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

Cozens, Daniel; Grahame, Edward; Sutherland, Erin; Taylor, Geraldine; Berry, Catherine C.; Davies, Robert L.

doi:10.1038/s41598-017-19079-y

Cited by 27 publications

(69 citation statements)

References 78 publications

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“…30 Of note, on thick scaffolds (over 300 µm), we did not see formation of mucociliary pseudostratified epithelium even after HA treatment. 31,32 Our results are in line with these reports. Previously, it was shown that the differentiation of the tracheal epithelium depends on the porosity of the scaffold used.…”

Section: The Role Of Matrix Thickness and Permeabilitysupporting

confidence: 93%

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Non‐woven bilayered biodegradable chitosan‐gelatin‐polylactide scaffold for bioengineering of tracheal epithelium

et al. 2019

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Objectives:The conversion of tissue engineering into a routine clinical tool cannot be achieved without a deep understanding of the interaction between cells and scaffolds during the process of tissue formation in an artificial environment. Here, we have investigated the cultivation conditions and structural features of the biodegradable non-woven material in order to obtain a well-differentiated human airway epithelium. Materials and methods:The bilayered scaffold was fabricated by electrospinning technology. The efficiency of the scaffold has been evaluated using MTT cell proliferation assay, histology, immunofluorescence and electron microscopy. Results:With the use of a copolymer of chitosan-gelatin-poly-l-lactide, a bilayered non-woven scaffold was generated and characterized. The optimal structural parameters of both layers for cell proliferation and differentiation were determined. The basal airway epithelial cells differentiated into ciliary and goblet cells and formed pseudostratified epithelial layer on the surface of the scaffold. In addition, keratinocytes formed a skin equivalent when seeded on the same scaffold. A comparative analysis of growth and differentiation for both types of epithelium was performed. Conclusions:The structural parameters of nanofibres should be selected experimentally depending on polymer composition. The major challenges on the way to obtain the well-differentiated equivalent of respiratory epithelium on non-woven scaffold include the following: the balance between scaffold permeability and thickness, proper combination of synthetic and natural components, and culture conditions sufficient for co-culturing of airway epithelial cells and fibroblasts. For generation of skin equivalent, the lack of diffusion is not so critical as for pseudostratified airway epithelium.

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Section: The Role Of Matrix Thickness and Permeabilitysupporting

confidence: 93%

“…32 Many of these factors are directly secreted by resident fibroblasts or are under fibroblast expressional control. 32 Many of these factors are directly secreted by resident fibroblasts or are under fibroblast expressional control.…”

Section: Co-cultivation Of Airway Epithelial Cells With Fibroblastsmentioning

confidence: 99%

Non‐woven bilayered biodegradable chitosan‐gelatin‐polylactide scaffold for bioengineering of tracheal epithelium

et al. 2019

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“…Cozens et al found an EGF concentration of 10 ng/mL most suitable for bovine bronchial epithelium 40 . As mAir was supplemented with EGF to a concentration of 10 ng/mL, our results support their findings.…”

Section: Discussionmentioning

confidence: 99%

Choosing the Right Differentiation Medium to Develop Mucociliary Phenotype of Primary Nasal Epithelial Cells In Vitro

Luengen

Kniebs

Buhl

et al. 2020

Sci Rep

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In vitro differentiation of airway epithelium is of interest for respiratory tissue engineering and studying airway diseases. Both applications benefit from the use of primary cells to maintain a mucociliated phenotype and thus physiological functionality. Complex differentiation procedures often lack standardization and reproducibility. To alleviate these shortfalls, we compared differentiation behavior of human nasal epithelial cells in four differentiation media. Cells were differentiated at the air-liquid interface (ALI) on collagen-coated inserts. Mucociliary differentiation status after five weeks was analyzed by electron microscopy, histology and immunohistochemistry. The amount of ciliation was estimated and growth factor concentrations were evaluated using ELISA. We found that retinoic-acidsupplemented mixture of DMEM and Airway Epithelial Cell Growth Medium gave most promising results to obtain ciliated and mucus producing nasal epithelium in vitro. We discovered the balance between retinoic acid (RA), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF) and fibroblast growth factor β (FGF-β) to be relevant for differentiation. We could show that low VEGF, EGF and FGF-β concentrations in medium correspond to absent ciliation in specific donors. Therefore, our results may in future facilitate donor selection and non-invasive monitoring of ALI cultures and by this contribute to improved standardization of epithelial in vitro culture.

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“…Bovine bronchial epithelial cells can be stimulated to differentiate into a more representative model of the in vivo microenvironment through exposure to the atmosphere [36, 37, 59]. The methodology for differentiation of bovine airway epithelial cells have been fully optimised [44] and the model well-characterised [45]. The differentiated BBEC model has been shown to replicate the hallmark defences of the respiratory tract, including active mucocilary clearance.…”

Section: Discussionmentioning

confidence: 99%

“…We have previously investigated the growth conditions required for optimal growth and differentiation of bovine bronchial epithelial cells at an ALI [44] and assessed the temporal differentiation of these cells to identify an optimum window suitable for infection studies [45]. The aim of the present study was to investigate the interactions of a panel of M. haemolytica isolates, representing virulent and commensal strains recovered from both cattle and sheep, with differentiated bovine bronchial epithelial cells grown at an ALI.…”

Section: Introductionmentioning

confidence: 99%

Interactions of pathogenic and commensal strains of Mannheimia haemolytica with differentiated bovine airway epithelial cells grown at an air-liquid interface

Cozens

Sutherland

Lauder

et al. 2017

Preprint

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18Mannheimia haemolytica serotype A2 is a common commensal species present in the 19 nasopharynx of healthy cattle. However, prior to the onset of bovine pneumonic 20 pasteurellosis, there is sudden increase in M. haemolytica serotype A1 within the upper 21 respiratory tract. The events during this selective proliferation of serotype A1 strains are 22 [19, 20] and can also be distinguished by differences in their outer membrane protein (OMP) 58 profiles [21], lipopolysaccharide types [21, 22] and nucleotide sequence variation in various 59 virulence-associated genes including lktA [23], ompA [24], tbpA and tbpB [25], plpE [26] as 60 well as a number of other genes [20]. The upper respiratory tract of healthy cattle is 61predominantly colonized by serotype A2 strains but, for reasons that are not clear (but 62 probably related to stress and/or viral infection), a transition occurs within this 63 4 microenvironment which leads to a sudden explosive proliferation in the number of serotype 64 A1/A6 bacteria present and subsequent colonization [1, 3]. This sudden and selective 65 explosion in the A1/A6 population within the upper respiratory tract leads to the inhalation of 66 bacteria-containing aerosol droplets into the trachea and lungs and the onset of pneumonic 67 pasteurellosis [27]. Crucially, the specific bacterial and host factors responsible for the 68 sudden shift from commensal serotype A2 to pathogenic serotype A1/A6 populations within 69 the upper respiratory tract are not clear. 70The leukotoxin (LktA) of M. haemolytica plays a central role in the pathogenesis of 71 pneumonic pasteurellosis and significant attention has been given to understanding the 72 molecular mechanisms associated with LktA activity within the lung [1, 3, 28, 29]. In 73 contrast, there has been far less focus on the interactions of M. haemolytica with respiratory 74 airway epithelial cells and events that might account for the sudden proliferation of serotype 75 A1/A6 bacteria within the upper respiratory tract. A contributing factor to our poor 76 understanding of early host-pathogen interactions associated with pneumonic pasteurellosis, 77 and indeed BRD in general, is the lack of physiologically-relevant and reproducible 78 methodologies with which to study the intricate molecular and immunological interactions 79 between pathogens and host. Traditionally, submerged, two-dimensional cultures of a single 80 cell type have been used to investigate interactions of M. haemolytic and other BRD 81 pathogens within the bovine respiratory tract [30-32] but these have numerous limitations: 82 they do not reflect the multicellular complexity of the parental tissue in vivo, they lack its 83 three-dimensional (3-D) architecture, and the physiological conditions are not representative 84 of those found within the respiratory tract. However, these characteristics that are lacking in 85 submerged cultures can be recapitulated using differentiated airway epithelial cells (AECs) 86 grown at an air-liquid interface (ALI) and, in recent years, s...

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

Cited by 27 publications

References 78 publications

Non‐woven bilayered biodegradable chitosan‐gelatin‐polylactide scaffold for bioengineering of tracheal epithelium

Non‐woven bilayered biodegradable chitosan‐gelatin‐polylactide scaffold for bioengineering of tracheal epithelium

Choosing the Right Differentiation Medium to Develop Mucociliary Phenotype of Primary Nasal Epithelial Cells In Vitro

Interactions of pathogenic and commensal strains of Mannheimia haemolytica with differentiated bovine airway epithelial cells grown at an air-liquid interface

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