Collagen-glycosaminoglycan flowable matrices (CGFM) are increasingly finding utility in a diversifying number of cutaneous surgical procedures. Cellular in-growth and vascularisation of CGFM remain rate-limiting steps, increasing cost and decreasing efficacy. Through in vitro and ex vivo culture methods, this study investigated the improvement of injectable CGFM by the incorporation of hyaluronan (HA) and viable human cells (primary human dermal fibroblasts (PHDFs) and bone marrow-derived mesenchymal stem cells (BM-MSCs)). Ex vivo investigations included the development and evaluation of a human cutaneous wound healing model for the comparison of dermal substitutes. Cells mixed into the Integra Flowable Wound Matrix (IFWM), a commercially available CGFM, were confirmed to be viable and proliferative through MTT assays (p < 0.05). PHDFs proliferated with greater rapidity than BM-MSCs up to 1 week in culture (p < 0.05), with PHDF proliferation further enhanced by HA supplementation (p < 0.05). After scaffold mixing, gene expression was not significantly altered (qRT-PCR). PHDF and BM-MSC incorporation into ex vivo wound models significantly increased re-epithelialisation rate, with maximal effects observed for BM-MSC supplemented IFWM. HA supplementation to PHDF populated IFWM increased re-epithelialisation but had no significant effect on BM-MSC populated IFWM. In conclusion, when combined with PHDF, HA increased re-epithelialisation in IFWM. BM-MSC incorporation significantly improved re-epithelialisation in ex vivo models over acellular and PHDF populated scaffolds. Viable cell incorporation into IFWM has potential to significantly benefit wound healing in chronic and acute cutaneous injuries by allowing a point-of-care matrix to be formed from autologous or allogenic cells and bioactive molecules.