Generación y caracterización de microtejidos funcionales para su utilización en protocolos de ingeniería tisular

Resum

Tissue Engineering (IT) has the objective of achieving the generation of artificial tissues that allow to repair, restore or even improve the functions of damaged tissues and organs. For this, it is essential that artificial tissues are biomimetic and highly biocompatible and, therefore, a large part of the efforts and research that arise in this field are focused on the design, generation and optimization of the basic elements of IT, such as biomaterials, cells and growth factors, which are combined for the biofabrication of artificial tissues for clinical use. In terms of biomaterials, the similarity in terms of the physical-chemical and structural properties of native tissues is sought, this are given by the extracellular matrix. Within the cell component it is sought to obtain, the behavior, function, viability and capacity for differentiation and maturation specific to a native tissue, from various cell populations. Finally, from growth factors or supplementation mediums, we seek to find those chemical stimuli that allow us to regulate cell behavior and differentiation to achieve the aforementioned objectives. As an alternative to the conventional biofabrication process, the microtissue technique (MT) has emerged, which allows generating highly functional biological units from cells and their capacity for proliferation, interaction and synthesis of extracellular matrix. For this purpose, different techniques have been developed, being the use of agarose microchips one of the simplest and most versatile techniques, which allows regulating the dimensions and geometry of the microtissues generated. In this Doctoral Thesis, the main objective is the generation of microtissues from two cellular sources, on the one hand Wharton jelly stem cells from umbilical cord (WJSC), and on the other hand human fibroblasts derived from oral mucosa (FIB) as a control group. For this purpose, the agarose microchip technique was used. The microtissues generated (MT-WJSC and MT-FIB) were evaluated and characterized during 28 days of ex vivo development, in order to understand the behavior of these cells, as well as their biological properties during the biofabrication process. The characterization of the MTs was carried out by a morphometric evaluation of the biofabrication process. The properties of cell viability and function were determined through morphofunctional and biochemical tests. Finally, a characterization of the cells and the extracellular matrix generated was performed using histological, histochemical and immunohistochemical techniques. Morphometric analysis revealed a process of progressive aggregation and compaction over time in both MT. MT-WJSC turned out to be considerably larger than MT-FIB. Assessment of cell viability and function showed that both MTs were composed of mostly metabolically active and viable cells, especially MT-WJSCs generated between days 4-21 of ex vivo development. Histological studies confirmed the findings through the morphometric analysis, such as the aggregation, compaction and circularity of the MT generated. In the case of MT-WJSC, histology revealed the progressive formation of a nucleus rich in various components of the extracellular matrix, which differs with the homogeneous distribution pattern observed in MT-FIB. The study of the extracellular matrix confirmed the great capacity for synthesis of various matrix molecules in both MTs, being considerably more abundant and complex in the MT-WJSC. Finally, this Doctoral Thesis project demonstrates the possibility of generating structurally stable, viable and functional MT from a new cellular source, the WJSC. These results demonstrate that the new MT generated from the WJSC have the biological and structural properties necessary for their use in various IT biofabrication protocols. However, further studies are needed to determine the potential therapeutic utility of these new advanced therapy products.