Abstract
Alveolar type 2 (AT2) cells play a pivotal role in maintaining lung homeostasis, and the generation of three-dimensional cultures, such as alveolospheres, provides a valuable model for studying lung development, pathologies, and drug responses. Here, we investigate the critical extracellular matrix (ECM) characteristics that influence AT2 alveolosphere formation and growth. By encapsulating AT2 cells in different extracellular matrix-based hydrogels, we identified laminin as a key ECM protein supporting robust alveolosphere formation akin to Matrigel. Our results show that laminin-rich hydrogels support alveolosphere formation across murine and human primary AT2 cells as well as induced human pluripotent stem cell derived AT2 cells (iAT2 cells). Notably, matrix stiffness strongly influenced alveolosphere formation. Hydrogels with a low Young's modulus and high compliance supported a greater number of alveolospheres, exhibiting a broader size distribution and a higher proportion of larger alveolospheres. Moreover, inhibition of matrix degradation and cellular contractility disrupted alveolosphere formation. Leveraging these insights, we developed a multicellular vascularized alveolosphere-on-a-chip model by integrating alveolospheres with endothelial cells and lung fibroblasts within a microfluidic device. Application of this model to assess the inflammatory effects of menthol, a common e-cigarette flavor, demonstrates its utility in evaluating the pulmonary effects of chemical exposures on alveolar cells. Our findings highlight the critical role of matrix physicochemical properties on alveolosphere formation and establish a versatile platform for advancing the study of lung biology, disease mechanisms, and drug discovery.