In vitro microfluidic 3D platform as a pathological model of the pancreatic acino-ductal unit

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest tumours that affect the exocrine pancreas and it has a poor survival rate. These unfortunate data are ascribed to the absence of symptoms in the early stages, invasive metastasis, and high resistance to the therapeutic options. For these reasons, in vitro models that can reproduce the microenvironment of the pancreatic tissue are required to develop therapies and to predict drug safety and efficacy in humans. Organ-on-chips can be a powerful technology to achieve these goals. In this thesis project, a microfluidic device for modelling the pancreatic acino-ductal unit, is developed. The function of the platform is to investigate the crosstalk between PDAC and the stromal components which plays a fundamental role in cancer progression. To this aim a multilayers device in Polydimethylsiloxane (PDMS) composed by two layers separated by a membrane: a bottom layer loaded with collagen hydrogel mimicking the stromal component and a top layer reproducing the tumour component were fabricated using replica moulding. The connection between the two components was realized by the microporous membrane in polycaprolactone and gelatin (PCL/Gel) that was obtained by the electrospinning technique. SU-8 photolithography and laser ablation techniques have been also implemented to produce the molds of the bottom and top layers, respectively. The surface characterization of the mold was carried out through the use of a profilometer while the morphological characterization of the microfluidic device was performed using optical microscopy and Field Emission Scanning Electron Microscopy (FESEM) proving how the geometries reproduced were of the desired dimensions. In addition, a fluidic characterization was carried out through capacity and diffusivity tests. In vitro studies were implemented to evaluate the cell proliferative capability and the cellular response of cells cultured in the device. Moreover, different cell lines, specific of the pancreatic tissue, were used as mono- and co-culture. Specifically, Pancreatic Stellate Cells (PSCs) and Human Pancreatic Duct Epithelial Cells with mutation in oncogene KRAS (HPDE-KRAS) have been employed to reproduce the stroma and PDAC tissue that characterize the tumoral microenvironment in vivo. Vitality assays and fluorescence imaging were performed to analyse the cellular activity and the morphological structure of cells seeded in the bottom layer, in the top layer, and in the assembled device. Results suggest that HPDE cells adhered and proliferated on PCL/Gel membrane and PSCs were vital when loaded into collagen hydrogel for the whole culture period.