3D-printed in vitro platform for Non-Small Cell Lung Cancer (NSCLC) modelling

Cancer is the first cause of death worldwide. Lung cancer, in particular, is characterized by the lowest life expectancy after diagnosis. Among the types of lung cancer, Non-Small Cell Lung Cancer (NSCLC) presents a complex and varied structure: it consists of several cell types (among all epithelial cells and fibroblasts) embedded in an extracellular matrix, stiffer than healthy tissue and with a dense and tortuous vasculature. Currently, mouse models are considered the gold standard in cancer research. However, due to ethical and economic issues, there is a trend in the direction of replicating these models in vitro. There are several types of in vitro models, which differ in their complexity that reflects the biomimicry of the model. Among them, bioprinting offers the possibility of fabricating 3D models of both physiological and pathological tissue/organs using biocompatible materials and cell-friendly processes. The technique chosen during this thesis work is extrusion bioprinting, whose main challenge was the choice of bioink. The bioink must have a specific viscosity to be extruded without losing the fidelity of the printed geometry, ensure biocompatibility and stiffness similar to the chosen organ, to provide the same matrix, and thus the same mechanical and topological stimuli, to the constituent cells. An excellent candidate for this purpose is gelatin methacrylate (GelMA), which shares with the gelatin whence derived biocompatibility and ease of availability at a relatively low cost. Moreover, it possesses better mechanical properties due to methacrylate groups that allow its stabilization by photocuring with UV light. The importance of vasculature in reproducing a bioprinted tumor model is widely demonstrated. One of the methods which aim to replicate a vascular-like channel inside a 3D model includes a sacrificial material as a printing bath to print the desired model into. The sacrificial material must exhibit elastic and shear-thinning rheological behavior so that it acts as a viscous fluid when the needle penetrates it for printing, and as a "solid-like" material at the end of printing to maintain the print fidelity of the printed sample. This "transition" must occur in the shortest possible time, showing rapid recovery after printing. One promising material for this role is Carbopol, a copolymer of acrylic acid that exhibits the rheological characteristics listed below. In addition, the sacrificial material had to be biocompatible, not- photoreactive at the wavelength used to photocure GelMA, and easy to remove after printing under conditions not aggressive for cells. In the first part of this work, the aim was to optimize the concentration and the degree of methacrylation of GelMA, to better mimick NSCLC microenvironment. A proliferation assay was performed on two cell lines present in the chosen model (A549, alveolar epithelium-derived, and MRC-5, fibroblasts) to select the most appropriate matrix for the bioink realization. In addition, the bioink was characterized for its swelling behaviour and bioprinted. Then, the thesis project focused on the development of a vascular-like structure using the method presented below. For this purpose, two formulations of Carbopol, in water and DMEM solution, were compared. As a result, the channel structure was performed using GelMA H10% w/v containing fibroblasts and DMEM-based Carbopol to realize the channel perfusable with epithelial cells, and to build a bioprinted NSCLC model.