Optimisation of growth conditions for ovine airway epithelial cell differentiation at an air-liquid interface

Respiratory tract infections are of significant concern in the agriculture industry. There is a requirement for the development of well-characterised in vitro epithelial cell culture models in order to dissect the diverse molecular interactions occurring at the host-pathogen interface in airway epithelia. We have analysed key factors that influence growth and differentiation of ovine tracheal epithelial cells in an air-liquid interface (ALI) culture system. Cellular differentiation was assessed at 21 days post-ALI, a time-point which we have previously shown to be sufficient for differentiation in standard growth conditions. We identified a dose-dependent response to epidermal growth factor (EGF) in terms of both epithelial thickening and ciliation levels. Maximal ciliation levels were observed with 25 ng ml-1 EGF. We identified a strict requirement for retinoic acid (RA) in epithelial differentiation as RA exclusion resulted in the formation of a stratified squamous epithelium, devoid of cilia. The pore-density of the growth substrate also had an influence on differentiation as high pore-density inserts yielded higher levels of ciliation and more uniform cell layers than low pore-density inserts. Differentiation was also improved by culturing the cells in an atmosphere of sub-ambient oxygen concentration. We compared two submerged growth media and observed differences in the rate of proliferation/expansion, barrier formation and also in terminal differentiation. Taken together, these results indicate important differences between the response of ovine tracheal epithelial cells and other previously described airway epithelial models, to a variety of environmental conditions. These data also indicate that the phenotype of ovine tracheal epithelial cells can be tailored in vitro by precise modulation of growth conditions, thereby yielding a customisable, potential infection model

Temporal dynamics of ovine airway epithelial cell differentiation at an air-liquid interface

The respiratory tract and lungs are subject to diverse pathologies with wide-ranging implications for both human and animal welfare. The development and detailed characterization of cell culture models for studying such forms of disease is of critical importance. In recent years the use of air-liquid interface (ALI)-cultured airway epithelial cells has increased markedly, as this method of culture results in the formation of a highly representative, organotypic in vitro model system. In this study we have expanded on previous knowledge of differentiated ovine tracheal epithelial cells by analysing the progression of differentiation over an extensive time course at an ALI. We observed a pseudo-stratified epithelium with ciliation and a concurrent increase in cell layer thickness from 9 days post-ALI with ciliation approaching a maximum level at day 24. A similar pattern was observed with respect to mucus production with intensely stained PAS-positive cells appearing at day 12. Ultrastructural analysis by SEM confirmed the presence of both ciliated cells and mucus globules on the epithelial surface within this time-frame. Trans-epithelial electrical resistance (TEER) peaked at 1049 Ω × cm2 as the cell layer became confluent, followed by a subsequent reduction as differentiation proceeded and stabilization at ~200 Ω × cm2. Importantly, little deterioration or de-differentiation was observed over the 45 day time-course indicating that the model is suitable for long-term experiments

Magnetically levitated mesenchymal stem cell spheroids cultured with a collagen gel maintain phenotype and quiescence

Multicellular spheroids are an established system for three-dimensional cell culture. Spheroids are typically generated using hanging drop or non-adherent culture; however, an emerging technique is to use magnetic levitation. Herein, mesenchymal stem cell spheroids were generated using magnetic nanoparticles and subsequently cultured within a type I collagen gel, with a view towards developing a bone marrow niche environment. Cells were loaded with magnetic nanoparticles, and suspended beneath an external magnet, inducing self-assembly of multicellular spheroids. Cells in spheroids were viable and compared to corresponding monolayer controls, maintained stem cell phenotype and were quiescent. Interestingly, core spheroid necrosis was not observed, even with increasing spheroid size, in contrast to other commonly used spheroid systems. This mesenchymal stem cell spheroid culture presents a potential platform for modelling in vitro bone marrow stem cell niches, elucidating interactions between cells, as well as a useful model for drug delivery studies

Differentiated ovine tracheal epithelial cells support the colonisation of pathogenic and non-pathogenic strains of Mannheimia haemolytica

Mannheimia haemolytica is the primary bacterial species associated with respiratory disease of ruminants. A lack of cost-effective, reproducible models for the study of M. haemolytica pathogenesis has hampered efforts to better understand the molecular interactions governing disease progression. We employed a highly optimised ovine tracheal epithelial cell model to assess the colonisation of various pathogenic and non-pathogenic M. haemolytica isolates of bovine and ovine origin. Comparison of single representative pathogenic and non-pathogenic ovine isolates over ten time-points by enumeration of tissue-associated bacteria, histology, immunofluorescence microscopy and scanning electron microscopy revealed temporal differences in adhesion, proliferation, bacterial cell physiology and host cell responses. Comparison of eight isolates of bovine and ovine origin at three key time-points (2 h, 48 h and 72 h), revealed that colonisation was not strictly pathogen or serotype specific, with isolates of serotype A1, A2, A6 and A12 being capable of colonising the cell layer regardless of host species or disease status of the host. A trend towards increased proliferative capacity by pathogenic ovine isolates was observed. These results indicate that the host-specific nature of M. haemolytica infection may result at least partially from the colonisation-related processes of adhesion, invasion and proliferation at the epithelial interface

Pathogenic Mannheimia haemolytica invades differentiated bovine airway epithelial cells

The Gram-negative bacterium Mannheimia haemolytica is the primary bacterial species associated with bovine respiratory disease (BRD) which is responsible for significant economic losses to the livestock industries worldwide. Healthy cattle are frequently colonised by commensal serotype A2 strains, but disease is usually caused by pathogenic strains of serotype A1. For reasons that are poorly understood, a transition occurs within the respiratory tract and a sudden explosive proliferation of serotype A1 bacteria leads to the onset of pneumonic disease. Very little is known about the interactions of M. haemolytica with airway epithelial cells of the respiratory mucosa which might explain the different abilities of serotype A1 and A2 strains to cause disease. In the present study, host-pathogen interactions in the bovine respiratory tract were mimicked using a novel differentiated bovine bronchial epithelial cell (BBEC) infection model. In this model, differentiated BBECs were inoculated with serotype A1 or A2 strains of M. haemolytica and the course of infection followed over a five-day period by microscopic assessment and measurement of key proinflammatory mediators. We have demonstrated that serotype A1, but not A2, M. haemolytica invades differentiated BBECs by transcytosis and subsequently undergoes rapid intracellular replication before spreading to adjacent cells and causing extensive cellular damage. Our findings suggest that the explosive proliferation of serotype A1 M. haemolytica that occurs within the bovine respiratory tract prior to the onset of pneumonic disease is potentially due to bacterial invasion of, and rapid proliferation within, the mucosal epithelium. The discovery of this previously unrecognised mechanism of pathogenesis is important because it will allow the serotype A1-specific virulence determinants responsible for invasion to be identified and thereby provide opportunities for the development of new strategies for combatting BRD aimed at preventing early colonisation and infection of the bovine respiratory tract

Temporal differentiation of bovine airway epithelial cells grown at an air-liquid interface

There is an urgent need to develop improved, physiologically-relevant in vitro models of airway epithelia with which to better understand the pathological processes associated with infection, allergies and toxicological insults of the respiratory tract of both humans and domesticated animals. In the present study, we have characterised the proliferation and differentiation of primary bovine bronchial epithelial cells (BBECs) grown at an air-liquid interface (ALI) at three-day intervals over a period of 42 days from the introduction of the ALI. The differentiated BBEC model was highly representative of the ex vivo epithelium from which the epithelial cells were derived; a columnar, pseudostratified epithelium that was highly reflective of native airway epithelium was formed which comprised ciliated, goblet and basal cells. The hallmark defences of the respiratory tract, namely barrier function and mucociliary clearance, were present, thus demonstrating that the model is an excellent mimic of bovine respiratory epithelium. The epithelium was fully differentiated by day 21 post-ALI and, crucially, remained healthy and stable for a further 21 days. Thus, the differentiated BBEC model has a three-week window which will allow wide-ranging and long-term experiments to be performed in the fields of infection, toxicology or general airway physiology

Development and optimization of a differentiated airway epithelial cell model of the bovine respiratory tract

Cattle are subject to economically-important respiratory tract infections by various bacterial and viral pathogens and there is an urgent need for the development of more realistic in vitro models of the bovine respiratory tract to improve our knowledge of disease pathogenesis. In the present study, we have optimized the culture conditions in serum-free medium that allow bovine bronchial epithelial cells (BBECs) grown at an air-liquid interface to differentiate into a three-dimensional epithelium that is highly representative of the bovine airway. Epidermal growth factor was required to trigger both proliferation and differentiation of BBECs whilst retinoic acid was also essential for mucociliary differentiation. Triiodothyronine was demonstrated not to be important for the differentiation of BBECs. Oxygen concentration had a minimal effect although optimal ciliation was achieved when BBECs were cultured at 14% oxygen tension. Insert pore-density had a significant effect on the growth and differentiation of BBECs; a high-pore-density was required to trigger optimum differentiation. The established BBEC model will have wide-ranging applications for the study of bacterial and viral infections of the bovine respiratory tract; it will contribute to the development of improved vaccines and therapeutics and will reduce the use of cattle in in vivo experimentation

Mesenchymal stem cell-derived extracellular vesicles may promote breast cancer cell dormancy

Disseminated breast cancer cells have the capacity to metastasise to the bone marrow and reside in a dormant state within the mesenchymal stem cell (MSC) niche. Research has focussed on paracrine signalling factors, such as soluble proteins, within the microenvironment. However, it is now clear extracellular vesicles (EVs) secreted by resident MSCs into this microenvironment also play a key role in the initiation of dormancy. Dormancy encourages reduced cell proliferation and migration, whilst upregulating cell adhesion, thus retaining the cancer cells within the bone marrow microenvironment. Here, MCF7 breast cancer cells were treated with MSC-derived EVs, resulting in reduced migration in 2D and 3D culture, with reduced cell proliferation and enhanced adhesion, collectively supporting cancer cell dormancy

Mesenchymal stem cell-derived extracellular vesicles may promote breast cancer cell dormancy

Disseminated breast cancer cells have the capacity to metastasise to the bone marrow and reside in a dormant state within the mesenchymal stem cell (MSC) niche. Research has focussed on paracrine signalling factors, such as soluble proteins, within the microenvironment. However, it is now clear extracellular vesicles (EVs) secreted by resident MSCs into this microenvironment also play a key role in the initiation of dormancy. Dormancy encourages reduced cell proliferation and migration, whilst upregulating cell adhesion, thus retaining the cancer cells within the bone marrow microenvironment. Here, MCF7 breast cancer cells were treated with MSC-derived EVs, resulting in reduced migration in 2D and 3D culture, with reduced cell proliferation and enhanced adhesion, collectively supporting cancer cell dormancy

Review of Orbiter Flight Boundary Layer Transition Data

In support of the Shuttle Return to Flight program, a tool was developed to predict when boundary layer transition would occur on the lower surface of the orbiter during reentry due to the presence of protuberances and cavities in the thermal protection system. This predictive tool was developed based on extensive wind tunnel tests conducted after the loss of the Space Shuttle Columbia. Recognizing that wind tunnels cannot simulate the exact conditions an orbiter encounters as it re-enters the atmosphere, a preliminary attempt was made to use the documented flight related damage and the orbiter transition times, as deduced from flight instrumentation, to calibrate the predictive tool. After flight STS-114, the Boundary Layer Transition Team decided that a more in-depth analysis of the historical flight data was needed to better determine the root causes of the occasional early transition times of some of the past shuttle flights. In this paper we discuss our methodology for the analysis, the various sources of shuttle damage information, the analysis of the flight thermocouple data, and how the results compare to the Boundary Layer Transition prediction tool designed for Return to Flight