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. 2020 Jan 8;10(1):1.
doi: 10.1038/s41598-019-56847-4.

Re-epithelialization and immune cell behaviour in an ex vivo human skin model

Ana Rakita  1 , Nenad Nikolić  1 , Michael Mildner  1 , Johannes Matiasek  2 , Adelheid Elbe-Bürger  3
Affiliations

Re-epithelialization and immune cell behaviour in an ex vivo human skin model

Ana Rakita et al. Sci Rep. .

Abstract

A large body of literature is available on wound healing in humans. Nonetheless, a standardized ex vivo wound model without disruption of the dermal compartment has not been put forward with compelling justification. Here, we present a novel wound model based on application of negative pressure and its effects for epidermal regeneration and immune cell behaviour. Importantly, the basement membrane remained intact after blister roof removal and keratinocytes were absent in the wounded area. Upon six days of culture, the wound was covered with one to three-cell thick K14+Ki67+ keratinocyte layers, indicating that proliferation and migration were involved in wound closure. After eight to twelve days, a multi-layered epidermis was formed expressing epidermal differentiation markers (K10, filaggrin, DSG-1, CDSN). Investigations about immune cell-specific manners revealed more T cells in the blister roof epidermis compared to normal epidermis. We identified several cell populations in blister roof epidermis and suction blister fluid that are absent in normal epidermis which correlated with their decrease in the dermis, indicating a dermal efflux upon negative pressure. Together, our model recapitulates the main features of epithelial wound regeneration, and can be applied for testing wound healing therapies and investigating underlying mechanisms.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Re-epithelialization of ex vivo-induced epidermal wounds upon culture. Type IV Collagen (Col-IV) staining in cryostat sections of freshly isolated unwounded (A) and suction blister wounded skin (B). Hematoxylin and eosin (H&E) stained paraffin sections show epidermis and dermis of a freshly wounded (C, insert) and 6 day-cultured wounded biopsy (E,G, inserts). Immunofluorescent labeling demonstrates that K14+ keratinocytes are present in the unwounded epidermis but absent in the wound bed before culture (day 0; D). The wound was closed by a one to three cell thick K14+ keratinocyte layer on day 6 upon culture (F). Several keratinocytes express the proliferation marker Ki67 above the initial wound bed (H). Black and yellow arrows indicate the wound edge. Nuclei are visualized with 4′,6-diamidino-2-phenylindole (DAPI, blue). One representative of six (C,E,G) or three (A,B,D,F,H) experiments is demonstrated. ep, epidermis; de, dermis. Scale bars = 50 µm.
Figure 2
Figure 2
Expression of epidermal differentiation proteins in re-epithelialized skin. As compared to normal skin (A,C), expression of Desmoglein (DSG-1) and Corneodesmosin (CDSN) is observed in lower epidermal layers at day 10 of culture in unwounded skin (B,D). They appear in low epidermal layers above the initial wound bed in wounded skin (b,d). Filaggrin is expressed at the beginning of the culture and at day 10 in untreated cultured biopsies (E,F). A layer, expressing filaggrin was not present in wounded skin (e) and emerged above the initial wound bed at 10 day after wounding (f). Yellow arrows indicate the wound edges. White dashed lines indicate the dermal-epidermal boundary. Nuclei are visualized with DAPI. One representative of three experiments is demonstrated. ep, epidermis; de, dermis. Scale bars = 50 µm.
Figure 3
Figure 3
Distinct MMP-9+ cells appear with culture. MMP-9+ cells are almost absent in freshly wounded biopsies (A), while many cell types appear in the epidermis and dermis upon longer culture (B–D). MMP-9 expression pattern differs in cells. Strong MMP-9 expression is present at wound edges on day 4 and is weaker on day 6 after wounding (B,C, upper inserts). In some cells MMP-9 expression appears in dots around cells (E,F, right insert), on the cell surface (G,H, arrowhead) or interconnecting cells forming a ´´vessel-like´´ structure (G,H, upper insert). Counterstaining with CD31 revealed that dermal cells express either MMP-9 or CD31 (E,F, left insert; G,H, inserts). Yellow arrows indicate the wound edge. White dotted line indicates the basement membrane. Nuclei are counterstained with DAPI. One representative of three experiments is indicated. ep, epidermis; de, dermis. Scale bars = 50 µm.
Figure 4
Figure 4
Influx of dermal immune cells into suction blister roof epidermis. A few T cells are visible in unwounded epidermis (A, insert; B), whereas in blister roof epidermis they are evenly distributed (C, arrows, insert) and appear also in clusters (D). Immunofluorescence labeling of unwounded sections shows T cells as single cells and clustered in the papillary and reticular dermis (E, insert). In wounded skin, some T cells are located along the wound bed and are consistently less abundant in dermal clusters (F, insert) compared to unwounded skin. CD207+CD11c− LCs are detected in normal and blister roof epidermal sheets (G–N), while CD207−CD11c+ cells with a dendritic morphology are only detected in blister roof epidermis often in close proximity to LCs (M,N, open arrows). In unwounded skin, CD207+ LCs are distributed throughout the basal and suprabasal epidermal layers and CD11c+ DCs are localized mostly in the papillary dermis (O, insert). In blister wounded skin, CD207+ LCs are absent in the wound bed (P). CD11c+ DCs are never present in the wounded area as compared to unwounded skin in the same section (P, inserts). Tryptase+ mast cells were found to be equally distributed throughout dermis and in close proximity to the basement membrane in unwounded controls (Q) and wounded sections immediately after wounding (R). Yellow arrows indicate the wound edge. Dotted white line demarcates the basement membrane. Nuclei are counterstained with DAPI (blue). One representative of three experiments is demonstrated. ep, epidermis; de, dermis. Scale bars = 50 µm.
Figure 5
Figure 5
Identification of viable immune cells in suction blister fluid. CD83+ cells in blister roof epidermis ex vivo (A–D) and in vivo (E–H) do not co-express CD207 (A,E) and RTNA1 (B,F). CD56+ cells co-express either RTN1A (C,G) or CD11c (H) or are single positive (C,D,G,H). Cell numbers were counted from ex vivo (I) and in vivo (M) induced suction blisters. Dead cells were determined by trypan blue exclusion (J, N; n = 3). Bars represent mean ± SD of investigated groups. Paired student t-test was used. *P < 0.05, ***P < 0.001. Immunofluorescence labeling shows CD3+ and CD83+ cells on adhesion slides obtained from ex vivo (K,L) and in vivo (O,P) suction blister fluid. Nuclei are counterstained with DAPI. One representative of three experiments is demonstrated. Scale bars = 50 µm.
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