A novel method for expansion and diferentiation of mouse tracheal epithelial cells in culture

Results

Serum free medium and the inhibition of Notch signaling enables basal cell expansion. Although the current methods of ALI culture represent the pseudostratifed airway epithelium in vitro, a limitation of this technique is the relatively low number of cells obtained from a trachea. We frst validated the existing culturing method of MTEC whereby isolated MTEC are cultured directly onto transwell inserts (Fig. S1). Tis culture method allowed us to plate approximately 4 to 5 transwell inserts (50,000 cells per insert) using two mice (±100,000 cells per trachea). Improving the expansion of MTEC will allow for multiple experiments to be performed with fewer mice. Ideally, following isolation from the trachea, cells can be expanded in culture for at least two passages to generate sufcient cells for multiple experiments (Fig. 1a). In previous studies, MTEC are isolated and plated on inserts in proliferation medium and upon reaching confuency, cultured at ALI in diferentiation medium (Table S1)13,14. Following expansion in proliferation medium, MTEC showed a limited proliferation capacity, with the presence of swollen cells with enlarged vacuoles (Fig. 1b). We were unable to successfully passage MTEC grown in proliferation medium. Because we considered the possibility that proliferation medium may be defcient in factors preventing senescence, we switched to another medium. Keratinocyte Serum Free medium supplemented with epithelial growth factor (EGF), Bovine pituitary extract (BPE) and isoproterenol (KSFM) allows for expansion of human airway epithelial cells in vitro15,16. Additionally, adding a selective inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK), Y-27632, to the culture medium has been shown to increase proliferation of airway epithelial cells17. Growing MTEC in KSFM with Y-27632 indeed resulted in improved cellular expansion, survival and morphology (Fig. 1b). However, despite improved cell morphology and survival, the expansion rate was very slow and still a large number of enlarged cells were present. Notch signaling has been shown to play a pivotal role in preserving the basal cell population and in subsequent diferentiation into specialized cell types. Furthermore, inhibition of Notch signaling has been shown to lead to an increased number of basal cells in mouse ALI cultures10–12,18,19. To evaluate whether inhibition of Notch signaling promotes the expansion of MTEC afer isolation, we added DAPT, a γ-secretase inhibitor and indirect Notch signaling inhibitor, to the KSFM medium containing Y-27632. Growing MTEC in the presence of DAPT and Y-27632 in KSFM medium resulted in a marked increase in proliferation rate with a slightly better morphology compared to cells grown in absence of DAPT; these diferences were already apparent afer 5 days of culture of P0 cells, but even more afer passaging whereby an increased number of transwell inserts could be obtained when DAPT was present during expansion (Fig. 1c). KSFM medium containing Y-27632 and DAPT will be further referred to as KSFM expansion medium. Notably, expanding MTEC from two mice in KSFM expansion medium resulted in 42.5 million cells, sufcient for 85 transwell inserts when plated at a density of 8×104 cells/cm2 , which is in stark contrast to the 200,000 cells required for 4–5 transwell inserts that are obtained if MTEC were isolated and cultured directly onto the transwell inserts. Also, no contaminating fbroblasts were observed during MTEC expansion. Taken together, we conclude that MTEC expansion is more efcient in the presence of the Notch signaling inhibitor DAPT than without

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