This work was supported by the Biomedical Research Council, Singapore through core support to the Institute of Medical Biology and grant SPF2013/005. M.R. was supported by a NUS Faculty Research Committee Grant (Engineering in Medicine) (M.R.) R-397-000-081-112, and the NUS Tissue Engineering Program (NUSTEP). The authors would like to thank Professor Irene Leigh for providing the collagen VII antibody. Electron microscopy work was carried out at the National University of Singapore Electron Microscopy Unit.

1. Macromolecular Crowding in 2D Skin Cell Cultures
<ol>
	<li>Seed 50,000 cells (primary fibroblasts or primary keratinocytes or co-culture of primary fibroblasts and keratinocytes) per well of a 24-well plate. Seed cells in 1 ml of the corresponding cell type growth media.</li>
	<li>Allow cells to adhere overnight, in a 37 &#176;C incubator at 5% CO2.</li>
	<li>Discard old media and replace with 1 ml fresh media containing macromolecular crowders and 100 &#181;M ascorbic acid. Use a crowder cocktail consisting of 37.5 mg/ml Ficoll 70 and 25 mg/ml Ficoll 400. Use fibroblast media (FM) consisting of DMEM supplemented with 10% FBS and 1% penicillin-streptomycin antibiotics. Grow keratinocytes in a serum-free media, KSFM or CnT-57. Maintain co-cultures in a mixture of 50% FM and 50% serum-free media.</li>
	<li>Incubate cultures for 6 days in a 37 &#176;C incubator at 5% CO2, with a media change every 2 days.</li>
</ol>
2. Fluorescent Immunostaining on Skin Cell Cultures
<ol>
	<li>Wash cell cultures twice with 1x PBS.</li>
	<li>Fix cell cultures with either methanol or paraformaldehyde (depending on the antibody specifications).
	<ol>
		<li>For Methanol Fixation
		<ol>
			<li>Aliquot 500 &#181;l of cold methanol into each well and incubate at -20 &#176;C for 10 min. Aspirate fixative and discard methanol waste. Air dry in a laminar fume hood.</li>
		</ol>
		</li>
		<li>For Paraformaldehyde Fixation
		<ol>
			<li>Aliquot 500 &#181;l of 2% paraformaldehyde solution into each well and incubate at room temperature for 15 min. Aspirate fixative and discard paraformaldehyde waste.</li>
			<li>Wash with 1x PBS, for three washes, 5 min per wash.</li>
			<li>Aliquot 1 ml of 1x PBS into each well.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Fluorescent Immunostaining Using Antibodies
	<ol>
		<li>Aliquot 500 &#181;l of 3% bovine serum albumin (diluted in 1x PBS) into each well. Incubate at room temperature for 30 min. Aspirate the blocking solution and discard.</li>
		<li>Primary Antibody
		<ol>
			<li>For imaging directly from plate: Aliquot 200 &#181;l of primary antibody (1:100 dilution in 10% goat serum) solution into each well and incubate for 90 min at room temperature.</li>
			<li>For coverslips: Aliquot 50 &#181;l of primary antibody (1:100 dilution in 10% goat serum) solution onto a flat piece of Parafilm. Place coverslip onto the Parafilm, with the cell-side (part of coverslip with cells) facing the solution. Cover the entire cell-side, with no bubbles. Incubate for 90 min at room temperature.</li>
		</ol>
		</li>
		<li>Aspirate primary antibody solution and discard.</li>
		<li>Wash with 1x PBS, for three washes, 5 min per wash.</li>
		<li>Secondary Antibody
		<ol>
			<li>For imaging directly from plate: Aliquot 200 &#181;l of secondary antibody (1:400 dilution in 10% goat serum) solution into each well and incubate for 30 min at room temperature, covering the plate with aluminum foil.</li>
			<li>For coverslips: Aliquot 50 &#181;l of secondary antibody (1:400 dilution in 10% goat serum) solution onto a flat piece of Parafilm. Place coverslip onto the Parafilm, with the cell-side (part of coverslip with cells) facing the solution. Cover the entire cell-side, with no bubbles. Incubate for 30 min at room temperature, covering the Parafilm with aluminum foil.</li>
		</ol>
		</li>
		<li>Aspirate secondary antibody solution and discard.</li>
		<li>Wash with 1x PBS, for three washes, 5 min per wash. Aliquot 1 ml of 1x PBS into each well.</li>
		<li>Mounting
		<ol>
			<li>For imaging directly from plate: do not mount. Proceed to microscope.</li>
			<li>For imaging of coverslips: Mount coverslips, in mounting media, onto a glass slide. Dry overnight in a container covered with aluminum foil. Proceed to microscope.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Acquire Images Using a Confocal Laser Scanning Microscope with a 40X/1.30 Oil Objective.</li>
</ol>
3. Western Blotting of Skin Cell Cultures
<ol>
	<li>Wash cell layers twice with 1x PBS.</li>
	<li>Aliquot 100 &#181;l of lysis buffer (see Materials List) into each well (6-well plate format). Scrape the cell layer with a cell scraper and transfer solution into a microcentrifuge tube.</li>
	<li>Centrifuge solution at 15,330 x g for 30 min at 4 &#176;C. Transfer the supernatant to another microcentrifuge tube and discard the pellet.</li>
	<li>Mix 13 &#181;l of each sample with 5 &#181;l of sample buffer and 2 &#181;l of reducing agent (see Materials List). Heat samples at 95 &#176;C for 10 min.</li>
	<li>Centrifuge samples at 15,330 x g for 1 min to collect condensation water. Load samples into a pre-cast PAGE gel and run at 100 V for approximately 1 hr.</li>
	<li>To transfer separated proteins to a nitrocellulose membrane, sandwich the gel and membrane between pieces of paper and sponges (of the same size). Ensure there are no bubbles between the gel, membrane, papers and sponges. Close the sandwich cassette and run the transfer at 70 V for at 2 hr.</li>
	<li>To check if the transfer was complete, incubate the membrane with 10 ml Ponceau S. Wash membrane with 0.1% PBS-Tween-20, three times, 10 min per wash.</li>
	<li>Block with 10 ml of 5% milk for 1 hr at room temperature. Wash membrane with 0.1% PBS-Tween-20, three times, 10 min per wash.</li>
	<li>Incubate with 10 ml primary antibody for 90 min (mouse anti-collagen VII, LH 7.2, 1:1,000 dilution in 5% milk). Wash membrane with 0.1% PBS-Tween-20, three times, 10 min per wash.</li>
	<li>Incubate with 10 ml secondary antibody for 30 min (goat anti-mouse-HRP, 1:2,000 dilution in 5% milk). Wash membrane with 0.1% PBS-Tween-20, three times, 10 min per wash.</li>
	<li>Transfer the membrane to a cassette and add 1 ml of ECL Detection Reagent. Place a clear, thin plastic sheet over the membrane.</li>
	<li>To detect chemiluminescence, place a light-sensitive film over the plastic sheet and close the cassette. After 2-5 min, remove the film from the cassette and place it into a developer.</li>
</ol>
4. Making and Using a Cell-derived Matrix
<ol>
	<li>Seed 50,000 cells (primary fibroblasts or primary keratinocytes or co-culture of primary fibroblasts and keratinocytes) per well of a 24-well plate.</li>
	<li>Allow cells to adhere overnight, in a 37 &#176;C incubator at 5% CO2.</li>
	<li>Discard old media and replace with fresh media containing macromolecular crowders and 100 &#181;M ascorbic acid. The crowder cocktail consists of 37.5 mg/ml Ficoll 70 and 25 mg/ml Ficoll 400. Fibroblast media (FM) consists of DMEM supplemented with 10% FBS and 1% pen-strep. Keratinocytes are grown in a serum-free media, KSFM or CnT-57. Maintain co-cultures in a mixture of 50% FM and 50% KSFM or CnT-57.</li>
	<li>Incubate cultures for 6 days in a 37 &#176;C incubator at 5% CO2, changing media every 2 days.</li>
	<li>Decellularize cell layers to obtain a cell-derived matrix (f-mat = fibroblast-derived matrix, k-mat = keratinocyte-derived matrix, co-mat = matrix derived from a co-culture of fibroblasts and keratinocytes).
	<ol>
		<li>Wash cell layers twice in 1x PBS.</li>
		<li>Add 250 &#181;l of 0.5% sodium deoxycholate. Incubate, on ice, for 10 min. Aspirate cell lysate.</li>
		<li>Repeat Step 4.5.2 thrice.</li>
		<li>Wash cell-derived matrix twice in distilled H2O. Keep cell-derived matrix in 1x PBS. Matrices can be stored for up to 1 week at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare Secondary Cell Suspension (For Example, Seeding Keratinocytes on Top of f-mat).
	<ol>
		<li>Aspirate 1x PBS.</li>
		<li>Seed 50,000 keratinocytes on top of the cell-derived matrix (f-mat). Allow cells to adhere overnight, in a 37 &#176;C incubator at 5% CO2. 
		Note: Assays may be performed directly on the adherent cells.</li>
	</ol>
	</li>
</ol>
5. Characterization of the Cell-derived Matrix
<ol>
	<li>Fluorescent immunostaining: Refer to Protocol 2.</li>
	<li>Interference Reflection Microscopy
	<ol>
		<li>Seed cells on to glass coverslips, following Protocol 4.1-4.5.</li>
		<li>After obtaining the cell-derived matrix, fix samples following Protocol 2.2. 
		Note: Antibody staining is optional. If required, refer to Protocol 2.3.</li>
		<li>Perform image acquisition by using a confocal laser scanning microscope with a 40X/1.30 oil objective. Set the pinhole at 74 mm.</li>
		<li>Use LP 505 for the interference re&#64258;ection microscopy (IRM) channel and BP 575-615 IR for the &#64258;uorescence red channel for filters. Use NT 80/20 for the IRM channel and HFT 405/488/561, NFT 565, plate for the &#64258;uorescence red channel for the beam splitters. Use the following lasers: DPSS 561-10 (wavelength 561 nm) at 1.1% power for the &#64258;uorescence red channel and HeNe633 (wavelength 633 nm) at 5.0% power for the IRM channel.</li>
	</ol>
	</li>
</ol>
6. 3D Organotypic Skin Co-culture
<ol>
	<li>Cast a Collagen Gel, with Fibroblasts Encapsulated, in a Cell-culture Insert (6-well plate format).
	<ol>
		<li>Aliquot 10 ml of rat tail collagen type I to a cold beaker.</li>
		<li>Add 1 ml of cold DMEM. Add 0.5 ml of 1 M NaOH to neutralize the acidic collagen. Add 0.5 ml of fibroblast suspension (containing 1,200,000 fibroblasts).</li>
		<li>Transfer the solution, in a cold pipette, to the cell-culture inserts within a 6-well plate. 12 ml of total solution is evenly divided into 6 cell-culture inserts.</li>
		<li>Incubate the gel in a 37 &#176;C incubator at 5% CO2, for 1 hr.</li>
		<li>After the gel has solidified, submerge the gel in FM. After 24 hr, discard the old media and replace with fresh media, containing macromolecular crowders. Add a volume of 2 ml to the interior of the cell-culture insert and 2 ml to the outside of the cell-culture insert.</li>
	</ol>
	</li>
	<li>After 24 hr, Seed Keratinocytes on top of the Collagen Gel.
	<ol>
		<li>Trypsinize keratinocytes using 0.125% trypsin/chelating agent for 5 min at 37 &#176;C. Tap sides of flask gently to dislodge cells and neutralize trypsin with serum containing media. Count cells and prepare keratinocyte suspension (300,000 keratinocytes per gel and suspended in 200 &#181;l media).</li>
		<li>Aspirate old media from cell-culture insert. Add keratinocytes on top of the gel.</li>
		<li>Incubate the gel in a 37 &#176;C incubator at 5% CO2, for 1 hr.</li>
		<li>After keratinocytes have attached to the gel surface, submerge the gel with 2 ml KSFM or CnT-57 to the inside of the cell-culture insert and 2 ml of FM to the outside of the cell-culture insert.</li>
	</ol>
	</li>
	<li>After 24 hr, discard the old media and replace with fresh media, containing macromolecular crowders.</li>
	<li>After 7 days of submerged culture, raise the culture to an air-liquid interface.
	<ol>
		<li>Transfer the cell-culture insert to a deep-well plate. Add 10 ml stratification media to the outside of the cell-culture insert. Keep the interior of the cell-culture insert dry.</li>
	</ol>
	</li>
	<li>Incubate cultures in a 37 &#176;C incubator at 5% CO2 and change media every 3 days, for the next 14 days. 
	Note: After 2 weeks at the air-liquid interface, the skin equivalent is mature and can be harvested (either snap frozen or formalin fixed).</li>
</ol>
7. Harvest and Characterization of Organotypic Skin Equivalents
<ol>
	<li>Aspirate stratification media.</li>
	<li>Using tweezers, transfer the culture inserts onto a paper towel and dab away remaining media from the base of the insert.</li>
	<li>Using a scalpel, cut along the inner circumference of the culture insert.</li>
	<li>Fix the organotypic skin co-culture (together with the PET membrane).
	<ol>
		<li>Snap Freeze
		<ol>
			<li>Transfer the gel construct to a cryomold. Cover the interior of the cryomold with OCT compound. Place the cryomold into liquid nitrogen for 10 sec.</li>
			<li>Using tweezers, remove the frozen cryomold from the liquid nitrogen, wrap in aluminum foil and store the frozen samples in a -80 &#176;C freezer.</li>
			<li>Section frozen samples with a cryostat, with a section thickness of 7 &#181;m.</li>
		</ol>
		</li>
		<li>Formalin Fixation
		<ol>
			<li>Place a &#39;biopsy sponge pad&#39; into the &#39;biopsy cassette&#39;. Transfer the gel construct onto the sponge pad. Place another sponge pad gently on top of the gel construct. Close the cassette.</li>
			<li>Fully submerge the cassette in 10% neutral buffered formalin for 48 hr, at room temperature. Remove the cassette and discard formalin waste.</li>
			<li>Embed the fixed sample into a wax block by first dehydrating with an ethanol series and then gradually infiltrating the sample with wax. Section the wax block with a microtome.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Characterization of the Skin Equivalent
	<ol>
		<li>Immunostaining
		<ol>
			<li>For frozen Sections:
			<ol>
				<li>Incubate sections in 10% goat serum (diluted in 1x PBS) for 1 hr at room temperature. Wash sections in 1x PBS to remove the goat serum.</li>
				<li>Incubate sections with primary antibody (1:100 dilution in 10% goat serum) for 90 min at room temperature. Wash sections in 1x PBS, three times, 5 min per wash.</li>
				<li>Incubate sections with secondary antibody (1:400 dilution in 10% goat serum) for 30 min at room temperature, in a container covered with aluminum foil. Wash sections in 1x PBS, three times, 5 min per wash.</li>
				<li>Mount slides, with mounting media, onto a coverglass. Cover samples with aluminum foil and allow to dry overnight.</li>
				<li>Image using a confocal microscope with a 40X/1.30 oil objective.</li>
			</ol>
			</li>
			<li>For Formalin Fixed Paraffin Embedded Sections:
			<ol>
				<li>Dewax sections (and rehydrate) in descending percentages of ethanol to water (Xylene - 100% ethanol - 90% ethanol - 80% ethanol - 70% ethanol - water). Fully submerge samples in solutions. Each dewaxing step should be 3 min.</li>
				<li>Incubate sections in 1% hydrogen peroxide for 30 min. Wash sections in 1x PBS for 5 min.</li>
				<li>Incubate sections in citrate solution (10 ml citrate solution is dissolved in 90 ml water) at 120 &#176;C for 20 min. Allow slides to cool overnight, at room temperature. Wash sections in 1x PBS, three times, 5 min per wash.</li>
				<li>Incubate sections in 10% goat serum (diluted in 1x PBS) for 1 hr at room temperature. Wash sections in 1x PBS to remove the goat serum.</li>
				<li>Incubate sections with primary antibody (1:100 dilution in 10% goat serum) for 90 min at room temperature. Wash sections in 1x PBS, three times, 10 min per wash.</li>
				<li>Incubate sections with HRP-labeled polymer (undiluted) for 30 min, at room temperature. Wash sections with 1x PBS, three times, 10 min per wash.</li>
				<li>To a microcentrifuge tube, combine 1 ml DAB Substrate (3,3&#39;-diaminobenzidine) with 1 drop of chromogen. Incubate the sections with this solution for 15 sec - 5 min (brown color = positive signal).</li>
				<li>To stop the reaction, wash the sections in running water.</li>
				<li>Counterstain nuclei with hematoxylin, for 5 min. Rinse sections in running water.</li>
				<li>Incubate sections in acid alcohol solution, for 1 min. Rinse sections in running water.</li>
				<li>Incubate sections in Scott&#39;s tap water solution, for 2 min. Rinse sections in running water.</li>
				<li>Optional: Incubate sections in eosin for 1 min and wash in running water.</li>
				<li>Dehydrate sections in ascending percentages of ethanol to xylene (70% ethanol - 80% ethanol - 90% ethanol - 100% ethanol - xylene). Ensure that samples are fully submerged for 3 min per stage.</li>
				<li>Mount slides, using mounting media, to a coverglass. Air dry overnight.</li>
				<li>Image using a confocal microscope with a 40X/1.30 oil objective.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Transmission Electron Microscopy (To Visualize Anchoring Fibrils)
		<ol>
			<li>Fix samples in 2.5% glutaraldehyde for 72 hr.</li>
			<li>Wash samples in 1x PBS, three times, 10 min per wash.</li>
			<li>Cut into small pieces (1 mm3). Post-fix samples in 1% osmium tetroxide, pH 7.4, for 1 hr at room temperature. Wash samples in distilled water. 
			Caution: Osmium tetroxide is poisonous and toxic and should be handled carefully in a fume hood.</li>
			<li>Dehydrate samples through an ascending ethanol series (25% ethanol - 50% ethanol - 75% ethanol - 95% ethanol - 100% ethanol - acetone) for 20 min per step.</li>
			<li>Infiltrate by soaking samples in 100% acetone: araldite resin (1:1) for 30 min at room temperature and then at 1:3 for 1 hr, followed by 1:6 overnight. Soak sample in fresh araldite resin for 2 hr at room temperature, followed by a second change of fresh araldite resin for 1 hr at 40 &#176;C. Leave the third fresh araldite resin change for 1 hr at 45 &#176;C and leave the fourth fresh araldite resin change for 1 hr at 50 &#176;C.</li>
			<li>Incubate samples at 60 &#176;C for 24 hr to allow polymerization.</li>
			<li>Section samples using a glass knife, to obtain semi-thin sections with a thickness of 1 &#181;m. Stain section with methylene blue to observe histology. Section the sample to obtain ultra-thin sections of 70 nm and collect each section onto a copper grid.</li>
			<li>Stain sections with 5% uranyl acetate solution for 15 min followed by 3% lead citrate solution for 10 min.</li>
			<li>Acquire images with a transmission electron microscope (100 kV), at a magnification of 30,000X.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Breitkreutz, D., Mirancea, N., Nischt, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membranes+in+skin:+unique+structures+with+diverse+functions.">Basement membranes in skin: unique structures with diverse functions.</a> Histochem Cell Biol. 132 (1), 1-10 (2009).</li><li>Lane, E. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mutation+in+the+conserved+helix+termination+peptide+of+keratin+5+in+hereditary+skin+blistering.">mutation in the conserved helix termination peptide of keratin 5 in hereditary skin blistering.</a> Nature. 356 (6366), 244-246 (1992).</li><li>Fuchs, E., Raghavan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+under+the+skin+of+epidermal+morphogenesis.">Getting under the skin of epidermal morphogenesis.</a> Nat Rev Genet. 3 (3), 199-209 (2002).</li><li>Niessen, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+junctions/adherens+junctions:+basic+structure+and+function.">Tight junctions/adherens junctions: basic structure and function.</a> J Invest Dermatol. 127 (11), 2525-2532 (2007).</li><li>Jarvelainen, H., Sainio, A., Koulu, M., Wight, T. N., Penttinen, R. <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+molecules:+potential+targets+in+pharmacotherapy.">Extracellular matrix molecules: potential targets in pharmacotherapy.</a> Pharmacol Rev. 61 (2), 198-223 (2009).</li><li>Schaefer, L., Schaefer, 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=Proteoglycans:+from+structural+compounds+to+signaling+molecules.">Proteoglycans: from structural compounds to signaling molecules.</a> Cell Tissue Res. 339 (1), 237-246 (2010).</li><li>Frantz, C., Stewart, K. M., Weaver, V. 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+extracellular+matrix+at+a+glance.">The extracellular matrix at a glance.</a> J Cell Sci. 123 (Pt 24), 4195-4200 (2010).</li><li>Ghalbzouri, A., Jonkman, M., Dijkman, R., Ponec, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basement+membrane+reconstruction+in+human+skin+equivalents+is+regulated+by+fibroblasts+and/or+exogenously+activated+keratinocytes.">Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes.</a> J Invest Dermatol. 124 (1), 79-86 (2005).</li><li>Lareu, R. R., Arsianti, I., Harve, K. S., Peng, Y. X., 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> Tiss Eng. 13 (2), 385-391 (2007).</li><li>Lareu, R. 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=Collagen+matrix+deposition+is+dramatically+enhanced+in+vitro+when+crowded+with+charged+macromolecules:+the+biological+relevance+of+the+excluded+volume+effect.">Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect.</a> FEBS Lett. 581 (14), 2709-2714 (2007).</li><li>Lareu, R. R., Harve, K. S., 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=Emulating+a+crowded+intracellular+environment+in+vitro.+dramatically+improves+RT-PCR+performance.">Emulating a crowded intracellular environment in vitro. dramatically improves RT-PCR performance.</a> Biophys Biochem Res Comm. 363 (1), 171-177 (2007).</li><li>Harve, K. S., Ramakrishnan, V., Rajagopalan, R., 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=Macromolecular+crowding+in+vitro+as+means+of+emulating+cellular+interiors:+when+less+might+be+more.">Macromolecular crowding in vitro as means of emulating cellular interiors: when less might be more.</a> Proc Natl Acad USA Sci. 105 (51), E119 (2008).</li><li>Chen, 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=The+Scar-in-a-Jar:+Studying+antifibrotic+lead+compounds+from+the+epigenetic+to+extracellular+level+in+a+single+well.">The Scar-in-a-Jar: Studying antifibrotic lead compounds from the epigenetic to extracellular level in a single well.</a> Br J Pharmacol. 158 (5), 1196-1209 (2009).</li><li>Harve, K. S., Lareu, R., Rajagopalan, R., 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=Understanding+how+the+crowded+interior+of+cells+stabilizes+DNA/DNA+and+DNA/RNA+hybrids-in+silico+predictions+and+in+vitro+evidence.">Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids-in silico predictions and in vitro evidence.</a> Nucleic Acids Res. 38 (1), 172-181 (2009).</li><li>Satyam, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding+meets+tissue+engineering+by+self-assembly:+A+paradigm+shift+in+regenerative+medicine.">Macromolecular crowding meets tissue engineering by self-assembly: A paradigm shift in regenerative medicine.</a> Adv Mat. 26 (19), 3024-3034 (2014).</li><li>Chen, C. Z. 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> Adv Drug Deliv Rev. 63 (4-5), 277-290 (2011).</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> Tiss Eng C. , (2014).</li><li>Dewavrin, J. Y., Hamzavi, N., Shim, V. P. W., 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=Tuning+the+Architecture+of+3D+Collagen+Hydrogels+by+Physiological+Macromolecular+Crowding.">Tuning the Architecture of 3D Collagen Hydrogels by Physiological Macromolecular Crowding.</a> Acta Biomaterialia. 10 (10), 4351-4359 (2014).</li><li>Dewavrin, J. Y., 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=Synergistic+Rate+Boosting+of+Collagen+Fibrillogenesis+in+Heterogeneous+Mixtures+of+Crowding+Agents.">Synergistic Rate Boosting of Collagen Fibrillogenesis in Heterogeneous Mixtures of Crowding Agents.</a> J Phys Chem B. 119 (12), 4350-4358 (2015).</li><li>Auger, F. A., Berthod, F., Moulin, V., Pouliot, R., Germain, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineered+skin+substitutes:+from+in+vitro+constructs+to+in+vivo+applications.">Tissue engineered skin substitutes: from in vitro constructs to in vivo applications.</a> Biotechnol Appl Biochem. 39, 263 (2004).</li><li>Chen, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+crowding+effect+on+cartilaginous+matrix+production:+a+comparison+of+two-dimensional+and+three-dimensional+models.">Macromolecular crowding effect on cartilaginous matrix production: a comparison of two-dimensional and three-dimensional models.</a> Tiss Eng Part C Methods. 19 (8), 586-595 (2013).</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), e37904 (2012).</li><li>Kumar, P., 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=Macromolecularly+crowded+in+vitro+microenvironments+accelerate+the+production+of+extracellular+matrix-rich+supramolecular+assemblies.">Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies.</a> Scientific Reports. 5, 8729 (2015).</li><li>Ang, X. 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=Macromolecular+crowding+amplifies+adipogenesis+of+human+bone+marrow-derived+MSCs+by+enhancing+the+pro-adipogenic+microenvironment.">Macromolecular crowding amplifies adipogenesis of human bone marrow-derived MSCs by enhancing the pro-adipogenic microenvironment.</a> Tiss Eng Part A. 20 (5-6), 966-981 (2014).</li><li>Peng, Y. X., 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=Human+Fibroblast+Matrices+Bioassembled+Under+Macromolecular+Crowding+Support+Stable+Propagation+Of+Human+Embryonic+Stem+Cells.">Human Fibroblast Matrices Bioassembled Under Macromolecular Crowding Support Stable Propagation Of Human Embryonic Stem Cells.</a> J Tiss Eng Regen Med. 6 (10), e74-e84 (2012).</li><li>Benny, P., Badowski, C., Lane, E. B., 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=Making+More+Matrix:+Enhancing+the+deposition+of+dermal-epidermal+junction+components+in+vitro+and+accelerating+organotypic+skin+culture+development,+using+macromolecular+crowding.">Making More Matrix: Enhancing the deposition of dermal-epidermal junction components in vitro and accelerating organotypic skin culture development, using macromolecular crowding.</a> Tiss Eng A. 21 (1-2), 182-192 (2015).</li></ol>

The authors have no conflicting interests to disclose.

Enhanced extracellular matrix is obtained upon introduction of macromolecular crowders to cell culture, owing to the excluded volume effect which increases the propeptide cleavage by proteinases. This results in extracellular matrix, in particular collagen, to be processed faster and deposited on the culture surface. While other groups have obtained thick fibroblast cell layers, it involved a culture time of several weeks20. In contrast, MMC described in this Protocol, dramatically shortens the culture time wh...

The skin forms a protective barrier by preventing water loss and pathogen entry. It is made up of three major components; the stromal rich dermis1, a stratified epidermis2,3,4 on top of it, and the dermo-epidermal junction in between5,6. The dermis is composed largely of collagen and elastic fibers and is sparsely populated with fibroblasts7. In contrast, the cell-rich epidermis is composed of multiple layers of keratinocytes. The keratinocytes of the inner-most layer are proliferative and provide new basal cells which renew and replace terminally differentiating keratinocytes that constantly move to the outer-most layer of the skin and have lost their nuclei and cytoplasmic material, resulting in a cornified layer which undergoes desquamation.
The dermal-epidermal junction, a specific type of basement membrane, is a complex structure composed of interconnecting matrix molecules, which tethers the epidermis to the dermis. Collagen I fibers of the dermis interlace with collagen VII anchoring fibrils which are anchored to the collagen IV rich lamina densa. Anchoring filaments (laminin 5, collagen XVII and integrins) in turn connect the lamina densa with hemidesmosomes of basal keratinocytes. Basal keratinocytes (stratum basale) have the capacity to proliferate and renew, as well as differentiate and stratify to form the suprabasal layers; stratum spinosum, stratum granulosum, and finally the stratum corneum, which represents the contact surface of the skin with the environment. En route from the basal layer to the cornified layer, keratinocytes switch expression patterns of cytokeratins and finally undergo apoptosis and encase themselves in cornified envelopes, hulls of specific proteins that are covalently crosslinked by transglutaminase activity.
Recreating skin and its layers in vitro, including the complex structures of the dermal-epidermal junction and dermal extracellular matrix, and to emulate the cornification process, has long intrigued scientists and bioengineers as a challenging task. There have been significant advances in skin tissue engineering, for example, the successful extraction of skin cells from patient biopsies and the generation of skin organotypic cultures using patient-derived skin cells8. However, unsolved problems remain pertaining to poor secretion of extracellular matrix proteins by the skin cells themselves and resulting in sub-optimal skin models. Furthermore, the time required to generate 3D organotypic skin co-culture using current protocols varies between four to eight weeks, a timeframe which could potentially be shortened with the incorporation of macromolecular crowders. Reducing culture time saves reagent cost, reduces the incidence of cell senescence and reduces the waiting time of the patient should the product be used in the clinic.
Macromolecular crowding (MMC) involves introducing specific macromolecules to the culture medium to generate excluded volume effects. These affect enzymatic reaction rates including the proteolytic cleavage of procollagen which is tardy under standard aqueous culture conditions9-13. Under MMC, enzymatic reactions are hastened without increasing the amount of reagents14,15 resulting in, in the case of procollagen cleavage, an increased amount of collagen I molecules in crowded cultures as compared to uncrowded controls10. As the conversion of procollagen to collagen allows the formation of collagen assemblies, fibroblasts cultured with MMC for 48 hours yielded significantly more collagen I as compared to uncrowded fibroblast cultures monitored for up to four weeks11,16,17. Besides effects on enzymatic activities that affect the formation, stabilization and remodeling of ECM, MMC also has been shown to directly enhance and modulate collagen fiber formation18,19.
We present here a method to enhance the extracellular matrix (ECM) production by skin cells, in particular, dermal fibroblasts and epidermal keratinocytes. In addition, we show that the enriched ECM produced under MMC in monolayer cultures can be decellularized and used as pure cell-derived matrix (CDM).
We use a non-conventional approach to visualize and fully appreciate the ECM deposited by skin cells cultured with MMC. Interference reflection microscopy is typically used for studying cell-matrix interactions or cell-to-glass contact points. This technique was utilized in our system to view the total amount of matrix deposited on the glass surface. Interference reflection microscopy was coupled with fluorescent immunostaining to obtain the most amount of information in terms of extracellular matrix composition and pattern, in the presence and absence of MMC.
Organotypic skin co-cultures is a classical method to model the skin in vitro in a three-dimensional context. While two-dimensional co-cultures may provide significant information, it is limited when translating this data and applying it back to an in vivo environment, which is inherently a three-dimensional structure. Skin keratinocytes, in particular, are polarized and contain apical and basal segments which are essential for homeostasis and cell adherence. In addition, the expression of typical suprabasal proteins in keratinocytes above the basal layer, such as keratin 1, keratin 10 and filaggrin is only present in the course of stratification and terminal differentiation of keratinocytes. As terminal differentiation is hardly present in typical monolayer cultures, suprabasal protein expression is normally not achieved in this culture system. Therefore, organotypic cultures start submerged in culture medium, but are then lifted to an air-liquid interface to drive keratinocyte differentiation. This results in the expression of stratification markers, even cornification and a generally better reflection of epidermal physiology. While other groups have previously generated organotypic skin co-cultures successfully, the establishment of a functional dermo-epidermal junction zone has been an issue. Here, we present a new method for culturing organotypic skin co-cultures with an enhanced basement membrane, in a condensed time frame and without compromising the maturity of these constructs. This would provide skin mimetics for in vitro modeling, the study of skin biology and an assortment of screening assays.

<table border="0" cellpadding="0" cellspacing="0" width="1154">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ascorbic acid</td>
			<td style="width:181px;">Wako</td>
			<td style="width:175px;">013-12061</td>
			<td style="width:499px;">Cell culture media additive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture inserts&#160; (6-well format)</td>
			<td style="width:181px;">Greiner Bio-One</td>
			<td style="width:175px;">657610</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citrate solution</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">S2369</td>
			<td style="width:499px;">DakoCytomation Target Retrieval Solution Citrate, pH 6 (x10)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Collagen (rat tail)</td>
			<td style="width:181px;">Corning</td>
			<td style="width:175px;">354236</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">CnT-57&#160;</td>
			<td style="width:181px;">CELLnTEC</td>
			<td style="width:175px;">CnT-57</td>
			<td>Cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">DAB Substrate + chromogen kit</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">K3468</td>
			<td>Histology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Deep-well plate (6-well format)</td>
			<td style="width:181px;">Corning</td>
			<td style="width:175px;">355467</td>
			<td>Organotypic cultures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">ECL detection reagent</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">RPN2106</td>
			<td>Amersham ECL Western Blotting Detection Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Electron microscope (TEM)</td>
			<td>&#160;JEOL&#160;</td>
			<td>&#160;JEM-1010 (100kV)</td>
			<td>Transmission electron microscopy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Fibroblast media (FM)</td>
			<td></td>
			<td></td>
			<td>High Glucose-DMEM + 10% Fetal Bovine Serum + 1% Penicillin Streptomycin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Ficoll PM70</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">17-0310-05</td>
			<td>Macromolecular crowder</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Ficoll PM400</td>
			<td style="width:181px;">GE Healthcare Life Sciences</td>
			<td style="width:175px;">17-0300-05</td>
			<td>Macromolecular crowder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Keratinocyte serum-free media (KSFM)</td>
			<td style="width:181px;">Life Technologies</td>
			<td style="width:175px;">17005-042</td>
			<td>Cell culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Lysis buffer</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">89900</td>
			<td style="width:499px;">Lysis buffer for protein extraction. A protease inhibitor (Roche, #11836170001) was added to this lysis buffer.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;">LSM510</td>
			<td style="width:499px;">Red, green and blue channels to visualize AF594, AF488 and DAPI fluorescent immunostainings (40X mag).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mounting media (Hydromount)</td>
			<td style="width:181px;">National Diagnostics</td>
			<td style="width:175px;">HS-106</td>
			<td>Fluorescent staining</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Mounting media (Cytoseal)</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">8310-16</td>
			<td>Histology (HRP)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">OCT compound (Tissue Tek)</td>
			<td style="width:181px;">Sakura</td>
			<td style="width:175px;">4583</td>
			<td>Embedding for cryotomy</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Penicillin-streptomycin antibiotics</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:175px;">A5955</td>
			<td>Cell culture media additive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Primary antibodies</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Collagen I antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:175px;">#ab6308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Collagen Type IV</td>
			<td style="width:181px;">Novocastra</td>
			<td style="width:175px;">#NCL-COLL-IV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-collagen VII</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>LH7.2 (in house)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Anti-Fibronectin antibody</td>
			<td style="width:181px;">Abcam</td>
			<td style="width:175px;">#ab2413</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Reducing agent&#160;</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">NP0009</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sample buffer</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">NP0008</td>
			<td>Western blot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Secondary antibodies (Immunostaining)</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI)</td>
			<td style="width:181px;">Sigma Aldrich</td>
			<td style="width:175px;">#D9542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 594 goat-anti-rabbit</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-11037&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 594 goat-anti-mouse</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">&#160;#A-11005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 488 chicken-anti-rabbit</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-21441</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">AlexaFluor 488 goat-anti-mouse</td>
			<td style="width:181px;">ThermoFisher Scientific</td>
			<td style="width:175px;">#A-11001</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">Secondary antibodies (HRP)</td>
			<td style="width:181px;">Dako</td>
			<td style="width:175px;">Envision + System - HRP Labelled Polymer Anti-Rabbit and Anti-Mouse</td>
			<td>undiluted</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Sodium deoxycholate</td>
			<td style="width:181px;">Prodotti Chimicie Alimentari</td>
			<td style="width:175px;"></td>
			<td>Decellularization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Stratification media</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Dulbecco&#39;s Modified Eagle&#39;s Medium&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Ham&#8217;s F12&#160;&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">10% fetal bovine serum&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">100U/mL penicillin and 100U/mL streptomycin&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">0.4mg/mL hydrocortisone&#160;&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">5mg/mL insulin</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">1.8&#183;10-4 M adenine&#160;&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">5mg/mL transferrin&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">2&#183;10- 11 M triiodothyronine&#160;</td>
			<td style="width:181px;"></td>
			<td style="width:175px;"></td>
			<td>Used during the air-liquid interface of organotypic cultures. This media is added to the outside of the cell-culture insert.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Trypsin</td>
			<td style="width:181px;">Biopolis Shared Facilities</td>
			<td style="width:175px;">0.125% Trypsin/Versene&#160;&#160; pH 7.0 + 0.3&#160;</td>
			<td>Used to trypsinize cells</td>
		</tr>
	</tbody>
</table>

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.

Macromolecular crowding was able to enhance ECM deposition, in particular, fibroblasts deposited more collagen I, IV and fibronectin as compared to control cultures (Figure 1, Cell layer; 1A, collagen I; 1B, collagen IV; 1C, fibronectin). Upon decellularization, it was evident that fibroblasts were the main depositors of collagen I, IV and fibronectin as compared to keratinocytes (Figure 1, Matrix).
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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.

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 ...

improving-2d-and-3d-skin-in-vitro-models-using-macromolecular-crowding

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

12.4K Views.  National University of Singapore. 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.

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

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology 1. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes 2, 3. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice 4, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast 5. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes 6, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.
In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.

Visualizing protein samples by negative stain electron microscopy (EM) has become a popular structural analysis method. It is useful for quantitative structural analysis, such as calculating a 3D reconstruction of the molecules being studied, and also for qualitative examination of the quality of protein preparations. In this article we present detailed protocols for preparing the EM grids, staining the sample and visualizing the sample in an electron microscope. Novice users can follow these protocols easily and to utilize negative stain EM as a routine assay, in addition to other biochemical assays, for evaluating their protein samples.

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural ...

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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.

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 ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

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

Summary

Explore More Videos

Chapters in this video

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion