A single mode fibre-optic evanescent wave biosensor.

This paper reports experimental developments in the construction and operation of a single-mode fibre-optic evanescent wave biosensor using an exposed core silica single-mode fibre embedded in a silica block. The device was able to monitor the concentration of a blue dye, Procion Blue MX-G, in overlayers of various refractive indices. The practicality of such a biosensor has been demonstrated with a colorimetric enzyme assay system. Penicillin G in the 0-0.4 mM concentration range was monitored at 633 nm by the decoloration of the starch-iodine reagent when Bacillus cereus penicillinase was immobilized over the exposed core of the monomode fibre.     

Poly(lactic-co-glycolic acid) hollow fibre membranes for use as a tissue engineering scaffold

Mass transfer limitations of scaffolds are currently hindering the development of 3-dimensional, clinically viable, tissue engineered constructs. We have developed a poly(lactide-co-glycolide) (PLGA) hollow fibre membrane scaffold that will provide support for cell culture, allow psuedovascularisation in vitro and provide channels for angiogenesis in vivo. We produced P(DL)LGA flat sheet membranes using 1, 4-dioxane and 1-methyl-2-pyrrolidinone (NMP) as solvents and water as the nonsolvent, and hollow fibre membranes, using NMP and water, by dry/wet- and wet-spinning. The resulting fibres had an outer diameter of 700 micro m and an inner diameter of 250 micro m with 0.2-1.0 micro m pores on the culture surface. It was shown that varying the air gap and temperature when spinning changed the morphology of the fibres. The introduction of a 50 mm air gap caused a dense skin of 5 micro m thick to form, compared to a skin of 0.5 micro m thick without an air gap. Spinning at 40 degrees C produced fibres with a more open central section in the wall that contained more, larger macrovoids compared to fibres spun at 20 degrees C. Culture of the immortalised osteogenic cell line 560pZIPv.neo (pZIP) was carried out on the P(DL)LGA flat sheets in static culture and in a P(DL)LGA hollow fibre bioreactor under counter-current flow conditions. Attachment and proliferation was statistically similar to tissue culture polystyrene on the flat sheets and was also successful in the hollow fibre bioreactor. The P(DL)LGA hollow fibres are a promising scaffold to address the size limitations currently seen in tissue engineered constructs.     

Reduction of extraction times in liquid-phase microextraction

Recently, we introduced a simple and inexpensive disposable device for liquid-phase microextraction (LPME) based on porous polypropylene hollow fibres. In the present paper, extraction times were significantly reduced by an increase in the surface of the hollow fibres. The model compounds methamphetamine and citalopram, were extracted from 2.5 ml of urine, plasma, and whole blood after dilution with water and alkalisation with 125 μl of 2 M NaOH though a porous polypropylene hollow fibre impregnated with hexyl ether and into an aqueous acceptor phase consisting of 0.1 M HCl. Two commercially available hollow fibres, which differed in surface area, wall thickness and internal diameter, were compared. An increase in the contact area of the hollow fibre with the sample solution by a factor of approximately two resulted in reduction in equilibrium times by approximately the same factor. Thus, the model compounds were extracted to equilibrium within 15 min from both urine and plasma, and within 30 min from whole blood. For the first time LPME was utilised to extract drugs from whole blood, and the extracts were comparable with plasma both with regard to sample clean-up and extraction recoveries. Extraction recoveries for methamphetamine and citalopram varied from 60 to 100% using the two fibres and the different matrices.     

Bioanalysis of drugs by liquid-phase microextraction coupled to separation techniques

The demand for automation of liquid-liquid extraction (LLE) in drug analysis combined with the demand for reduced sample preparation time has led to the recent development of liquid-phase microextraction (LPME) based on disposable hollow fibres. In LPME, target drugs are extracted from aqueous biological samples, through a thin layer of organic solvent immobilised within the pores of the wall of a porous hollow fibre, and into an microl volume of acceptor solution inside the lumen of the hollow fibre. After extraction, the acceptor solution is subjected directly to a final analysis either by high performance liquid chromatography (HPLC), capillary electrophoresis (CE), mass spectrometry (MS), or capillary gas chromatography (GC) without any further treatments. Hollow fibre-based LPME may provide high enrichment of drugs and excellent sample clean-up, and probably has a broad application potential within the area of drug analysis. This review focuses on the principle of LPME, and recent applications of three-phase, two-phase, and carrier mediated LPME of drugs from plasma, whole blood, urine, and breast milk.     

Production of recombinant human interleukin-2 with BHK cells in a hollow fibre and a stirred tank reactor with protein-free medium.

For the production of recombinant human interleukin-2 (IL-2) two different culture processes, a 1-2 liter homogeneous stirred bubble-free aerated system and a dense cell hollow fibre bioreactor were compared. Cultivations were carried out with serum- or protein-free medium formulations. In the stirred culture 0.75 mg IL-2 were produced with 1 l of perfused medium at a maximum cell number of 3 X 10(10). The product yield in the hollow fibre module was only 0.23 mg l-1 at a maximum cell number of 6 X 10(10). In contrast to results with hybridoma or EBV-transformed cell lines, in which hollow fibre bioreactors showed comparable efficiency to perfused stirred tank reactors, the tissue-like cell density is disadvantageous as adherent cells tend to stick together leaving insufficient intercellular space for removal of product.     

Immobilisation of pig liver esterase in hollow fibre membranes

Different methods were evaluated to immobilise Pig Liver Esterase (PLE) in hollow fibre membranes. Four covalent bonding techniques (using epoxy, imidazol, amino and carboxylic acid terminal groups) were tested to link the enzyme to microporous nylon membranes. Physical immobilisation was also studied, by entrapment of the enzyme inside the microporous structure of a polysulfone asymmetric ultrafiltration membrane. The entrapment method lead to a higher retention of enzymatic activity for a longer period of time. This technique was selected to be used in a biphasic membrane bioreactor where the microporous hydrophilic membrane, containing the enzyme, is used to separate an aqueous from an organic phase, in which the substrate is dissolved. Different enzyme loading procedures were studied in the biphasic reactor and the resulting axial and radial enzyme distribution in the hollow fibre module were related to the global enzymatic activity.     

The purification of recombinant hepatitis B surface antigen by hollow fibre ultrafiltration membrane diafiltration

Summary Recombinant hepatitis B surface antigen (HBsAg) broth was purified by diafiltration with a hollow fibre ultrafiltration membrane (cut off 100 kDa), by pre—treating the HBsAg broth with enzyme, most of the contaminating proteins was removed and very high recovery ratio of HBsAg was achieved. When HBsAg solution was concentrated using hollow fibre ultrafiltration membrane to 10% of its original volume, the HBsAg was recovered almost completely. Accordingly, membrane purification of HBsAg is a high yield, fast method.     

A long-term stable and adjustable osmotic pump for small volume flow based on principles of phloem loading

An adjustable pump for microfluidics employing principles of osmoregulation analogous to those of phloem loading in plant leaves has been constructed and tested. Volume flow arises in a hollow fibre with vapour-permeable hydrophobic membrane. The fibre is connected to a source chamber filled with salt crystals and saturated salt solution. The source chamber takes up water through a relatively small membrane area and delivers saturated salt solution to one end of the capillary flow path within the hollow fibre. A stationary osmotic gradient is sustained in the hollow fibre lumen by constant input of saturated salt solution and radial osmotic water absorption. The strong temperature dependence of isothermal membrane distillation enables adjustment of the flow rate up to 20 nL/s. The pump provides pulse-free flow of any liquid with constant rate for at least 26 days without recharging the source chamber. Backpressures up to 1 bar decrease the flow rate by less than 4%. The volume delivered at a constant rate is more than 40 times larger than the volume of the source chamber. Osmoregulatory pumps of the described type may be useful for microinfusion, microdialysis and analytical microsystems.     

Reactive extraction of penicillin G in hollow-fiber and hollow-fiber fabric modules

Penicillin G (Pen G) can be rapidly extracted in hollow-fiber liquid-liquid contactors using N-lauryl-N-trialkylmethylamine (Amberlite LA-2) as the extractant. n-Butylacetate is much better than decanol as a diluent for such an extraction, although decanol can give a partition coefficient four times larger. The overall mass transfer coefficient found is a function of aqueous flow on the lumen side of the fiber, and is less dependent on shell-side flow. In backextraction, the overall mass transfer coefficient determined is only one tenth that of the forward extraction, primarily because the hydrophobic hollow fibers used have a high mass transfer resistance under these conditions. The mass transfer experiments show that hollow-fiber extraction of Pen G is competitive with centrifugal extraction. The prospects for extraction of other fermentation products with hollow fibers can be estimated based on the present study.     

A novel design of bioartificial kidneys with improved cell performance and haemocompatibility

Treatment with bioartificial kidneys had beneficial effects in animal experiments and improved survival of critically ill patients with acute kidney injury in a Phase II clinical trial. However, a Phase II b clinical trial failed. This and other results suggested various problems with the current design of bioartificial kidneys. We propose a novel design to improve various properties of device, including haemocompatibility and cell performance. An important feature of the novel design is confinement of the blood to the lumina of the hollow fibre membranes. This avoids exposure of the blood to the non‐haemocompatible outer surfaces of hollow fibre membranes, which usually occurs in bioartificial kidneys. We use these outer surfaces as substrate for cell growth. Our results show that commercial hollow fibre membranes can be directly applied in the bioreactor when human primary renal proximal tubular cells are grown in this configuration, and no coatings are required for the formation of robust and functional renal epithelia. Furthermore, we demonstrate that the bioreactor unit produces significant amounts of interleukins. This result helps to understand the immunomodulatory effects of bioartificial kidneys, which have been observed previously. The novel bioartificial kidney design outlined here and the results obtained would be expected to improve the safety and performance of bioartificial kidneys and to contribute to a better understanding of their effects.     

Preparation of a mesoporous silica quorum quenching medium for wastewater treatment using a membrane bioreactor

Abstract

Various quorum quenching (QQ) media have been developed to mitigate membrane biofouling in a membrane bioreactor (MBR). However, most are expensive, unstable and easily trapped in hollow fibre membranes. Here, a sol-gel method was used to develop a mesoporous silica medium entrapping a QQ bacterial strain (Rhodococcus sp. BH4). The new silica QQ medium was able to remove quorum sensing signalling molecules via both adsorption (owing to their mesoporous hydrophobic structure) and decomposition with an enzyme (lactonase), preventing MBR biofouling without affecting the water quality. It also demonstrated a relatively long life span due to its non-biodegradability and its relatively small particle size (<1.0?mm), which makes it less likely to clog in a hollow fibre membrane module.     

Ultrafiltration of enzyme solutions by means of blood dialyzers

Hollow fibres with a symmetrical pore structure as used in artifical kidneys are tested for other purposes. The modules are shown to be suitable for the processing of enzymes such as thermitase, penicillin acylase, uratoxidase, and phosphatase by means of ultrafiltration and diafiltration. These hollow fibre modules are compared with other modules used in laboratory or in small technical devices. Similar values and material transfer coefficients are calculated.     

Hollow fibre bioreactor for ethanol production: Application to the conversion of lactose by Kluyveromyces fragilis

Kluyveromyces fragilis cells have been packed into the shell side of an industrial size hollow fibre module. The feed was pumped through the tube side under pressure. During continuous, single-pass operation with a synthetic lactose medium containing 50 g l^?1lactose, ethanol productivity was 30–60 g l^?1h^?1at dilution rates of 1–4 h^?1. With 150 g l^?1lactose concentration, the productivity was 100–135 g l^?1h^?1. Productivity was generally lower when cottage cheese whey permeate (45 g l^?1lactose) was used as the feed. Long-term stability of the hollow fibre bioreactor was good, provided adequate care was taken to bleed the gas generated and restrict cell concentration in the shell side.     

Hollow-fibre fermenter using ultrafiltration

Summary A novel bioreactor with hollow fibres was developed to facilitate substrate transfer across membrane walls as well as to retain a continuous cell growth in the shell side. Ultrafiltration was induced through membrane by pressurizing feed solution to the inside of a hollow fibre with inlet and outlet pumps. The ultrafiltrate accumulated outside the hollow fibres was recirculated through a reservoir where a part of solution containing cells and substrate was removed to keep the level of reservoir solution constant. Ethanol production by Saccharomyces cerevisiae was carried out to test the feasibility of this reactor. The productivity of this reactor was compared with that of a continuous stirred tank reactor (CSTR).     

Monitoring of the production of monoclonal antibodies by hybridomas. Part II: Characterization and purification of acid proteases present in cell culture supernatant.

An acid proteolytic activity has been found in cell culture supernatants from long-term cultivations of hybridoma cells in hollow fibre bioreactors using serum free medium. The proteolytic activity has now been further characterized and the main results were: (1) the proteolytic activity showed a maximum around pH 3 and declined essentially to zero at pH 8; (2) the activity was specifically inhibited by pepstatin A; (3) the acid proteases consisted of two sets of closely spaced bands with apparent molecular weights of 40-45K and 90-105K, respectively; (4) the protease bands (40-45K and 90-105K) were reactive with anti-human cathepsin D; (5) the IEP values of the acid proteases ranged from pH 4.55-6.5. Furthermore, IgG incubation with the acid proteases isolated from hybridoma cells yielded fragments similar to those found in serum-free hollow fibre cell culture supernatants. These results indicated that the IgG fragments are the result of degradation by cathepsin D like proteases released after cell death or cell lysis.     

Ultra‐low background Raman sensing using a negative‐curvature fibre and no distal optics

Measuring Raman spectra through an optical fibre is usually complicated by the high intrinsic Raman scatter of the fibre material. Common solutions such as the use of multiple fibres and distal optics are complex and bulky. We demonstrate the use of single novel hollow‐core negative‐curvature fibres (NCFs) for Raman and surface‐enhanced Raman spectroscopy (SERS) sensing using no distal optics. The background Raman emission from the silica in the NCF was at least 1000× smaller than in a conventional solid fibre, while maintaining the same collection efficiency. We transmitted pump light from a 785‐nm laser through the NCF, and we collected back the weak Raman spectra of different distal samples, demonstrating the fibre probe can be used for measurements of weak Raman and SERS signals that would otherwise overlap spectrally with the silica background. The lack of distal optics and consequent small probe diameter (<0.25 mm) enable applications that were not previously possible.      

Equilibrium sampling through membrane based on a single hollow fibre for determination of drug-protein binding and free drug concentration in plasma

The determination of drug-protein binding and free drug concentration in plasma applying the equilibrium sampling through membrane (ESTM) technique has been studied using supported liquid membrane extraction in a single hollow fibre without any membrane carrier. In the extraction setup, the donor phase (plasma or buffer) was placed in the vial, into which was immersed the hollow fibre with the acceptor phase situated in the lumen. This proposed technique was applied to study the drug-protein binding of five local anaesthetics and two antidepressants as model substances, and the influence of the total drug concentration on the drug-protein binding was investigated. The brief theoretical background for determination of the drug-protein binding under equilibrium conditions is described. The developed method shows a new, improved and simple procedure for determination of free drug concentration in plasma and extent of drug-protein binding.     

Culture of primary rat hepatocytes within a flat hollow fibre cassette for potential use as a component of a bioartificial liver support system

Primary rat hepatocytes were cultured in a flat, hollow-fibre cassette, `The Tecnomouse', which provided direct oxygenation and a homogeneous environment for cells within the cassette. Most hollow fibre systems utilise media oxygenators to provide O₂ to cells; in the Tecnomouse cassette, cells are provided with direct oxygenation via gas channels in the silicone membrane surrounding the hollow fibres. Hepatocyte functionality was monitored by following urea production, albumin production and cytochrome P-450 enzyme activities. The system could maintain cells in a viable state and the presence of specific hepatocyte functions including albumin production and cytochrome P-450 activity. Electron microscopy showed aggregated spherical hepatocytes and apparent high extent of necrosis.     

Chromosome organization revealed upon the decondensation of telophase chromosomes inAllium

Chromosomes of root tip cells ofAllium cepa andAllium sativum were studied in early, middle and late telophase to examine the organization of mitotic chromosomes, taking advantage of the naturally occurring chromosome dispersion during the process of decondensation in telophase. Longitudinal and transverse sections of telophase chromosomes viewed under the transmission electron microscope showed that mitotic chromosomes inAllium were composed of helically coiled 400–550 nm chromatin fibres. In some regions of the longitudinal sections, these chromatin fibres were seen to be orientated parallel to one another but formed roughly a right angle to the long axis of the chromosome. In transverse sections, the telophase chromosome appeared to have a hollow centre encircled by the 400–550 nm chromatin fibre which in turn was a hollow tube structure formed by the coiling of a thinner fibre of 170–200 nm. In addition, cross views of chromatin fibres of 170–200 nm and 50–70 nm were also identified in telophase chromosome preparations. These two organizational levels of chromatin fibres also showed a hollow centre. The process of decondensation of telophase chromosomes is described, and some morphological characteristics associated with the activities of chromosome decondensation are analysed. Based on the observations made onAllium chromosomes in this study, various models of chromosome organization are discussed.     

Performance of a β-d-galactosidase hollow fibre reactor

β-d-Galactosidase was immobilized in a hollow fibre ultrafiltration module. The hydrolysis of 2-nitrophenyl β-d-galactopyranoside (ONPG) was significantly affected by enzyme loading, flow rate and substrate concentration. Pretreatment of hollow fibres with a protein was necessary to minimize enzyme inactivation. Residence time distribution studies indicated that the product of the reaction (ONP) was significantly adsorbed by the fibres, which resulted in the reactor taking 10–30 h to achieve steady-state. An equation based on Michaelis-Menten kinetics and a plug-flow model adequately described the performance of the reactor with regard to operating variables, even though some diffusion effects were observed.