Name: In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding (Video) | JoVE Journal
Uploaded: 2020-05-01T15:00:00.000+00:00
Duration: 8 min 20 s
Description: Learn macromolecular crowding techniques to create collagen-rich in vitro models of human hypertrophic scarring using skin fibroblasts. This method mimics in vivo tissue environments for studying fibrotic disease mechanisms.

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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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<li>Linge, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hypertrophic+Scar+Cells+Fail+to+Undergo+a+Form+of+Apoptosis+Specific+to+Contractile+Collagen%5Bmdash%5DThe+Role+of+Tissue+Transglutaminase%5BTitle%5D)+AND+%22Journal+of+Investigative+Dermatology%22%5BJournal%5D)">Hypertrophic Scar Cells Fail to Undergo a Form of Apoptosis Specific to Contractile Collagen[mdash]The Role of Tissue Transglutaminase.</a> Journal of Investigative Dermatology. 125 (1), 72-82 (2005).</li>
<li>Penn, J. W., Grobbelaar, A. O., Rolfe, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+the+TGF-beta+family+in+wound+healing%2C+burns+and+scarring%3A+a+review%5BTitle%5D)+AND+%22International+Journal+of+Burns+and+Trauma%22%5BJournal%5D)">The role of the TGF-beta family in wound healing, burns and scarring: a review.</a> International Journal of Burns and Trauma. 2 (1), 18-28 (2012).</li>
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<li>Kuznetsova, I. M., Turoverov, K. K., Uversky, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((What+macromolecular+crowding+can+do+to+a+protein%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">What macromolecular crowding can do to a protein.</a> International Journal of Molecular Sciences. 15 (12), 23090-23140 (2014).</li>
<li>Chen, C., Loe, F., Blocki, A., Peng, Y., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Applying+macromolecular+crowding+to+enhance+extracellular+matrix+deposition+and+its+remodeling+in+vitro+for+tissue+engineering+and+cell-based+therapies%5BTitle%5D)+AND+%22Advanced+Drug+Delivery+Reviews%22%5BJournal%5D)">Applying macromolecular crowding to enhance extracellular matrix deposition and its remodeling in vitro for tissue engineering and cell-based therapies.</a> Advanced Drug Delivery Reviews. 63 (4-5), 277-290 (2011).</li>
<li>Zeiger, A. S., Loe, F. C., Li, R., Raghunath, M., Van Vliet, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macromolecular+Crowding+Directs+Extracellular+Matrix+Organization+and+Mesenchymal+Stem+Cell+Behavior%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Macromolecular Crowding Directs Extracellular Matrix Organization and Mesenchymal Stem Cell Behavior.</a> PloS one. 7 (5), 37904(2012).</li>
<li>Chen, C. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Scar-in-a-Jar%3A+studying+potential+antifibrotic+compounds+from+the+epigenetic+to+extracellular+level+in+a+single+well%5BTitle%5D)+AND+%22British+Journal+of+Pharmacology%22%5BJournal%5D)">The Scar-in-a-Jar: studying potential antifibrotic compounds from the epigenetic to extracellular level in a single well.</a> British Journal of Pharmacology. 158 (5), 1196-1209 (2009).</li>
<li>Fan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+%22macromolecular+crowding%22+in+vitro+to+investigate+the+naphthoquinones+shikonin%2C+naphthazarin+and+related+analogues+for+the+treatment+of+dermal+scars%5BTitle%5D)+AND+%22Chemico-Biological+Interactions%22%5BJournal%5D)">Application of "macromolecular crowding" in vitro to investigate the naphthoquinones shikonin, naphthazarin and related analogues for the treatment of dermal scars.</a> Chemico-Biological Interactions. 310, 108747(2019).</li>
<li>Canty, E. G., Kadler, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Procollagen+trafficking%2C+processing+and+fibrillogenesis%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">Procollagen trafficking, processing and fibrillogenesis.</a> Journal of Cell Science. 118, Pt 7 1341-1353 (2005).</li>
<li>Minton, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Models+for+Excluded+Volume+Interaction+between+an+Unfolded+Protein+and+Rigid+Macromolecular+Cosolutes%3A+Macromolecular+Crowding+and+Protein+Stability+Revisited%5BTitle%5D)+AND+%22Biophysical+Journal%22%5BJournal%5D)">Models for Excluded Volume Interaction between an Unfolded Protein and Rigid Macromolecular Cosolutes: Macromolecular Crowding and Protein Stability Revisited.</a> Biophysical Journal. 88 (2), 971-985 (2005).</li>
<li>Ernst, O., Zor, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Linearization+of+the+bradford+protein+assay%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Linearization of the bradford protein assay.</a> Journal of Visualized Experiments. (38), e1918(2010).</li>
<li>Beltrame, C. O., Cortes, M. F., Bandeira, P. T., Figueiredo, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+the+RNeasy+Mini+Kit+to+obtain+high-quality+total+RNA+from+sessile+cells+of+Staphylococcus+aureus%5BTitle%5D)+AND+%22Brazilian+Journal+of+Medical+and+Biological+Research%22%5BJournal%5D)">Optimization of the RNeasy Mini Kit to obtain high-quality total RNA from sessile cells of Staphylococcus aureus.</a> Brazilian Journal of Medical and Biological Research. 48 (12), 1071-1076 (2015).</li>
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<li>Rohani, M. G., Parks, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix+remodeling+by+MMPs+during+wound+repair%5BTitle%5D)+AND+%22Matrix+Biology%22%5BJournal%5D)">Matrix remodeling by MMPs during wound repair.</a> Matrix Biology. 44-46, 113-121 (2015).</li>
<li>Ulrich, D., Ulrich, F., Unglaub, F., Piatkowski, A., Pallua, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix+metalloproteinases+and+tissue+inhibitors+of+metalloproteinases+in+patients+with+different+types+of+scars+and+keloids%5BTitle%5D)+AND+%22Journal+of+Plastic%2C+Reconstructive+%26+Aesthetic+Surgery%22%5BJournal%5D)">Matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with different types of scars and keloids.</a> Journal of Plastic, Reconstructive & Aesthetic Surgery. 63 (6), 1015-1021 (2010).</li>
<li>Ghazizadeh, M., Tosa, M., Shimizu, H., Hyakusoku, H., Kawanami, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+Implications+of+the+IL-6+Signaling+Pathway+in+Keloid+Pathogenesis%5BTitle%5D)+AND+%22Journal+of+Investigative+Dermatology%22%5BJournal%5D)">Functional Implications of the IL-6 Signaling Pathway in Keloid Pathogenesis.</a> Journal of Investigative Dermatology. 127 (1), 98-105 (2007).</li>
<li>Wilgus, T. A., Ferreira, A. M., Oberyszyn, T. M., Bergdall, V. K., Dipietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regulation+of+scar+formation+by+vascular+endothelial+growth+factor%5BTitle%5D)+AND+%22Laboratory+Investigation%22%5BJournal%5D)">Regulation of scar formation by vascular endothelial growth factor.</a> Laboratory Investigation. 88 (6), 579-590 (2008).</li>
<li>Rashid, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+use+for+polyvinylpyrrolidone+as+a+macromolecular+crowder+for+enhanced+extracellular+matrix+deposition+and+cell+proliferation%5BTitle%5D)+AND+%22Tissue+Engineering+Part+C%3A+Methods%22%5BJournal%5D)">Novel use for polyvinylpyrrolidone as a macromolecular crowder for enhanced extracellular matrix deposition and cell proliferation.</a> Tissue Engineering Part C: Methods. 20 (12), 994-1002 (2014).</li>
<li>Lago, J. C., Puzzi, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+effect+of+aging+in+primary+human+dermal+fibroblasts%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">The effect of aging in primary human dermal fibroblasts.</a> PloS one. 14 (7), 0219165(2019).</li>
<li>Cigognini, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Macromolecular+crowding+meets+oxygen+tension+in+human+mesenchymal+stem+cell+culture+-+A+step+closer+to+physiologically+relevant+in+vitro+organogenesis%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Macromolecular crowding meets oxygen tension in human mesenchymal stem cell culture - A step closer to physiologically relevant in vitro organogenesis.</a> Scientific Reports. 6, 30746(2016).</li>
<li>Cuttle, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+porcine+deep+dermal+partial+thickness+burn+model+with+hypertrophic+scarring%5BTitle%5D)+AND+%22Burns%22%5BJournal%5D)">A porcine deep dermal partial thickness burn model with hypertrophic scarring.</a> Burns. 32 (7), 806-820 (2006).</li>
<li>Fan, C., Xie, Y., Dong, Y., Su, Y., Upton, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Investigating+the+potential+of+Shikonin+as+a+novel+hypertrophic+scar+treatment%5BTitle%5D)+AND+%22Journal+of+Biomedical+Science%22%5BJournal%5D)">Investigating the potential of Shikonin as a novel hypertrophic scar treatment.</a> Journal of Biomedical Science. 22, 70(2015).</li>
<li>Fan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shikonin+reduces+TGF-beta1-induced+collagen+production+and+contraction+in+hypertrophic+scar-derived+human+skin+fibroblasts%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Medicine%22%5BJournal%5D)">Shikonin reduces TGF-beta1-induced collagen production and contraction in hypertrophic scar-derived human skin fibroblasts.</a> International Journal of Molecular Medicine. 36 (4), 985-991 (2015).</li>
<li>Werner, S., Krieg, T., Smola, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Keratinocyte-Fibroblast+Interactions+in+Wound+Healing%5BTitle%5D)+AND+%22Journal+of+Investigative+Dermatology%22%5BJournal%5D)">Keratinocyte-Fibroblast Interactions in Wound Healing.</a> Journal of Investigative Dermatology. 127 (5), 998-1008 (2007).</li>
</ol>

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

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's post-hoc test were applied to analyze statistical differences (*p ˂ 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 Fig...

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 ...

in-vitro-model-human-cutaneous-hypertrophic-scarring-using

Research

JoVE Journal

Bioengineering

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Learn macromolecular crowding techniques to create collagen-rich in vitro models of human hypertrophic scarring using skin fibroblasts. This method mimics in vivo tissue environments for studying fibrotic disease mechanisms.

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

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