| dc.description.abstract | Intracellular delivery of extracellular cargo into stem cells is an active area of research with enormous potential in field of regenerative medicine. Different types of cargo can be delivered, including genetic materials (DNA, RNA, siRNA), proteins, and small molecules that are not permeable to the cell membrane. Based on the macromolecule delivered, it is possible to directly control stem cell gene expression, activate specific intracellular pathways, or induce the secretion of therapeutic growth factors. Over the years, several strategies have been investigated to promote efficient internalization of external cargos, and they can be subdivided into two main groups according to the method of delivery. In the first category, we commonly include carrier-based approaches where the physical and chemical properties of the carrier dictate the route of internalization and the delivery efficiency. Aside from the carrier, the cell microenvironment can also impact the process of internalization when using these strategies. Additionally, several parameters such as chemistry, stiffness, and topography of the cell-substrate need to be properly selected for efficient delivery of the external cargo. The second group consists of all the other possible carrier-free based strategies that aim to promote intracellular delivery by increasing the permeability of the cell membrane. Transient pores can be induced in the cell membrane by using different physical and mechanical methods that enable the passive diffusion of extracellular cargo. In this case, the method of permeabilization should not be harmful to the cell and should not elicit any permanent damage to the plasma membrane. Based on these scientific premises, which will be discussed in detail in Chapter 1, the aim of this thesis was to investigate both types of delivery methods to identify novel approaches for effective intracellular delivery of genes as well as small polar molecules into stem cells using human adipose-derived stem cells (hASCs) as a model cell line. The overall work of this thesis is iv subdivided into two separate sections based on the strategy adopted for the delivery of the external cargo. The first part of the thesis, discussed in Chapter 2, aimed to investigate the modulatory role of substrate stiffness in the transfection of stem cells by using conventional lipid-based carriers complexed with plasmid DNA (lipoplexes). Precisely, we investigated whether the changes in cellular morphology and cytoskeletal rearrangement, induced by a variation in the stiffness of the cell-substrate, had any significant impact on the process of transfection. The findings of this work are important to advance the understanding of how the physical properties of the cell microenvironment can be controlled to enhance the efficacy of conventional carrier-based strategies for the transfection of stem cells. The second part of the thesis, discussed in Chapter 3 and 4, was focused on the design of a novel carrier-free approach for the intracellular delivery of small polar molecules, such as trehalose. Trehalose is a cryoprotecting agent (CPA) that could be used as a replacement of more toxic molecules such as dimethylsulfoxide (DMSO). However, the poor permeability of trehalose through cell membranes limits its applicability as a CPA for stem cell banking applications. Therefore, to promote efficient intracellular delivery of trehalose, we fabricated and optimized a microfluidic device that could create temporary pores in the cell membranes to enable the uptake of extracellular cargo without the use of any carrier. The safety and efficiency of this new platform were tested by studying several parameters, including cell viability, apoptosis, cellular morphology, and differentiation potential of hASCs loaded with trehalose after cryopreservation. Overall, the results and findings of this final section of the thesis provide a novel valuable strategy for the internalization of trehalose to promote efficient cryopreservation of stem cells without the use of DMSO. | |
