Development of a 3D in vitro model of the pancreatic acino-ductal unit using melt electrospinning writing.

Pancreatic ductal adenocarcinoma, PDAC, the main neoplastic disease of the pancreas (90% of cases), is a malignant tumour with a typically negative outcome and, indeed, it is the fourth cause of cancer death in the world, despite being the twelfth most common cancer in terms of incidence. The 5-year survival rate is below 8% and this aggressiveness is related to the rapid development without specific symptoms, the absence of screening tests, the consequent late diagnosis (85% of patients show metastases when the disease is discovered), and the inefficiency of current therapies, with just 10% of patients operable at the time of diagnosis. Despite numerous studies, the causes and early steps of PDAC onset are currently unclear. However, the tumour microenvironment is known to play a central and active role in the development and progression of pathology and it constitutes a major limitation to treatment potential. In this context, the development of a 3D in vitro model of the pancreatic acino-ductal unit, the fundamental unit of the exocrine pancreas, could allow to study of both physiological and pathological tissues and to understand the development of PDAC and its interactions with the extracellular matrix (ECM). To achieve these goals, a PCL scaffold was fabricated via a promising additive manufacturing technology, melt electrospinning writing (MEW). MEW uses the electric potential difference between a needle and a collector to generate a molten polymer filament that, appropriately deposited on the collector thanks to the relative motion for the needle, solidifies and allows to obtain the required 3D geometry. Thanks to this process, it is possible to obtain filaments with diameters of a few micrometers that allow to create highly porous structures with interconnected pores suitable for cell infiltration and highly biomimetic of the ECM structure. Nonetheless, there are currently no publications attesting to MEW use applied to the exocrine pancreas and PDAC, making this work the first example of a scaffold-assisted in vitro model realised by MEW technology for the study of pancreatic cancer. Specifically, this thesis project involved both the design and manufacturing of scaffolds and cellular testing on them. The design started from a CAD drawing and parallelepiped geometries with a square base and a central cavity in the shape of a hemisphere or cylinder were tested to mimic the central part of the acino-ductal unit. For the scaffold production step, several parameters and combinations of them were tested and optimized to achieve adequate fiber and pore size and printing fidelity. Sizes were assessed by analyzing images obtained by SEM. Cellular assays were performed using the HPDE-KRAS, HFF-1, and PSC cell lines and studying viability at different time-steps using the CellTiter-Blue assay. The seeded scaffolds were also observed under a confocal microscope, labeling the nuclei and actin filaments. Future developments of the work should include the insertion of a channel from the central cavity of the scaffold, to better mimic the real geometry, and cocultures to observe the interaction between the different cells on the scaffolds.