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Biomedical subjects

P Dunnill

Publications and source records attributed to P Dunnill.

At least 19 recordsLinked to original sources
PubMed2004

Doppler optical coherence tomography for measuring flow in engineered tissue.

The engineering of human tissue represents a major paradigm shift in clinical medicine. Early embodiments of tissue engineering are currently being taken forward to the clinic by production methods that are essentially extensions of laboratory manual procedures. However, to achieve the status of routine large-scale clinical practice, automation and scale-out processes are required. This in turn will require the development of reliable on-line monitoring and control systems. This paper examines one demand of crucial importance, namely the real time in vitro monitoring of the flow characteristics through growing tissue since this has a complex interrelationship. Doppler optical coherence tomography (DOCT) is a recently developed imaging technique for studying the rheological properties of tissues in vivo. Capable of non-invasive imaging in real time with high resolution, it is potentially ideal for the continuous monitoring of engineered tissues in vitro. As a base line, the current status of DOCT in vivo is therefore reviewed. This paper also reports the first preliminary use of DOCT in tissue engineering. The application described involves the imaging of a fully developed laminar flow through a combined tissue fabrication/bioreactor with a tissue-engineered construct (substitute blood vessel) in situ.

Bioreactors↗
PubMed2004

The potential of optical coherence tomography in the engineering of living tissue.

The better repair of human tissue is an urgent medical goal and in order to achieve a safe outcome there is a parallel need for sensitive, non-invasive methods of assessing the quality of the engineered tissues and organs prior to surgical implantation. Optical coherence tomography (OCT) can potentially fulfil this role. The current status of OCT as an advanced imaging tool in clinical medicine, developmental biology and material science is reviewed and the parallels to the engineering of living tissue and organs are discussed. Preliminary data are also presented for a tissue engineering bioreactor with in situ OCT imaging. The data suggest that OCT can be utilized as a real time, non-destructive, non-invasive tool to critically monitor the morphology of tissue-engineered constructs during their fabrication and growth.

Animals↗
PubMed2002

Engineering and cell attachment properties of human fibronectin-fibrinogen scaffolds for use in tissue engineered blood vessels.

Tissue engineered constructs reported to date have been prepared primarily from poly(glycolic) acid or collagen scaffolds onto which cells are grown and matured. In this paper we report experimental data to demonstrate the use of a natural, human protein, as a tubular scaffold for vascular grafting. Using a manual and a scalable dip-coating technique we prepared fibronectin-based tubes up to 12 cm in length and up to 3 mm in diameter. The tubes were flexible and their mechanical properties, measured in terms of tensile strength and burst pressure as a function of humidity, demonstrated their suitability as scaffolds for use in vascular grafting, e.g. coronary artery by pass grafting. In vitro tests involved the attachment of endothelial cells pumped under laminar flow conditions through the tube lumen and the adherence of smooth muscle cells on the outer surface of the tubes. These tests, carried out in multiwells, showed that the scaffolds had excellent cell attachment and guidance characteristics.

Journal Article↗
PubMed2002

A comparison of the process issues in expressing the same recombinant enzyme periplasmically in Escherichia coli and extracellularly in Streptomyces lividans.

The choice of a host for the production of a biological molecule will have a significant effect on isolation and purification procedures employed. This paper makes a comparison between the production of a single enzyme, a recombinant alpha-amylase, in Escherichia coli and Streptomyces lividans, on a small scale. It defines the differences in the cultivation and in the isolation stages and also describes the impact of the expression system on later downstream processing steps. At the cultivation stage, the specific productivity of the E. coli in units per gram per hour is four times that of the S. lividans while the total biomass yields are of the same order. The initial volume for downstream processing of S. lividans is six-fold larger and the total protein released into the extracellular medium is three times greater than E. coli, however, the recoverable yield from the E. coli is a fifth of that obtained from the S. lividans and requires three additional stages prior to chromatography. Even with these stages the final specific activity is 64% of the S. lividans. The results indicate the need to consider the whole process when making such comparisons.

Biomass↗
PubMed2001

Prediction of the pilot-scale recovery of a recombinant yeast enzyme using integrated models.

This article describes the rapid prediction of recovery process performance for a new recombinant enzyme product on the basis of a broad portfolio of computer models and highly targeted experimentation. A process model for the recombinant system was generated by linking unit operation models in an integrated fashion, with required parameter estimation and physical property determination accomplished using data from scale-down studies. This enabled the generic modeling framework established for processing of a natural enzyme from bakers' yeast to be applied. An experimental study of the same operations at the pilot scale showed that the process model gave a conservative prediction of recombinant enzyme recovery. The model successfully captured interactions leading to a low overall product yield and indicated the need for further study of precipitate breakage in the feed zone of a disc stack centrifuge in order to improve performance. The utility of scale-down units as an aid to fast model generation and the advantage of integrating computer modeling and scale-down studies to accelerate bioprocess development are highlighted.

Alcohol Dehydrogenase↗
PubMed2001

Wet extrusion of fibronectin-fibrinogen cables for application in tissue engineering.

A method for the wet extrusion of human plasma-derived fibronectin-fibrinogen cables is described. Solutions of fibronectin and fibrinogen with and without sodium alginate and carboxymethylcellulose (CMC) are tested. The rheological properties of the protein solutions changed from Newtonian to shear thinning non-Newtonian in the presence of small quantities of these additives, the apparent viscosity increased, and the extrusion properties of the protein solutions improved. Cables were prepared using a capillary with a diameter of 1 mm and overall length of 18 mm. Cable diameter was reduced to about 0.5 mm by drawing using a series of rollers. Cables prepared with sodium alginate were found to have suitable properties, and those made with CMC were sticky and difficult to handle. Solutions containing no sodium alginate required a minimum total protein concentration of about 70 mg/mL for extrusion. Extruded cables were prepared with solutions containing 140 mg/mL total protein with 12.9 mg/mL alginate (high protein), and 46 mg/mL total protein with 47.6 mg/mL of sodium alginate (high alginate). The mechanical strength of the extruded cables was within the range suitable for application in tissue engineering. Extrusion of the protein solutions into cables was achieved in a coagulation bath. Cables with a mechanical strength of approximately 30 N/mm(2), suitable for wound repair and nerve regeneration applications, were prepared with a coagulation bath containing 0.25 M HCl, 2% CaCl(2) at a pH of <0.9. These cables also had a large average elongation at break of 52%, and showed an increase in cable length after breakage (permanent set) of 20%, demonstrating the potential for drawing the cables down to a fine diameter.

Alginates↗
PubMed2001

Purification of essentially RNA free plasmid DNA using a modified Escherichia coli host strain expressing ribonuclease A.

Regulatory agencies have stringent requirements for the large-scale production of biotherapeutics. One of the difficulties associated with the manufacture of plasmid DNA for gene therapy is the removal of the host cell-related impurity RNA following cell lysis. We have constructed a modified Escherichia coli JM107 plasmid host (JMRNaseA), containing a bovine pancreatic ribonuclease (RNaseA) expression cassette, integrated into the host chromosome at the dif locus. The expressed RNaseA is translocated to the periplasm of the cell, and is released during primary plasmid extraction by alkaline lysis. The RNaseA protein is stable throughout incubation at high pH ( approximately 12-12.5), and subsequently acts to hydrolyse host cell RNA present in the neutralised solution following alkaline lysis. Results with this strain harbouring pUC18, and a 2.4 kb pUC18DeltalacO, show that sufficient levels of ribonuclease (RNase) activity are produced to hydrolyse the bulk of the host RNA. This provides a suitable methodology for the removal of RNA, whilst avoiding the addition of exogenous animal sourced RNase and its associated regulatory requirements.

Animals↗
PubMed2000

The enzymatic transformation of water-insoluble reactants in nonaqueous solvents. Conversion of cholesterol to cholest-4-ene-3-one by a Nocardia sp. Reprinted from Biotechnology and Bioengineering, Vol. XVII, Pages 815-826 (1975).

The rapid conversion of cholesterol to cholestenone by Nocardia in the presence of high proportions of water-immiscible solvent has been demonstrated. At high agitator speeds, the reaction rate was not limited by the rates of transfer of oxygen or cholesterol to the microorganisms. Using 100 g of thawed cells in 200 ml of carbon tetrachloride containing 16% (w/v) cholesterol, at 20 degrees C cholestenone was formed at 7 g/hr. Cells could be separated easily from the organic solvent and reused. After 7 runs (69 hr) the reaction rate had fallen only to half the value for the first run.

Cholesterol↗
PubMed2000

The use of laboratory centrifugation studies to predict performance of industrial machines: studies of shear-insensitive and shear-sensitive materials.

A method for using a bench-top centrifuge is described in order to mimic the recovery performance of an industrial-scale centrifuge, in this case a continuous-flow disc stack separator. Recovery performance was determined for polyvinyl acetate particles and for biological process streams of yeast cell debris and protein precipitates. Recovery of polyvinyl acetate particles was found to be well predicted for these robust particles. The laboratory centrifugation scale-down technique again predicted the performance of the disc stack centrifuge for the recovery of yeast cell debris particles although there was some suggestion of over-prediction at high levels of debris recovery due to the nature of any cell debris aggregates present. The laboratory centrifuge scale-down technique also proved to be an important investigative probe into the extent of shear-induced breakup of shear-sensitive protein precipitate aggregates during recovery in continuous high speed centrifuges. Such breakup can lead to over 10-fold reduction in separator capacity.

Acceleration↗
PubMed2000

Removal of contaminant nucleic acids by nitrocellulose filtration during pharmaceutical-grade plasmid DNA processing.

Pharmaceutical-grade plasmid DNA for use in vaccines and gene therapy requires the development of reproducible and scaleable downstream processes. Shearing of chromosomal DNA at the commencement of the purification results in fragments that are difficult to separate from supercoiled plasmid DNA. Regulatory standards will probably require that the level of chromosomal DNA contamination is kept below 0.01 mg mg(-1) plasmid DNA. This work reports the use of nitrocellulose membranes to decrease chromosomal DNA contamination in plasmid DNA preparations derived from a 450-l bioreactor. Clarified lysates, resuspended PEG precipitates and anion exchange chromatography elutes were filtered through nitrocellulose. In all the cases, chromosomal DNA was selectively retained by the membrane while most supercoiled plasmid DNA was recovered in the filtrate. Contamination levels dropped from over 27% to below 1% as measured by Southern analysis. Under ionic strength conditions equal to or above 1.5 M NaCl, a fraction of the contaminant RNA was also retained by the nitrocellulose membrane.

Blotting, Southern↗
PubMed2000

Biochemical engineering approaches to the challenges of producing pure plasmid DNA.

Plasmid-based genes offer promise for a new generation of vaccines and for gene therapy, but the size and character of plasmids pose new challenges to biochemical engineers. By acknowledging these and using bioprocess-design information based on fundamental studies of the system's properties, it will be possible to create efficient and consistent processes for these materials. This review addresses the purity required, the key issue of the sensitivity of the chromosomal DNA contaminant and larger plasmids to hydrodynamic forces, and the impact of this and other characteristics of plasmids on the recovery and purification of DNA for pharmaceutical purposes.

Biotechnology↗
PubMed1999

Metabolic modeling as a tool for evaluating polyhydroxyalkanoate copolymer production in plants.

The production of polyhydroxyalkanoates in plants is an interesting commercial prospect due to lower carbon feedstock costs and capital investments. The production of poly-(3-hydroxybutyrate) has already been successfully demonstrated in plant plastids, and the production of more complex polymers is under investigation. Using a mathematical simulation model this paper outlines the theoretical prospects of producing the copolymer poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-3HV)] in plant plastids. The model suggests that both the 3HV/3HB ratio and the copolymer production rate will vary considerably between dark and light conditions. Using metabolic control analysis we predict that the beta-ketothiolase predominately controls the copolymer production rate, but that the activity of all three enzymes influence the copolymer ratio. Dynamic simulations further suggest that controlled expression of the three enzymes at different levels may enable desirable changes in both the copolymer production rate and the 3HV/3HB ratio. Finally, we illustrate that natural variations in substrate and cofactor levels may have a considerable impact on both the production rate and the copolymer ratio, which must be taken into account when constructing a production system.

Acetyl-CoA C-Acyltransferase↗
PubMed1998

Time course of SDS-alkaline lysis of recombinant bacterial cells for plasmid release.

SDS-alkaline lysis of recombinant Escherichia coli cell suspensions was carried out in a coaxial cylinder rheometer, and the data were used to establish the time course of lysis reaction. The results of the experiments showed that cell lysis reaction time depended on cell strain but was unaffected by plasmid size and plasmid copy number. The high molecular weight globular proteins and chromosomal DNA were denatured, and the resulting changes in rheometric measurements characterised the denaturation time.

Escherichia coli↗
PubMed1998

Rational engineering of the TOL meta-cleavage pathway

The meta-cleavage pathway of Pseudomonas putida mt-2 was simulated using a biochemical systems simulation developed by Regan (1996). A non-competitive inhibition term for catechol-2,3-dioxygenase (C23O) by 2-OH-pent-2,4-dienoate (Ki = 150 μM) was incorporated into the model. The simulation predicted steady state accumulation levels in the μM range for metabolites pre-meta-cleavage, and in the mM range for metabolites post-meta-cleavage. The logarithmic gains L[V-i, Xj] and L[X-i, Xj] clearly indicated that the pathway was most sensitive to the concentration of the starting substrate, benzoate, and the first enzyme of the pathway, toluate-1, 2-dioxygenase (TO). The simulation was validated experimentally; it was found that the amplification of TO increased the steady state flux from 0.024 to 0.091 (mmol/g cell dwt)/h. This resulted in an increased accumulation of a number of the pathway metabolites (intra- and extracellularly), especially cis-diol, 4-OH-2-oxovalerate, and 4-oxalocrotonate. Metabolic control analysis indicated that C23O was, in fact, the major controling enzymic step of the pathway with a scaled control coefficient of 0.83. The amplification of TO resulted in a shift of some of the control away from C23O. Catechol-2,3-dioxygenase, however, remained as the major controling element of the pathway. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗
PubMed1997

Factors determining more efficient large-scale release of a periplasmic enzyme from E. coli using lysozyme.

Large scale use of lysozyme for periplasmic release has been impeded by the cost of the pure enzyme and its subsequent presence as a contaminant in later downstream processing steps. In this paper, we discuss the use of lysozyme for pilot scale recovery of a periplasmic enzyme from E. coli. The effects of concentration of sucrose, lysozyme and cells on periplasmic enzyme release were examined. Lysozyme concentration can be reduced 5-fold from previous reports and a reduction in sucrose concentration from 20 to 15% (w/v) allows an improvement in centrifugal harvesting by reducing viscosity. High levels of release were still achieved using this technique and further improvements in yield were obtained by optimising other components of the releasing mixture. Results show that some release is still achieved in circumstances where no lysozyme use is possible. Results also indicate that a substantial proportion (up to 70%) of lysozyme remains bound to the cellular debris after its action and is removed with this material.

Escherichia coli↗
PubMed1997

Characterisation of non-porous magnetic chelator supports and their use to recover polyhistidine-tailed T4 lysozyme from a crude E. coli extract.

The use of high capacity micron-sized non-porous magnetic metal chelator adsorbents for the direct recovery of a recombinant metal-binding protein from crude liquors is described. Selectivity and interaction strength of magnetic chelator particles were assessed using a set of native proteins with known behaviour towards commercially available immobilised metal chelate adsorbents. Particles charged with Cu2+ were highly effective in recovering a recombinant histidine-tailed T4 lysozyme fusion protein directly from crude E. coli extracts in a single step. Levels of recovery and purity were high and compared favourably with those achieved by chromatography of pre-clarified extracts on Cu(2+)-IDA Sepharose. The magnetic approach offers advantages such as the avoidance of clarification to prevent fouling of chromatography columns, steps that become especially significant at large scale. By detailed characterisation of the magnetic chelators the practical use of tailed T4 lysozyme for repeated production of periplasmic products is a realistic prospect.

Bacteriophage T4↗
PubMed1996

High speed centrifugal separator for rapid on-line sample clarification in biotechnology.

Sample clarification is a common operation in biochemical analytical methods for removing interfering or unwanted particulates from an analyte sample. Filtration provides one option for removal of particulates. However, in many cases the loss of soluble protein due to filter adsorption is unacceptable and an alternative must be sought. In this paper a microcentrifuge designed to automatically sample, spin, deliver supernatant to an analyser and wash out solids from the bowl is described. The performance of the system is assessed in terms of its clarification efficiency and the time required to achieve satisfactory clarification. Additionally, the effects of different protein precipitating agents on yeast homogenate samples separated using the microcentrifuge are studied where the system is used to deliver supernatant to a flow injection analyser. The paper demonstrates that the microcentrifuge may be used to separate rapidly such samples on a time scale between 10-60 s depending upon the type and size of sample and be successfully used as a component of an at-line monitoring system.

Biotechnology↗