Morgan Denyer

Imaging of the cell surface interface using objective coupled widefield surface plasmon microscopy

We report on the development and on the first use of the widefield surface plasmon (WSPR) microscope in the examination of the cell surface interface at submicron lateral resolutions. The microscope is Kohler illuminated and uses either a 1.45 numerical aperture (NA) oil immersion lens, or a 1.65 NA oil immersion lens to excite surface plasmons at the interface between a thin gold layer and a glass or sapphire cover slip. Like all surface plasmon microscope systems the WSPR has been proven in previous studies to also be capable of nanometric z-scale resolutions. In this study we used the system to image the interface between HaCaT cells and the gold layer. Imaging was performed in air using fixed samples and the 1.45 NA objective based system and also using live cells in culture media using the 1.65 NA based system. Imaging in air enabled the visualisation of high resolution and high-contrast submicron features identified by vinculin immunostaining as component of focal contacts and focal adhesions. In comparison, imaging in fluid enabled cell surface interfacial interactions to be tracked by time-lapse video WSPR microscopy. Our results indicate that the cell surface interface and thus cell signalling mechanisms may be readily interrogated in live cells without the use of labelling techniques.

Contact guidance in human dermal fibroblasts is modulated by population pressure.

Morphogenesis is underpinned by orientated cell division, motility and growth. The substratum for migrating cells in vivo comprises either extracellular matrix or the surfaces of adjacent cells and both are believed to inform the dynamic behaviour of adherent cells through contact guidance. Collisions between migrating cells in vitro can induce the phenomena of contact inhibition of locomotion and division, suggesting that their sensitivity to substratum‐derived cues may also be influenced by population density. In the present study dermal fibroblasts, which are known to be motile in culture and are fundamental to the organization of the extracellular matrix, were used to examine the influence of population pressure on the ability of substratum topography to induce contact guidance. The findings suggest that sensitivity to substratum‐derived morphogenetic guidance cues, as revealed by alignment of cells to microtopography, is modulated by population pressure.

Development of a novel liquid crystal based cell traction force transducer system.

Keratinocyte traction forces play a crucial role in wound healing. The aim of this study was to develop a novel cell traction force (CTF) transducer system based on cholesteryl ester liquid crystals (LC). Keratinocytes cultured on LC induced linear and isolated deformation lines in the LC surface. As suggested by the fluorescence staining, the deformation lines appeared to correlate with the forces generated by the contraction of circumferential actin filaments which were transmitted to the LC surface via the focal adhesions. Due to the linear viscoelastic behavior of the LC, Hookes equation was used to quantify the CTFs by associating Youngs modulus of LC to the cell induced stresses and biaxial strain in forming the LC deformation. Youngs modulus of the LC was profiled by using spherical indentation and determined at approximately 87.1±17.2kPa. A new technique involving cytochalasin-B treatment was used to disrupt the intracellular force generating actin fibers, and consequently the biaxial strain in the LC induced by the cells was determined. Due to the improved sensitivity and spatial resolution (∼1μm) of the LC based CTF transducer, a wide range of CTFs was determined (10-120nN). These were found to be linearly proportional to the length of the deformations. The linear relationship of CTF-deformations was then applied in a bespoke CTF mapping software to estimate CTFs and to map CTF fields. The generated CTF map highlighted distinct distributions and different magnitude of CTFs were revealed for polarized and non-polarized keratinocytes.

Biophysical characteristics of cells cultured on cholesteryl ester liquid crystals

This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and β1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material.

Imaging via widefield surface plasmon resonance microscope for studying bone cell interactions with micropatterned ECM proteins

The widefield surface plasmon resonance microscope has recently been used to monitor label free antibody/antigen binding events and focal contacts in HaCaT cells at high special resolutions. Thus the aim of this study was to examine MG63 bone cell attachment and alignment to microcontact printed extracellular matrix proteins. Collagen, fibronectin and laminin were stamp patterned onto glass slides using templates consisting of 5‐, 10‐, 25‐, 50‐ and 100‐μm‐wide repeat grating. MG63 bone cells were seeded at 50 000 cells per 25 cm2 and cell alignment was determined from micrographs taken at time‐points 2, 5 and 18 h after cell seeding. Cells on the fibronectin pattern attached and elongated at early stages after seeding. In the case of collagen and laminin, cells did not adhere readily and appeared more rounded until 18 h after seeding. This indicated MG63 cells attach mostly via fibronectin specific integrins. The cells aligned well on the fibronectin‐patterned cover slips especially to the 50‐ and 100‐μm‐wide patterns, although in this case cells did not position themselves in the middle of each fibronectin‐coated region, but instead aligned to the small features associated with the edges of the fibronectin‐coated regions. Patterned and un‐patterned cells also had quite different morphologies. The un‐patterned cells had a more rounded morphology and lengths of 25 to 35 μm, whereas patterned cells elongated in the direction of the pattern and had lengths of 50–70 μm. The widefield surface plasmon resonance imaging indicated that cells on un‐patterned surfaces had a rounded morphology in which the focal contacts were evenly distributed around the periphery of the cell. However, MG63 bone cells on fibronectin‐patterned substrates organized most of their focal contacts along the periphery of the cell distal to the edge of the fibronectin patterns. This suggests that the interaction between the cell and the edge of the pattern induces a reorganization of focal contacts such that the region of the cell guided by the edge of the fibronectin pattern is relatively loosely coupled to the cell culture substrate, but the region of the cell positioned away from that edge is quite tightly coupled to the fibronectin‐coated region of the culture substrate. This in turn suggests that guidance is not necessarily associated with enhanced cell substrate coupling along the guidance cue, but may be more associated with a decreased coupling at the guidance cue. Such an arrangement may influence cytoplasmic streaming and as such modulate cell extension. Verification of this finding is required; as such a response to a guidance cue is quite unexpected because it is believed that cells cluster their focal contacts along a guidance cue.

Effects of different transforming growth factor beta (TGF-β) isomers on wound closure of bone cell monolayers.

This study aimed at determining the role of the transforming growth factor-beta (TGF-β) isomers and their combinations in bone cell behaviour using MG63 cells. The work examined how TGF-β1, 2 and 3 and their solvent and carrier (HCl and BSA, respectively) effected cell morphology, cell proliferation and integrin expression. This study also aimed at examining how the TGF-βs and their solvent and carrier influenced wound closure in an in vitro wound closure model and how TGF-βs influence extracellular matrix (ECM) secretion and integrin expression. The wound healing response in terms of healing rate to the TGF-βs and their solvent/carrier was investigated in 300 μm ± 10-30 μm SD wide model wounds induced in fully confluent monolayers of MG63 bone cells. The effect of different TGF-β isomers and their combinations on proliferation rate and cell length of human bone cells were also assessed. Immunostaining was used to determine if TGF-βs modifies integrin expression and ECM secretion by the bone cells. Imaging with WSPR allowed observation of the focal contacts without the need for immunostaining. The wound healing results indicated that TGF-β3 has a significant effect on the wound healing process and its healing rate was found to be higher than the control (p < 0.001), TGF-β1 (p < 0.001), TGF-β2 (p < 0.001), BSA/HCl (p < 0.001) and HCl (p < 0.001) in ascending order. It was also found that TGF-β1 and TGF-β2 treatment significantly improved wound closure rate in comparison to the controls (p < 0.001). All TGF-β combinations induced a faster healing rate than the control (p < 0.001). It was expected that the healing rate following treatment with TGF-β combinations would be greater than those healing rates following treatments with TGF-β isomers alone, but this was not the case. The results also suggest that cell morphological changes were observed significantly more in cells treated with TGF-β(2 + 3) and TGF-β(1 + 3) (p < 0.001). Any cell treated with TGF-β1, TGF-β(1 + 2) and TGF-β(1 + 2 + 3) showed significantly less elongation compared to the control and other TGF-β isomers. In terms of proliferation rate, TGF-β3 and TGF-β(2 + 3) increased cell numbers more than TGF-β1, TGF-β2 and other combinations. TGF-β1 and its combinations did not show significant proliferation and attachment compared to the control. Immunostaining indicated that treatment with TGF-β3 significantly enhanced the secretion of collagen type I, fibronectin and integrins α3 and β1. The WSPR experiments also indicated that TGF-βs influenced the distribution of focal contacts. In conclusion, combining TGF-β3 with any other TGF-β isomer resulted in a faster model wound closure rate (p < 0.001), while treatment with TGF-β1 in any TGF-β combination reduced the healing rate (p < 0.001). It can therefore be concluded that the presence of TGF-β1 has an inhibitory effect on bone wound healing while TGF-β3 had the opposite effect and increased the rate of wound closure in a 2 dimensional cell culture environment.

Development of an intestinal cell culture model to obtain smooth muscle cells and myenteric neurones.

This paper reports on the development of an entirely new intestinal smooth muscle cell (ISMC) culture model using rat neonates for use in pharmacological research applications. Segments of the duodenum, jejunum and ileum were obtained from Sprague‐Dawley rat neonates. The cell extraction technique consisted of ligating both ends of the intestine and incubating (37 ºC) in 0.25% trypsin for periods of 30–90 min. Isolated cells were suspended in DMEM‐HEPES, plated and allowed to proliferate for 7 days. Cell culture quality was assessed via a series of viability tests using the dye exclusion assay. In separate experiments, tissues were exposed to trypsin for varying durations and subsequently histological procedures were applied. Cell purification techniques included differential adhesion technique for minimizing fibroblasts. Selective treatments with neurotoxin scorpion venom (30 µg mL−1) and anti‐mitotic cytosine arabinoside (6 µm) were also applied to purify respectively ISMC and myenteric neurones selectively. The different cell populations were identified in regard to morphology and growth characteristics via immunocytochemistry using antibodies to smooth muscle α‐actin, α‐actinin and serotonin‐5HT3 receptors. Based on both viability and cell confluence experiments, results demonstrated that intestinal cells were best obtained from segments of the ileum dissociated in trypsin for 30 min. This provided the optimum parameters to yield highly viable cells and confluent cultures. The finding was further supported by histological studies demonstrating that an optimum incubation time of 30 min is required to isolate viable cells from the muscularis externae layer. When cell cultures were treated with cytosine arabinoside, the non‐neuronal cells were abolished, resulting in the proliferation of cell bodies and extended neurites. Conversely, cultures treated with scorpion venom resulted in complete abolition of neurones and proliferation of increasing numbers of ISMC, which were spindle‐shaped and uniform throughout the culture. When characterized by immunocytochemistry, neurones were stained with antibody to 5HT3 receptors but not with antibodies to α‐smooth muscle actin and α‐actinin. Conversely, ISMC were stained with antibodies to α‐smooth muscle actin and α‐actinin but not with antibody to 5HT3 receptors. The present study provides evidence that our method of dissociation and selectively purifying different cell populations will allow for pharmacological investigation of each cell type on different or defined mixtures of different cell types.

Interfacial study of cell adhesion to liquid crystals using widefield surface plasmon resonance microscopy

Widefield surface plasmon resonance (WSPR) microscopy provides high resolution imaging of interfacial interactions. We report the application of the WSPR imaging system in the study of the interaction between keratinocytes and liquid crystals (LC). Imaging of fixed keratinocytes cultured on gold coated surface plasmon substrates functionalized with a thin film of liquid crystals was performed in air using a 1.45NA objective based system. Focal adhesion of the cells adhered to glass and LC were further studied using immunofluorescence staining of the vinculin. The imaging system was also simulated with 2×2 scattering matrix to investigate the optical reflection of the resonant plasmonic wave via the glass/gold/cell and glass/gold/LC/cell layers. WSPR imaging indicated that keratinocytes are less spread and formed distinct topography of cell-liquid crystal couplings when cultured on liquid crystal coated substrates. The simulation indicates that glass/LC shifted the surface plasmon excitation angle to 75.39° as compared to glass/air interface at 44°. The WSPR microcopy reveals that the cells remodelled their topography of adhesion at different interfaces.

Motogenic substrata and chemokinetic growth factors for human skin cells

Extracellular matrix remodelling and accurate spatio‐temporal coordination of growth factor expression are two factors that are believed to regulate mitoses and cell migration in developing and regenerating tissues. The present quantitative videomicroscopical study examined the influence of some of the principal components of extracellular matrix and several growth factors that are known to be expressed in dermal wounds on three important facets of human skin cell behaviour in culture. Keratinocytes, melanocytes and dermal fibroblasts (and myofibroblast controls) exhibited varying degrees of substrate adhesion, division and migration depending on the composition of the culture substrate. Substrates that are recognized components of transitional matrices generally accentuated cell adhesion and proliferation, and were motogenic, when compared with serum‐treated control surfaces, whereas components of more stable structures such as basement membrane had less influence. Platelet‐derived growth factor (PDGF), epidermal growth factor (EGF) and α fibroblastic growth factor (αFGF) all promoted cell proliferation and were chemokinetic to dermal fibroblasts, but not keratinocyte growth factor (KGF) or transforming growth factor β (TGFβ). PDGF, EGF and KGF, but not TGFβ or αFGF, all enhanced proliferation of dermal keratinocytes. The same growth factors, and in addition KGF, all stimulated motility in keratinocytes, but TGFβ and αFGF again had no effect. Developing a better understanding of the interdependency of factors that control crucial cell behaviour may assist those who are interested in the regulation of histogenesis and also inform the development of rational therapeutic strategies for the management of chronic and poorly healed wounds.

Interactions of Cells with Elastic Cholesteryl Liquid Crystals

Exploitation of elastic property of the liquid crystal to sense cells mechanics is a novel application. Both quantitative and qualitative analysis of cholesteryl liquid crystal has shown flexibility and elasticity of the material to sense cell contraction and relaxation over a period of time. Importantly, cells adhesion was precluded with the use of extracellular matrix proteins in this technique. The optimum operating range with linear elasticity of the cholesteryl liquid crystal is ≤ 0.1s− 1 which has a good correlation with the shear rate of the cells originated from the focal adhesions. Localized contraction was observed with a good resolution. In addition, clear definition of deformation lines between two cells has shown their interacting path through active mechano-sensing.