The distributions of methylene and ether bridges have been shown to impact the mechanical propert... more The distributions of methylene and ether bridges have been shown to impact the mechanical properties of phenolic resin. This work demonstrates the ability of the novel SEM based technique, secondary electron hyperspectral imaging (SEHI), to characterise and map methylene and ether bridges within phenolic resin at the nanoscale. † Electronic supplementary information (ESI) available. See
The evaluation of novel photosensitizers (PSs) for photodynamic therapy (PDT) is difficult due to... more The evaluation of novel photosensitizers (PSs) for photodynamic therapy (PDT) is difficult due to the limitations of two-dimensional cell culture and multiple parameters (dose, light intensity, uptake time), which complicate progression to in vivo experiments and clinical translation. Three-dimensional (3D) cell culture models like multicellular cancer tumor spheroids (MCTS) show great similarities to in vivo avascular tumor conditions, improving the speed and accuracy of screening novel compounds with various treatment combinations. In this study, we utilize C8161 human melanoma spheroids to screen PDT treatment combinations using protoporphyrin IX (PpIX) and drug-loaded carbon dot (CD) conjugates PpIX-CD and PpIX@CD at ultralow fluence values (<10 J/cm 2). Conjugates show equivalent light-induced damage to PpIX from 1 μg/mL with significantly less dark cytotoxicity up to 72 h after exposure, shown by LDH release and dsDNA content. Fractionated treatments, carried out by dividing light exposure with 24 h intervals, demonstrate an enhanced PDT effect compared to single exposure at equal concentrations. Light sheet fluorescence microscopy combined with live/dead staining demonstrates that spheroids sustain extensive damage after PDT, with PpIX and PpIX-CD showing improved uptake compared to PpIX@CD. We show that PDT parameter screening can be carried out using a low-cost and convenient combination of assays to improve the efficiency of evaluating novel compounds.
Two-photon active Graphene Quantum Dots (GQDs) are obtained from extracts of the neem root. These... more Two-photon active Graphene Quantum Dots (GQDs) are obtained from extracts of the neem root. These biocompatible GQDs are found to be suitable for structured illumination microscopy. Two-photon microscopy ensured lysosome...
Enhanced image contrast in biological second harmonic imaging microscopy (SHIM) has previously be... more Enhanced image contrast in biological second harmonic imaging microscopy (SHIM) has previously been reported via quantitative assessments of forward- to epi-generated signal intensity ratio and by polarization analysis. Here we demonstrate a new form of contrast: the material-specific, wavelength-dependence of epi-generated second harmonic generation (SHG) excitation efficiency, and discriminate collagen and myosin by ratiometric epi-generated SHG images at 920 nm and 860 nm. Collagen shows increased SHG intensity at 920 nm, while little difference is detected between the two for myosin; allowing SHIM to characterize different SHG-generating components within a complex biological sample. We propose that momentum-space mapping of the second-order non-linear structure factor is the source of this contrast and develop a model for the forward and epi-generated SHG wavelength-dependence. Our model demonstrates that even very small changes in the assumed material fibrillar structure can p...
International Journal of Polymeric Materials and Polymeric Biomaterials
To see the final version of this work please visit the publisher's website. Access to the publish... more To see the final version of this work please visit the publisher's website. Access to the published online version may require a subscription.
Alterations in quantity or architecture of elastin and collagen fibres are associated with some b... more Alterations in quantity or architecture of elastin and collagen fibres are associated with some blood vessel pathologies. Also some medical interventions such as endovascular catheterization have the potential to damage blood vessels. This study reports the use of porcine aorta as a model system for studying the physical impact of catheters on vasculature, in conjunction with non-invasive imaging techniques to analyse collagen and elastin fibre organization and assess load-induced changes. Porcine aorta was exposed to frictional trauma and elastin and collagen fibre orientation evaluated by destructive, histochemical methods and non-invasive imaging. The latter allowed the immediate impact of force on fibre orientation and fibre recovery to be evaluated longitudinally. In normal aorta, elastin fibres are aligned at the surface, but become less aligned with increasing depth, showing no alignment by ~30 µm. Collagen fibres meanwhile appear aligned down to a depth of 35 µm. Changes in collagen and elastin fibre orientation in healthy pig aorta were detected by conventional destructive histology within 5 minutes of application of a sliding 10N load, while lesser loads had less impact. Good recovery of fibre orientation was observed within 20 minutes. Non-invasive imaging of ex vivo aorta tissue provides a good indication of the extent of fibre reorganization following frictional stress, at loads similar to those encountered during medical interventions such as catheterization. These results indicate that tissue deformation can occur from these procedures, even in healthy tissue, and highlight the potential for the development of an in vivo probe capable of monitoring vascular changes in patients.
Solid tumours display varied oxygen levels and this characteristic can be exploited to develop ne... more Solid tumours display varied oxygen levels and this characteristic can be exploited to develop new diagnostic tools to determine and exploit these variations. Oxygen is an efficient quencher of emission of many phosphorescent compounds, thus oxygen concentration could in many cases be derived directly from relative emission intensity and lifetime. In this study, we extend our previous work on phosphorescent, low molecular weight platinum(II) complex as an oxygen sensing probe to study the variation in oxygen concentration in a viable multicellular 3D human tumour model. The data shows one of the first examples of non-invasive, real-time oxygen mapping across a melanoma tumour spheroid using one-photon phosphorescence lifetime imaging microscopy (PLIM) and a small molecule oxygen sensitive probe. These measurements were quantitative and enabled real time oxygen mapping with high spatial resolution. This combination presents as a valuable tool for optical detection of both physiological and pathological oxygen levels in a live tissue mass and we suggest has the potential for broader clinical application. The use of metal complexes as dyes and probes for emission-based cellular imaging has developed into a vibrant area of research over the past decade 1-3. This emerging class of probes offers photo-physical properties that differ to a range of commercially available organic probes, and is enabling a new range of technologies to be explored. Notably, recent advances in optics and electronics, combined with the long emission lifetimes typical for transition metal complexes (from hundreds of nanoseconds to microseconds), has resulted in the emergence of multiphoton microsecond lifetime mapping techniques, such as Phosphorescence Lifetime Imaging Microscopy (PLIM) 4, 5 and Time-Resolved Emission Imaging Microscopy (TREM) 4. Luminescent transition metal complexes typically emit from a triplet excited state. Although transitions between states of a different spin are formally forbidden, intersystem crossing to the triplet state and subsequent relaxation to the ground state via phosphorescence are facilitated in transition metal complexes by the high spin orbit coupling constant associated with the heavy metal atom. The forbidden nature of the phosphorescence transition, results in a slow rate of emission; hence phosphorescence lifetimes are typically the order of hundreds of nanoseconds to microseconds. It is well documented that molecular oxygen quenches such triplet emitters and that the rate of quenching is dependent on oxygen concentration 5-10. One application of phosphorescent emitters in biological imaging is in oxygen detection, where more sensitive, non-invasive methods are in demand. The combination of phosphorescence quenching and high-resolution lifetime imaging is a powerful approach for non-invasive, real-time hypoxia detection and oxygen quantification. Oxygen quantification in biological systems is primarily focused on the use of large platinum and palladium porphyrins. These compounds display long emission lifetimes (typically 40-60 µs), which typically decreases
A recombinant Chinese hamster ovary (CHO) cell line making human interferon-? (IFN-y) was grown i... more A recombinant Chinese hamster ovary (CHO) cell line making human interferon-? (IFN-y) was grown in 12-L stirred tank fermentors in three batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition t o cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate structures attached to each glycosylation site of IFN-y w a s achieved using matrix-assisted laser desorption m a s s spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. Complex biantennary oligosaccharides (particularly Gal,GlcNAc,Man, which was core 1x1-6 fucosylated at Asn,, but not at Asn,,) were most prevalent at both glycosylation sites. However, considerable microheterogeneity arising from the presence of triantennary and truncated glycan structures w a s also observed. The proportion of t h e d o m i n a n t core glycan structure (Gal,GlcNAc,Man, 2 Fuc,) decreased by 1 5 2 6 % during batch culture, with increases in the proportion of oligomannose and truncated glycans over the s a m e time period. Prolonged culture resulting from an extended lag phase led to further accumulation of oligomannose and truncated structures, reaching up to 52% of total glycans attached t o Asn,, by 240 h of culture. The implications of these glycosylation changes for optimizing the time for harvesting cell cultures, and for the clearance of recornbinant therapeutic products in vivo are discussed.
EPITOPE DETERMINATION FOR ANTIBODIES RAISED AGAINST RECOMBINANT HUMAN INTERFERON-GAMMA Andrew D. ... more EPITOPE DETERMINATION FOR ANTIBODIES RAISED AGAINST RECOMBINANT HUMAN INTERFERON-GAMMA Andrew D. Hooker', Nicola H. Green1, David C. James, 1 Philip G. Strange1, Anthony J. Baines1, Alan T. Bull1 and Nigel Jenkins2. 'Dept of Biosciences, ...
Background Polypropylene mesh used as a mid-urethral sling is associated with severe clinical com... more Background Polypropylene mesh used as a mid-urethral sling is associated with severe clinical complications in a significant minority of patients. Current in vitro mechanical testing shows that polypropylene responds inadequately to mechanical distension and is also poor at supporting cell proliferation. Aims and Objectives Our objective therefore is to produce materials with more appropriate mechanical properties for use as a sling material but which can also support cell integration. Methods Scaffolds of two polyurethanes (PU), poly-L-lactic acid (PLA) and co-polymers of the two were produced by electrospinning. Mechanical properties of materials were assessed and compared to polypropylene. The interaction of adipose derived stem cells (ADSC) with the scaffolds was also assessed. Uniaxial tensiometry of scaffolds was performed before and after seven days of cyclical distension. Cell penetration (using DAPI and a fluorescent red cell tracker dye), viability (AlamarBlue assay) and total collagen production (Sirius red assay) were measured for ADSC cultured on scaffolds. Results Polypropylene was stronger than polyurethanes and PLA. However, polypropylene mesh deformed plastically after 7 days of sustained cyclical distention, while polyurethanes maintained their elasticity. Scaffolds of PU containing PLA were weaker and stiffer than PU or polypropylene but were significantly better than PU scaffolds alone at supporting ADSC.
This work reports the synthesis and characterization of thermoresponsive, stretchable, biodegrada... more This work reports the synthesis and characterization of thermoresponsive, stretchable, biodegradable and biocompatible poly(glycerol sebacate)-based polyurethane hydrogels.
This book is focused on the optical techniques that can be applied to regenerative medicine, incl... more This book is focused on the optical techniques that can be applied to regenerative medicine, including optical coherence tomography, acousto-optic imaging, Raman spectroscopy, machine vision, polarized light imaging, fibre optic sensors, second harmonic generation, multi-photon microscopy, coherent anti-Stokes Raman scattering, and polarized light imaging. It covers applications both in fundamental research and in the regenerative medicine industry for tissue engineering products. Each chapter gives an overview of a particular technique, its advantages and limitations in terms of structural and functional information provided, and examples of applications in regenerative medicine. The book provides a practical guide to the most appropriate techniques for a given application. It also offers a summary of major recent advances, such as use of machine vision for tracking growth of three-dimensional constructs in bioreactors and polarization sensitive optical coherence tomography for mon...
EPITOPE DETERMINATION FOR ANTIBODIES RAISED AGAINST RECOMBINANT HUMAN INTERFERON-GAMMA Andrew D. ... more EPITOPE DETERMINATION FOR ANTIBODIES RAISED AGAINST RECOMBINANT HUMAN INTERFERON-GAMMA Andrew D. Hooker', Nicola H. Green1, David C. James, 1 Philip G. Strange1, Anthony J. Baines1, Alan T. Bull1 and Nigel Jenkins2. 'Dept of Biosciences, ...
A recombinant Chinese hamster ovary (CHO) cell line making human interferon-? (IFN-y) was grown i... more A recombinant Chinese hamster ovary (CHO) cell line making human interferon-? (IFN-y) was grown in 12-L stirred tank fermentors in three batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition t o cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate structures attached to each glycosylation site of IFN-y w a s achieved using matrix-assisted laser desorption m a s s spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. Complex biantennary oligosaccharides (particularly Gal,GlcNAc,Man, which was core 1x1-6 fucosylated at Asn,, but not at Asn,,) were most prevalent at both glycosylation sites. However, considerable microheterogeneity arising from the presence of triantennary and truncated glycan structures w a s also observed. The proportion of t h e d o m i n a n t core glycan structure (Gal,GlcNAc,Man, 2 Fuc,) decreased by 1 5 2 6 % during batch culture, with increases in the proportion of oligomannose and truncated glycans over the s a m e time period. Prolonged culture resulting from an extended lag phase led to further accumulation of oligomannose and truncated structures, reaching up to 52% of total glycans attached t o Asn,, by 240 h of culture. The implications of these glycosylation changes for optimizing the time for harvesting cell cultures, and for the clearance of recornbinant therapeutic products in vivo are discussed.
Invasion of melanoma cells from the primary tumor involves interaction with adjacent tissues and ... more Invasion of melanoma cells from the primary tumor involves interaction with adjacent tissues and extracellular matrix. The extent of this interaction is not fully understood. In this study Raman spectroscopy was applied to cryo-sections of established 3D models of melanoma in human skin. Principal component analysis was used to investigate differences between the tumor and normal tissue and between the peri-tumor area and the normal skin. Two human melanoma cells lines A375SM and C8161 were investigated and compared in 3D melanoma models. Changes were found in protein conformations and tryptophan configurations across the entire melanoma samples, in tyrosine orientation and in more fluid lipid packing only in tumor dense areas, and in increased glycogen content in the peri-tumor areas of melanoma. Raman spectroscopy revealed changes around the perimeter of a melanoma tumor as well as detecting differences between the tumor and the normal tissue.
The distributions of methylene and ether bridges have been shown to impact the mechanical propert... more The distributions of methylene and ether bridges have been shown to impact the mechanical properties of phenolic resin. This work demonstrates the ability of the novel SEM based technique, secondary electron hyperspectral imaging (SEHI), to characterise and map methylene and ether bridges within phenolic resin at the nanoscale. † Electronic supplementary information (ESI) available. See
The evaluation of novel photosensitizers (PSs) for photodynamic therapy (PDT) is difficult due to... more The evaluation of novel photosensitizers (PSs) for photodynamic therapy (PDT) is difficult due to the limitations of two-dimensional cell culture and multiple parameters (dose, light intensity, uptake time), which complicate progression to in vivo experiments and clinical translation. Three-dimensional (3D) cell culture models like multicellular cancer tumor spheroids (MCTS) show great similarities to in vivo avascular tumor conditions, improving the speed and accuracy of screening novel compounds with various treatment combinations. In this study, we utilize C8161 human melanoma spheroids to screen PDT treatment combinations using protoporphyrin IX (PpIX) and drug-loaded carbon dot (CD) conjugates PpIX-CD and PpIX@CD at ultralow fluence values (<10 J/cm 2). Conjugates show equivalent light-induced damage to PpIX from 1 μg/mL with significantly less dark cytotoxicity up to 72 h after exposure, shown by LDH release and dsDNA content. Fractionated treatments, carried out by dividing light exposure with 24 h intervals, demonstrate an enhanced PDT effect compared to single exposure at equal concentrations. Light sheet fluorescence microscopy combined with live/dead staining demonstrates that spheroids sustain extensive damage after PDT, with PpIX and PpIX-CD showing improved uptake compared to PpIX@CD. We show that PDT parameter screening can be carried out using a low-cost and convenient combination of assays to improve the efficiency of evaluating novel compounds.
Two-photon active Graphene Quantum Dots (GQDs) are obtained from extracts of the neem root. These... more Two-photon active Graphene Quantum Dots (GQDs) are obtained from extracts of the neem root. These biocompatible GQDs are found to be suitable for structured illumination microscopy. Two-photon microscopy ensured lysosome...
Enhanced image contrast in biological second harmonic imaging microscopy (SHIM) has previously be... more Enhanced image contrast in biological second harmonic imaging microscopy (SHIM) has previously been reported via quantitative assessments of forward- to epi-generated signal intensity ratio and by polarization analysis. Here we demonstrate a new form of contrast: the material-specific, wavelength-dependence of epi-generated second harmonic generation (SHG) excitation efficiency, and discriminate collagen and myosin by ratiometric epi-generated SHG images at 920 nm and 860 nm. Collagen shows increased SHG intensity at 920 nm, while little difference is detected between the two for myosin; allowing SHIM to characterize different SHG-generating components within a complex biological sample. We propose that momentum-space mapping of the second-order non-linear structure factor is the source of this contrast and develop a model for the forward and epi-generated SHG wavelength-dependence. Our model demonstrates that even very small changes in the assumed material fibrillar structure can p...
International Journal of Polymeric Materials and Polymeric Biomaterials
To see the final version of this work please visit the publisher's website. Access to the publish... more To see the final version of this work please visit the publisher's website. Access to the published online version may require a subscription.
Alterations in quantity or architecture of elastin and collagen fibres are associated with some b... more Alterations in quantity or architecture of elastin and collagen fibres are associated with some blood vessel pathologies. Also some medical interventions such as endovascular catheterization have the potential to damage blood vessels. This study reports the use of porcine aorta as a model system for studying the physical impact of catheters on vasculature, in conjunction with non-invasive imaging techniques to analyse collagen and elastin fibre organization and assess load-induced changes. Porcine aorta was exposed to frictional trauma and elastin and collagen fibre orientation evaluated by destructive, histochemical methods and non-invasive imaging. The latter allowed the immediate impact of force on fibre orientation and fibre recovery to be evaluated longitudinally. In normal aorta, elastin fibres are aligned at the surface, but become less aligned with increasing depth, showing no alignment by ~30 µm. Collagen fibres meanwhile appear aligned down to a depth of 35 µm. Changes in collagen and elastin fibre orientation in healthy pig aorta were detected by conventional destructive histology within 5 minutes of application of a sliding 10N load, while lesser loads had less impact. Good recovery of fibre orientation was observed within 20 minutes. Non-invasive imaging of ex vivo aorta tissue provides a good indication of the extent of fibre reorganization following frictional stress, at loads similar to those encountered during medical interventions such as catheterization. These results indicate that tissue deformation can occur from these procedures, even in healthy tissue, and highlight the potential for the development of an in vivo probe capable of monitoring vascular changes in patients.
Solid tumours display varied oxygen levels and this characteristic can be exploited to develop ne... more Solid tumours display varied oxygen levels and this characteristic can be exploited to develop new diagnostic tools to determine and exploit these variations. Oxygen is an efficient quencher of emission of many phosphorescent compounds, thus oxygen concentration could in many cases be derived directly from relative emission intensity and lifetime. In this study, we extend our previous work on phosphorescent, low molecular weight platinum(II) complex as an oxygen sensing probe to study the variation in oxygen concentration in a viable multicellular 3D human tumour model. The data shows one of the first examples of non-invasive, real-time oxygen mapping across a melanoma tumour spheroid using one-photon phosphorescence lifetime imaging microscopy (PLIM) and a small molecule oxygen sensitive probe. These measurements were quantitative and enabled real time oxygen mapping with high spatial resolution. This combination presents as a valuable tool for optical detection of both physiological and pathological oxygen levels in a live tissue mass and we suggest has the potential for broader clinical application. The use of metal complexes as dyes and probes for emission-based cellular imaging has developed into a vibrant area of research over the past decade 1-3. This emerging class of probes offers photo-physical properties that differ to a range of commercially available organic probes, and is enabling a new range of technologies to be explored. Notably, recent advances in optics and electronics, combined with the long emission lifetimes typical for transition metal complexes (from hundreds of nanoseconds to microseconds), has resulted in the emergence of multiphoton microsecond lifetime mapping techniques, such as Phosphorescence Lifetime Imaging Microscopy (PLIM) 4, 5 and Time-Resolved Emission Imaging Microscopy (TREM) 4. Luminescent transition metal complexes typically emit from a triplet excited state. Although transitions between states of a different spin are formally forbidden, intersystem crossing to the triplet state and subsequent relaxation to the ground state via phosphorescence are facilitated in transition metal complexes by the high spin orbit coupling constant associated with the heavy metal atom. The forbidden nature of the phosphorescence transition, results in a slow rate of emission; hence phosphorescence lifetimes are typically the order of hundreds of nanoseconds to microseconds. It is well documented that molecular oxygen quenches such triplet emitters and that the rate of quenching is dependent on oxygen concentration 5-10. One application of phosphorescent emitters in biological imaging is in oxygen detection, where more sensitive, non-invasive methods are in demand. The combination of phosphorescence quenching and high-resolution lifetime imaging is a powerful approach for non-invasive, real-time hypoxia detection and oxygen quantification. Oxygen quantification in biological systems is primarily focused on the use of large platinum and palladium porphyrins. These compounds display long emission lifetimes (typically 40-60 µs), which typically decreases
A recombinant Chinese hamster ovary (CHO) cell line making human interferon-? (IFN-y) was grown i... more A recombinant Chinese hamster ovary (CHO) cell line making human interferon-? (IFN-y) was grown in 12-L stirred tank fermentors in three batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition t o cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate structures attached to each glycosylation site of IFN-y w a s achieved using matrix-assisted laser desorption m a s s spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. Complex biantennary oligosaccharides (particularly Gal,GlcNAc,Man, which was core 1x1-6 fucosylated at Asn,, but not at Asn,,) were most prevalent at both glycosylation sites. However, considerable microheterogeneity arising from the presence of triantennary and truncated glycan structures w a s also observed. The proportion of t h e d o m i n a n t core glycan structure (Gal,GlcNAc,Man, 2 Fuc,) decreased by 1 5 2 6 % during batch culture, with increases in the proportion of oligomannose and truncated glycans over the s a m e time period. Prolonged culture resulting from an extended lag phase led to further accumulation of oligomannose and truncated structures, reaching up to 52% of total glycans attached t o Asn,, by 240 h of culture. The implications of these glycosylation changes for optimizing the time for harvesting cell cultures, and for the clearance of recornbinant therapeutic products in vivo are discussed.
EPITOPE DETERMINATION FOR ANTIBODIES RAISED AGAINST RECOMBINANT HUMAN INTERFERON-GAMMA Andrew D. ... more EPITOPE DETERMINATION FOR ANTIBODIES RAISED AGAINST RECOMBINANT HUMAN INTERFERON-GAMMA Andrew D. Hooker', Nicola H. Green1, David C. James, 1 Philip G. Strange1, Anthony J. Baines1, Alan T. Bull1 and Nigel Jenkins2. 'Dept of Biosciences, ...
Background Polypropylene mesh used as a mid-urethral sling is associated with severe clinical com... more Background Polypropylene mesh used as a mid-urethral sling is associated with severe clinical complications in a significant minority of patients. Current in vitro mechanical testing shows that polypropylene responds inadequately to mechanical distension and is also poor at supporting cell proliferation. Aims and Objectives Our objective therefore is to produce materials with more appropriate mechanical properties for use as a sling material but which can also support cell integration. Methods Scaffolds of two polyurethanes (PU), poly-L-lactic acid (PLA) and co-polymers of the two were produced by electrospinning. Mechanical properties of materials were assessed and compared to polypropylene. The interaction of adipose derived stem cells (ADSC) with the scaffolds was also assessed. Uniaxial tensiometry of scaffolds was performed before and after seven days of cyclical distension. Cell penetration (using DAPI and a fluorescent red cell tracker dye), viability (AlamarBlue assay) and total collagen production (Sirius red assay) were measured for ADSC cultured on scaffolds. Results Polypropylene was stronger than polyurethanes and PLA. However, polypropylene mesh deformed plastically after 7 days of sustained cyclical distention, while polyurethanes maintained their elasticity. Scaffolds of PU containing PLA were weaker and stiffer than PU or polypropylene but were significantly better than PU scaffolds alone at supporting ADSC.
This work reports the synthesis and characterization of thermoresponsive, stretchable, biodegrada... more This work reports the synthesis and characterization of thermoresponsive, stretchable, biodegradable and biocompatible poly(glycerol sebacate)-based polyurethane hydrogels.
This book is focused on the optical techniques that can be applied to regenerative medicine, incl... more This book is focused on the optical techniques that can be applied to regenerative medicine, including optical coherence tomography, acousto-optic imaging, Raman spectroscopy, machine vision, polarized light imaging, fibre optic sensors, second harmonic generation, multi-photon microscopy, coherent anti-Stokes Raman scattering, and polarized light imaging. It covers applications both in fundamental research and in the regenerative medicine industry for tissue engineering products. Each chapter gives an overview of a particular technique, its advantages and limitations in terms of structural and functional information provided, and examples of applications in regenerative medicine. The book provides a practical guide to the most appropriate techniques for a given application. It also offers a summary of major recent advances, such as use of machine vision for tracking growth of three-dimensional constructs in bioreactors and polarization sensitive optical coherence tomography for mon...
EPITOPE DETERMINATION FOR ANTIBODIES RAISED AGAINST RECOMBINANT HUMAN INTERFERON-GAMMA Andrew D. ... more EPITOPE DETERMINATION FOR ANTIBODIES RAISED AGAINST RECOMBINANT HUMAN INTERFERON-GAMMA Andrew D. Hooker', Nicola H. Green1, David C. James, 1 Philip G. Strange1, Anthony J. Baines1, Alan T. Bull1 and Nigel Jenkins2. 'Dept of Biosciences, ...
A recombinant Chinese hamster ovary (CHO) cell line making human interferon-? (IFN-y) was grown i... more A recombinant Chinese hamster ovary (CHO) cell line making human interferon-? (IFN-y) was grown in 12-L stirred tank fermentors in three batch fermentations under conditions of constant temperature, pH, and dissolved oxygen tension. In addition t o cell growth, metabolite, and productivity data, a detailed analysis of the carbohydrate structures attached to each glycosylation site of IFN-y w a s achieved using matrix-assisted laser desorption m a s s spectrometry (MALDI-MS) in combination with exoglycosidase array sequencing. Complex biantennary oligosaccharides (particularly Gal,GlcNAc,Man, which was core 1x1-6 fucosylated at Asn,, but not at Asn,,) were most prevalent at both glycosylation sites. However, considerable microheterogeneity arising from the presence of triantennary and truncated glycan structures w a s also observed. The proportion of t h e d o m i n a n t core glycan structure (Gal,GlcNAc,Man, 2 Fuc,) decreased by 1 5 2 6 % during batch culture, with increases in the proportion of oligomannose and truncated glycans over the s a m e time period. Prolonged culture resulting from an extended lag phase led to further accumulation of oligomannose and truncated structures, reaching up to 52% of total glycans attached t o Asn,, by 240 h of culture. The implications of these glycosylation changes for optimizing the time for harvesting cell cultures, and for the clearance of recornbinant therapeutic products in vivo are discussed.
Invasion of melanoma cells from the primary tumor involves interaction with adjacent tissues and ... more Invasion of melanoma cells from the primary tumor involves interaction with adjacent tissues and extracellular matrix. The extent of this interaction is not fully understood. In this study Raman spectroscopy was applied to cryo-sections of established 3D models of melanoma in human skin. Principal component analysis was used to investigate differences between the tumor and normal tissue and between the peri-tumor area and the normal skin. Two human melanoma cells lines A375SM and C8161 were investigated and compared in 3D melanoma models. Changes were found in protein conformations and tryptophan configurations across the entire melanoma samples, in tyrosine orientation and in more fluid lipid packing only in tumor dense areas, and in increased glycogen content in the peri-tumor areas of melanoma. Raman spectroscopy revealed changes around the perimeter of a melanoma tumor as well as detecting differences between the tumor and the normal tissue.
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