Name: generation of a three-dimensional full thickness skin equivalent and automated wounding
Uploaded: 2015-02-26T15:00:00.000+00:00
Duration: 8 min 35 s
Description: Watch this Scientific Journal Video about Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding at JoVE.com

The authors thank the Fraunhofer ISC for the collaboration concerning the construction of the automated wounding device. The project was founded by Fraunhofer internal project &#8220;M&#228;rkte von &#220;bermorgen&#8221; (SkinHeal).

NOTE: The protocol is designed for the production of 24 full thickness skin equivalents. Human dermal fibroblasts and epidermal keratinocytes were isolated from skin biopsies according to a previously published protocol.21,22 Informed consent was obtained beforehand and the study was approved by the institutional ethics committee on human research of the Julius-Maximilians-University W&#252;rzburg (vote 182/10).
1. Production of Dermal Component
<ol>
	<li>Dissolve collagen with 0.1% acetic acid to a final concentration of 6&#160;mg/ml. For the gel neutralization solution, mix 232.5&#160;ml 2x DMEM, 7.5 ml fetal calf serum, 7.5&#160;ml 3 M&#160;HEPES, 2.5&#160;ml chondroitin sulfate (5&#160;mg/ml). Keep gel neutralization solution and collagen solution on ice. Calculate 500&#160;&#181;l of gel per insert/skin equivalent. 
	NOTE: As the gel neutralization solution will be mixed with two parts of collagen solution prepare double amount of collagen.</li>
	<li>Place 24 inserts into a 24 well plate.</li>
	<li>Resuspend human dermal fibroblasts (hDF) in 10&#160;ml DMEM, centrifuge for 5 min at 270 x g and count the cells. Extract the needed amount of hDF (5 x 104&#160;cells per 500&#160;&#956;l collagen gel) and centrifuge the cells for 5 min at 270 x g.</li>
	<li>Remove the supernatant and carefully resuspend the hDF in 4 ml cooled gel neutralization solution without producing air bubbles. From this step make sure to work as fast as possible to avoid premature solidifying of the collagen gels.</li>
	<li>Resuspend the solution with 8 ml of collagen solution.</li>
	<li>Take the collagen-cell-mixture with a multistep-pipette and fill quickly 500&#160;&#956;l of mixture in each insert. Incubate the gels for 20 min in an incubator (37 &#176;C, 5% CO2) to jell the gels.</li>
	<li>Submerge the gels in 2 ml Dulbecco&#8217;s Modified Eagle Medium (DMEM) + 10% fetal calf serum (FCS) per well and incubate it for 24 hr in an incubator (37 &#176;C, 5% CO2).</li>
</ol>
2. Addition of Human Epidermal Keratinocytes (hEK)
<ol>
	<li>Remove medium after 24 hr. Dissolve fibronectin human protein with ultrapure water to a final concentration of 50&#160;&#181;g/ml. Cover each dermal equivalent with 25&#160;&#956;l of fibronectin solution. Incubate the gels for 30 min in an incubator (37 &#176;C, 5% CO2).</li>
	<li>In the meantime, resuspend the hEK in 10&#160;ml KGM-2 and set cell concentration to 1 x 106 cells/ml with KGM-2 ready + 5% FCS. Seed 1 x 105 hEK on each gel. Incubate gels for 45 min in the incubator (37 &#176;C, 5% CO2) to allow cells to adhere.</li>
	<li>Submerse the gels with KGM-2 ready + 5% FCS and culture them in the incubator (37 &#176;C, 5%&#160;CO2).</li>
</ol>
3. Culture of Full Thickness Skin Equivalents (ftSE)
<ol>
	<li>Culture ftSE for 6-7 days in descending FCS-concentration (5%, 2%, and 0% FCS). Change the medium every 2-3 days with the next lower FCS concentration. For this, completely remove the medium and add 1.6 ml of fresh medium.</li>
	<li>On day 7, remove medium completely. 
	NOTE: Do not touch the ftSE surface with the tip of pipette while doing so.</li>
	<li>Place every insert into 1 well of a 6 well plate with sterile forceps. Add 1.5&#160;ml airlift medium per well, ensure not to wet the ftSE surface. Change medium every 2-3 days for additional 14 days. 
	NOTE: The filling level of the medium is up to the meniscus of the ftSE.</li>
</ol>
4. Histological Analysis
<ol>
	<li>Fixing and paraffin-embedding of the ftSE
	<ol>
		<li>Remove culture medium, transfer inserts in fresh 24 well plates and fix tissues by adding 1.6&#160;ml of 4%&#160;paraformaldehyde to each well and incubate the tissues for 2 hr at RT. Caution! Paraformaldehyd is toxic, manipulate under fume hood. Transfer the ftSE to a tissue-embedding cassette. Remove remaining fixative by washing with water and dehydrate the tissue using ascending ethanol concentrations.</li>
		<li>Divide the skin equivalent by a vertical cut in the middle. Place the two pieces in a paraffin-filled metal base mold with cut surfaces downwards. Add the tissue cassette on top of the mold as a backing.</li>
		<li>Cut 3-5&#160;&#181;m slices and float them on a 40 &#176;C water bath for straitening, mount slides onto suitable microscope slides. Dry slices thoroughly.</li>
	</ol>
	</li>
	<li>Hematoxylin &#38; Eosin (H&#38;E) and immunohistochemical (IHC) staining
	<ol>
		<li>Place slides into rack and incubate for 1 hr at 60 &#176;C. Make sure that paraffin is melted. Stain rehydrated slices with H&#38;E as a standardized overview staining.</li>
		<li>For H&#38;E staining prepare 4x glass staining cuvettes with xylol, 3x with ethanol 96%, 2x with ethanol 70%, 1x with ethanol 50%, 4x deionized water, 1x hematoxylin, 1x HCl-ethanol (13.7&#160;ml 1&#160;M HCl ad 200 ml 50% ethanol), 1x tab water, 1x eosin (1&#160;g eosin in 100&#160;ml deionized water) and 2x 2-propanol.</li>
		<li>Place slides 10 min in xylol I and 10 min in xylol II to deparaffinize and rehydrate slides. Dip slides 3x in ethanol 96% I, 3x in ethanol 96% II, 3x in ethanol 70% and 3x in ethanol&#160;50%. Rotate slides in deionized water. To differentiate hematoxylin dye, dip 2x in HCl-ethanol. Rinse in deionized water. For blueing, place slides 5 min in tab water. For eosin staining, place slides 1 min in eosin and wash afterwards in deionized water. For dehydration, dip slides 2x in ethanol 70%, 2x in ethanol 96% and place them for 5 min in 2-propanol I, for 5 min in 2-propanol II, 5 min in xylol I and 5 min in xylol II. After H&#38;E staining, cell nuclei are stained in blue, cytoplasm and extracellular matrix are stained in red.</li>
		<li>For antigen retrieval, remove paraffin with xylene and rehydrate slices for staining. Place slices in steam cooker and cook deparaffinized and rehydrated slices for 20 min in pre-heated 10&#160;mM citrate buffer (pH&#160;6).</li>
		<li>Place slides in washing buffer (PBS buffer + 0.5% Polysorbat&#160;20) and circle slices with a liquid repellent slide marker pen or diamond stylus.</li>
		<li>Place slides in a moisture chamber. Block endogenous peroxidase, by adding 100&#160;&#181;l 3%&#160;hydrogen peroxide solution onto each slice. Caution! Very hazardous in case of skin and eye contact. Handle with personal protective equipment. Wash slides in washing buffer.</li>
		<li>Apply primary antibody and incubate for 1 hr at RT. Wash slides three times with washing buffer. From here on, samples should be kept in the dark.</li>
		<li>Use biotin-streptavidin detection system for detection of specific antigen-antibody binding.</li>
		<li>Counterstain nuclei with Hematoxylin for 1 min. Dehydrate and mount slides.</li>
		<li>Image samples using an inverse microscope.</li>
	</ol>
	</li>
</ol>
5. Injury by Use of the Automated Wounding Device (aWD)
<ol>
	<li>Prepare the aWD by disinfecting the working area with 70% ethanol and ultraviolet light. Make sure the drilling head of the desired size (e.g., 1 mm) is attached.</li>
	<li>Place skin equivalents into designated areas of the autoclaved sample carrier plate using sterile forceps. Place the sample carrier plate into the socket below the drilling head.</li>
	<li>Use the control software to set wounding parameters as follows: spin velocity: 15,000 rpm; penetration: 1.5 mm; propulsion: 100 Hz. These parameters need to be defined for each sample type individually. Transfer wounding parameters to the aWD.</li>
	<li>Start wounding procedure. Remove sample carrier plate from the socket. Transfer wounded ftSE back into inserts used for culture using sterile forceps.</li>
	<li>Proceed with culture of wounded ftSE in the incubator (37 &#176;C, 5% CO2) for investigation of regeneration. Alternatively, use the models for histological analysis at any time.</li>
</ol>

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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+separation+of+the+epidermis+from+the+dermis.">A method for the separation of the epidermis from the dermis.</a> J Invest Dermatol. 15 (1), 9-10 (1950).</li><li>El Ghalbzouri, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblasts+facilitate+re-epithelialization+in+wounded+human+skin+equivalents.">Fibroblasts facilitate re-epithelialization in wounded human skin equivalents.</a> Lab Invest. 84 (1), 102-112 (2004).</li><li>Vaughan, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reproducible+laser-wounded+skin+equivalent+model+to+study+the+effects+of+aging+in+vitro.">A reproducible laser-wounded skin equivalent model to study the effects of aging in vitro.</a> Rejuvenation Res. 7 (2), 99-110 (2004).</li><li>Geer, D. J., Swartz, D. D., Andreadis, S. 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The authors have nothing to disclose.

In vitro cells are usually expanded in two-dimensional cell cultures, in which cells adhere to plastic surfaces. However, these culture conditions do not reflect the physiological three-dimensional conditions in which cells grow in vivo. Under three-dimensional conditions, cells can form natural cell-cell and cell-matrix attachments and migrate in three-dimensions. Especially in the cutaneous wound healing the resemblance of the in vivo situation is pivotal to generate meaningful data, as cell migration and matrix generation are key elements of wound healing.
In order to mimic these conditions we developed ftSE that are composed of human primary cells and reflect both the dermal and the epidermal layer of the skin. During two weeks of culture at the air-liquid interface, the hEK form an epidermis composed of several layers of keratinocytes in different differentiation phases and a stratum corneum. Furthermore, the dermal layer is composed of hDF, which are embedded in a collagen I hydrogel. During the culture time, the hDF remodel the dermal component, which results in a significant shrinkage of the models (Figure 2B).
To prepare reproducible and standardized wounds, we developed an aWD. The computer controlled machine can set defined and precise cutaneous injuries (Figure 4). Depending on the intended research, depth, shape and size of the injury can be adapted. However, due to the technical specifications of the aWD and the morphology of the ftSE, it is recommended to set a minimal penetration of 100 &#181;m reproducibility. The aWD is operating under sterile conditions in a closed box-system and all components can be sterilized by autoclaving, disinfection solutions or ultraviolet light. In preparation for the wounding procedure the samples need to be transferred to a sample carrier plate. This plate facilitates an exact position of the samples. Furthermore the adhesion between sample and the sample carrier plate is sufficient to keep the samples in place during the wounding procedure. The wounding procedure itself can be monitored optically via a front window or by a high resolution camera that records all steps from a close-up position. Unlike other systems that use lasers to excavate tissue to generate a wound, the aWD employs a rotating drill. Thus, no heat is applied during the procedure, which is a vital perquisite to investigate mechanical wounds. Additionally, specific shape of the drilling head ensures a material removal from the wound channel. Using empirically determined wounding parameters mentioned above the wounding procedure can be adjusted to meet specific physiological properties of either the ftSE or native skin samples. This way any undesirable side effects like unintentional detachment of epidermal layers can be minimized and reproducible wounding performed with a success rate of 90%.
This study demonstrates that the developed ftSE partly mimic the in vivo situation of skin accurately and thus can be employed as a model for wound healing studies. Though important aspects of the wound healing process such as the effect of the vasculature, the blood clot and the immune system are lacking in the model, the epidermal-mesenchymal and cell-matrix interaction is recapitulated in a three-dimensional highly standardized environment. In addition, we could show that the aWD generates standardized wounds in skin models. In combination, both technologies can be used to investigate wound healing and the effect of substances and drugs that support the healing process. In order to mimic the in vivo situation more closely, the ftSE can be extended by other cells such as melanocytes, skin tumor cells or micro-vascular endothelial cells.

The skin is the largest organ of the body. It creates a barrier between the external environment and the internal organs. Moreover, the skin protects the body from fluid loss, environmental influences, injuries and infections and helps to regulate body temperature1. Due to its exposed location, the skin is often affected by mechanical, thermal or chemical trauma. Though the skin is generally capable of self-repair, multiple local factors such as infection, oxygenation, and venous sufficiency can lead to impaired wound healing. Wound healing can also be interfered by systemic factors as obesity, alcoholism, smoking, medication, nutrition and diseases such as diabetes.2
The process of wound healing can be divided into 3 phases: (i) the inflammatory phase, (ii) the proliferative and the (iii) remodeling phase. Upon injury to the skin, a complex signal-cascade starts, leading to the closure of the wound.3 After injury, the wound is bleeding and a blood clot is formed. Fibroblasts move into the blood clot and replace it with new tissue which is subsequently remodeled over years.
The current understanding of the biological processes underlying cutaneous repair is limited. Small animal and pig models have been used to study wound healing. However, these results cannot be directly transferred to humans due to species-specific differences. In addition to these in vivo models, some aspects of wound healing can be studied by simulating a wound situation via scratching of in vitro monolayer cultures based on immortalized cell lines or primary cells.4 These scratching models are highly standardized but do not sufficiently reflect the complex in vivo physiology.5 Besides two-dimensional models, three-dimensional human skin equivalents have been developed for dermatological research. The dermal part of these models are generated using various scaffolds including decellularized dermis,6 collagen hydrogels,7,8 glycosaminoglycans9 or synthetic materials.10 Employing these skin equivalents, the role of epithelial-mesenchymal interactions11, the re-epithelialization, the cellular crosstalk between fibroblasts and keratinocytes and the influence of different growth factors can be studied. Moreover, these models are useful to gain new knowledge about how fibroblasts migrate into the wounded area and how chemotactic factors influence tissue regeneration.12
Not only generation of the wound healing model itself is challenging, but also to establish a highly standardized wound in a model is problematic. Common techniques to create wounds are scratch tests,13 burns,14 tape abrasion,15 thermal injury,16 suction blisters,17 liquid nitrogen,18 lasers,19 scalpels,18 meshers6 and biopsy punches.20 Most of these methods have the same pitfalls. Injuries implemented manually are difficult to standardize and to reproduce between multiple tests. Size, shape and depth of the wound vary between studies and thus impair the quality of research data. The use of laser for defined skin wounding can be relatively easily standardized but leads to a situation mimicking burn wounds. Heat applied by laser can cause protein denaturation, platelets aggregation or vessels constriction, which can lead to necrotic tissue.
In an alternative approach, we developed an automated wounding device (aWD), which allows us to generate defined and precise cutaneous wounds under sterile conditions. Wounding parameters, like depth and speed of penetration as well as revolutions of the drill head can be regulated. In this study, we combined the aWD with in house developed full thickness skin equivalents (ftSE) that are comparable to a protocol published by Gangatirkar et al. 8 The dermal layer of the skin equivalent is composed of human dermal fibroblasts (hDF), which are embedded in a collagen I hydrogel. On the dermal layer, human epidermal keratinocytes (hEK) are seeded. Within two weeks at the air-liquid interface the hEK build up an epidermis composed of several vital cell layers and a stratum corneum. Besides the generation of this model, this study shows the use of the aWD to create defined and precise wounds in ftSE.

<table><tbody><tr><td>Trypsin EDTA (1:250) 0.5% in DPBS</td><td>PAA</td><td>L11-003</td><td>0.05%</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Collagen (6 mg/ml in 0.1% acetic acid)</td><td>Produced in house</td><td></td><td></td></tr><tr><td>Fibronectin Human Protein, Plasma (50&amp;nbsp;&amp;micro;g/ml)</td><td>Life Technologies</td><td>33016-015</td><td></td></tr><tr><td>Inserts</td><td>Nunc</td><td>140627</td><td></td></tr><tr><td>6 well plate&amp;nbsp;</td><td>Nunc</td><td>140685</td><td></td></tr><tr><td>24 well plate</td><td>Nunc</td><td>142485</td><td></td></tr><tr><td>Microscope slides</td><td>R. Langenbrinck</td><td>03-0070</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Fibroblasts culturing (500 ml):&lt;/strong&gt;</td></tr><tr><td>DMEM, high glucose</td><td>Life Technologies</td><td>11965-092</td><td>89% (445 ml)</td></tr><tr><td>Fetal bovine serum</td><td>Bio &amp;amp; Sell</td><td>FCS.ADD.0500</td><td>10% (50 ml)</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td>1% (5 ml)</td></tr><tr><td colspan="4">&lt;strong&gt;Keratinozyten culture medium (500 ml):&lt;/strong&gt;</td></tr><tr><td>Keratinocyte Growth Medium 2</td><td>Promocell&amp;nbsp;</td><td>C-20111</td><td>89% (445 ml)</td></tr><tr><td>Keratinocyte Growth Medium 2 SupplementPack</td><td>Promocell&amp;nbsp;</td><td>C-39011</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td>1% (5 ml)</td></tr><tr><td colspan="4">&lt;strong&gt;Gel neutralization solution (250 ml):&lt;/strong&gt;</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium, high Glucose Powder with L-Glutamine</td><td>PAA</td><td>G0001,3010</td><td>93% (232.5 ml)</td></tr><tr><td>Chondroitin sulfate sodium salt from shark cartilage</td><td>Sigma</td><td>C4384-1g</td><td>1% (2.5 ml)</td></tr><tr><td>Fetal bovine serum</td><td>Bio &amp;amp; Sell</td><td>FCS.ADD.0500</td><td>3% (7.5 ml)</td></tr><tr><td>HEPES</td><td>Sigma&amp;nbsp;</td><td>H3375-1kg</td><td>3% (7.5 ml)</td></tr><tr><td colspan="4">&lt;strong&gt;Skin model submers medium (500&amp;nbsp;ml):&lt;/strong&gt;</td></tr><tr><td>Keratinocyte Growth Medium 2</td><td>Promocell&amp;nbsp;</td><td>C-20111</td><td></td></tr><tr><td>Keratinocyte Growth Medium 2 SupplementPack</td><td>Promocell&amp;nbsp;</td><td>C-39011</td><td></td></tr><tr><td>Fetal calf serum</td><td>Bio &amp;amp; Sell</td><td>FCS.ADD.0500</td><td>5%-2% (25 ml-10 ml)&amp;nbsp;</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td>1% (5 ml)</td></tr><tr><td colspan="4">&lt;strong&gt;Skin model air-liquid interface medium (500 ml):&lt;/strong&gt;</td></tr><tr><td>Keratinocyte Growth Medium 2</td><td></td><td></td><td></td></tr><tr><td>Keratinocyte Growth Medium 2 Supplement Pack</td><td>Promocell&amp;nbsp;</td><td>C-39011</td><td>adding only supplements: insulin, hydrocortisone, epinephrine, transferrin, CaCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Penicillin-Streptomycin (10,000 U/ml)</td><td>Life Technologies</td><td>15140-122</td><td>1% (5 ml)</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; (300 mM)</td><td>Sigma&amp;nbsp;</td><td>C7902-500g</td><td>0.62% (3.1 ml)</td></tr><tr><td colspan="4">&lt;strong&gt;Histology:&lt;/strong&gt;</td></tr><tr><td>IHC-Kit DCS SuperVision 2 HRP</td><td>DCS</td><td>PD000KIT</td><td></td></tr><tr><td>Vimentin antibody</td><td>Abcam</td><td>ab92547</td><td></td></tr><tr><td>CK14 antibody</td><td>Sigma&amp;nbsp;</td><td>HPA023040-100&amp;micro;l</td><td></td></tr><tr><td>CK10 antibody</td><td>Dako</td><td>M7002</td><td></td></tr><tr><td>Filaggrin antibody</td><td>Abcam</td><td>ab81468</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;H&amp;amp;E staining:&lt;/strong&gt;</td></tr><tr><td>Mayer&amp;acute;s Haemalaun</td><td>AppliChem</td><td>A0884,2500</td><td></td></tr><tr><td>Xylol</td><td>Sigma Aldrich</td><td>296325-4X2L</td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>32205-4X2.5L</td><td></td></tr><tr><td>HCl</td><td>Sigma Aldrich</td><td>H1758-500ML</td><td></td></tr><tr><td>Eosin</td><td>Sigma Aldrich</td><td>E4009-5G</td><td></td></tr><tr><td>2-Propanolol</td><td>Sigma Aldrich</td><td>I9516-500ML</td><td></td></tr><tr><td>Mounting Medium</td><td>Sigma Aldrich</td><td>M1289-10ML</td><td></td></tr></tbody></table>

generation of a three-dimensional full thickness skin equivalent and automated wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The isolated hDF and hEK differ both in morphology and in expression of typical markers. The hDF showed typical spindle-shape morphology, whereas morphology of hEK can be described by a cobblestone morphology. The cells were characterized by immunohistochemical staining (Figure 1) before using them for ftSE. The hDF are positive for vimentin (Figure 1A), a marker for fibroblasts. Primary hEK highly express early differentiation protein cytokeratin-14 (Figure 1B) but nearly no late keratinocyte differentiation protein cytokeratin-10 (Figure 1C).
Following primary culture, we detached the cells from the cell culture flasks and used them for the generation of ftSE. The ftSE were cultured for 7 days under submers medium conditions (Figure 2A) followed by a 14 days culture at the air-liquid interface (Figure 2B). During this process, the ftSE contract significantly. Within 21 days the hEK are proliferating (Figure 2C-E). By changing the medium level in such way that the surface of the models is exposed to air and by increasing calcium concentration, the hEK are stimulated to differentiate into a multi-cellular epidermis that is composed of several vital cell layers of keratinocytes in different differentiation states and a stratum corneum (Figure 2E).
Comparing the Haematoxylin &#38; Eosin staining of ftSE (Figure 2E) with native human skin (Figure 2F) it becomes obvious that ftSE mimics histological architecture of the skin. This can be further demonstrated by immunohistochemical stainings (Figure 3). The hDF in the collagen&#160;I hydrogel can be stained with primary antibodies against vimentin (Figure 3A, E). Keratinocytes form an epidermal layer composed of cytokeratin-14 positive cells (Figure 3B, F) and the late differentiation marker cytokeratin-10 is expressed in the supra-basal layers (Figure 3C, G). Furthermore, the stratum corneum is positive for filaggrin (Figure 3D, H).
Following 21 day culture period, the aWD was used to generate cutaneous wounds in the ftSE. As shown in Figure 4A, the aWD is constructed as a closed box-system to ensure sterile conditions. In Figure 4B, a schematic drawing of the wounding process is demonstrated. The ftSE are fixed on a sample carrier plate. The whole wounding process, including the setting of parameters such as lowering speed and revolutions of the drill head (Figure 4D) as well as the depth of penetration is controlled computer assisted. The wounding procedure can be monitored optically via a front window or by a high resolution camera that records all steps during the wounding procedure. Size, shape and depth of the wound injury can be adjusted individually (Figure 4C).
<img alt="Figure 1" src="/files/ftp_upload/52576/52576fig1.jpg" /> 
Figure 1. Characterization of cells used for the construction of full thickness skin equivalents. Immunohistochemical staining of primary human dermal fibroblasts for vimentin (A) and of epidermal keratinocytes for the early differentiation keratin filament cytokeratin-14 (B) and the late differentiation filament cytokeratin-10 (C). Positive staining is shown by brown color. Scale bars indicate 100&#160;&#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52576/52576fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52576/52576fig2.jpg" /> 
Figure 2. Changes of culture conditions and maturation of full thickness skin equivalents. Macroscopic pictures of full thickness skin equivalents cultured for 7 days under submers conditions (A) and 14 days at the air-liquid interface (B). Comparison of Haematoxylin &#38; Eosin staining of full thickness skin equivalents in different developmental stages: After 7 days (C), 14 days (7 days at the air-liquid interface, D) and 21 days (14 days at the air-liquid interface, E). Haematoxylin &#38; Eosin staining of native human skin (F). Scale bars indicate 100&#160;&#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52576/52576fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52576/52576fig3.jpg" /> 
Figure 3. Characterization of full thickness skin equivalents. Comparison of immunohistochemical staining of full thickness skin equivalents (A-D) and native human skin (E-H) for vimentin (A, E), cytokeratin-14 (B, F), cytokeratin-10 (C, G) and filaggrin (D, H). Scale bars indicate 100&#160;&#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52576/52576fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52576/52576fig4.jpg" /> 
Figure 4. Automatic Wounding Device for controlled wounding on full-thickness skin equivalents. The automated wounding device (aWD) provides sterile conditions during the controlled wounding procedure (A). An attached computer (CPU) controls the aWD. The full thickness skin equivalents (ftSEs) are placed on the sample carrier plate (SCP), deviations in sample morphology are equalized by a light barrier (LB), which triggers the wounding procedure. The wound shape depends on the used drilling head (DH). The diameter ranges from 1&#160;mm (C, upper image) to 2&#160;mm (C, lower image). The wounding procedure is monitored and can be recorded for documentation and quality management (D). Hematoxylin &#38; Eosin stained cross section of a full thickness skin equivalent wounded with the automated wounding device using a 1 mm drilling head (E). All scale bars indicate 2&#160;mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52576/52576fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

generation-three-dimensional-full-thickness-skin-equivalent-automated

Nanyang Technological University

Research

JoVE Journal

Bioengineering

18.7K Views.  University Hospital Würzburg. The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility. 

Video: Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Developmental Biology

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Fabrication and Characterization of a Conformal Skin-like Electronic System for Quantitative, Cutaneous Wound Management

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of cutaneous wound healing on the skin. Existing methods, however, still rely on visual inspections through various microscopic tools and devices that normally include high-cost, sophisticated systems and require well trained personnel for operation and data analysis. Here, we describe methods and protocols to fabricate a conformal, skin-like electronics system that enables conformal lamination to the skin surface near the wound tissues, which provides recording of high fidelity electrical signals such as skin temperature and thermal conductivity. The methods of device fabrication provide details of step-by-step preparation of the microelectronic system that is completely enclosed with elastomeric silicone materials to offer electrical isolation. The experimental study presents multifunctional, biocompatible, waterproof, reusable, and flexible/stretchable characteristics of the device for clinical applications. Protocols of clinical testing provide an overview and sequential process of cleaning, testing setup, system operation, and data acquisition with the skin-like electronics, gently mounted on hypersensitive, cutaneous wound and contralateral tissues on patients.

This article presents methods to fabricate and characterize a conformal, skin-like electronic system and protocols for the use in clinical applications, particularly on cutaneous wound management.

Recent advances in the development of electronic technologies and biomedical devices offer opportunities for non-invasive, quantitative assessment of ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Efficient 3D Skin Models for Flexible Research Application

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

Author Spotlight: Enhancing Skin Model Diversity with Cost-Effective 3D Cellular Models

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

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