Generation of a Full-Thickness Human Skin Equivalent on an Immunodeficient Mouse

Abstract

Human skin equivalents composed of epidermal cells and fibroblasts are important for modeling human epidermal development, testing new therapeutics, and designing novel treatment strategies for human skin diseases. Here, we describe a procedure for the generation of an in vivo fullthickness human skin equivalent on an immunodeficient mouse using a grafting chamber system. The protocol involves mixing human epidermal cells and fibroblasts in a silicone grafting chamber that is surgically inserted onto the muscle fascia of a recipient immunodeficient mouse. Following the removal of the silicone chamber, the graft area is exposed to air to induce stratification of developing epidermis, resulting in the reconstitution of full-thickness human skin tissue on a live mouse. This grafting system provides a straightforward approach to study human skin diseases in an animal model and has been previously used to determine the ability of both mouse and human primary epidermal cells and cells derived from pluripotent stem cells to regenerate functional skin in vivo.

Telomerase mRNA Enhances Human Skin Engraftment for Wound Healing

“…To evaluate the effect of hTERT mRNA DOTAP‐A LNP on hSCS engraftment, a partial thickness wound healing model was developed in immunocompromised mice. [ 38 ] The overall flow of the experiment is summarized in Figure a. hSCS was isolated using the RECELL Device which includes enzymatic and mechanical processing steps, resulting in a cell suspension containing >50% viable cells, mainly keratinocyte and fibroblasts.…”

“…An in vivo humanized partial‐thickness wound model was used to study human skin cell engraftment after transfection with hTERT mRNA LNP (Figure 5), modifying previously reported models. [ 38,46,47 ] Our studies have shown that hSCS engraftment was significantly improved by pretreatment with hTERT mRNA DOTAP‐A LNP as compared to untreated hSCS or hSCS pretreated with EGFP mRNA LNP. The following markers were assessed: Lamin A, Ki67, p21, and 53BP1 (Figure 7).…”

“…In Vivo Assessment of Healing in Humanized Wound Model: Athymic immunodeficient mice (Foxn1 nu , Strain #:0 02019, Jackson Laboratory) 8-12 weeks old were used in accordance with protocols approved by the HMRI Institutional Animal Use and Care Committee (IACUC #ISO00006024). To create the humanized full-thickness wound skin model, under isoflurane anesthesia a grafting polymeric dome (3D printed in house, d outer = 24 mm; d inner = 14 mm; h = 10 mm with two 2 mm holes for gas exchange) was inserted in 1 cm vertical surgical incision on the back of the animals as described previously, [38] Fibroblast-fibrin hydrogel (200 μL) (10 6 fibroblasts, 1 mg fibrinogen, 0.4 U thrombin, 2.6 mm CaCl 2 , 1% FBS in DMEM) was placed into the grafting dome following by 200 μL of hSCS (10 6 cells) untreated or pretreated with mRNA LNP.…”

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

“…Several human xenograft models have been previously published, including a skin flap assay ( Qiao et al, 2008 ), human skin transplantation-like approaches for grafting dermal-epidermal equivalents onto opened wounds of recipient mice ( Escamez et al, 2004 ; Martinez-Santamaria et al, 2012 ; Yanez et al, 2015 ; Jorgensen et al, 2020 ), and chamber grafting assay ( Wang et al, 2000 ; Diette et al, 2020 ). While promising, all these assays suffer from several limitations.…”

“…They include (1) the complexity of the procedure that requires the generation of 3D skin equivalents in cell culture conditions before the transplantation onto a mouse, and (2) the limited availability and proliferative capacity of primary human cells, especially cells derived from patients with rare inherited skin diseases. A chamber grafting assay ( Wang et al, 2000 ; Diette et al, 2020 ) provides a simplified procedure for grafting primary human skin cells since the assay promotes the self-assembly of fibroblasts and keratinocytes in an in vivo environment without the need to produce skin equivalents in a dish. However, successful production of human skin equivalents in the grafting assay still requires at least 5 × 10 6 primary keratinocytes per 1.4 cm 2 of the wound area, which is a significant number when the availability of primary patient cells is limited.…”