Inhibitory effects of persistent apoptotic cells on monoclonal antibody production in vitro:simple removal of non-viable cells improves antibody productivity by hybridoma cells in culture

Cells undergoing apoptosis in vivo are rapidly detected and cleared by phagocytes. Swift recognition and removal of apoptotic cells is important for normal tissue homeostasis and failure in the underlying clearance mechanisms has pathological consequences associated with inflammatory and auto-immune diseases. Cell cultures in vitro usually lack the capacity for removal of non-viable cells because of the absence of phagocytes and, as such, fail to emulate the healthy in vivo micro-environment from which dead cells are absent. While a key objective in cell culture is to maintain viability at maximal levels, cell death is unavoidable and non-viable cells frequently contaminate cultures in significant numbers. Here we show that the presence of apoptotic cells in monoclonal antibody-producing hybridoma cultures has markedly detrimental effects on antibody productivity. Removal of apoptotic hybridoma cells by macrophages at the time of seeding resulted in 100% improved antibody productivity that was, surprisingly to us, most pronounced late on in the cultures. Furthermore, we were able to recapitulate this effect using novel super-paramagnetic Dead-Cert Nanoparticles to remove non-viable cells simply and effectively at culture seeding. These results (1) provide direct evidence that apoptotic cells have a profound influence on their non-phagocytic neighbors in culture and (2) demonstrate the effectiveness of a simple dead-cell removal strategy for improving antibody manufacture in vitro.

Ultrashort pulse generation from diode laser devices

This thesis presents a detailed, experiment-based study of generation of ultrashort optical pulses from diode lasers. Simple and cost-effective techniques were used to generate high power, high quality optical short pulses at various wavelength windows. The major achievements presented in the thesis is summarised as follows. High power pulses generation is one of the major topics discussed in the thesis. Although gain switching is the simplest way for ultrashort pulse generation, it proves to be quite effective to deliver high energy pulses on condition that the pumping pulses with extremely fast rising time and high enough amplitude are applied on specially designed pulse generators. In the experiment on a grating-coupled surface emitting laser (GCSEL), peak power as high as 1W was achieved even when its spectral bandwidth was controlled within 0.2nm. Another experiment shows violet picosecond pulses with peak power as high as 7W was achieved when the intensive electrical pulses were applied on optimised DC bias to pump on InGaN violet diode laser. The physical mechanism of this phenomenon, as we considered, may attributed to the self-organised quantum dots structure in the laser. Control of pulse quality, including spectral quality and temporal profile, is an important issue for high power pulse generation. The ways to control pulse quality described in the thesis are also based on simple and effective techniques. For instance, GCSEL used in our experiment has a specially designed air-grating structure for out-coupling of optical signals; hence, a tiny flat aluminium mirror was placed closed to the grating section and resulted in a wavelength tuning range over 100nm and the best side band suppression ratio of 40dB. Self-seeding, as an effective technique for spectral control of pulsed lasers, was demonstrated for the first time in a violet diode laser. In addition, control of temporal profile of the pulse is demonstrated in an overdriven DFB laser. Wavelength tuneable fibre Bragg gratings were used to tailor the huge energy tail of the high power pulse. The whole system was compact and robust. The ultimate purpose of our study is to design a new family of compact ultrafast diode lasers. Some practical ideas of laser design based on gain-switched and Q-switched devices are also provided in the end.

Assessment of cell viability in a three-dimensional enzymatically cross-linked collagen scaffold

Microbial transglutaminase (mTGase) is an enzyme that introduces a covalent bond between peptide bound glutamine and lysine residues. Proteins cross-linked in this manner are often more resistant to proteolytic degradation and show increased tensile strength. This study evaluates the effects of mTGase mediated cross-linking of collagen on the cellular morphology, behaviour and viability of murine 3T3 fibroblasts following their seeding into collagen scaffolds. Additionally, cell mediated scaffold contraction, porosity and level of cross-linking of the scaffold has been analysed using image analysis software, scanning electron microscopy (SEM), colorimetric assays, and Fourier transform infrared spectroscopy (FTIR). We demonstrate that the biocompatibility and cellular morphology, when comparing cultures of fibroblasts integrated in mTGase cross-linked collagen scaffolds with the native collagen counterparts, remained unaffected. It has been also elicited that the structural characteristics of collagen have been preserved while introducing enzymatically resistant covalent bonds.

Evolution of non-uniformly seeded warm clouds in idealized turbulent conditions

We present a mean-field model of cloud evolution that describes droplet growth due to condensation and collisions and droplet loss due to fallout. The model accounts for the effects of cloud turbulence both in a large-scale turbulent mixing and in a microphysical enhancement of condensation and collisions. The model allows for an effective numerical simulation by a scheme that is conservative in water mass and keeps accurate count of the number of droplets. We first study the homogeneous situation and determine how the rain-initiation time depends on the concentration of cloud condensation nuclei (CCN) and turbulence level. We then consider clouds with an inhomogeneous concentration of CCN and evaluate how the rain initiation time and the effective optical depth vary in space and time. We argue that over-seeding even a part of a cloud by small hygroscopic nuclei, one can substantially delay the onset and increase the amount of precipitation.

A novel silicon membrane-based biosensing platform using distributive sensing strategy and artificial neural networks for feature analysis

A novel biosensing system based on a micromachined rectangular silicon membrane is proposed and investigated in this paper. A distributive sensing scheme is designed to monitor the dynamics of the sensing structure. An artificial neural network is used to process the measured data and to identify cell presence and density. Without specifying any particular bio-application, the investigation is mainly concentrated on the performance testing of this kind of biosensor as a general biosensing platform. The biosensing experiments on the microfabricated membranes involve seeding different cell densities onto the sensing surface of membrane, and measuring the corresponding dynamics information of each tested silicon membrane in the form of a series of frequency response functions (FRFs). All of those experiments are carried out in cell culture medium to simulate a practical working environment. The EA.hy 926 endothelial cell lines are chosen in this paper for the bio-experiments. The EA.hy 926 endothelial cell lines represent a particular class of biological particles that have irregular shapes, non-uniform density and uncertain growth behaviour, which are difficult to monitor using the traditional biosensors. The final predicted results reveal that the methodology of a neural-network based algorithm to perform the feature identification of cells from distributive sensory measurement has great potential in biosensing applications.

The microbial ecology of cow slurry in vermiculture beds

The suitability of cow slurry as a substrate for vermicomposting by Eisenia fetida was investigated. Particular attention was given to the effects of the earthworm on the decomposition and stabilisation of the slurry; and to the interactions between E. fetida and the microflora of the substrate. Assessment of the chemical and microbiological changes in cow slurry stored under forced aeration, and subsequently in shallow trays, showed that neither method was suitable for the treatment of slurry. A comparison of two methods of vermicomposting showed that top-feeding of slurry was more efficient in promoting earthworm growth and cocoon production than the mixing of slurry with solid materials. Management practices were found to have an important influence on the efficiency of the process. An investigation o:f the effect of E. fetida. on the decomposition of slurry indicated that the presence of this earthworm enhanced the stabilisation of the substrate and increased the plant-available nitrogen content. Specific nutritional interactions were observed between E. fetida and micro-organisms in sand/cellulose microcosms. The earthworms were found to be feeding directly upon the cells of certain micro-organisms. Other species were found to be toxic to E. fetida.. A technique was developed :for the production of axenic E. fetida., and the use of such earthworms in :feeding experiments confirmed the importance of some micro-organisms in earthworm nutrition. The seeding of vermiculture beds with one such micro-organism stimulated earthworm growth and consumption of the substrate. Vermicomposted mixtures of cow slurry and spent mushroom compost were shown to have potential application as casing materials in mushroom cultivation. The findings of this study indicate the suitability of vermicomposting as a method for the stabilisation of intensively-produced cow slurry, and give some indication of the importance of micro-organisms in the nutrition of E. fetida.

Aspects of signal transduction in the gastrointestinal tract

This study was undertaken to increase knowledge of the mechanisms of inter- and intracellular signalling in the gastrointestinal tract. Specific aims were: to use cell lines to elucidate factors affecting growth of gastric cells, to investigate the distribution and aspects of function of isoforms of protein kinase C in a gastric cell line and in the rat gastrointestinal tract and to determine the presence and regulation of nitric oxide synthase in gastrointestinal tissues from the rat and in cell lines. The gastric cancer cell line HGT-1 was used to investigate control of growth. Increases in cell number were found to be dependent on the seeding density of the cells. In cells plated at low density insulin, epidermal growth factor and gastrin all increased cell number. Gastrin produced a bell-shaped dose response curve with a maximum activity at 5nM. No effect of gastrin was apparent in cells plated at high density. α and β isoforms of protein kinase C were found, by immunoblotting procedures, to be widespread in the gastrointestinal tract of the rat, but protein kinase Cε was confined to the gastric mucosa and gastrointestinal smooth muscle. HGT-1 cells contained protein kinase C α and ε but β or γ were not detected. Preincubation of HGT-1 cells for 24h with 1μM phorbol-12,13-dibutyrate down-regulated protein kinase C α but not ε. The inhibition by the activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate (TPA) of the histamine-stimulated increase in cAMP in HGT-1 cells was down regulated by phorbol-12,13-dibutyrate. Inhibition of histamine-stimulation of adenylate cyclase by TPA was Ca2+-dependent and inhibited by the addition of an antibody to protein kinase C α. A role for protein kinase C α in modulating the effect of histamine on adenylate cyclase in HGT-1 cells is suggested. No nitric oxide synthase activity was detected in the gastrointestinal cell lines HGT-l, MKN-45 or CaCo-2. Ca2+-dependent nitric oxide synthase activity was observed in the gastric mucosa and the gastrointestinal smooth muscle from stomach to colon. The gastric: mucosal enzyme was soluble and showed half-maximal activity at 400nM Ca2+. Pretreatment of rats with endotoxin (3mg/kg body weight) induced nitric oxide synthase activity in both jejunal, ileal and colonic mucosa and muscle. A major portion of the induced activity in ileal and colonic mucosa was Ca2+-independent. Nitric oxide synthase activity in a high-density fraction of gastric mucosal cells was inhibited in a dose-dependent fashion by L-nitroarginine, NG-monomethyl-L-arginine, trifluoperazine and L-canavanine (in descending order of potency). Preincubation with okadaic acid and addition of ATPlMg2+ to the homogenisation buffer inhibited enzyme activity, which implies that phosphorylation inhibits gastric mucosal nitric oxide synthase.

Optimization of protein crystallization:the OptiCryst project

Protein crystallization has gained a new strategic and commercial relevance in the postgenomic era due to its pivotal role in structural genomics. Producing high quality crystals has always been a bottleneck to efficient structure determination, and this problem is becoming increasingly acute. This is especially true for challenging, therapeutically important proteins that typically do not form suitable crystals. The OptiCryst consortium has focused on relieving this bottleneck by making a concerted effort to improve the crystallization techniques usually employed, designing new crystallization tools, and applying such developments to the optimization of target protein crystals. In particular, the focus has been on the novel application of dual polarization interferometry (DPI) to detect suitable nucleation; the application of in situ dynamic light scattering (DLS) to monitor and analyze the process of crystallization; the use of UV-fluorescence to differentiate protein crystals from salt; the design of novel nucleants and seeding technologies; and the development of kits for capillary counterdiffusion and crystal growth in gels. The consortium collectively handled 60 new target proteins that had not been crystallized previously. From these, we generated 39 crystals with improved diffraction properties. Fourteen of these 39 were only obtainable using OptiCryst methods. For the remaining 25, OptiCryst methods were used in combination with standard crystallization techniques. Eighteen structures have already been solved (30% success rate), with several more in the pipeline.

3D culture of bone-derived cells immobilised in alginate following light-triggered gelation

Photoreactive liposomes have been exploited as a means of developing 3D tissue constructs. Liposomes formulated using the photosensitive lipid 1,2-bis(4-(n-butyl)phenylazo-4′-phenylbutyroyl)phosphatidylcholine (Bis Azo PC), which undergoes conformational change on stimulation with long wavelength ultraviolet light, were prepared with entrapped CaCl2 before being incorporated into a 4% alginate solution. It was shown that stimulation of the photosensitive lipid using a light emitting diode (LED) (peak emission at 385 nm, dose equivalent to 9 mJ/cm2) caused the release of liposome-entrapped CaCl2, resulting in cross-linking of the alginate solution and immobilisation of bone-derived cells over a range of seeding densities, approximately 97% of which remained viable for periods of up to 14 days in culture. Entrapment volumes of a variety of liposome types were evaluated and interdigitating fusion vesicles were identified as having the highest payload (24%), however the inclusion of cholesterol as a means of shifting Bis Azo PC sensitivity into the visible light wavelengths resulted in an approximately 10-fold reduction in calcium entrapment. This application of light-sensitised liposomes offers the potential to create complex tissue engineering substrates containing cells immobilised in precise locations, in contrast with substrates onto which cells are seeded post-production. © 2007 Elsevier B.V. All rights reserved.

Case based design of knitwear

In the developed world we are surrounded by man-made objects, but most people give little thought to the complex processes needed for their design. The design of hand knitting is complex because much of the domain knowledge is tacit. The objective of this thesis is to devise a methodology to help designers to work within design constraints, whilst facilitating creativity. A hybrid solution including computer aided design (CAD) and case based reasoning (CBR) is proposed. The CAD system creates designs using domain-specific rules and these designs are employed for initial seeding of the case base and the management of constraints. CBR reuses the designer's previous experience. The key aspects in the CBR system are measuring the similarity of cases and adapting past solutions to the current problem. Similarity is measured by asking the user to rank the importance of features; the ranks are then used to calculate weights for an algorithm which compares the specifications of designs. A novel adaptation operator called rule difference replay (RDR) is created. When the specifications to a new design is presented, the CAD program uses it to construct a design constituting an approximate solution. The most similar design from the case-base is then retrieved and RDR replays the changes previously made to the retrieved design on the new solution. A measure of solution similarity that can validate subjective success scores is created. Specification similarity can be used as a guide whether to invoke CBR, in a hybrid CAD-CBR system. If the newly resulted design is suffciently similar to a previous design, then CBR is invoked; otherwise CAD is used. The application of RDR to knitwear design has demonstrated the flexibility to overcome deficiencies in rules that try to automate creativity, and has the potential to be applied to other domains such as interior design.

Fabrication and characterization of macroporous poly(ethylene glycol) hydrogels generated by several types of porogens

Polymer scaffolds play an important role in tissue engineering applications. Poly(ethylene glycol) based hydrogels have received a lot of attention in this field because of their high biocompatibility and ease of processing. However, in many cases they do not exhibit proper tissue invasion and nutrient transport because of their dense structure. In the present work, several approaches were developed and compared to each other to produce interconnected macroporous poly(ethylene glycol) hydrogels by including different types of porogens in the photocrosslinking reaction. The swelling capacity of the resulting hydrogels was analyzed and compared to non-porous hydrogel samples. Moreover, the obtained materials were characterized by means of mechanical properties and porosity using rheometry, scanning electron microscopy, and mercury intrusion porosimetry. Results showed that interconnected and uniform pores were obtained when a porogen template was used during hydrogel fabrication by photocrosslinking. On the other side, when the porogen particles were dispersed into the macromer solution before matrix photocrosslinking the interconnexion was negligible. The templates must be dissolved before the hydrogel's cell-seeding in vitro, while the dispersed porogen can be used in situ in the in vitro seeding tests. Copyright © 2013 Taylor & Francis Group, LLC.

Novel bioactive scaffolds incorporating nanogels as potential drug eluting devices

Big advances are being achieved in the design of new implantable devices with enhanced properties. For example, synthetic porous three-dimensional structures can mimic the architecture of the tissues, and serve as templates for cell seeding. In addition, polymeric nanoparticles are able to provide a programmable and sustained local delivery of different types of biomolecules. In this study novel alternative scaffolds with controlled bioactive properties and architectures are presented. Two complementary approaches are described. Firstly, scaffolds with nanogels as active controlled release devices incorporated inside the three-dimensional structure are obtained using the thermally induced phase separation (TIPS) method. Secondly, a novel coating method using the spraying technique to load these nanometric crosslinked hydrogels on the surface of two-dimensional (2D) and three-dimensional (3D) biodegradable scaffolds is described. The scanning electron microscopy (SEM) images show the distribution of the nanogels on the surface of different substrates and also inside the porous structure of poly-a-hydroxy ester derivative foams. Both of them are compared in terms of manufacturability, dispersion and other processing variables.

Decellularization of bovine corneas for tissue engineering applications

Scaffolds derived from processed tissues offer viable alternatives to synthetic polymers as biological scaffolds for regenerative medicine. Tissue-derived scaffolds provide an extracellular matrix (ECM) as the starting material for wound healing and the functional reconstruction of tissues, offering a potentially valuable approach for the replacement of damaged or missing tissues. Additionally, acellular tissue may provide a natural microenvironment for host-cell migration and the induction of stem cell differentiation to contribute to tissue regeneration. There are a number of processing methods that aim to stabilize and provide an immunologically inert tissue scaffold. Furthermore, these tissue-processing methods can often be applied to xenogenic transplants because the essential components of the ECM are often maintained between species. In this study, we applied several tissue-processing protocols to the cornea in order to obtain a decellularized cornea matrix that maintained the clarity and mechanical properties of the native tissue. Histology, mechanical testing and electron microscopy techniques were used to assess the cell extraction process and the organization of the remaining ECM. In vitro cell seeding experiments confirmed the processed corneas’ biocompatibility.

Human single-donor composite skin substitutes based on collagen and polycaprolactone copolymer

The development and characterization of an enhanced composite skin substitute based on collagen and poly(e-caprolactone) are reported. Considering the features of excellent biocompatibility, easy-manipulated property and exempt from cross-linking related toxicity observed in the 1:20 biocomposites, skin substitutes were developed by seeding human single-donor keratinocytes and fibroblasts alone on both sides of the 1:20 biocomposite to allow for separation of two cell types and preserving cell signals transmission via micro-pores with a porosity of 28.8 ± 16.1 µm. The bi-layered skin substitute exhibited both differentiated epidermis and fibrous dermis in vitro. Less Keratinocyte Growth Factor production was measured in the co-cultured skin model compared to fibroblast alone condition indicating a favorable microenvironment for epidermal homeostasis. Moreover, fast wound closure, epidermal differentiation, and abundant dermal collagen deposition were observed in composite skin in vivo. In summary, the beneficial characteristics of the new skin substitutes exploited the potential for pharmaceutical screening and clinical application.

Multi-wavelength switchable fibre ring laser based on polarisation selective tilted fibre gratings capable of strain and temperature sensing

Using three fibre gratings with excessively tilted structures in the cavity, we have experimentally demonstrated a multiwavelength switchable erbium-doped fibre ring laser system. The three tilted gratings act as in-fibre polariser and polarisation dependent loss filters to induce the polarisation hole burning effect in the cavity for the operation of the laser at single, double, triple and quadruple wavelengths. The laser system has demonstrated good stability under room temperature conditions and also achieved a high degree of polarization (~30dB), high optical signal to noise ratio (up to 63dB) and high side mode suppression (~50dB). The system has also been investigated for temperature and strain sensing by subjecting the seeding fibre Bragg gratings (FBG) to temperature and strain variations. Since the loss band of the polarisation dependent loss filter is broader than the bandwidth of the seeding FBG, the laser output shifts in wavelength with the applied temperature and strain. The fibre ring laser has shown good responses to the temperature and strain, providing sensitivities of approximately 11.7 pm/°C and 0.85pm/µe respectively.