Impact of mouse tracheal basal cell expansion medium on formation and metabolic characteristics of tracheospheres

Airway basal cells have been shown to play important roles in lung disease, prompting their use in research involving 3-dimensional (3D) organoid culture. Mouse tracheal basal cells (MTBC) represent a key model in understanding the differentiation of airway basal cells in normal development and diseased states. The isolation, expansion, and culture media of basal cells can affect their capacity to maintain stemness and properly differentiate, thereby introducing unknown experimental variables. As MTBC use in 3D organoid culture proceeds, there is an increasing need for comparative studies to identify unknown experimental variables. In this study, we examined the effects of MTBC expansion media on the differentiation and tracheosphere-forming ability in 3D culture. MTBC were isolated from mouse tracheas, and three commonly used airway basal cell media were compared during MTBC expansion in cell culture (hereafter referred to as Media 1, 2, and 3). Following expansion, MTBC were cultured identically in 3D conditions to analyze tracheosphere-forming capability. Image analyses were performed to characterize tracheosphere count, size, and morphology, while metabolic and transcriptomic signatures were analyzed to assess MTBC differentiation in 3D culture. Expansion of MTBC in the three distinct media resulted in subsequent 3D tracheospheres that displayed unique metabolite and gene expression profiles, accompanied by overall changes in tracheosphere count and size. MTBC expanded in Medium 1 displayed lower levels of TCA cycle intermediates, along with lowered expression of differentiation markers Foxj1 and Scgb1a1, compared to MTBC expanded in Media 2 or 3. Pathway analysis showed an upregulation of idiopathic pulmonary fibrosis signaling pathways in tracheospheres cultured from MTBC expanded in Media 2 or 3. This study identified several key differences in tracheosphere-forming ability of MTBC resulting from MTBC expansion media and highlights the importance of transparency in cell culture protocols, particularly those involving stem cells or 3D culture.