Aim: We investigated the healing effects of conditioned medium (CM) derived from a physiological 3D culture system engineered to use an extracellular matrix/stromal vascular fraction (SVF) gel enriched for adipose on diabetic wounds in rats. This CM (Gel-CM) was compared with that from a 2D culture system that used SVF cells (SVF-CM). Materials & methods: Keratinocytes, fibroblasts and wounds were treated with Gel-CM and SVF-CM, and cytokine levels in the CM types were quantified. Results: Proliferation and migration of keratinocytes and fibroblasts were significantly higher after treatment with Gel-CM than with SVF-CM. Collagen secretion by fibroblasts and wound closure were highly stimulated by Gel-CM. Proteomic analyses revealed a higher concentration of growth factors in Gel-CM than in SVF-CM. Conclusion: Gel-CM is a promising therapeutic option for treating diabetic wounds.