Autologous cell therapy uses patients’ own cells to deliver precise and ideal treatment through a personalized medicine approach. Isolation of patients’ cells from residual tissue extracted during surgery involves specific planning and lab steps. In the present manuscript, a path from isolation to in vitro research with human mesenchymal stem cells (MSCs) obtained from residual bone tissues is described as performed by a medical unit in collaboration with a research center. Ethical issues have been addressed by formulating appropriate harvesting protocols according to European regulations. Samples were collected from 19 patients; 10 of them were viable and after processing resulted in MSCs. MSCs were further differentiated in osteoblasts to investigate the biocompatibility of several 3D scaffolds produced by electrospinning and 3D printing technologies; traditional orthopedic titanium and nanostructured titanium substrates were also tested. 3D printed scaffolds proved superior compared to other substrates, enabling significantly improved response in osteoblast cells, indicating that their biomimetic structure and properties make them suitable for synthetic tissue engineering. The present research is a proof of concept that describes the process of primary stem cells isolation for in vitro research and opens avenues for the development of personalized cell platforms in the case of patients with orthopedic trauma. The demonstration model has promising perspectives in personalized medicine practices.