There are two major types of stem cells with potential application in tissue engineering and regenerative medicine, namely, embryonic stem cells and adult stem cells. Embryonic stem cells (ESCs) offer exciting possibilities as they can give rise to cell types of the three germ layers, depending on culturing conditions. However it has been difficult to establish their culturing methodologies and the use of human ESCs involves ethical and legal considerations that are different in each country. Therefore, adult stem cells, which are present in several different tissues and organs, have been widely investigated. Many sources of these stem cells have been explored, such as blood, adipose tissue, umbilical cord, amniotic fluid, synovium, periosteum, and others, but the majority of the studies have focused on bone marrow. Nevertheless, adipose tissue has gained great attention as a source of adult stem cells since this is probably the most abundant and easily accessible source of stem cells for regenerative medicine applications. Adipose tissue can be found in all clinical relevant species and adipose-derived stromal/stem cells can differentiate along multiple lineage pathways. Moreover, the common procedure to harvest adipose tissue -the liposuction- is performed most frequently as elective surgery and is less traumatic and painful than that associated to the harvesting of bone marrow, for example. This also facilitates their use in autologous approaches. Therefore, the potential advantages of using human adipose-derived stem cells (hASCs) in tissue engineering and regenerative medicine are well recognized. Nevertheless, the transplantation of these cells when exposed to animal-derived reagents presents severe risks, such as cell-mediated immune response, rejection, severe anaphylaxis, viral or bacterial infections, prions and as yet unidentified zoonoses. For this reason, it is essential to standardize isolation and culture procedures, and also to assess the potential of ASCs in pre-clinical models, documenting their safety and efficiency in specific regenerative strategies. With this in mind, the main goals proposed for this work were as follows: - Determine optimal parameters for storage and processing of adipose tissue samples; - Eliminate the use of xenogenic products while isolating and culturing ASCs, finding alternatives that will not change yield or regenerative potential of these cells; - Assess any significant difference in cells isolated from different fat depots; - Evaluate in vivo the regenerative potential of these cells, combined with adequate scaffold materials, in a bone tissue engineering strategy. For this purpose, it was firstly analyzed the optimal window of time/storage conditions to process lipoaspirate upon harvesting of the samples. Lipoaspirate samples were kept at room temperature and processed in consecutive days (up to 4 days). Results demonstrated that cell isolation within the first 24 hours after tissue harvesting is crucial for the success of this procedure. Subsequently, alternatives to animal-derived products were evaluated for processing adipose tissue and passaging the cells, namely collagenase and trypsin products respectively. In both cases, the alternatives tested maintained total numbers of cells isolated and recovered, as well as their differentiation potential, revealing no changes in the expression of cell surface markers after treatment with each of the products studied. In a subsequent study it was evaluated the ratio between ASC and immune cells found in subcutaneous (SQ) and infrapatellar fat pads (IPFP) from the knee in osteoarthritic patients, and compared the immunophenotype and differentiation potential of ASCs isolated from subcutaneous fat depots from these patients and healthy donors. No significant differences were found in either of the comparisons; therefore IPFP provides a potential source of ASCs for tissue engineering and regenerative medical applications comparable to that of subcutaneous adipose tissue. Finally, the in vivo regenerative potential of ASCs was evaluated in a tissue engineering approach, i.e. combined with a scaffold material. Cells were seeded onto SPCL scaffolds, which were developed at 3B’s research group and for which there are reported evidences of suitability for bone regeneration strategies. Critical size defects were created in a nude mice calvaria model and scaffolds with and without ASCs were implanted. Results clearly demonstrated that SPCL scaffolds exhibit adequate porous structure to support cell adhesion and proliferation and also tissue ingrowth upon implantation of the construct. These outcomes showed the potential of SPCL scaffolds to be used in tissue engineering strategies to regenerate bone tissue defects, and moreover, that ASCs enhance the regeneration of bone tissue providing enhanced tissue formation. In summary, these studies allowed the optimization of ASCs isolation and culture methodologies, and demonstrated the importance of these cells in tissue engineering and regenerative medicine approaches, namely in bone tissue regeneration. These outcomes have direct implication on the broader implementation and application of ASCs in clinical practice, establishing these cells as a promising alternative for tissue engineering therapy.