BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) 3D CULTURE MODELS: IN VITRO TOOLS TO STUDY CETACEAN PATHOLOGY

Cetaceans’ health is threatened by several natural and anthropic factors. Given their role in the marine food chain and their long lifespan, they can be considered important indicators of environmental conditions such as pollution and viral spread. Despite the rising interest and demand for information, there are still considerable gaps in our understanding of cetacean biology and medicine, largely due to the limitations of studies conducted in their natural habitats. Consequently, there is an urgent need to develop cetaceans’ study models. Marine mammal cell culture systems are considered unique multifunctional tools for studying the cell physiology and biochemistry, as well as pathology, of these animals. Regarding cell culturing, three-dimensional (3D) cultures are in vitro models in which cells grow in complex interactions with the extracellular matrix (ECM), using a scaffold/matrix or in a scaffold-free manner. Compared to the traditional Two-dimensional (2D) cultures, these systems better resemble the condition of cells in vivo and have been established both in human and veterinary medicine. However, only a few attempts have been carried out for the establishment of cetacean 3D culture. Therefore, the purpose of the present thesis was to assess and develop cetacean 3D in vitro models. We used immortalized fibroblasts of bottlenose dolphin (Tursiops truncatus) which is one of the most numerous and charismatic cetaceans in the Mediterranean Sea. The strandings of this protected species are homogeneously distributed along the Italian coastline, giving to the scientific community the opportunity to use these animals as bioindicators of the marine environmental health In collaboration with the Università Campus Bio-medico (UCBM, Rome) we developed for the first time a bottlenose dolphin skin on chip, a preliminary attempt to use a micro-physiological platform to grow our fibroblasts. Meanwhile in collaboration with the Department of Industrial Engineering of Padua we tested different 3D supports for the same cell line: novel collagen-free scaffolds comparing them with Matrigel, one of the most used 3D matrices in human and veterinary medicine. Additionally, to obtain biological material for other future in vitro studies, we performed sampling activities at Jungle Park, Aqualand Costa Adeje (Tenerife, Spain) and Palmitos Park (Gran Canaria, Spain) collecting plasma and buffy coat from under human care (UHC) bottlenose dolphins and liver and skin biopsies of a leopard. Lastly, In the context of wildlife conservation efforts by Zoos, this thesis explores several ex-situ conservation projects, carried out during my PhD mobility, that can benefit UHC cetaceans and other protected species.