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

Area of Science:

• Regenerative Medicine
• Dermatology
• Biotechnology

Background:

• Human skin reconstruction in mice is crucial for research and therapy development.
• Sustainable and prolonged skin equivalents require reproducible models with patient-derived cells.
• Current methods often need high cell numbers, limiting studies with scarce patient cells.

Purpose of the Study:

• To present a novel, step-by-step protocol for grafting human skin cells onto immunocompromised mice.
• To develop a reproducible method that requires a low number of starting cells, including frozen cells.
• To optimize conditions for efficient and robust skin graft formation.

Main Methods:

• Sequential transplantation of fibroblasts and keratinocytes into a fibrin-based hydrogel within a silicone chamber.
• Optimization of fibrin gel formulation, in vivo gel polymerization timing, cell culture conditions, and seeding density.
• Utilizing a modified skin chamber assay for layer-by-layer engraftment.

Main Results:

• Successful engraftment of as few as 1.0 × 10^6 fresh or 2.0 × 10^6 cryopreserved keratinocytes per 1.4 cm^2.
• Demonstrated feasibility of using frozen-then-thawed keratinocytes and fibroblasts.
• Achieved successful layer-by-layer engraftment without complex or costly techniques like bioprinting.

Conclusions:

• The developed protocol offers a robust and efficient method for generating high-quality human skin grafts in vivo.
• This technique is valuable for studying skin conditions using limited patient-derived cells and enables the use of cryopreserved cells.
• The system shows potential for evaluating induced pluripotent stem cell-derived skin cells and modeling various skin phenotypes.