucuronyltransferase to the corresponding glucuronides. During this process the lipophilic compounds are converted to hydrophilic substances, which are not able to rediffuse through the lipophilic membrane into the donor side of the hollow fiber module. The produced glucuronide is separated by means of a coupled dialysis with a module of hydrophilic surface (cellulose acetate), while the enzyme protein is retained.On the stripping side of the dialysing module the glucuronide can be separated by solid phase extraction (Lichroprep RP-18) while a continuous substitution of cofactor into this compartment is possible. UDPGA follows its own concentration gradient and migrates into the enzymatic mixture, where it is utilized. This new technique using hollow fiber modules offers completely new possibilities for long-term high-capacity, highly specific glucuronidation of phenolic compounds. Fields of application are not only the economical production of special glucuronides, but also the specific elimination of phenols from waste fluids or from serum and blood of patients.For the production of glucuronides by this technique the use of highly purified enzymes is not essential. Cheap crude enzyme preparations are quite adequate for an optimal reaction. Using a crude enzyme preparation with a specific batch activity of 13 nMol/min per mg of protein, the activity in the reactor system was observed to be 4.6 nMol/min of 2-naphtol glucuronide formed per mg of protein. This corresponds to 3.6 nMol/h of product formed per mg of protein per cm² of hollow fiber surface.Using a membrane surface of 0.5 m² the production of 18 mMol of glucuronide per h and mg protein can be achieved.     

Membrane transport of anandamide through resealed human red blood cell membranes

The use of resealed red blood cell membranes (ghosts) allows the study of the transport of a compound in a nonmetabolizing system with a biological membrane. Transmembrane movements of anandamide (N-arachidonoylethanolamine, arachidonoylethanolamide) have been studied by exchange efflux experiments at 0 degrees C and pH 7.3 with albumin-free and albumin-filled human red blood cell ghosts. The efflux kinetics is biexponential and is analyzed in terms of compartment models. The distribution of anandamide on the membrane inner to outer leaflet pools is determined to be 0.275 +/- 0.023, and the rate constant of unidirectional flux from inside to outside is 0.361 +/- 0.023 s(-1). The rate constant of unidirectional flux from the membrane to BSA in the medium ([BSA]o) increases with the square root of [BSA]o in accordance with the theory of an unstirred layer around ghosts. Anandamide passed through the red blood cell membrane very rapidly, within seconds. At a molar ratio of anandamide to BSA of <1, membrane binding of anandamide increases with increasing temperatures between 0 degrees C and 37 degrees C, and the equilibrium dissociation constants are in the nanomolar range. The nature of membrane binding and the mechanism of membrane translocation are discussed.     

Blocking phenomena and charge transport through membranes

Summary Substances which uncouple oxidative phosphorylation in mitochondrial membranes usually increase the electrical conductivity of synthetic bimolecular phospholipid membranes. Among these uncouplers is a group characterized chemically as weak acids. For this group the conductivity of synthetic membranes, when measured versus pH at fixed uncoupler concentration, shows a maximum at a pH approximately equal to the pK value of the uncoupler used. Corresponding maxima in membrane electrical potential arising from ion concentration gradients are also observed. To explain such phenomena a model is proposed which assumes charge transport by the direct transfer of either protons or anions of the uncoupler between binding sites located on the membrane boundaries. A fixed surface density of such sites is assumed. The transfer of an ion requires both its presence on an initiating site and the availability of a terminal site which is not already occupied by an ion of the same species. Failure to satisfy both criteria leads to blockage of current flow at both low and high concentrations of the transported ion.On sabbatical leave for the academic year 1968–69 from the University of California, Riverside, California, USA.     

Model of oxygen transport limitations in hollow fiber bioreactors

Axial and radial oxygen depletion are believed to be critical scale-limiting factors in the design of cell culture hollow fiber bioreactors. A mathematical analysis of oxygen depletion has been performed in order to develop effectiveness factor plots to aid in the scaling of hollow fiber bioreactors with cells immobilized in the shell-side. Considerations of the lumen mass transport resistances and the axial gradients were added to previous analyses of this immobilization geometry. An order of magnitude analysis was used to evaluate the impact of the shell-side convective fluxes on the oxygen transport. A modified Thiele modulus and a lumen and membrane resistance factor have been derived from the model. Use of these terms in the effectiveness factor plots results in a considerable simplification of the presentation and use of the model. Design criteria such as fiber dimensions and spacing, reactor lengths, and recycle flow rates can be selected using these plots. Model predictions of the oxygen limitations were compared to experimental measurements of the axial cell distributions in a severely oxygen limited hollow fiber bioreactor. Despite considerable uncertainty in our parameters and nonidealities in hollow fiber geometry, the cell distribution correlated well with the modeling results.     

Secretome analysis using a hollow fiber culture system for cancer biomarker discovery

Secreted proteins, collectively referred to as the secretome, were suggested as valuable biomarkers in disease diagnosis and prognosis. However, some secreted proteins from cell cultures are difficult to detect because of their intrinsically low abundance; they are frequently masked by the released proteins from lysed cells and the substantial amounts of serum proteins used in culture medium. The hollow fiber culture (HFC) system is a commercially available system composed of small fibers sealed in a cartridge shell; cells grow on the outside of the fiber. Recently, because this system can help cells grow at a high density, it has been developed and applied in a novel analytical platform for cell secretome collection in cancer biomarker discovery. This article focuses on the advantages of the HFC system, including the effectiveness of the system for collection of secretomes, and reviews the process of cell secretome collection by the HFC system and proteomic approaches to discover cancer biomarkers. The HFC system not only provides a high-density three-dimensional (3D) cell culture system to mimic tumor growth conditions in vivo but can also accommodate numerous cells in a small volume, allowing secreted proteins to be accumulated and concentrated. In addition, cell lysis rates can be greatly reduced, decreasing the amount of contamination by abundant cytosolic proteins from lysed cells. Therefore, the HFC system is useful for preparing a wide range of proteins from cell secretomes and provides an effective method for collecting higher amounts of secreted proteins from cancer cells. This article is part of a Special Issue entitled: An Updated Secretome.     

An intercalated-spiral wound hollow fiber bioreactor for the culture of mammalian cells

Summary A dual circuit bioreactor is developed; it consists of two intercalated-spiral sets of hollow fibers with alternated dead-ends. The construction is relatively simple, but guarantees uniform distribution of the arterial and venous circuits. Data are presented to illustrate the ability of the bioreactor to culture a human lymphoblastoid B-cell line.     

Protein A immunoaffinity hollow fiber membranes for immunoglobulin G purification: experimental characterization

Immunoaffinity adsorption is increasingly used for protein purification and medical applications. Synthetic membranes have advantages as support matrices in comparison to conventional bead supports because they are not compressible and they eliminate internal diffusion limitations. The goal of this study was to explore in detail the performance of microporous hollow fibers composed of modified polysulfone to which protein A was immobilized for adsorption of human IgG. The internal matrix was characterized by scanning electron microscopy. The binding equilibrium constant was measured using both static and dynamic methods. Break-through curves up to ligand saturation were measured and used to study the effects of IgG concentration, presence of contaminant albumin, flow direction, flow mode, and especially filtrate flow rate and maximum IgG binding capacity. The highest binding capacities studied were comparable with that attainable with bead matrices. All of the breakthrough curves could be represented on a single figure when plotted versus the dimensionless relative throughput (the mass of IgG loaded on the membrane divided by the mass that would be bound when the entire fiber is in equilibrium with the feed concentration), and the effect of operating variables on the position and shape of the individual breakthrough curves could be understood in terms of a dimensional performance parameter (the product of membrane volume and maximum binding capacity divided by the filtrate flow rate). The best breakthrough curves were obtained with the highest values of the performance parameter. Based on the results, membranes as solid supports for immunoadsorption can be a useful alternative to the use of traditional columns for protein separations. (c) 1995 John Wiley & Sons, Inc.