Re-epithelialization and immune cell behaviour in an ex vivo human skin model methods
Aim. Evidence-backed execution summary for Re-epithelialization and immune cell behaviour in an ex vivo human skin model methods from Re-epithelialization and immune cell behaviour in an ex vivo human skin model.
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human
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Immunofluorescent staining
reagent used in the protocol.
- Use
- Freshly isolated and cultivated skin samples were harvested at indicated time-points, embedded in optimum cutting tissue compound (Tissue-plus; Scigen Scientific, Gardena, CA, USA), snap frozen in liquid nitrogen and stored at -80 °C until further processing. Frozen tissues were sectioned (5 &...
A multi-layered epidermis expressing epidermal differentiation markers forms after wound closure
reagent used in the protocol.
- Use
- We next tested whether keratinocytes upon wound closure can also differentiate and form multiple epidermal layers upon prolonged culture. Indeed, several K14 + keratinocyte layers were observed upon 8 days of culture above the initial wound bed (Fig. ). During the continued culture period of 12 days, multiple...
Increased frequency of matrix metalloproteinase 9 (MMP-9) expressing cells in wounded skin upon prolonged culture
reagent used in the protocol.
- Use
- MMPs become activated during wound repair. In particular, MMP-9 influences the detachment of basal keratinocytes from the basement membrane thus improving their migration,. We noticed scarce MMP-9 + cells directly under the wound bed and in unwounded skin areas (Fig. ). In contrast, strong MMP-9 expression w...
Negative pressure leads to an influx of dermal cell populations into blister roof epidermis
reagent used in the protocol.
- Use
- The suction blister method has been used in several studies to investigate LCs in human epidermis in vivo such as effects of ultraviolet B irradiation, as well as a calcineurin inhibitor and corticosteroids. To determine, whether application of negative pressure affects LCs in ex vivo -induced blister roof epiderm...
Negative pressure leads to an influx of dermal cell populations into blister roof epidermis
reagent used in the protocol.
- Use
- To test whether the dendritically shaped cells may belong to the nerve network in human skin, we employed an antibody directed against the neural cell adhesion molecule-1 (NCAM-1)/CD56. In the dermis, terminal nerve endings are enveloped by single CD56 + Schwann cells. Schwann cells constitute a substantial cell p...
Hematoxilin and eosin (H&E) staining
reagent used in the protocol.
- Use
- Freshly isolated and cultivated skin samples were harvested at time points, as indicated in the figures, and fixed immediately in 7.5% paraformaldehyde (SAV Liquid Production, Flintsbach am Inn, Germany). Fixed tissues were embedded in paraffin, sectioned (5 µm) (Microtome HM 335 E-Microm, GMI, USA)...
Suction blister-induced wound model
reagent used in the protocol.
- Use
- A negative pressure instrument (Electronic Diversities, Finksburg, MD, USA) constructed to produce standard suction blisters upon application of negative pressure, was used on healthy skin ( ex vivo: abdominal skin; in vivo: lower forearm). Subcutaneous fat was partially removed from ex vivo skin using a scissor. Su...
Statistical analysis
Data was analysed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Paired t-test was used for comparing means. The results were considered significant with P -values smaller than 0.05.
- Use
- Data was analysed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Paired t-test was used for comparing means. The results were considered significant with P -values smaller than 0.05.
Suction blister-induced wound model
A negative pressure instrument (Electronic Diversities, Finksburg, MD, USA) constructed to produce standard suction blisters upon application of negative pressure, was used on healthy skin ( ex vivo: abdominal skin; in vivo: lower forearm). Subcutaneous fat was partially removed from ex vivo skin using a scissor. Su...
- Use
- A negative pressure instrument (Electronic Diversities, Finksburg, MD, USA) constructed to produce standard suction blisters upon application of negative pressure, was used on healthy skin ( ex vivo: abdominal skin; in vivo: lower forearm). Subcutaneous fat was partially removed from ex vivo skin using a scissor. Su...
Statistical analysis
Software used for acquisition, scoring, statistics, or reporting.
- Use
- Data was analysed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Paired t-test was used for comparing means. The results were considered significant with P -values smaller than 0.05.
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A multi-layered epidermis expressing epidermal differentiation markers forms after wound closure
We next tested whether keratinocytes upon wound closure can also differentiate and form multiple epidermal layers upon prolonged culture. Indeed, several K14 + keratinocyte layers were observed upon 8 days of culture above the initial wound bed (Fig. ). During the continued culture period of 12 days, multiple K14 + layers have formed (Fig. ). In normal, unwounded skin, K10 is exclusively localized in suprabasal keratinocytes (Rakita, unpublished observation). In wounded skin, a few K10 + basal keratinocytes, in particular at the wound edge, were observed in addition to the conventional staining pattern in suprabasal keratinocytes at day 8 of culture (Fig. ). K10 expression appeared in all layers except the basal layer at later time points (Fig. ). Next, expression kinetics of particular epidermal differentiation markers in unwounded skin and blister-induced wou...
Increased frequency of matrix metalloproteinase 9 (MMP-9) expressing cells in wounded skin upon prolonged culture
MMPs become activated during wound repair. In particular, MMP-9 influences the detachment of basal keratinocytes from the basement membrane thus improving their migration,. We noticed scarce MMP-9 + cells directly under the wound bed and in unwounded skin areas (Fig. ). In contrast, strong MMP-9 expression was observed at the leading wound edges at day 4 and already decreased 6 days after wounding (Fig., inserts). MMP-9 + cells greatly accumulated in the dermis at day 10 of culture (Fig., arrowheads). Of note, different MMP-9 expression patterns were observed in dermal cells implying that distinct cell types upregulate this enzyme. MMP-9 appeared in dots around the nucleus (Fig., right insert), on the cell surface (Fig. arrowhead) or interconnecting cells forming a "vessel-like" shape (Fig., upper insert). Inspired by the MMP-9 ...
Methods
Healthy skin was obtained from routine reduction surgeries from anonymous donors and experiments on ex vivo skin were typically performed within 1-2 hours (h) after surgery. The study was approved by the ethics committee of the Medical University of Vienna and conducted according to the Declaration of Helsinki principles. Written informed consent from all participants was obtained.
Suction blister-induced wound model
A negative pressure instrument (Electronic Diversities, Finksburg, MD, USA) constructed to produce standard suction blisters upon application of negative pressure, was used on healthy skin ( ex vivo: abdominal skin; in vivo: lower forearm). Subcutaneous fat was partially removed from ex vivo skin using a scissor. Subsequently, skin (10 × 10 cm 2 ) was placed (not fixed, not kept in medium) on a styrofoam lid that was covered with aluminium foil to provide (at least partial) backpressure. Suction chambers with 5 openings (Ø = 5 mm) on the orifice plate were attached to skin, topped with a styrofoam lid and pressed with 1 kg weight in order to avoid movement of the plate. A pressure of 200-250 millimeter (mm) mercury (Hg) ( ex vivo ) or 150-200 mm Hg ( in vivo ) caused the skin to be drawn through the openings creatin...
Measurement outputs
What raw and processed outputs should exist?
Data was analysed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Paired t-test was used for comparing means. The results were considered significant with P -valu...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
We next tested whether keratinocytes upon wound closure can also differentiate and form multiple epidermal layers upon prolonged culture. Indeed, several K14 + keratinocyte laye...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
MMPs become activated during wound repair. In particular, MMP-9 influences the detachment of basal keratinocytes from the basement membrane thus improving their migration,. We...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
The suction blister method has been used in several studies to investigate LCs in human epidermis in vivo such as effects of ultraviolet B irradiation, as well as a calcineurin...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
Data was analysed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
from paperScoring or quantification
Quantify the primary readouts for this experiment: Data was analysed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Paired t-test was used for comparing means. The results were considered significant with P -valu...; We next tested whether keratinocytes upon wound closure can also differentiate and form multiple epidermal layers upon prolonged culture. Indeed, several K14 + keratinocyte laye...; MMPs become activated during wound repair. In particular, MMP-9 influences the detachment of basal keratinocytes from the basement membrane thus improving their migration,. We...; The suction blister method has been used in several studies to investigate LCs in human epidermis in vivo such as effects of ultraviolet B irradiation, as well as a calcineurin....
from paperStatistical comparison
Data was analysed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Paired t-test was used for comparing means. The results were considered significant with P -valu...; Next, the activation marker CD83 was used to further characterize the CD11c + cell population in blister roof epidermis. While CD83 + cells were indeed found in the blister roof...
from paperReporting output
Report representative outputs alongside summary comparisons for Data was analysed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Paired t-test was used for comparing means. The results were considered significant with P -valu..., We next tested whether keratinocytes upon wound closure can also differentiate and form multiple epidermal layers upon prolonged culture. Indeed, several K14 + keratinocyte laye..., MMPs become activated during wound repair. In particular, MMP-9 influences the detachment of basal keratinocytes from the basement membrane thus improving their migration,. We..., The suction blister method has been used in several studies to investigate LCs in human epidermis in vivo such as effects of ultraviolet B irradiation, as well as a calcineurin....
inferred from protocolStructured statistical methods
Data was analysed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). Paired t-test was used for comparing means. The results were considered significant with P -valu...; Next, the activation marker CD83 was used to further characterize the CD11c + cell population in blister roof epidermis. While CD83 + cells were indeed found in the blister roof...
source structuredSource and audit
What supports the facts on this page?
Evidence quotes (4)
We next tested whether keratinocytes upon wound closure can also differentiate and form multiple epidermal layers upon prolonged culture. Indeed, several K14 + keratinocyte layers were observed upon 8 days of culture above the initial wound bed (Fig. ). During the continued culture period of 12 days, multiple K14 + layers have formed (Fig. ). In normal, unwounded skin, K10 is exclusively localized in suprabasal keratinocytes (Rakita, unpublished observation). In wounded skin, a few K10 + basal keratinocytes, in particular at the wound edge, were observed in addition to the conventional staining pattern in suprabasal keratinocytes at day 8 of culture (Fig. ). K10 expression appeared in all layers except the basal layer at later time points (Fig. ). Next, expression kinetics of particular epidermal differentiation markers in unwounded skin and blister-induced wounds were assessed. Desmoglein-1 (DSG-1), a cell adhesion structure playing a role in maintaining the structure of the epidermis through its adhesive function, is located above the basal layer in normal skin. Corneodesmosin (CDSN) is synthesized in lower granular keratinocytes and represents an epid...
MMPs become activated during wound repair. In particular, MMP-9 influences the detachment of basal keratinocytes from the basement membrane thus improving their migration,. We noticed scarce MMP-9 + cells directly under the wound bed and in unwounded skin areas (Fig. ). In contrast, strong MMP-9 expression was observed at the leading wound edges at day 4 and already decreased 6 days after wounding (Fig., inserts). MMP-9 + cells greatly accumulated in the dermis at day 10 of culture (Fig., arrowheads). Of note, different MMP-9 expression patterns were observed in dermal cells implying that distinct cell types upregulate this enzyme. MMP-9 appeared in dots around the nucleus (Fig., right insert), on the cell surface (Fig. arrowhead) or interconnecting cells forming a "vessel-like" shape (Fig., upper insert). Inspired by the MMP-9 "vessel-like" staining pattern, we tested whether they co-express CD31 which in normal human skin is expressed primarily in endothelial cells lining the interior surface of blood and lymphatic vessels. Surprisingly, we found that dermal cells expressing MMP-9 and CD31 are mutually exclusiv...
Healthy skin was obtained from routine reduction surgeries from anonymous donors and experiments on ex vivo skin were typically performed within 1-2 hours (h) after surgery. The study was approved by the ethics committee of the Medical University of Vienna and conducted according to the Declaration of Helsinki principles. Written informed consent from all participants was obtained.
A negative pressure instrument (Electronic Diversities, Finksburg, MD, USA) constructed to produce standard suction blisters upon application of negative pressure, was used on healthy skin ( ex vivo: abdominal skin; in vivo: lower forearm). Subcutaneous fat was partially removed from ex vivo skin using a scissor. Subsequently, skin (10 × 10 cm 2 ) was placed (not fixed, not kept in medium) on a styrofoam lid that was covered with aluminium foil to provide (at least partial) backpressure. Suction chambers with 5 openings (Ø = 5 mm) on the orifice plate were attached to skin, topped with a styrofoam lid and pressed with 1 kg weight in order to avoid movement of the plate. A pressure of 200-250 millimeter (mm) mercury (Hg) ( ex vivo ) or 150-200 mm Hg ( in vivo ) caused the skin to be drawn through the openings creating typical suction blisters of different size within 6-8 h ( ex vivo ) and 1-2 h ( in vivo ). Suction blister fluid (~110 µl/5 blisters) was collected using a syringe with a needle. Cells within the fluid were counted and placed on adhesion slides for staining and a...
Machine-readable layer
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