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

Abstract

17The respiratory epithelium is exposed to assault by toxins and pathogens through the process 18 of inhalation, which has numerous implications on both human and animal health. As such, 19 there is a need to develop and characterise an in vitro model of the airway epithelium to study 20 respiratory pathologies during infection or toxicology experiments. This has been achieved 21 by growing airway epithelial cells at an air-liquid interface (ALI). Characterisation of ALI 22 models are not well-defined for airway epithelial cells derived from non-human species. In 23 this study we have fully characterised a bovine airway epithelial cell models (AECM) grown 24 at an ALI in relation to ex vivo tissue. The morphology of the model was monitored at three 25 day intervals, to identify the time-period at which the culture was optimally differentiated. derived from donor animals, our bovine AECM was shown to be highly representative of the 35 in vivo bovine bronchial epithelium and can be utilised in the study of respiratory 36 pathologies. 37 peer-reviewed)

Air‐liquid interface cell culture: From airway epithelium to the female reproductive tract

“…Until day 21, the cell layer differentiates into high columnar shape, and the overall topography remains consistent afterwards (Figure ). The cellular height increases from ≤5 μm on day 3 to 15–40 μm (depending on cell origin) on day 21 (Chen, Einspanier, & Schoen, 2013b; Cozens et al., ; Gualtieri et al., ; Munson, Wilkinson, & Schlafer, ; O'Boyle, Sutherland, Berry, & Davies, ).…”

“…One hallmark of differentiation in airway and oviduct epithelia is the presence of motile cilia, which are responsible for clearance and transport functions. Under ALI conditions, a confluent cell layer is formed approximately 3–7 days after seeding; however, cultured cells lack cilia at that time point (Cozens et al., ; Gualtieri et al., ; O'Boyle et al., ; You et al., ). A few days later, patches of short cilia become visible, reflecting the early stages of ciliogenesis.…”

“…A few days later, patches of short cilia become visible, reflecting the early stages of ciliogenesis. Long, motile cilia could be observed from day 9–10 onwards, which progressively increase to maximum ciliation by around week 3 and remain relatively constant thereafter (Chen et al., 2013b; Cozens et al., ; O'Boyle et al., ; Prince, Krunkosky, & Krause, ; You et al., ). In ALI cultures of human airway epithelial cells, a steady increase in the proportion of ciliated cells has been observed for up to 5–6 months (Wiszniewski et al., ).…”

“…A steady decline in TEER is observed after introducing the ALI. This reflects the formation of a leaky to moderate leaky epithelium with vivid basal‐apical transport and secretion functions (Abraham et al., ; Chen et al., 2013b, ; Cozens et al., ; O'Boyle et al., ; Rajagopal, Tollner, Finkbeiner, Cherr, & Widdicombe, ; Wang et al., ).…”

“…Similarly, ALI cultures are currently the most commonly used approach to study the biology of airway epithelia. Tracheal and bronchial epithelial cells cultured at the ALI differentiate and form pseudostratified cell layers with tight junctions, cilia, and mucin production, faithfully recapitulating the characteristics of the upper airway epithelium in vivo (Cozens et al., ; Pezzulo et al., ; Whitcutt et al., ). Compared to the in vivo situation, ALI cultured cells show certain differences in the cell population, cell cycle, and proliferation as well as immune competence.…”