The Development of an Advanced Model for Multilayer Human Skin Reconstruction In Vivo.

Human skin reconstruction on immune-deficient mice has become indispensable for in vivo studies performed in basic research and translational laboratories. Further advancements in making sustainable, prolonged skin equivalents to study new therapeutic interventions rely on reproducible models utilizing patient-derived cells and natural three-dimensional culture conditions mimicking the structure of living skin. Here, we present a novel step-by-step protocol for grafting human skin cells onto immunocompromised mice that requires low starting cell numbers, which is essential when primary patient cells are limited for modeling skin conditions. The core elements of our method are the sequential transplantation of fibroblasts followed by keratinocytes seeded into a fibrin-based hydrogel in a silicone chamber. We optimized the fibrin gel formulation, timing for gel polymerization in vivo, cell culture conditions, and seeding density to make a robust and efficient grafting protocol. Using this approach, we can successfully engraft as few as 1.0 × 10 fresh and 2.0 × 10 frozen-then-thawed keratinocytes per 1.4 cm of the wound area. Additionally, it was concluded that a successful layer-by-layer engraftment of skin cells in vivo could be obtained without labor-intensive and costly methodologies such as bioprinting or engineering complex skin equivalents. Key features • Expands upon the conventional skin chamber assay method (Wang et al., 2000) to generate high-quality skin grafts using a minimal number of cultured skin cells. • The proposed approach allows the use of frozen-then-thawed keratinocytes and fibroblasts in surgical procedures. • This system holds promise for evaluating the functionality of skin cells derived from induced pluripotent stem cells and replicating various skin phenotypes. • The entire process, from thawing skin cells to establishing the graft, requires 54 days. Graphical overview.