Skin Substitute Grafting in Nude Mice

TES-4 samples were grafted onto the backs of CD1-Foxn1 nude mice (Charles River Laboratories, Wilmington, NC, USA), as previously described [32 (link),33 (link)]. Briefly, a portion of the mid-back skin of the mouse was removed. Then, the fascicular panniculus was removed with forceps to expose muscle, and a custom-made silicone Fusenig’s chamber was inserted around the wound to prevent contraction of the mouse skin. The Fusenig’s chamber was secured in place by four intramuscular sutures (Prolene 4-0, Ethicon, Raritan, NJ, USA). A 5 cm² biopsy was taken from the skin substitutes cultured for 10 days at the air–liquid interface, mounted on a non-adhering dressing (AdapticTM, Acelity, San Antonio, TX, USA), and grafted into the aperture. To prevent graft displacement, sterile gauzes were placed over the substitute inside the Fusenig’s chamber and served as a bolus tie-over pressure dressing. The gauzes were taken out after 7 days. The silicone chambers were removed 21 days after grafting. Three to four mice per condition at each time point were euthanized 3 weeks, 3 months, and 6 months after grafting for tissue collection and analysis.