Development of Human In-vitro 3D Models for Idiopathic Pulmonary Fibrosis (IPF)

Abstract

Idiopathic Pulmonary Fibrosis (IPF) is a chronic and deadly respiratory disease. It affects primarily individuals aged between 45 and 70 years, and most of them are diagnosed in the late stage. A major limitation is the incomplete understanding of its mechanism of IPF disease and the lack of effective treatment options. So, there is a need to find potential drugs to treat the IPF disease, and to address these challenges, it is necessary to develop IPF disease-specific advanced 3D in-vitro model. Furthermore, in recent decades, many studies have been performed and found that 3D in-vitro models mimic the human microenvironment. Recent research trends show a shift from conventional 2D cell cultures to 3D cell culture systems, as 2D cell models fail to accurately replicate cell morphology and pathophysiology, particularly in fibrotic diseases. In this project, the main focus was on developing 3D in-vitro human models for idiopathic pulmonary fibrosis to enable high-throughput drug screening and target identification, which will help to find potential drugs for the treatment of IPF disease. Optimized the cell culture protocols and other in-vitro assay protocols, namely RNA isolation and immunocytochemistry protocols, for both 2D plates with different stiffness levels and the 3D lung fibrosis spheroid model. Several optimization steps were performed, including determining the appropriate titration of the cell seeding density and TGF-β concentrations for in-vitro 2D and 3D models. In addition, cell culture protocols were optimized for Matrigel-based and Growdex-based organoid models. For plates with different stiffness levels, a comparison study was performed between CytoSoft plates (product purchased from Advanced Biomatrix) and Softwell plates(product purchased from Matrigen). Based on immunocytochemistry imaging results, Softwell plates were selected due to their differential expression of α-SMA in response to stiffness. The 3D lung spheroid model was chosen for further experiments due to its ease of handling and gene expression analysis showed that ACTA2 expression was lower in spheroid models compared to tissue culture-treated phenoplates. Moreover, spheroids overexpressed fibronectin-1 and matrix metalloproteinases-2 compared to tissue culture-treated phenoplates, which indicates excessive deposition of extracellular matrix proteins, which is a hallmark of idiopathic pulmonary fibrosis. Organoids based on Matrigel and Growdex were excluded due to technical handling difficulties encountered during organoid culture. Taking into account these findings for future experiments, it is necessary to carry out comprehensive research by performing RNA-Seq and proteomics studies to demonstrate that the 3D lung fibrosis spheroid model closely resembles the human microenvironment.