3D bioprinted models of colorectal cancer for the development of a stimuli-responsive nanomedicine therapy

Advanced therapies are developed every day to find alternative procedures to life-threatening diseases. In the field of cancer research, tissue-engineered 3D cancer models have been developed to achieve more accurate biomedical representations. Although state-of-the-art 3D models still lack in the modelling of the complexity of the native tissue, they represent a first step towards reaching the goal in terms of the 3R (replacement, reduction, and refinement) framework. In the present Master Thesis, the purpose is to develop two systems enabling the study of a 3D model of colorectal cancer (CRC) to evaluate the evolution of the tumour and eventually the efficacy of a combined treatment of acoustic external stimuli and biomimetic stimuli-responsive hybrid nanoparticles. The first part of the thesis project aims to obtain a hydrogel bioink for microfluidic bioprinting of hollow conduits to recreate intestinal tissue. A coaxial needle is used to extrude through the external conduit the bioink, composed of ionically crosslinked sodium alginate and enzyme-crosslinked bovine gelatine. A crucial step of the study has been the optimization of all the parameters used during the extrusion such as bioink components percentages, extrusion temperature, tube diameters, which enabled to obtain a solid and long-lasting system subsequently implemented with the introduction of tumour cells. Concerning this last stage, two different approaches have been explored: in the first case tumour cells from HT-29 cell-line have been seeded inside the empty conduits, while in the second one the same type of cells has been included in the bioink and extruded, to obtain a perfusable network. Since stable and reliable 3D conduits seeded with colorectal cancer cells were accurately implemented, a combined treatment, consisting of an ultrasound (US) therapy and zinc oxide nanoparticles coated with a lipid formulation and a targeting peptide, was tested to validate the bioprinted tumour models. In the second part of the project a high-throughput screening device has been designed with 3D Rhino and then fabricated through the casting of PDMS on a tridimensional negative mold, previously 3D printed. Pre-grown CRC spheroids obtained from HT-29 cell line were loaded in the chip and kept in place by an array of specifically fabricated traps. The microfluidic device outlined the development of a very versatile and high-throughput platform, which can be employed to analyse the effects of anti-cancer treatments in the drug screening research. Both the described solutions represent a valid alternative to a classic, poorly representative 2D culture, offering innovative approaches to achieve and study more realistic cancer models.