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Background: Inhaled corticosteroids (ICSs) are the main prophylactic treatment for asthma and are used in other diseases, including chronic pulmonary obstructive disease, yet the interaction of ICS...

Background Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies followed by ciliated differentiation at the air–liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that can be expanded from such biopsies. Methods We describe an immunofluorescence screening method, enabled by extensive expansion of basal cells from PCD patients and the directed differentiation of these cells into ciliated epithelium in miniaturised 96-well transwell format ALI cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia), in this case caused by a homozygous nonsense mutation in the MCIDAS gene. Results Initial analyses of ciliary ultrastructure, beat pattern and beat frequency in the 96-well transwell format ALI cultures indicate that a range of different PCD defects can be retained in these cultures. The screening system in our proof-of-principal investigation allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. We observed restoration of basal body formation but not the generation of cilia in the patient's nasal epithelial cells in vitro. Conclusion Our study provides a platform for higher throughput analyses of airway epithelia that is applicable in a range of settings and suggests novel avenues for drug evaluation and development in PCD caused by nonsense mutations., Primary cell culture of nasal epithelial cells (including differentiation to multiciliated cells) from patients with primary ciliary dyskinesia enabled immunofluorescence-based screening in miniaturised air–liquid interface cultures https://bit.ly/3rjoxBF

Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies, followed by ciliated differentiation at air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique’s broader utility, including in pre-clinical PCD research, has been limited by the number of basal cells that it is possible to expand from such biopsies. Here, we describe a high-content, imaging-based screening method, enabled by extensive expansion of PCD patient basal cells and their culture into differentiated human respiratory epithelium in miniaturised 96-well transwell format ALI cultures. Analyses of ciliary beat pattern, beat frequency and ultrastructure indicate that a range of different PCD defects are retained in these cultures. We perform a proof-of-principle personalized investigation in reduced generation of motile cilia (RGMC), a rare and very severe form of PCD, in this case caused by a homozygous nonsense mutation (c.441C>A; p.Cys147*) in theMCIDASgene. The screening system allowed multiple drugs inducing translational readthrough to be evaluated alone or in combination with inhibitors of nonsense-mediated decay. Restoration of basal body formation in the patient’s nasal epithelial cells was seenin vitro, suggesting a novel avenue for drug evaluation and development in PCD.SummaryWe describe primary cell culture of nasal epithelial cells from patients with primary ciliary dyskinesia including differentiatiation of these to a ciliary phenotype and high-content screening in miniaturised air-liquid interface cultures.

The human nasal epithelium contains basal stem/progenitor cells that produce differentiated multiciliated and mucosecretory progeny. Basal epithelial cells can be expanded in cell culture and instructed to differentiate at an air-liquid interface using transwell membranes and differentiation media. For basal cell expansion, we have used 3T3-J2 co-culture in epithelial culture medium containing EGF, insulin, and a RHO-associated protein kinase (ROCK) inhibitor, Y-27632 (3T3 + Y). Here we describe our protocols for ciliated differentiation of these cultures at air-liquid interface and compare four commercially available differentiation media, across nine donor cell cultures (six healthy, two patients with chronic obstructive pulmonary disease (COPD), and one with primary ciliary dyskinesia (PCD)). Bright-field and immunofluorescence imaging suggested broad similarity between differentiation protocols. Subtle differences were seen in transepithelial electrical resistance (TEER), ciliary beat frequency, mucus production, and the extent to which basal cells are retained in differentiated cultures. Overall, the specific differentiation medium used in our air-liquid interface culture protocol was not a major determinant of ciliation, and our data suggest that the differentiation potential of basal cells at the outset is a more critical factor in air-liquid interface culture outcome. Detailed information on the constituents of the differentiation media was only available from one of the four manufacturers, a factor that may have profound implications in the interpretation of some research studies.

Respiratory syncytial virus (RSV) is a highly contagious pathogen with a huge global health impact. It is a major cause of hospital-acquired infection; a large number of those exposed develop infection. Those infected in hospital are at increased risk of a severe clinical course. Prevention of nosocomial spread currently focuses on spread by hand and large droplets. There is little research evidence to determine if aerosol spread of infectious RSV is possible.To determine if the air surrounding infants with RSV-positive bronchiolitis contains RSV in aerosolized particles that remain capable of causing infection.The amount of RSV contained in aerosolized particles produced by infants with bronchiolitis due to RSV was measured using viable impactor sampling. The ability of RSV contained in these particles to infect healthy and chronic obstructive pulmonary disease (COPD) human ciliated respiratory epithelium was determined.We showed for the first time that infants with RSV-positive bronchiolitis nursed in a ward setting or ventilated in intensive care produced large numbers of aerosol particles containing RSV that remained infectious and were capable of infecting healthy and COPD human ciliated epithelium. A significant amount of RSV was found in particles with aerodynamic diameters less than 5 μm.Many of the aerosolized particles that contained RSV in the air surrounding infants with bronchiolitis were sufficiently small to remain airborne for a significant length of time and small enough to be inhaled and deposited throughout the respiratory tract. It is likely that this leads to spread of infection to others, with dissemination of infection throughout the respiratory tract.

Current methods to replace damaged upper airway epithelium with exogenous cells are limited. Existing strategies use grafts that lack mucociliary function, leading to infection and the retention of secretions and keratin debris. Strategies that regenerate airway epithelium with mucociliary function are clearly desirable and would enable new treatments for complex airway disease. Here, we investigated the influence of the extracellular matrix (ECM) on airway epithelial cell adherence, proliferation and mucociliary function in the context of bioengineered mucosal grafts. In vitro, primary human bronchial epithelial cells (HBECs) adhered most readily to collagen IV. Biological, biomimetic and synthetic scaffolds were compared in terms of their ECM protein content and airway epithelial cell adherence. Collagen IV and laminin were preserved on the surface of decellularised dermis and epithelial cell attachment to decellularised dermis was greater than to the biomimetic or synthetic alternatives tested. Blocking epithelial integrin α2 led to decreased adherence to collagen IV and to decellularised dermis scaffolds. At air–liquid interface (ALI), bronchial epithelial cells cultured on decellularised dermis scaffolds formed a differentiated respiratory epithelium with mucociliary function. Using in vivo chick chorioallantoic membrane (CAM), rabbit airway and immunocompromised mouse models, we showed short-term preservation of the cell layer following transplantation. Our results demonstrate the feasibility of generating HBEC grafts on clinically applicable decellularised dermis scaffolds and identify matrix proteins and integrins important for this process. The long-term survivability of pre-differentiated epithelia and the relative merits of this approach against transplanting basal cells should be assessed further in pre-clinical airway transplantation models., Collagen IV- and laminin-rich decellularised dermis scaffolds support a mucociliary airway epithelial graft but in vivo transplantation in pre-clinical models is challenging http://bit.ly/2IdQp5d

Background: Influenza virus is a major human pathogen, yet surprisingly little data is available on the earliest stage of infection. We have developed a novel method to study natural transmission influenza infection by aerosol and to observe the effects of early infection on the ciliated airway epithelium using high-speed video microscopy. Methods: Primary human ciliated epithelial cultures were infected with influenza A (H1N1), delivered either by aerosol or by liquid immersion. Cells were stained for viral antigens and the level of inflammatory mediators, and the number of motile ciliated cells and ciliary beat frequency and pattern was measured. Results: Infection by aerosol and liquid inoculums of influenza virus was shown to be trophic for ciliated cells. Infection by both methods also led to a significant decrease in the number of cells with motile cilia over the first 24 hours; however, the ciliary beat frequency and beat pattern of the remaining cilia was maintained over 24 hours. Conclusions: Influenza virus aerosols readily infect human ciliated nasal epithelial cells resulting in early loss of motile ciliated cells. Delivery of the virus by aerosol elicited an anti-inflammatory Th2 response, which was distinct from cells exposed to virus by liquid immersion delivery. This suggests our aerosol model may provide a more clinically relevant model for studying the early effects of influenza infection.

Background Inhaled Fluticasone Propionate (FP) is widely used in the treatment of patients with asthma and COPD. Little is known of the initial interaction of inhaled FP particles with the ciliated respiratory epithelium. In this study we demonstrate a novel interaction between FP particles and rapidly moving cilia. Methods Ciliated epithelial cell cultures from healthy volunteers were grown at air-liquid interface (ALI). FP 2mg/ml was nebulized on to the apical surface of ciliated cell cultures using a bespoke system that allowed observation in real time by high-speed video microscopy. Results Flixotide particles were observed to impact on the ciliated cultures. Particles were transported around the culture well by the motile cilia. A number of FP particles bound to the tips of rapidly moving cilia. Binding occurred as early as 5 minutes after nebulization but continued to occur over time. Although the number of particles bound to cilia reduced over time some were still present at 48 hours. Binding of FP particles significantly (P Conclusion Binding of FP to cilia may result in retention of steroid particles in ciliated epithelial airways.

Stem cell-based tracheal replacement represents an emerging therapeutic option for patients with otherwise untreatable airway diseases including long-segment congenital tracheal stenosis and upper airway tumors. Clinical experience demonstrates that restoration of mucociliary clearance in the lungs after transplantation of tissue-engineered grafts is critical, with preclinical studies showing that seeding scaffolds with autologous mucosa improves regeneration. High epithelial cell-seeding densities are required in regenerative medicine, and existing techniques are inadequate to achieve coverage of clinically suitable grafts.To define a scalable cell culture system to deliver airway epithelium to clinical grafts.Human respiratory epithelial cells derived from endobronchial biopsies were cultured using a combination of mitotically inactivated fibroblasts and Rho-associated protein kinase (ROCK) inhibition using Y-27632 (3T3+Y). Cells were analyzed by immunofluorescence, quantitative polymerase chain reaction, and flow cytometry to assess airway stem cell marker expression. Karyotyping and multiplex ligation-dependent probe amplification were performed to assess cell safety. Differentiation capacity was tested in three-dimensional tracheospheres, organotypic cultures, air-liquid interface cultures, and an in vivo tracheal xenograft model. Ciliary function was assessed in air-liquid interface cultures.3T3-J2 feeder cells and ROCK inhibition allowed rapid expansion of airway basal cells. These cells were capable of multipotent differentiation in vitro, generating both ciliated and goblet cell lineages. Cilia were functional with normal beat frequency and pattern. Cultured cells repopulated tracheal scaffolds in a heterotopic transplantation xenograft model.Our method generates large numbers of functional airway basal epithelial cells with the efficiency demanded by clinical transplantation, suggesting its suitability for use in tracheal reconstruction.

Background Stem-cell-based tracheal replacement has been used to treat patients with end-stage airway disease in compassionate cases. Clinical experience suggests that restoration of an epithelial barrier is a priority to improve outcomes, but recent data suggest that a substantial number of autologous epithelial cells are required for effective engraftment. Existing cell culture methods fail to expand the airway epithelial progenitor pool, presenting a considerable hurdle for progression to clinical trials. We aimed to assess feeder layers and a Rho-associated kinase inhibitor as a method to expand human respiratory epithlieum for airway bioengineering. Methods Human epithelial cells from endobronchial biopsy samples were cultured on mouse 3T3-J2 fibroblast feeder layers in medium containing Y-27632, a Rho-associated kinase inhibitor (3T3+Y). Air-liquid interface and tracheosphere assays were done to determine differentiation capacity, and high-speed video microscopy to observe ciliary behaviour. Telomerase expression was measured with immunofluorescence and western blotting and telomere length with real-time PCR. Cells were karyotyped and engraftment potential tested in ex-vivo and in-vivo xenograft models. Findings 3T3+Y allowed for rapid and sustained expansion of airway epithelial basal cells. Population doublings demonstrated that cultures derived from donor biopsy samples could provide sufficient cell numbers to recellularise human tracheal scaffolds. At clinically applicable passages, cells were capable of airway differentiation in vitro, forming both ciliated and goblet lineages. Ciliary beat frequency and beat pattern were within normal range. Cells were karyotypically normal despite extensive expansion. Protein expression of telomerase was increased in 3T3+Y compared with that in conventional growth medium, and telomere length appeared to stabilise. Cultured cells repopulated a decellularised tracheal scaffold ex vivo and restored a differentiated epithelium in an in-vivo tracheal transplantation xenograft model. Interpretation The findings show that 3T3+Y generates large numbers of airway basal epithelial stem cells that retain qualities desirable for clinical transplantation. These data have important implications for personalised autologous airway epithelial cell therapy because cells could be obtained rapidly and in an appropriate number. Preclinical and clinical validation is required. Funding Wellcome Trust.

Background Tracheal reconstruction relies on the use of a split skin graft to re-epithelialise the mucosal layer. Since split skin grafts are made up of a keratinising stratified epithelial layer, sloughing occurs within the airway with mucus retention and subsequent airway obstruction. The delivery of a graft with the same mucociliary function as the native airway would overcome these limitations and greatly improve the safety and effectiveness of this type of surgery. We aimed to generate a transplantable tissue-engineered respiratory epithelial graft with mucociliary function. Methods Cadaveric human skin was decellularised and the epidermal layer removed. Human bronchial epithelial cells were seeded with human respiratory fibroblasts onto the dermis at densities of 1 × 10 6 per cm 2 and 1 × 10 4 per cm 2 , respectively, and cultured at air–liquid interface in a transwell system. At 3 weeks, the constructs were transplanted onto a decellularised trachea that had been prevascularised within a muscle wrap in an immunosuppressed New Zealand White rabbit. Findings After 3 weeks of air–liquid interface culture, high-speed video microscopy showed beating cilia on the surface of the dermis, and the epithelial layer stained positively for the ciliated cell marker acetylated α-tubulin, the secretory cell marker MUC5AC, and the epithelial cell marker pan-cytokeratin on top-down whole-mount confocal microscopy. Staining with haematoxylin and eosin (H&E) demonstrated a pseudostratified mucociliary layer along the length of the dermis. 24 h after transplantation, a pseudostratified, ciliated layer could be observed on H&E staining of sections of trachea. At 5 days, the respiratory epithelial layer consisted of a single layer of cytokeratin 5-positive epithelial cells. Interpretation This study is the first, to our knowledge, to report the delivery of a transplantable tissue-engineered respiratory epithelial graft with mucociliary function. 24 h after transplantation the mucociliary layer was preserved although only a basal layer was demonstrated by 5 days, possibly due to the loss of the air–liquid interface within the muscle wrap. Funding Medical Research Council.