Developing a Combined Calu-3 Barrier and Smooth Muscle Model of the Airways Utilising a Perfused Microphysiological System
Adeel A. Ahmed1, Emily L. Richardson2, Tomasz Kostrzewski2 & Darragh Murnane1
1University of Hertfordshire, Hatfield, AL10 9AB, UK
2CN Bio Innovations Limited, Cambridge, CB4 0WN, UK
Summary
Successfully incorporating medium perfusion into the standard insert-based air-liquid (ALI) Calu-3 model has resulted in a more physiologically representative pre-clinical In-Vitro model. The perfusion led to a tight epithelial monolayer working as an effective barrier to paracellular permeability and was maintained for up to 28 days. This tight barrier was established 7 days quicker than the static equivalent. qPCR analysis showed an increase in ZO1 and Claudin-1 expression in the perfused model between days 7 and day 14, showing barrier formation was still occurring between the two timepoints. Using immunofluorescent staining, ZO1 was also shown to be significantly increased in expression in the perfused model at day 7 compared to the static model. Significantly, at day 7 the expression of the OCT1 cation transporter was greater in the perfused model than in the static model. Concurrently, airway smooth muscle cells were cultured in the basolateral face of an insert membrane to examine the feasibility of a micro-physiological airways tissue co-culture model. Cell population expansion and functionality was demonstrated using alpha-smooth muscle actin staining with microscopy revealing the formation of a differentiated smooth muscle architecture. Culturing Calu-3 under perfused conditions resulted in faster formation of a permeability barrier than static. Furthermore, bronchial smooth muscle cells were successfully cultured on both the underside of the insert and the base of the well plate. Together, the construction of these models will allow more advanced and predictive models suitable for bioavailability studies and disease modelling.