3D bioprinting of a GelMA-based lung adenocarcinoma in vitro model

Lung cancer is one of the most diagnosed and deadly cancer worldwide. It is classified in two main groups according to the cell of origin and histopathological properties: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). The focus of this thesis is put on the most common subtype of NSCLC, the adenocarcinoma (ADC), which grown in the alveoli from type 2 pneumocytes, the epithelial cells involved in interstitial mucus production. Beyond tumor cells, the aggressiveness of ADC is linked to the microenvironment which mainly consists in the extracellular matrix (ECM), which provides mechanical and biological support to the cellular components of the stroma. Over the years many efforts have been put on the study of ADC heterogeneity using in vivo animal models. However increased awareness on ethical issues and limitations linked to differences with the human system led to the development of innovative in vitro culture models. Initially 2D culture systems have been employed because of ease handling and low cost, but the lack of the physiological ECM structure and of complex geometry promote the studying of 3D bioprinting techniques, especially the extrusion-based, to design 3D in vitro models that better mimic the physiological organization of the tissue. The aim of this thesis is the printing a lung adenocarcinoma model with a multi-material approach. For the fabrication of the model, A549 adenocarcinoma cells were chosen as cell line and they were embedded in a matrix of Gelatin Methacryloyl (GelMA), a biocompatible hydrogel widely used in extrusion bioprinting due to its viscoelastic properties. The optimized formulation is a GelMA with high degree of functionalization (around the 90%), derived from Type A gelatin dissolved at 10% w/v in cell culture medium. The methacryloyl added groups allow the crosslinking through UV-visible light irradiation in the presence of a photoinitiator (i.e., LAP), which triggers the polymerization through radical species formation. The obtained formulations were characterized by rheological analysis to select the optimal printing parameter. Further tests demonstrated the good printability of the obtained bioinks and the maintenance of high cell viability after the process. As show in various studies, the presence of channels perfused with culture medium is essential for providing cells with nutrients, growth factors, and oxygen. Indeed, in the absence of an appropriate nourishment, constructs could develop necrotic regions, which can negatively affect the reliability of the developed model. To this purpose, during printing process, a channel was created inside the GelMA scaffold through multimaterial approach. Specifically, Pluronic F-127, a copolymer chosen for its thermosensitive properties, was used as a sacrificial ink and removed from the construct by lowering the temperature after GelMA photopolymerization. Subsequently, the samples were placed into commercial microfluidic chambers by aligning the obtained channel with the inlet and the outlet. Then, the culture system was connected to a peristaltic pump that enabled the continuous movement of culture medium within the fabricated channel. Interestingly, although the dynamic condition showed higher viability compared to the static ones, general cell death was still detected demonstrating that nutrient and oxygen supply must be implemented by optimizing channel configuration.