Development of 3D Cancer Cell Models to Test Metabolic Interventions as an Anticancer Strategy

In recent years, it has become evident that the role of mitochondria in the metabolic rewiring is essential for cancer development and progression. The metabolic profile during tumorigenesis has been performed mainly in traditional 2D cell models, including cell lines of various lineages and phenotypes. Although useful in many ways, their relevance can be often debatable, as they lack the interactions between different cells of the tumour microenvironment and/or interaction with the extracellular matrix 1,2. Improved models are now being developed using 3D cell culture technology, contributing with increased physiological relevance 3,4. In this work, we improved a method for the generation of 3D models from healthy and tumour colon tissue, based on organoid technology, and performed their molecular and biochemical characterization and validation. Further, in-plate cryopreservation was applied to these models, and optimal results were obtained in terms of cell viability and functionality of the cryopreserved models. We also cryopreserved colon fibroblasts with the aim to introduce them in a co-culture cryopreserved model with organoids. This technology allows the conversion of cell models into “plug and play” formats. Therefore, cryopreservation in-plate facilitates the accessibility of specialized cell models to cell-based research and application, in cases where otherwise such specialized models would be out of reach. Finally, we briefly explored the field of bioprinting, by testing a new matrix to support the growth of colon tumour organoids, which revealed promising preliminary results. To facilitate the reader, we organized this thesis into chapters, divided by the main points of work which include development, characterization and validation of the model, commercial output, and associated applications. Each chapter has a brief introduction, followed by results and discussion and a final conclusion. The thesis has also a general discussion and conclusion section in the end, which covers the main results obtained during this work.

Gross and Microscopic Morphological Anatomical Study of the Guinea Pig (Cavia porcellus) and the Capybara (Hydrochoerus hydrochaeris), Aimed at the Preparation of a Comparative Anatomical Atlas of the Different Systems

In recent years, there has been an exponential increase in the so-called “new pets”, including the domestic guinea pig (Cavia porcellus) and the capybara (Hydrochoerus hydrochaeris), two closely related Caviid rodents native to South America. Both historically bred for food purposes, they have more recently become increasingly popular as pets in the European and American continents, respectively. This led to an increasing veterinary interest in deepening the knowledge regarding their normal anatomy, as a basic contribution to other fields of veterinary medicine, including diagnostic imaging, surgery, and pathological anatomy. Being part of a bilateral framework co-tutelage agreement leading to a joint Doctoral Degree between the Alma Mater Studiorum of Bologna, Italy and the Universidad Nacional del Litoral of Santa Fe, Argentina, this research project was partly carried out in Italy (study of guinea pigs) and partly in Argentina (study of capybaras). It consisted in the macroscopic study, through anatomical dissections of carcasses of both species as well as the use of anatomical casts, and in the histological study of the various systems in the two species, and was aimed at creating a gross and microscopic comparative anatomical atlas. From the gross and microscopic morphological and morphometrical anatomical study of the different system of the guinea pig and capybara, several analogies and differences emerged. The creation of a comparative anatomical atlas of gross and microscopic anatomy of the capybara and the guinea pig might prove useful for clinical, zootechnical and research purposes.