This work was supported by funding from Singapore&#39;s Agency for Science, Technology and Research &#8220;SPF 2013/004: Skin Biology Basic Research&#8221; and the &#8220;Wound Care Innovation for the Tropics&#8221; IAF-PP/2017 (HBMS) H17/01/a0/009. The authors gratefully acknowledge advice and assistance from Dr. Paula Benny and Dr. Michael Raghunath.

1. Cell culture
<ol>
	<li>Maintain hHSFs and normal dermal fibroblasts derived from non-pathological tissue (hNSFs) in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) containing 10% fetal calf serum (FCS) and 1% v/v penicillin/streptomycin solution (P/S) at 37&#176;C in an incubator with 5% CO2/95% air.</li>
	<li>Purchase Ficoll 70, Ficoll 400, and ascorbic acid from the appropriate companies.</li>
</ol>
2. Construction of MMC hypertrophic scar model
<ol>
	<li>Seed the hHSFs or hNSFs (50,000/well) into a 24 well plate containing 1 mL of media in each well.</li>
	<li>Place in a 37&#176;C incubator at 5% CO2 and leave overnight.</li>
	<li>Prepare the MMC media. Based on the total volume required for the experiment, produce 10% FCS/DMEM media by mixing Ficoll 70 (18.75 mg/mL), Ficoll 400 (12.5 mg/mL), and ascorbic acid (100 &#956;M).</li>
	<li>Place the mixture into a 37&#176;C water bath for 1 h to disperse the crowders into the solution, then sterilize the MMC media using a 0.2 &#956;m filter.</li>
	<li>Aspirate the spent media and replace with the freshly made MMC media.</li>
	<li>Incubate the cells for 6 days at 37&#176;C and 5% CO2, changing the media every 3 days.</li>
</ol>
3. Expression of the total amount of collagen
<ol>
	<li>Prepare Sirius red solution (0.1% w/v). Dissolve 0.2 g of Direct Red 80 powder in 200 mL of distilled deionized water (ddH2O) with 1 mL of acetic acid.</li>
	<li>Aspirate the MMC media and add 300 &#956;L of Sirius Red solution into each well. Incubate at 37 &#176;C for 90 min.</li>
	<li>Gently rinse the Sirius Red solution with tap water and allow the plate to air-dry overnight.</li>
	<li>Extract the Sirius Red stain by adding 200 &#956;L of sodium hydroxide (0.1 M) into each well. Place the plate onto an orbital shaker for 5&#8211;10 min to fully extract the Sirius Red stain.</li>
	<li>Transfer 100 &#956;L of the extracted Sirius Red stain into a 96 well transparent plate and measure the absorbance at 620 nm using a microplate reader.</li>
</ol>
4. Expression of collagen I (immunostaining)
<ol>
	<li>Wash the wells using 200 &#956;L of phosphate-buffered saline (PBS, pH = 7.35).</li>
	<li>Fix the cells using methanol (500 &#956;L/well) at 4 &#176;C for 10 min.</li>
	<li>Block nonspecific interactions with 3% bovine serum albumin for 30 min at room temperature (RT).</li>
	<li>Aspirate the blocking solution and incubate with 200 &#956;L of anti-collagen I antibody (10 &#956;g/mL) for 90 min at RT.</li>
	<li>Aspirate the primary antibody and wash 3x with PBS for 5 min each.</li>
	<li>Incubate with 200 &#956;L of Goat anti-Rabbit-FITC secondary antibody (1:400 dilution) and 4&#8217;,6-diamidino-2-phenylindole (DAPI; 1: 2000 dilution) for 30 min at RT. Cover the plate with aluminum foil.</li>
	<li>Discard both the secondary antibody and DAPI and wash 3x with PBS for 5 min each.</li>
	<li>Visualize the fluorescence staining directly under a microscope.</li>
</ol>
5. Western blotting
<ol>
	<li>Wash the cells 2x with PBS.</li>
	<li>Add 40 &#956;L of lysis buffer into each well and scrape the cell layer with a pipette tip. The lysis buffer contains RIPA buffer, protease inhibitor cocktail (PIC), 2 mM sodium vanadate, and 10 mM sodium fluoride.</li>
	<li>Transfer the protein lysate into microcentrifuge tubes and measure the protein concentration using a protein assay as per the manufacturer&#39;s instructions16.</li>
	<li>Load 10 &#956;g of protein of each group into the wells of 4%&#8211;12% Bis-Tris protein gels. Perform sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) at 200 V for 30 min.</li>
	<li>Transfer the protein to nitrocellulose membrane by running a western blot at 90 V for 90 min. Avoid formation of air bubbles between the gel and nitrocellulose membrane.</li>
	<li>Block the membrane with 10 mL of blocking buffer.</li>
	<li>Incubate with primary antibodies at 4&#176;C overnight. Primary antibodies are: anti-collagen I, anti-collagen III, anti-collagen IV, anti-&#945;SMA, anti-MMP-1, anti-MMP-2, anti-MMP-9, anti-MMP-13, and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH).</li>
	<li>Wash the membrane with 0.1% TBS-Tween 20 (5x for 5 min each). TBS/Tween 20 (1 L) contains the following: 50 mL of 1 M Tris (pH = 7.4), 30 mL of 5 M sodium chloride, 1 mL of Tween 20, and 920 mL of ddH2O.</li>
	<li>Incubate with a species appropriate secondary antibody at RT for 1 h.</li>
	<li>Repeat step 5.8 and visualize fluorescence using an imaging system.</li>
</ol>
6. RT-PCR
<ol>
	<li>Collect the total RNA using the lysis buffer mixed with 2-mercaptoethanol included in the RNA extraction assay kit.</li>
	<li>Purify the RNA as per the manufacturer&#39;s instructions17.</li>
	<li>Measure the RNA concentration using a microvolume spectrophotometer.</li>
	<li>Perform first strand cDNA synthesis using a cDNA synthesis kit as per the manufacturer&#39;s instructions.</li>
	<li>RT-PCR oligonucleotide primers are provided (precoated) in custom 96 well plates. Mix 100 ng of the cDNA samples with 10 &#956;L of SYBR green supermix into the customized plate.</li>
	<li>Increase the total volume to 20 &#956;L/well using ddH2O.</li>
	<li>Run RT-PCR using a thermal cycler following the manufacturer&#39;s instructions: 40 cycles of denaturing at 98&#176;C for 15 s and annealing/extension at 60 &#176;C for 60 s. The genes tested in RT-PCR include: COL1A1, COL3A1, ACTA2, SMAD2, SMAD3, SMAD4, SMAD7, IL1A, IL1B, IL6, IL8, MMP1, MMP2, MMP3, TGFB1, and VEGF.</li>
</ol>

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The authors have no conflicts of interest.

This protocol aims to optimize and authenticate an improved :scar-in-a-jar&#8221; in vitro model for human cutaneous scar tissue. Previous studies have reported the application of MMC technique to human lung fibroblasts12, human bone marrow mesenchymal stem cells23, and human dermal fibroblasts23 using dextran12, Ficoll12, and PVP23 as crowders. In the study reported here, the pr...

Scar tissue represents the endpoint of tissue repair. However, in many individuals, especially those suffering from burns or trauma1, scarring can be excessive and impose undesirable effects on the morphology and functioning of healed skin. Although the exact mechanisms of pathological (hypertrophic scars and keloids) scar formation are not fully understood, excessive deposition of collagen during wound healing has been demonstrated to be an essential contribution2.
It is well-established that transforming growth factor beta 1 (TGF-&#946;1) and alpha smooth muscle actin (&#945;SMA) play key roles in the formation of hypertrophic scars. Evidence suggests that elevated TGF-&#946;1 directly stimulates excessive deposition of collagen via regulating the SMAD signaling pathway3. In addition, &#945;SMA has been found to contribute to hypertrophic scar formation by regulating cell contraction and reepithelialization in the wound healing process4. The lack of suitable in vitro and in vivo models is a major impediment towards developing and evaluating interventions and therapies for scar remediation. The aim of this study is to utilize the existing MMC technique to construct an &#8220;in vivo-like&#8221; hypertrophic scar model that is suitable for evaluating novel and emerging scar-related interventions.
Reproducing living tissue outside of the body has been a goal for years in the scientific community. The development of in vitro techniques in the early twentieth century partly achieved this goal. Current in vitro techniques have slightly evolved from Roux&#39;s original demonstration that embryonic cells can survive ex vivo for several days in warm saline5. However, in vitro methodologies are mostly limited to single cell types cultivated in 2-D and do not accurately recapitulate tissues in vivo. While useful for examining cell biochemistry, physiology, and genetics, native tissues are 3-D and incorporate multiple cell types. Simple 2-D in vitro systems subject mammalian cells to highly artificial environments in which native tissue-specific architecture is lost6. In turn, this affects intracellular and extracellular events, resulting in abnormal cell morphology, physiology, and behaviour7.
The interest behind this protocol lies in the development and clinical management of hypertrophic scars and keloids. While it is well-established that dermal fibroblasts are largely responsible for the abundant production of collagens present in scar tissue, cultivating dermal fibroblasts using 2-D in vitro systems fails to reproduce the turnover of collagen observed in vivo8. Contemporary in vitro methods still essentially use &#8220;warm saline&#8221;, an environment completely different from that in living tissues. Tissues in vivo are extremely crowded, with proteins, nucleic acids, ribonucleoproteins, and polysaccharides, occupying 5%&#8211;40% of the total volume. As no two molecules can occupy the same space at the same time, there is little free space available and an almost complete absence of free water9.
The MMC technique imposes constraints affecting the thermodynamic properties of cytosol and interstitial fluids. Molecular interactions, receptor-ligand signaling complexes, enzymes, and organelles are confined and restricted from interacting freely9. Interactions within the pericellular environment (i.e., interstitium) are also constrained. Recent evidence confirms that high concentrations of inert macromolecules in crowded solutions perturb diffusion, physical association, viscosity and hydrodynamic properties10.
Interestingly, several popular crowding agents (i.e., Ficoll, dextran, polyvinylpyrrolidone [PVP], and sodium 4-styrenesulfonate [PSS]) are not equivalent when applied to different cell types and in different settings. In one previous study, Ficoll was reported to be less cytotoxic for mesenchymal stem cells compared to PVP. These results were interpreted to be the consequence of its neutral charge and relatively small hydrodynamic radius11. In contrast, a second study found that dextran is more effective in stimulating collagen I deposition by human lung fibroblasts compared to Ficoll12. Data from our own study suggests that Ficoll enhances collagen deposition by hypertrophic scar-derived fibroblasts, whereas PVP is toxic to these cells13.
It has been demonstrated that the conversion of procollagen to collagen is faster in a highly crowded in vivo environment14, while the rate of biological reactions is delayed in a diluted 2-D culture system15. We have optimized the in vitro protocol here, incorporating MMC to show the cultivation of dermal fibroblasts serving as a more &#8220;in vivo-like&#8221; model for dermal fibrosis and scar formation. In contrast to the common 2-D culture system, cultivating hHSFs with MMC stimulates the biosynthesis and deposition of collagen significantly13. Notably, other characteristics of fibrosis (i.e., increased expression of matrix metalloproteinases [MMPs] and proinflammatory cytokines) are also evident under this optimized MMC protocol13. When cultivated using this method, it is shown that dermal cells recapitulate the physiological, biochemical, and functional parameters measured in vivo.
The optimized MMC in vitro protocol has been used to evaluate the expression of collagen and other ECM proteins by dermal fibroblasts isolated from hypertrophic scar dermis and uninvolved adjacent dermis. When cultivated in MMC environments in vitro, it has been observed that hHSFs express certain characteristics (i.e., mRNA, biochemistry, physiology, and phenotype) similar to dermal hypertrophic scar tissue in vivo. The evidence indicates that physical and chemical properties are important considerations when selecting crowders and optimizing MMC conditions for cultivation in vitro.
For proof-of-principle, the MMC protocol is applied here to qualitatively and quantitatively evaluate the ability of Shikonin and its analogues to induce apoptosis. This allows evaluation of the potential applications of these naturally-derived Traditional Chinese Medicine (TCM) compounds for managing dermal scarring13. Notwithstanding, the simplicity, cost-effectiveness, and timeliness of this in vitro MMC protocol also satisfies recent regulations to eliminate experimentation in mammals by the EU Directive 2010/63/EU and U.S. Environmental Protection Agency (EPA).

<table><tbody><tr><td>0.2 &amp;mu;m filter</td><td>Sartorius</td><td>16534</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)</td><td>Thermo Fisher Scientific</td><td>P36962</td><td></td></tr><tr><td>Alexa Fluor 680</td><td>Thermo Fisher Scientific</td><td>A-21076</td><td></td></tr><tr><td>Alexa Fluor 800</td><td>Thermo Fisher Scientific</td><td>A-11371</td><td></td></tr><tr><td>alpha smooth muscle actin (&amp;alpha;SMA) primary antibody</td><td>Abcam</td><td>ab5694</td><td></td></tr><tr><td>Applied Biosystems 7500 Fast Real-Time PCR System (thermal cycler )</td><td>Thermo Fisher Scientific</td><td>4351106</td><td></td></tr><tr><td>Ascorbic acid</td><td>Wako</td><td>#013-12061</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>#A2153</td><td></td></tr><tr><td>Bradford protein assay</td><td>Bio-Rad</td><td>500-0006</td><td></td></tr><tr><td>Collagen I primary antibody (for immunostaining)</td><td>Abcam</td><td>6308</td><td></td></tr><tr><td>Collagen I primary antibody (for western blot)</td><td>Abcam</td><td>ab21286</td><td></td></tr><tr><td>Collagen III primary antibody</td><td>Abcam</td><td>ab7778</td><td></td></tr><tr><td>Collagen IV primary antibody</td><td>Abcam</td><td>ab6586</td><td></td></tr><tr><td>Direct Red 80</td><td>Sigma-Aldrich</td><td>2610108</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Life Technologies</td><td>11996-065</td><td></td></tr><tr><td>Fetal calf serum (FCS)</td><td>Life Technologies</td><td>6000-044</td><td></td></tr><tr><td>Ficoll 400</td><td>GE HealthCare</td><td>#17-0300-10</td><td></td></tr><tr><td>Ficoll 70</td><td>GE HealthCare</td><td>#17-0310-10</td><td></td></tr><tr><td>GAPDH primary antibody</td><td>Sigma-Aldrich</td><td>G8795</td><td></td></tr><tr><td>Goat Anti-Rabbit secondary antibody</td><td>Abcam</td><td>ab97050</td><td></td></tr><tr><td>Human hypertrophic scar/normal fibroblasts (hHSF/hNSF)</td><td>Cell Research Corporation</td><td>106, 107, 108</td><td></td></tr><tr><td>iScript cDNA Synthesis Kit</td><td>Bio-Rad</td><td>#1708890</td><td></td></tr><tr><td>MMP-1 primary antibody</td><td>Abcam</td><td>ab38929</td><td></td></tr><tr><td>MMP-13 primary antibody</td><td>Abcam</td><td>ab39012</td><td></td></tr><tr><td>MMP-2 primary antibody</td><td>Abcam</td><td>ab37150</td><td></td></tr><tr><td>MMP-9 primary antibody</td><td>Abcam</td><td>ab38898</td><td></td></tr><tr><td>NanoDrop Microvolume Spectrophotometers</td><td>Thermo Fisher Scientific</td><td>N/A</td><td></td></tr><tr><td>Nitrocellulose membrane</td><td>Bio-Rad</td><td>10484060</td><td></td></tr><tr><td>NuPAGE 4-12% Bis-Tris Protein Gels</td><td>Thermo Fisher Scientific</td><td>NP0321BOX</td><td></td></tr><tr><td>Odyssey blocking buffer</td><td>LI-COR Biosciences</td><td>927&amp;ndash;40000</td><td></td></tr><tr><td>Odyssey Fc Imaging System</td><td>LI-COR Biosciences</td><td>N/A</td><td></td></tr><tr><td>Olympus IX-81 HCS microscope (for immunostaining)</td><td>Olympus</td><td>N/A</td><td></td></tr><tr><td>Penicillin/streptomycin solution (P/S)</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>PrimePCR Assays</td><td>Bio-Rad</td><td></td><td>Customized primers pre-coated in 96-well plates based on requirement</td></tr><tr><td>Protease inhibitor cocktail (PIC)</td><td>Sigma-Aldrich</td><td>11697498001</td><td></td></tr><tr><td>PVP 360</td><td>Sigma</td><td>#PVP360</td><td></td></tr><tr><td>PVP 40</td><td>Sigma</td><td>#PVP40</td><td></td></tr><tr><td>RIPA buffer</td><td>Merck</td><td>R0278</td><td></td></tr><tr><td>RNeasy Plus Mini Kit</td><td>QIAGEN</td><td>#74134</td><td></td></tr><tr><td>Sodium vanadate</td><td>Sigma-Aldrich</td><td>450022</td><td></td></tr><tr><td>Sodium vanadate</td><td>Sigma-Aldrich</td><td>450243</td><td></td></tr><tr><td>SpectraMax M5 Multi-Mode microplate reader</td><td>Molecular Devices</td><td>N/A</td><td></td></tr><tr><td>SsoAdvanced universal SYBR green supermix</td><td>Bio-Rad</td><td>#172-5270</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P9416</td><td></td></tr></tbody></table>

in vitro model of human cutaneous hypertrophic scarring using macromolecular crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

Triplicate samples were performed in each experiment, and each experiment was repeated 3x using cells from three individual patients. Data are expressed as percentages of the control group. One-way ANOVA and Tukey&#39;s post-hoc test were applied to analyze statistical differences (*p &#706; 0.05).
MMC using Ficoll at 9% FVO (fractional volume occupancy) enhances the total amount of collagen and collagen I deposition in hHSF13. As illustrated in <strong class="...

Watch this Scientific Journal Video about In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding at JoVE.com

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

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Research

JoVE Journal

Bioengineering

6.7K Views.  Agency for Science, Technology and Research (A*STAR). We creëerden een individueel collageenrijk model om de omgeving na te bootsen die cellen in vivo ervaren met behulp van de macromoleculaire verdringingstechniek. Huid is een drukke omgeving, in tegenstelling tot de vloeibare omgeving die we al gebruiken om cellen in vitro te kweken. Vergeleken met de traditionele monolayer cel cultuur systeem, dit model reconstrueert de drukke moleculaire omgeving die cellen ervaren in vivo, vooral in littekenweefsel waar overvloedige extracellulaire matrix ...

Video: In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

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 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 ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

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 ...

Developmental Biology

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 ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

Medicine

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 ...

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo

The present protocol describes how to graft human skin onto non-obese diabetic (NOD)-scid interleukin-2 gamma chain receptor (NSG) mice. A detailed description of the preparation of human skin for transplant, preparation of mice for transplant, transplantation of split-thickness human skin, and post-transplantation recovery procedure are included in the report.

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational ...

Immunology and Infection

A Mouse Model of Mechanotransduction-driven, Human-like Hypertrophic Scarring

Hypertrophic scarring (HTS) is an abnormal process of wound healing that results in excessive scar tissue formation. Over the past decade, we have demonstrated that mechanotransduction&#8212;the conversion of mechanical stimuli into cellular responses&#8212;drives excessive fibrotic scar healing. A mouse model to assess human-like hypertrophic scarring would be an essential tool for examining various therapeutics and their ability to reduce scarring and improve healing. Specifically, our laboratory has developed a murine wound model that increases mechanical strain to promote human-like HTS. This&#160;protocol utilizes biomechanical loading devices, made from modified 13 mm palatal expanders, whose arms are placed on either side of the incision and distracted incrementally apart in order to apply continuous tension across the wound bed during healing. Over nearly two decades of use, this model has been significantly advanced to improve efficacy and reproducibility. Using the murine HTS model, significant dermal fibrotic scars can be induced to be histologically comparable to human hypertrophic scars. This murine model provides an environment to develop biologics involved in the treatment of HTS and mechanotransduction-related conditions such as foreign body response.

This protocol will explain how to establish a hypertrophic scarring murine model that increases mechanotransduction signaling to simulate human-like scarring. This method involves increasing mechanical tension across a healing incision in a mouse and using a specialized device to create reproducible, excessive scar tissue for detailed histological and bioinformatic analyses.

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

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