Pressure injuries/ulcers are frequent complications in elderly, paraplegic, and quadriplegic patients that account for considerable cost to the international health care economy and remain refractory to current treatment options. Autologous or allogeneic adult stromal/stem cells represent an alternative therapeutic approach. The current study extends prior findings by exploring the safety and efficacy of human adipose-derived stromal/stem cells (ASC) therapy in an established immunocompetent murine skin pressure ulcer model where dermal fibroblast cells (DFC) served as a control. Human adipose tissue was processed using a closed system device designed for point-of-care use in the operating room and on file with the Food and Drug Administration. Cell characterization was performed using colony forming unit-fibroblast (CFU-F), differentiation, and immunophenotypic assays in vitro. Wound healing was assessed over a 20-day period based on photomicrographs, histology, and immunohistochemistry. The isolated human ASC displayed significantly greater colony formation relative to DFC while both populations exhibited comparable immunophenotype and differentiation potential. Both fresh and cryopreserved human ASC significantly accelerated and enhanced wound healing in young (2 month) mice of both sexes relative to DFC controls based on tissue architecture and CD68+ cell infiltration. In contrast, while injection of either fresh or cryopreserved human ASC was safe in older mice, the fresh ASC significantly enhanced wound closure relative to the cryopreserved ASC. Overall, these findings support the safety and efficacy of human ASC isolated using a closed-system device designed for clinical procedures in the future treatment of pressure injuries.