C70-08 Establishment of a Patient-Derived Airway Organoid Model From Fresh and Frozen Samples

Abstract Rationale Organoid models are three-dimensional multicellular stem-cell-derived constructs mimicking in vivo tissue and thus offer new insights in cell mechanisms. The respiratory mucosa plays a key role in many lung disorders, such as chronic obstructive pulmonary disease (COPD) and asthma, which may be driven by type 2 inflammation. Airway organoids are useful to model disease pathogenesis as well as therapeutic screening and hence make progress in precision medicine. Our aim is to establish a patient-derived airway organoid culture model to investigate individual subtypes of airway inflammation in COPD and asthma. Methods We collected bronchial mucosa tissue, either form bronchoscopy biopsies or lungs received from lung transplantation patients. All patients agreed to the broad consent of the University Hospital Schleswig-Holstein. Tissue samples were collected in sterile medium and either directly processed or cryopreserved. Further, the tissue was minced into smallest possible pieces and enzymatically digested. After filtering the digested tissue solution through a cell strainer, the cell suspension was centrifuged and resuspended in medium. The human bronchial epithelial cells were seeded on collagen I coated plates to grow in a monolayer. When sufficiently grown, these cells were detached, palleted and counted. Finally, the cells were transferred in matrigel and seeded as domes. For four days, the organoids were treated with seeding medium before changing to differentiation medium. Quantitative reverse-transcription polymerase chain reaction (qPCR) from extracted organoid RNA and immunofluorescence microscopy of the organoids were performed to detect differentiated cell types. Results We successfully cultivated organoids from patients suffering from obstructive airway disease or not. Microscopically, cilia movement of the organoids was initially observed on day 14 of differentiation. Over the course of differentiation, qPCR experiments revealed increasing relative expression of CC10, FOXJ1 and MUC5AC and decreasing P63 and KRT5, indicating the differentiation of the cells in club, ciliated and goblet cells. These results could be validated with immunofluorescence microscopy. Conclusions With this protocol, we were able to demonstrate a method to reconstruct patient-derived bronchial epithelium with different cell types in a three-dimensional structure. Further experiments will focus on mimicking type 2 inflammation by interleukin stimulation and co cultures experiments. This abstract is funded by: None