Development and characterization of an organ-on-a-chip model resembling exocrine pancreas for studying pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer due to the lack of early diagnosis, rapid progression, early metastasis, and limited response to treatment. No test for early detection exists, and nearly all patients are asymptomatic in early stages. As a result, 90% of cases are diagnosed at an advanced stage, making surgery—the gold standard—too risky or ineffective. PDAC cells develop genetic and epigenetic changes that make them resistant to chemotherapy and radiation, complicating the search for new therapeutic strategies. There is no effective treatment for PDAC. Although immunotherapy is showing promise, there is an urgent need to better understand the limitations of current treatments. Improving knowledge of the mechanisms behind the poor response to therapy in PDAC is essential to enhancing drug efficacy and improving patient outcomes. In vitro models that better replicate tumor behavior are crucial for advancing personalized therapies. 3D models offer advantages over traditional 2D and animal models, enabling spatial organization and tissue-like structure. In PDAC, mimicking the tumor microenvironment (TME) is key to understanding disease progression and treatment responses. However, organoids and spheroids have limitations, including oversimplification of complex tissues, lack of nutrient exchange, and standardization issues. Microfluidic technology addresses these challenges by creating a controlled environment that optimizes factors such as temperature, pH, nutrient and oxygen supply, and waste removal. It also replicates physiological conditions like perfusion and nutrient gradients, vital for cell viability. This thesis presents an organ-on-chip model to replicate the pancreatic acino-ductal unit in vitro. The microfluidic device consists of three PDMS layers: a reservoir for culture media, a top layer where two types of human pancreatic duct epithelial (HPDE) cells were seeded,HPDE-WT to simulate a healthy environment and HPDE-KRAS to simulate a pathological condition, and a bottom layer containing human foreskin fibroblast (HFF-1) cells suspended in collagen hydrogel to mimic the stromal component. Each layer was fabricated using 3D printing and replica molding techniques. A microporous membrane made from polycaprolactone and gelatin (PCL/Gel) separates the top and bottom layers. The microfluidic device was characterized through various tests. PDMS replicas were analyzed using optical microscopy, while membrane morphology was studied with Scanning Electron Microscopy (SEM). Hydrophobicity was measured by contact angle, and membrane permeability was evaluated using fluorescein isothiocyanate-dextran (FICT-dextran). A preliminary drug delivery test using bortezomib validated the model for drug delivery within the PDAC-chip. Cell proliferation and viability on the chip were tested under different conditions. Viability was tested using live/dead assays and fluorescence microscopy. Immunofluorescence verified the proper distribution and morphology of cells. These studies demonstrate the successful optimization of a microfluidic device capable of replicating the pancreatic acino-ductal unit, providing a valuable platform for studying PDAC and stromal interactions. This system holds potential for drug development and improved understanding of PDAC biology.