Background: Pressure injuries (PIs) remain a formidable challenge in patients with spinal cord injury (SCI) due to their reduced regenerative capacity. This study established an in vitro platform for a minimally manipulated autologous cell-based approach that integrated adipose-derived stromal cells (ADSCs), platelet-rich plasma (PRP), and collagen scaffolds to accelerate the healing of PIs. Methods: ADSCs were isolated through mechanical processing of lipoaspirates, yielding distinct fat fractions—namely “Microfat”, “emulsified Microfat”, and “Nanofat”—which were subsequently characterised via flow cytometry and trilineage differentiation assays. Three-dimensional ADSC constructs using various collagen scaffolds were assessed for their ability to support adipogenic differentiation of ADSCs. The influence of PRP at different concentrations was evaluated on ADSCs proliferation, cellular senescence, and in co-culture with human umbilical vein endothelial cells (HUVECs). Additionally, the impact of ADSCs-derived exosomes on stemness maintenance was investigated. Results: Quantitative analyses demonstrated that “emulsified Microfat” enhanced ADSC delivery and reduced cellular senescence. Cell constructs using stiffer scaffolds demonstrated improved adipogenic differentiation. Supplementation with 5 % PRP effectively promoted ADSC proliferation and supported ADSC-HUVEC co-culture, showing results comparable to standard growth factor conditions in cultures. Although exosome treatment did not modify ADSCs proliferation rates, it was associated with an increased expression of stem cell markers. Conclusions: Altogether, these findings provide a preclinical foundational framework for future regenerative treatment strategies aimed at improving tissue repair following surgical intervention for PIs in patients with SCI.