Resumen de Cell derived-extracellular matrix scaffolds with polylactic acid microcarriers for tissue engineering and cell therapy
Organ shortage for transplantation, or the need of new therapies for the treatment of tissular damages have driven the development of an exciting field in research, called tissue engineering (TE). Its potential entails future strategies that will allow the development of functional substitutes for damaged tissues, obtained in vitro under risk-free environments, using autologous cells that will integrate within the host aiding in the regeneration and restore of the lost function. However, still current efforts have to deal with several restraints in order to achieve that paradigm.The aim of this thesis focuses in the development of in vitro tissue analogs with millimetric size (microtissues), using the inherent ability of cells to secrete their own extracellular matrix (ECM) when they are seeded on a biocompatible scaffold. In this case, we have used polylactic acid (PLA) microcarriers (MCs) (80 -120 ¿m in diameter) as scaffold. PLA has been an extensively used as a biomaterial applied in medicine, as it is a biodegradable and biocompatible synthetic polymer. Moreover, we have used a green, non-toxic method for the preparation of PLA microcarriers that allows a high control over size and distribution. The use of these microcarriers provides cells with an ideal three-dimensional environment for proliferation and secretion of ECM components, which are different than when exposed to conventional tissue culture plates. Likewise, the use of MCs in the formation of microtissues allows their aggregation (as building blocks) into bigger constructs or macrotissues, with high interconnectivity and porosity, as well as the feasibily to adapt to different shapes. Firstly, in this thesis we have studied different methodologies for the seeding of cells on microcarriers, and the latter formation of microtissues to define the best parameters for a homogeneous seeding and extensive ECM deposition. For that purpose, we have used a spinner flask bioreactor promoting a more uniform cell-MC colonization, and ECM deposition. After optimization, we have evaluated the obtained ECM microtissues, evaluating their components and possible applications. In that case, we introduced the use of commercially available-gelatine microcarriers and a comparative was performed with PLA microcarriers. We assessed whether the secreted ECM differed when using different microcarriers and could confirm that the scaffold choice influences cellular behaviour and the secreted matrix, favouring osteogenic with gelatine MCs or potentiating angiogenic capacity with a mixture of gelatine and PLA MCs. One of the biggest hurdles that halt the introduction of TE constructs into clinical applications is the vascularization process for the survival of cells, once implanted. The arrival of nutrients and oxygen must be favoured by a rapid in vivo vascularization. To aid in this process, we have studied the formation of co-cultured microtissues with mesenchymal stem and endothelial cells, together with PLA microcarriers. We were able to confirm the presence of both cells types in the microtissues, but there were no clear evidences that the presence of endothelial cells enhanced vascularization in mice models. Finally, we used cell-derived ECM microtissues as a platform for the introduction and survival of therapeutic cells in an anti-tumoral model. Microtissues acted like reservoir for these cells, allowing cell migration towards the tumour to provoke their bystander therapeutic effect. The results of this study demonstrated the efficiency of PLA microtissues obtained from therapeutic cells in stopping tumour progression. Moreover, we could appreciate a rapid microtissue vascularization, favouring cell survival. To summarise, this thesis describes the fabrication of cell-derived microtissues, created from seeding cells on PLA microcarriers as a favourable strategy in tissue engineering, as well as a tool for the delivery and survival of therapeutic cells for anti-tumoral applications.
