Optimizing New In Vitro Methodologies For Assessing The Effects Of Mechanical Stimuli On Lung Cells: Implications For Lung Biology And Disease

Chronic lung diseases affect millions of people in the United States and are a leading cause of both morbidity and mortality. Studying how environmental factors affect lung cell biology and function is being increasingly recognized as a critical step in understanding lung disease pathogenesis and the development of new therapeutic approaches that combat lung diseases. These factors include lung extracellular matrix (ECM) composition and the mechanobiological factors of stiffness and cyclic mechanical strain, which during breathing, act on cells during the normal expansion and contraction of the lung. However, current methodologies for studying these factors have significant limitations and new approaches are necessary, particularly for investigating how these factors are altered in diseased lungs. As such, the goal of this thesis was to develop and optimize new in vitro methodologies that can be used to assess the effects of these three factors (ECM composition, stiffness, and cyclic mechanical strain (stretch)) on lung cells under both normal and diseased conditions, where idiopathic pulmonary fibrosis served as the disease model. I present work from two independent sets of studies that were designed to investigate the effects of these factors individually and in combinatorial fashion. The first study focused on how stiffness and ECM composition, alone and in combination, can cause changes in a representative relevant human lung cell type, human lung fibroblasts (HLF). A novel approach utilized ECM protein (hydrogel) solutions derived from decellularized normal or diseased human lungs (composition factor) coated onto the surface of CytoSoft® stiffness specific plates (2 kPa, 8 kPa, or 16 kPa) (stiffness factor). Endpoint assessments included cell morphology, growth, metabolism, and relevant gene expression. These results demonstrate that ECM composition and substrate stiffness, both alone and in combination affect HLF behavior. In the second series of studies, the goal was to assess how cyclic mechanical stretch impact function of an important lung cell type: alveolar epithelial type II (AT2) cells, critical for surfactant production. Utilizing AT2s derived from induced pluripotent stem cells (iAT2s) as a model system, the cells underwent biaxial stretch using a FlexCell® FX-5000™ Tension System for either 2 or 24 hours. Endpoint assessments included imaging and gene expression compared to un-stretched cells. The results show unique morphological changes in the stretched samples, while the gene expression data prove to be more variable. Collectively, this work shows how environmental and mechanical factors, including matrix composition, stiffness, and stretch, impact lung cell function and provides optimized methods to study such interactions. The unique methodology utilized should enable further investigations into both normal physiology and lung pathologies