Development of a acino-ductal unit 3D in vitro model through additive manifacturing technologies

Sviluppo di un modello 3D in vitro dell’unità acino-duttale pancreatica, attraverso tecnologie di additive manufacturing In the last decade a vast number of pathologies connected to both endocrine and exocrine components of pancreas have been observed to arise with an increasing rate in their occurrence; in particular the pancreatic ductal adenocarcinoma (PDAC) stands out as the main neoplastic disease of the pancreas and as an extremely lethal malignancy with an increasing frequency in the occurrence. PDAC has a low survival rate, which is connected to the late diagnosis due to lack of specific and clear symptoms before late stages and the resistance to current treatments ascribed to the structure and composition of the tumor microenvironment. In spite of the fact that research on PDAC has grown in the last decade , the knowledge regarding its causes and specific pathological pathways is still lacking, even though it is clear that the dense fibrotic bioarchitecture of the mass and the heterogeneity of the tumor itself have a central role in the observed resistance to treatments and the consequent progression of the pathology. In this scenario the conventional 2D cell cultures used to study the PDAC have been found to be inconsistent to perform reliable drug screening and biotechnological studies since the tridimensional conformation of the tissue is highly relevant in its in vivo condition, hence 3D in vitro models acquire a crucial role as a preclinical and as a research tool getting closer to the physiological situation. Against this background the objective of this thesis project is based on the development of a 3D in vitro model of the fundamental unit of the exocrine pancreas, the adenomere, in order to possibly supply an instrument in the study of the pathological pancreatic tissue. The device development has been carried out using an interesting and innovating additive manufacturing technique called Melt Electro Writing (MEW) which is an emerging tool in the fibrous scaffolds manufacturing. Benefiting from the high precise deposition at the microscale of this technology, a methodical approach has been applied to comprehensively control the material deposition during the printing procedure using an innovating software called FullControl based on Python language that produces the G-code needed by the printer. The device has been designed in order to mimic the morphology of the adenomere and the structure proposed overcomes some intrinsic restriction of 3D print ing in the manufacturing of hollow caves by splitting in two halves the geometry, with the goal of form the correct structure again during the cell culture. The actual production has been then conducted optimizing several parameter combinations until the development of a structure with adequate fiber diameter and a sufficient precision in the deposition, assessed by the investigation through optical microscopy techniques and scanning electronic microscopy (SEM). Cellular cultures have been then conducted using Human Foreskin Fibroblasts (HFF-1) and Human Pancreatic Duct Epithelial (HPDE) cells in order to mimic the fibrotic nature of the physiological tissue and to grant a tumor-stroma communication. The in vitro cell tests have been then characterized through confocal imaging, labeling nuclei, cytoskeleton and E-cadherin proteins, and also through SEM.