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 hN...

<ol>
	<li>vander Veer, W. M., 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=Potential+cellular+and+molecular+causes+of+hypertrophic+scar+formation.">Potential cellular and molecular causes of hypertrophic scar formation.</a> Burns. 35 (1), 15-29 (2009).</li><li>Linge, C., 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=Hypertrophic+Scar+Cells+Fail+to+Undergo+a+Form+of+Apoptosis+Specific+to+Contractile+Collagen[mdash]The+Role+of+Tissue+Transglutaminase.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+TGF-beta+family+in+wound+healing,+burns+and+scarring:+a+review.">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><li>Wang, X. Q., Kravchuk, O., Winterford, C., Kimble, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+correlation+of+in+vivo+burn+scar+contraction+with+the+level+of+alpha-smooth+muscle+actin+expression.">The correlation of in vivo burn scar contraction with the level of alpha-smooth muscle actin expression.</a> Burns. 37 (8), 1367-1377 (2011).</li><li>Eitan, E., Zhang, S., Witwer, K. W., Mattson, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicle-depleted+fetal+bovine+and+human+sera+have+reduced+capacity+to+support+cell+growth.">Extracellular vesicle-depleted fetal bovine and human sera have reduced capacity to support cell growth.</a> Journal of Extracellular Vesicles. 4, 26373 (2015).</li><li>Pampaloni, F., Reynaud, E. G., Stelzer, E. H. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+dimension+bridges+the+gap+between+cell+culture+and+live+tissue.">The third dimension bridges the gap between cell culture and live tissue.</a> Nature Reviews Molecular Cell Biology. 8 (10), 839-845 (2007).</li><li>Kapalczynska, M., 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=2D+and+3D+cell+cultures+-+a+comparison+of+different+types+of+cancer+cell+cultures.">2D and 3D cell cultures - a comparison of different types of cancer cell cultures.</a> Archives of Medical Science. 14 (4), 910-919 (2018).</li><li>Lareu, R. R., Arsianti, I., Subramhanya, H. K., Yanxian, P., Raghunath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+enhancement+of+collagen+matrix+formation+and+crosslinking+for+applications+in+tissue+engineering:+a+preliminary+study.">In vitro enhancement of collagen matrix formation and crosslinking for applications in tissue engineering: a preliminary study.</a> Tissue Engineering. 13 (2), 385-391 (2007).</li><li>Kuznetsova, I. M., Turoverov, K. K., Uversky, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+macromolecular+crowding+can+do+to+a+protein.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applying+macromolecular+crowding+to+enhance+extracellular+matrix+deposition+and+its+remodeling+in+vitro+for+tissue+engineering+and+cell-based+therapies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+Crowding+Directs+Extracellular+Matrix+Organization+and+Mesenchymal+Stem+Cell+Behavior.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Scar-in-a-Jar:+studying+potential+antifibrotic+compounds+from+the+epigenetic+to+extracellular+level+in+a+single+well.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+"macromolecular+crowding"+in+vitro+to+investigate+the+naphthoquinones+shikonin,+naphthazarin+and+related+analogues+for+the+treatment+of+dermal+scars.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procollagen+trafficking,+processing+and+fibrillogenesis.">Procollagen trafficking, processing and fibrillogenesis.</a> Journal of Cell Science. 118, 1341-1353 (2005).</li><li>Minton, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+for+Excluded+Volume+Interaction+between+an+Unfolded+Protein+and+Rigid+Macromolecular+Cosolutes:+Macromolecular+Crowding+and+Protein+Stability+Revisited.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linearization+of+the+bradford+protein+assay.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+RNeasy+Mini+Kit+to+obtain+high-quality+total+RNA+from+sessile+cells+of+Staphylococcus+aureus.">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><li>Xue, M., Jackson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Matrix+Reorganization+During+Wound+Healing+and+Its+Impact+on+Abnormal+Scarring.">Extracellular Matrix Reorganization During Wound Healing and Its Impact on Abnormal Scarring.</a> Advances in wound care (New Rochelle). 4 (3), 119-136 (2015).</li><li>Rohani, M. G., Parks, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+remodeling+by+MMPs+during+wound+repair.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases+and+tissue+inhibitors+of+metalloproteinases+in+patients+with+different+types+of+scars+and+keloids.">Matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with different types of scars and keloids.</a> Journal of Plastic, Reconstructive &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Implications+of+the+IL-6+Signaling+Pathway+in+Keloid+Pathogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+scar+formation+by+vascular+endothelial+growth+factor.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+use+for+polyvinylpyrrolidone+as+a+macromolecular+crowder+for+enhanced+extracellular+matrix+deposition+and+cell+proliferation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+aging+in+primary+human+dermal+fibroblasts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding+meets+oxygen+tension+in+human+mesenchymal+stem+cell+culture+-+A+step+closer+to+physiologically+relevant+in+vitro+organogenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+porcine+deep+dermal+partial+thickness+burn+model+with+hypertrophic+scarring.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+potential+of+Shikonin+as+a+novel+hypertrophic+scar+treatment.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shikonin+reduces+TGF-beta1-induced+collagen+production+and+contraction+in+hypertrophic+scar-derived+human+skin+fibroblasts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte-Fibroblast+Interactions+in+Wound+Healing.">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 border="0" cellpadding="0" cellspacing="0" style="width:1337px;" width="1336">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">0.2 &#956;m filter</td>
			<td style="width:195px;">Sartorius</td>
			<td style="width:119px;">16534</td>
			<td style="width:537px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">2-Mercaptoethanol</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">M6250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">4&#8217;,6-diamidino-2-phenylindole (DAPI)</td>
			<td style="width:195px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">P36962</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Alexa Fluor 680</td>
			<td style="width:195px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A-21076</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Alexa Fluor 800</td>
			<td style="width:195px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">A-11371</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">alpha smooth muscle actin (&#945;SMA) primary antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:119px;">ab5694</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Applied Biosystems 7500 Fast Real-Time PCR System (thermal cycler )</td>
			<td style="width:195px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">4351106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Ascorbic acid</td>
			<td style="width:195px;">Wako</td>
			<td style="width:119px;">#013-12061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Bovine serum albumin</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">#A2153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Bradford protein assay</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:119px;">500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagen I primary antibody (for immunostaining)</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:119px;">6308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagen I primary antibody (for western blot)</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:119px;">ab21286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagen III primary antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:119px;">ab7778</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Collagen IV primary antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:119px;">ab6586</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Direct Red 80</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td>2610108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td style="width:195px;">Life Technologies</td>
			<td style="width:119px;">11996-065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Fetal calf serum (FCS)</td>
			<td style="width:195px;">Life Technologies</td>
			<td style="width:119px;">6000-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Ficoll 400</td>
			<td style="width:195px;">GE HealthCare</td>
			<td style="width:119px;">#17-0300-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Ficoll 70</td>
			<td style="width:195px;">GE HealthCare</td>
			<td style="width:119px;">#17-0310-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">GAPDH primary antibody</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8795</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Goat Anti-Rabbit secondary antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:119px;">ab97050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Human hypertrophic scar/normal fibroblasts (hHSF/hNSF)</td>
			<td style="width:195px;">Cell Research Corporation</td>
			<td style="width:119px;">106, 107, 108</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">iScript cDNA Synthesis Kit</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:119px;">#1708890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">MMP-1 primary antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:119px;">ab38929</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">MMP-13 primary antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:119px;">ab39012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">MMP-2 primary antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:119px;">ab37150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">MMP-9 primary antibody</td>
			<td style="width:195px;">Abcam</td>
			<td style="width:119px;">ab38898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">NanoDrop Microvolume Spectrophotometers</td>
			<td style="width:195px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Nitrocellulose membrane</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:119px;">10484060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td style="width:195px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">NP0321BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Odyssey blocking buffer</td>
			<td style="width:195px;">LI-COR Biosciences</td>
			<td style="width:119px;">927&#8211;40000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Odyssey Fc Imaging System</td>
			<td style="width:195px;">LI-COR Biosciences</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Olympus IX-81 HCS microscope (for immunostaining)</td>
			<td style="width:195px;">Olympus</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Penicillin/streptomycin solution (P/S)</td>
			<td style="width:195px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PrimePCR Assays</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td>Customized primers pre-coated in 96-well plates based on requirement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Protease inhibitor cocktail (PIC)</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">11697498001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PVP 360</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">#PVP360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">PVP 40</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">#PVP40</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">RIPA buffer</td>
			<td style="width:195px;">Merck</td>
			<td style="width:119px;">R0278</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">RNeasy Plus Mini Kit</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">#74134</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium vanadate</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">450022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Sodium vanadate</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">450243</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">SpectraMax M5 Multi-Mode microplate reader</td>
			<td style="width:195px;">Molecular Devices</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">SsoAdvanced universal SYBR green supermix</td>
			<td style="width:195px;">Bio-Rad</td>
			<td style="width:119px;">#172-5270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:487px;">Tween 20</td>
			<td style="width:195px;">Sigma-Aldrich</td>
			<td style="width:119px;">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 ...

We created an individual collagen-rich model to mimic the environment that cells experience in vivo using the macromolecular crowding technique. Skin is a crowded environment, unlike the liquid environment that we already use to grow cells in vitro. Compared to the traditional monolayer cell culture system, this model recreates the crowded molecular environment that cells experience in vivo, especially in scar tissues where abundant extracellular matrix displaces liquid water.Besides hypertrophic scars, this macromolecular crowding technique can be used for studies where the extracellular matrix is abundant, such as pulmonary fibrosis. Grow scar-derived and normal fibroblasts according to manuscript directions. Then seed them into 24 well plates at 50, 000 cells per well in one milliliter of medium.Incubate the plate overnight at 37 degrees Celsius and 5%carbon dioxide. Prepare macromolecular crowding or MMC media by mixing Ficoll 70, Ficoll 400 and ascorbic acid into 10%FCS DMEM. Incubate the media in a water bath to disperse the crowders into the solution.Then sterilize it with a 0.2 micrometer filter. Aspirate the spent media from the fibroblasts and replace it with freshly made MMC media. Incubate the cells at 37 degrees Celsius for six days, changing the MMC media every three days.Prepare a Sirius Red solution by dissolving 0.2 grams of Direct Red 80 powder in 200 milliliters of distilled deionized water with one milliliter of acetic acid. Aspirate the MMC media from the cells and add 300 microliters of Sirius Red solution to each well. Then return the cells to the incubator for 90 minutes.After the incubation, gently rinse the Sirius Red solution with tap water and allow the plate to air dry overnight. On the next day, extract the Sirius Red by adding 200 microliters of 0.1 molar sodium hydroxide into each well and shaking the plate for five to 10 minutes. Transfer 100 microliters of the extracted stain into a transparent 96 well plate and measure the absorbance at 620 nanometers with a micro plate reader.Wash each well with 200 microliters of PBS and fix the cells with methanol at four degrees Celsius for 10 minutes. Then add 3%BSA to the wells and incubate the plate for 30 minutes at room temperature. Aspirate the blocking solution and add 200 microliters of anti-collagen one antibody to each well.Incubate the plate for 90 minutes. Then aspirate the antibody And wash the wells three times with PBS for five minutes per wash. Add 200 microliters of Goat Anti-Rabbit-FITC Secondary Antibody and DAPI to each well.Cover the plate with aluminum foil and incubate it for 30 minutes at room temperature. Discard the secondary antibody and DAPI and repeat the washes with PBS. Then visualize the fluorescence directly under a microscope.After washing the cells twice with PBS, add 40 microliters of lysis buffer into each well and scrape the cell layer with a pipette tip. Then transfer the protein lysate into micro centrifuge tubes to measure the protein concentration. Load 10 micrograms of protein into each well of a four to 12%Bis-Tris protein gel and perform SDS-PAGE at 200 volts for 30 minutes.When finished, transfer the protein to a nitrocellulose membrane by running a western blot at 90 volts for 90 minutes, taking care to avoid bubble formation between the gel and the membrane. Block the membrane with 10 milliliters of blocking buffer. Then incubate it with primary antibodies at four degrees Celsius overnight.On the next day, wash the membrane five times with 0.1 percent TBS Tween 20 for five minutes per wash. Then incubate the membrane with a species-appropriate secondary antibody for one hour at room temperature. Repeat the washes and visualize fluorescence with an imaging system.After purifying the total RNA with a commercial RNA extraction kit, measure RNA concentration using a micro volume spectrophotometer. Then use a cDNA synthesis kit to perform a first strand DNA synthesis. Mix the cDNA with 10 microliters of SYBR Green Supermix and transfer the samples to a custom 96 well plate pre-coated with oligonucleotide primers.Adjust the total volume to 20 microliters per well with water and run RT-PCR according to manuscript directions. Cell density of hypertrophic scar-derived human skin fibroblasts significantly increased after culturing with Ficoll compared to using PVP or the control. Macromolecular crowding with Ficoll at 9%fractional volume occupancy significantly enhanced the deposition of collagen, including collagen one, compared to other formulations.This was further demonstrated with quantitative analysis. Scar-derived fibroblasts and normal dermal fibroblasts cultivated in MMC environments were found to regulate the expression of ECM species in addition to collagen. Notably, an elevated expression of matrix metalloproteinases or MMPs was found to accumulate in hypertrophic scar tissues compared to native tissues.It was observed that the expression of MMP2, nine and 13 were significantly up-regulated in both cell cultures cultivated in macromolecular crowding conditions. MMPs play an important role during wound healing and scar formation, regulating ECM assembly and remodeling. Synthesis of interleukin six and vascular endothelial growth factor was undetectable in a western blot.But RT-PCR analysis revealed that the expression of interleukin six was significantly up-regulated, while the expression of vascular endothelial growth factor was down-regulated in fibroblasts cultivated in MMC conditions. When attempting this protocol, it is very important to choose dermal fibroblasts that have very few CO pathogens. We also have to keep in mind that ascorbic acid is an essential ingredient in the MMC medium.We are particularly interested in further detecting the collagen deposition and alignment in this MMC model. We plan to use advanced microscopic techniques to compare this in vitro model with native scar tissues in vivo. We encourage others to use this macromolecular crowding technique to improve the in vitro authenticity of the ancillary modal tissues.Of course, macromolecular crowding can also be used to recalculate models of disease and pathology in vitro.

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JoVE Journal

Bioengineering

6.5K Görüntüleme.  Agency for Science, Technology and Research (A*STAR). Makromoleküler kalabalık tekniğini kullanarak hücrelerin in vivo deneyim çevresini taklit etmek için bireysel kollajen açısından zengin bir model oluşturduk. Cilt zaten in vitro hücreleri büyümek için kullandığımız sıvı ortamın aksine, kalabalık bir ortamdır. Geleneksel monolayer hücre kültür sistemi ile karşılaştırıldığında, bu model hücrelerin in vivo deneyim kalabalık moleküler ortamı yeniden oluşturur, özellikle bol hücre dışı matris sıvı su y...