A co-culture model of the bovine alveolus

Abstract

The epithelial lining of the lung is often the first point of interaction between the host and inhaled pathogens, allergens and medications. Epithelial cells are therefore the main focus of studies which aim to shed light on host-pathogen interactions, to dissect the mechanisms of local host immunity and study toxicology. If these studies are not to be conducted exclusively in vivo, it is imperative that in vitro models are developed with a high in vitro- in vivo correlation. We describe here a co-culture model of the bovine alveolus, designed to overcome some of the limitations encountered with mono-culture and live animal models. Our system includes bovine pulmonary arterial endothelial cells (BPAECs) seeded onto a permeable membrane in 24 well Transwell format. The BPAECs are overlaid with immortalised bovine alveolar type II epithelial cells and cultured at air-liquid interface for 14 days before use; in our case to study host-mycobacterial interactions. Characterisation of novel cell lines and the co-culture model have provided compelling evidence that immortalised bovine alveolar type II cells are an authentic substitute for primary alveolar type II cells and their co-culture with BPAECs provides a physiologically relevant in vitro model of the bovine alveolus. The co-culture model may be used to study dynamic intracellular and extracellular host-pathogen interactions, using proteomics, genomics, live cell imaging, in-cell ELISA and confocal microscopy. The model presented in this article enables other researchers to establish an in vitro model of the bovine alveolus that is easy to set up, malleable and serves as a comparable alternative to in vivo models, whilst allowing study of early host-pathogen interactions, currently not feasible in vivo. The model therefore achieves one of the 3Rs objectives in that it replaces the use of animals in research of bovine respiratory diseases.

Modelling early events in Mycobacterium bovis infection using a co-culture model of the bovine alveolus

“…Initial studies utilised the co-culture model grown at air–liquid interface, as previously published by the authors 36 , 37 . By rigorous washing of the membrane and recovery by centrifugation of BCG from the apical and basolateral compartments, the time-dependent tracking of migrating BCG was determined to be possible over a period of 48 h.p.i.…”

“…BPAECs were cultured for 5–7 days, replacing EGM-2 in the basolateral chamber and removing apical medium which had seeped through from the basolateral side of the membrane. Bovine Alveolar Type II (BATII) cells were derived from primary ATII cells isolated and immortalised as described previously 36 , 80 . For the current study, passages 14–18 were used.…”

“…Prior to infection, all medium was removed, and co-cultures were gently washed once with Dulbecco’s Phosphate Buffered Saline (DPBS) (Thermo Fisher Scientific, Waltham, MA) as previously described 36 . EGM-2 (600 μL) was added to the basolateral chamber.…”

“…We recently reported the development of the first co-culture model of the bovine alveolus 36 . This consists of a layer of bovine pulmonary arterial endothelial cells (BPAECs) onto which bovine alveolar type II (BATII) epithelial cells are laid.…”

“…This consists of a layer of bovine pulmonary arterial endothelial cells (BPAECs) onto which bovine alveolar type II (BATII) epithelial cells are laid. BATII cells are a novel cell line generated by the lentiviral transduction of particles encoding the catalytic subunit of human telomerase (hTERT) and Simian Virus 40 large T antigen (SV40) onto primary ATII cells from adult bovine lung 36 , 37 . The epithelial-endothelial co-culture is cultured at air–liquid interface on permeable membranes, to recapitulate rudimentary aspects of the primary bovine alveolus.…”