Diabetic foot ulcers (DFUs) are associated with a high risk of amputations and a 50% 5-year survival rate due at least in part to the limited angiogenic and wound healing capacity of patients with diabetes. Cell therapy via intramuscular injection of peripheral blood mononuclear cells showed encouraging but limited results. Such limitations may arise from the limited ability of therapeutic cells to adhere to the target tissue. The development of a methodology able to support the targeted delivery of viable angiogenic cells would improve cell therapy outcomes in DFU. Here, we optimized a protocol for the production of autologous extracellular matrix (ECM)-rich pericyte-based cell sheets for cell delivery. Pericytes were isolated from skeletal muscle biopsies of DFU patients and non-diabetic controls and characterized by flow cytometry and immunofluorescence. Human umbilical vein endothelial cells used for the optimization of collagen IV deposition showed a positive correlation with seeding density and a negative one with sub-culture passaging (P < 0.05). Macromolecular crowding significantly increased collagen IV deposition both in human umbilical vein endothelial cells and in patient-derived pericytes (P < 0.01) without affecting proliferation (P > 0.05). Finally, DFU patient-derived pericytes effectively deposited ECM supporting their use for autologous cell sheet production.