ESTABLISHMENT AND MORPHOLOGICAL CHARACTERISATION OF 3D LUNG CELLULAR MODELS WITH APPLICATIONS IN MODELLING PULMONARY FIBROSIS IN VITRO

Idiopathic pulmonary fibrosis (IPF) is a chronic respiratory disease characterized by progressive fibrotic scarring with a very low survival rate after its diagnosis. Despite its severity, its pathophysiological mechanism is still unclear due to the involvement of a heterogeneous population of lung cells that interact during its pathogenesis. To understand this complex interaction of cells, a well-characterized model is the Bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model. But in accordance with the 3Rs recommendations, alternative in-vitro three-dimensional (3D) lung cellular models can better mimic some aspect of PF pathogenesis. Although a lot of pluripotent stem cells (PSCs)-derived 3D models, along with some diseased or immortalized adult lung cells-derived models, are available but the healthy and adult primary mouse lung (PML) cells-derived organoids model for PF modelling is still lacking. Therefore, the current research aimed at establishing a heterogeneous population containing 3D models for PF modelling. We successfully established in vitro culture conditions for spheroids and organoids derived from PML. This achievement allowed us to obtain 3D structures that closely mimic key aspects of the lung microarchitecture, including cell–cell and cell–matrix interactions. The use of mixed cellular populations enabled the spontaneous organization of epithelial and stromal components within the same structure, providing a more physiologically relevant model compared to traditional two-dimensional (2D) cultures. To obtain these models, we utilized stepwise optimization, which included optimization of 2D culture followed by simple 3D spheroids in EPCM5% medium and lastly complex 3D organoids optimization with healthy and adult PML-derived cells in specific medium conditions. After 10 days of culture in maintenance medium (MM) (MM-D10 condition), well-structured lung organoids characterized by a large central lumen and a complex three-dimensional organization were obtained. These organoids exhibited a high proportion of epithelial cells (78.4%) along with fibronectin (FNC) positive (28.04%) and ECM-producing cells (14.69%). The low apoptotic activity (Caspase3 1.17%) together with moderate proliferative activity indicated by Ki67 expression (14.28%) suggests a high degree of cellular stability and viability within the organoid microenvironment. Following differentiation induction up to day 14 (4 days total) in differentiation medium (DM) (MM-DM-D14 condition), the proportion of epithelial cells slightly decreased (67.40%), while Caspase3 activity increased (12.85%), likely reflecting enhanced apoptosis due to the spontaneous withdrawal of growth factors and medium changes. Consistently, Ki67 expression declined (4.40%), indicating reduced proliferative capacity as cells underwent differentiation. Although the proportion of FNC remained relatively stable, ECM-producing cells markedly increased (40.42%), suggesting enhanced extracellular matrix deposition and tissue remodelling during the differentiation phase. Overall, these findings indicate that the established culture conditions support the formation of structurally stable and viable lung organoids that undergo controlled differentiation and extracellular matrix maturation over time. Then we utilized the established 3D organoid models for modelling PF through an injury induced by an optimized dose of BLM (1µg/ml). BLM was mixed with DM and dispensed in a culture dish at day 10 for 4 days (BLM-D14 condition). Upon BLM-injury, we noticed the hallmarks of cellular damage, e.g. higher P53 level (37.86%) in the BLM-D14 condition as compared to the control MM-DM-D14 condition (9.63%), along with high Caspase3 activity (29.8%). Moreover, we also found a non-significant slight increase in Vimentin (VIM) level from 19.69% to 35.26% and FNC level from 29.2.% to 35.26%. Furthermore, a decrease in bronchial epithelial cells from 67.40% to 56.68% was also observed after BLM injury. Unexpectedly, we also observed a non-significant slight increase in SFTPC-positive AT2 cells from 3.42% to 7.58%. Moreover, we also observed the resolution of injury after BLM removal and growing cells in only DM medium for an additional 3 days (BLM-D17 condition). We observed that the level of P53 in the control MM-DM-D17 (4.69%) condition and the BLM-D17 condition (5.33%) was not statistically different. We also didn’t observe a significant difference in the level of Caspase3 activity between the MM-DM-D17 condition (4.94%) and the BLM-D17 condition (6.4%). Moreover, a slight but non-significant increase in the level of VIM was observed in the BLM-D17 condition (40.6%) as compared to the MM-DM-D17 condition (22.2%). While the level of FNC remained stable in both conditions. Finally, a slight increase in bronchial epithelial cells in the BLM-D17 condition (60.5%) as compared to the MM-DM-D17 condition (57.7%) was observed, along with a slight decrease in AT2 cells in the BLM-D17 condition (5.16%) as compared to the MM-DM-D17 condition (6.83%). These findings successfully recapitulate the findings of the gold standard pre-clinical BLM-induced PF mouse model. Therefore, in line with the 3Rs recommendation, we established and characterized in vitro 3D organoid model derived from a heterogeneous population of lung cells as a model for PF research.