Contribution of resident immune cells in a human autologous 3D skin model

Skin wound healing results in hemostasis, inflammation, proliferation, and remodeling. The early phases require the recruitment of several immune cells from the blood. However, the role of these cells is not well defined. In addition to monocytes-derived macrophages, known to regulate the outcome of wound healing, resident macrophages in the injured skin may play a distinct role in the healing process. The aim was to develop a 3D autologous skin model containing immune and endothelial cells, to investigate their contribution during wound healing. A cell extraction technique was firstly developed to isolate skin resident cells from the same donor (keratinocytes, fibroblasts, immune and endothelial cells). Analysis by flow cytometry revealed the presence of resident skin macrophages (CD45+ CD14+ CD163+); lymphocytes (CD45+ CD3+); dendritic cells (CD45+ CD14- CD1a+) and endothelial cells (CD45- CD31+) in freshly isolated skin cells. From this isolation, an autologous in vitro 3D human skin model was developed. This approach was applied to 4 donors. Immunofluorescence staining confirmed the presence of previously described immune cells in this model. Endothelial cells self-assembled to form capillary-like networks and immune cells did not exfiltrate after 14 days in culture and are able to synthesize TNFα. Moreover, characterization of the proliferation rate in the epidermal basal layer by Ki67 staining revealed a difference between reconstructed skin supplemented with resident immune and endothelial cells compared to the model containing only fibroblasts and keratinocytes. These results indicate that this innovative skin model provides a great tool to study the interaction between resident immune cells such as macrophages with other skin cells and their contribution to healing in a wounded model.