There is an increasing demand to not only accelerate the development of advanced therapy tissue engineered medicines, but to also eliminate xenogeneic materials from their development cycle. With these in mind, herein we first assessed the influence of carrageenan as macromolecular crowding agent to enhance and accelerate extracellular matrix deposition in xeno-free human umbilical cord mesenchymal stromal cell cultures and we developed and characterised a non-animal sourced chitosan scaffold. Following appropriate in vitro experimentation, a splinted nude mouse wound healing model was used to assess wound closure and scar size of non-treated control, non-animal sourced chitosan scaffold, non-animal sourced chitosan scaffold loaded with xeno-free human umbilical cord mesenchymal stromal cells and non-animal sourced chitosan scaffold loaded with xeno-free human umbilical cord mesenchymal stromal cells cultured under macromolecular crowding conditions groups. Across all three donors, carrageenan supplementation significantly increased collagen deposition at day 5, day 8 and day 11 without affecting cell morphology, viability, DNA concentration and metabolic activity. Through freeze drying, a non-animal sourced chitosan sponge was developed with appropriate structural and mechanical properties for wound healing applications. In vitro biological analysis made apparent that neither the scaffold nor macromolecular crowding negatively impacted xeno-free human umbilical cord mesenchymal stromal cell metabolic activity and proliferation. In vivo biological analysis revealed no significant differences between the groups in wound closure and scar size, raising question about the suitability of the model. In any case, this work sets the foundations for the development of completely xeno-free tissue engineered medicines.