Methods:

Axin2 CreERT2-tdT mice were used to provide a model for the AEP phenotype. Axin2 CreERT2-tdT epithelial cells were sorted using fluorescence-activated cell sorting (FACS). Cells were seeded in culture along with mice lung fibroblasts cocultured in Matrigel& small airway epithelial cell growth medium 50/50mix. Cells were monitored for organoid development with media changes every 2 days. Organoids were harvested after 3 weeks. Laboratory standard organoid retrieval protocol was used to extract organoids. Samples were stored in organoid washing buffer for storage for later use. Samples underwent several protocols for imaging. Sample set #1 underwent cryofreezing then staining via H&E protocol. Sample set #2 underwent whole mount immunohistochemistry, those organoids were stained for DAPI, RAGE, SPC1 markers for cell nuclei, AT1, AT2 respectively.

Results:

Three plates were successfully seeded with Axin2 CreERT2-tdT epithelial cells. However, only two plates developed successful cell density evident by organoid development to be experimentally viable. This was determined by spheroids visualized by the naked eye and under bright field microscope. All staining protocol were successful thus visualized via microscopy. Images depict spherical organoids meeting the definition of self-organizing strutures. Immunostained organoids depicts expression of SPC and RAGE markers highlighting the differentiation process of AEPs into AT2 and AT1 cells.

Conclusion:

The AEP model of lung organoids is a viable model to study alveolar structure. An ideal framework to conduct further experiments to elucidate the signaling mechanisms that stimulate the regenerative process of AEPs. Especially in the presence of lung injury, one hopes to find out how the signaling occurs in epithelial cells without endothelial influence.Lung injury provides a source of signifcant morbidity in the United States. Whether acute in nature e.g. SARS-CoV-2 or chronic e.g. Idiopathic Pulmonary Fibrosis. Both causes significant lung injury particularly in the alveolus affecting gas exchange thus respiration. In order to understand the mechanisms of lung injury in turn the regenerative process. Researchers have developed a model for development and differentiation of the lung, particularly the alveolus. This model called lung organoids. Organoids are defined as self-organizing, 3-D structure derived from progenitor cells in culture. In the alveoli, the alveolar epithelial progenitor cells (AEPs) initially form AT2 cells responsible for surfactant production that reduces surface tension. Later, AT1cells develop, the cells are responsible for gas exchange. In order to understand the molecular processes behind the stabilization of AEPs along with regeneration. This project hopes to elucidate genetic factors that favor the regenerative phenotype in this population within the alveoli.