Abstract Rationale Survival after lung transplantation continues to lag behind that of other solid organ transplants, primarily due to high rates of rejection. To expand our ability to study lung transplant rejection using primary human cells, we previously developed airway and alveolar organoids from lung transplant patients. Organoids are grown from bronchoalveolar lavage (BAL) performed in a lobar segment of the transplanted lung; cells are cultured under conditions that favor either airway or alveolar epithelial cell expansion. To advance our organoid models, we asked whether these cells originate from the lung transplant donor or recipient. Methods Under our IRB-approved Lung Transplant Biorepository, we obtained BAL fluid from lung transplant recipients undergoing surveillance bronchoscopies at the University of Wisconsin-Madison. Using our established methods, we derived airway and alveolar organoids from each BAL specimen. Organoids were passaged 2-3 times, until at least 200,000 cells were obtained. Cells were then collected and analyzed by short tandem repeat (STR) analysis. For each patient, we obtained specimens of their native lung tissue, removed at the time of transplant, as a control containing only recipient cells. By comparing STR profiles of organoids with that of the recipient’s native lung tissue, we determined the percentage of organoid cells that originated from the transplant donor and recipient. Results We analyzed five lines of airway organoids and found a range of results. Two lines contained 100% donor-derived cells, while two others were mixtures of donor and recipient cells (60% donor and 43% donor). One line contained 100% recipient cells. In contrast, three lines of alveolar organoids all contained 100% donor cells; no recipient cells were detected. For airway and alveolar organoids that contained 100% donor cells, we also showed that, for the same patient, the result was the same 3 months and 9 months post-transplant. Conclusions Airway and alveolar organoids derived from the BAL of lung transplant recipients can serve as a source of donor lung epithelial cells. Alveolar organoids are fully donor-derived, while airway organoids may include recipient cells, likely due to tracheal basal cells captured during bronchoscopy. We are now developing methods to isolate pure populations of donor and recipient airway cells, and we are determining the origin of immune cells in BAL. This will provide a platform for co-culturing donor epithelial cells with recipient immune cells to model human lung transplant rejection in vitro, using only cells from the living patient. This abstract is funded by: American Thoracic Society Unrestricted Research Grant 24-25U4; NIH KL2TR002374; UW Letters & Science STEM Scholars Fund
Liu et al. (Fri,) studied this question.