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. 2024 Jan 20;14(2):e4919.
doi: 10.21769/BioProtoc.4919.

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

Maryna Pavlova  1 , Velmurugan Balaiya  1 , Jocelyn C Flores  1 , Michael Ferreyros  1 , Katie Bush  2 , Amy Hopkin  2 , Igor Kogut  1 , Dennis R Roop  1 , Ganna Bilousova  1
Affiliations

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

Maryna Pavlova et al. Bio Protoc. .

Abstract

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 × 106 fresh and 2.0 × 106 frozen-then-thawed keratinocytes per 1.4 cm2 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.

Keywords: Fibrin-based hydrogel; Human skin equivalent; In vivo skin model; Multilayered skin graft; Regenerative medicine.

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Conflict of interest statement

Competing interestsDRR has an equity interest in AVITA Medical. KB is an employee of AVITA Medical, and AH was an employee at the time the research was conducted. AVITA Medical may potentially benefit from the research findings presented here. Other authors do not have any potential conflicts of interest to declare.

Figures

Figure 1.
Figure 1.. Representative picture of keratinocyte and fibroblast cultures ready to harvest for grafting
. Keratinocytes (A) and fibroblasts (B) are shown at the appropriate confluency for grafting. Scale bar, 50 µm.
Figure 2.
Figure 2.. Visual representation of the grafting procedure.
(A) Dimensions of the silicone chamber are shown. (B) The mouse skin is cut to create a hole with a circumference of approximately 1 cm for chamber implantation. The chamber is then inserted into the wound (C–D), and a fibroblast-comprised fibrin hydrogel is delivered to form the dermal layer (E). Following the polymerization of the dermal layer, a keratinocyte-comprised fibrin hydrogel is delivered on top (F).
Video 1.
Video 1.. Procedure for chamber insertion
Video 2.
Video 2.. Procedure for chamber removal
Figure 3.
Figure 3.. Representative images of grafts at different stages of the healing process.
The fibrin-based grafting was performed with (A, E) 2.0 × 106 freshly cultured keratinocytes together with 2.0 × 106 of freshly cultured fibroblasts; (B, F) 1.0 × 106 freshly cultured keratinocytes together with 1.0 × 106 freshly cultured fibroblasts, and (C, G) 2 × 106 frozen-then-thawed keratinocytes together with 2 × 106 frozen-then- thawed fibroblasts. As a control, grafts generated using a classical grafting chamber assay with 5.0 × 106 freshly cultured keratinocytes and 5.0 × 106 freshly cultured fibroblasts are shown (D, H). Representative images of forming grafts on the day of chamber removal (day 7) are shown on top and corresponding fully established grafts at 5 weeks post-grafting are shown on the bottom. OW–opened wound after chamber removal, and CW–closed wound at the day of harvest of the skin graft.
Figure 4.
Figure 4.. Histological examination of human skin xenografts.
Representative images of fibrin-based grafts generated with 2.0 × 106 freshly cultured keratinocytes together with 2.0 × 106 freshly cultured fibroblasts (A–C), 1.0 × 106 freshly cultured keratinocytes together with 1.0 × 106 freshly cultured fibroblasts (D–F), as well as with 2.0 × 106 frozen-then-thawed keratinocytes together with 2.0 × 106 frozen-then-thawed fibroblasts (G–I) are shown. As a control, grafts generated using a classical grafting chamber assay with 5.0 × 106 freshly cultured keratinocytes and 5.0 × 106 freshly cultured fibroblasts were also analyzed (J–L). H & E staining of the grafts (A, D, G, J). Immunofluorescence staining using mouse-specific Keratin (K)1 (moK1; green) and human/mouse-specific K14 (red). Note that the moK1 antibody stains only the mouse epidermis (B, E, H, K). Immunofluorescence staining using human-specific Loricrin (Lor; green) detects the human epidermis, while human-specific Vimentin (Vim, red) detects the human dermis (C, F, I, L). Nuclei stained with DAPI (blue). Scale bar, 100 μm.
Figure 5.
Figure 5.. Digital planimetry.
Individual plots for a wound contraction rate are shown in A. The length of grafts (B), the thickness of the dermis (C), and the thickness of the epidermis (D) were determined by analyzing immunofluorescence images of grafts developed at 6 weeks after surgery (****p < 0.0001; ***p < 0.002; **p < 0.0094; *p < 0.02).
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