We thank the Wistar Institute Animal Facility, Microscopy Facility, Histotechnology Facility and Research Supply Center. This study was funded in part by grants from the National Institutes of Health CA 076674, CA 098101, CA 025874 and CA 10815.

1. The Three-Dimensional Human Skin Reconstruct Model:
<ol>
 <li>Preparation of acellular layer: Mix the following reagents in a 50 mL tube on ice: 0.59 mL 10X EMEM, 50 &mu;l 200mM L-glutamine, 0.6 mL FBS, 120 &mu;l 7.5% Sodium bicarbonate and 4.6 mL bovine collagen I. Add 1 mL of the mixture into each insert of tissue culture trays. Incubate for 30 min at room temperature and allow the gel to solidify. Color of the mixture should be from straw-yellow to pink.</li>
 <li>Trypsinize human fibroblasts from culture flasks with 0.25% trypsin/EDTA, add DMEM containing 10% FBS to neutralize. Collect cells by centrifugation and resuspend 0.45 x 106 cells in 1.5 mL DMEM with 10% FBS. For dermal stem cells, collect 6600 dermal spheres (when dermal stem cells grow in stem cell medium, they form spheres in a similar manner to neurospheres) by tapping flask to detach the spheres, centrifugation.</li>
 <li>Preparation of cellular layer: Mix the following in a 50 mL tube on ice: 1.65 mL 10X EMEM, 150 &mu;l 200 mM L-glutamine, 1.85 mL FBS, 350 &mu;l 7.5% Sodium bicarbonate, 14 mL bovine collagen I and 1.5 mL fibroblasts suspension from step 2 (alternatively, use combination of 0.45 x 106 fibroblasts and 6600 dermal spheres in 1.5 mL of skin reconstruct medium I for dermal stem cell reconstructs), mix well. Add 3 mL of the mixture to each acellular layer-coated insert. Incubate for 45 min at 37 &deg;C in a 5 % CO2 tissue culture incubator. Color of the mixture should be from straw yellow to pink. After the gel solidifies, add DMEM containing 10% FBS (2 mL inside and 10 mL outside of insert in each well of skin reconstruct trays). Incubate for 4 days and make sure that the gel contracts.</li>
 <li>Aspirate medium from both inside and outside of each insert. Add washing medium (HBSS with 1% dialyzed FBS) 2 mL to the inside and 10 mL to the outside of insert in order to wash off regular serum. Incubate for 1 hour at 37 &deg;C.</li>
 <li>Preparation of skin reconstruct medium:
 <ol>
 <li>For normal melanocyte and dermal stem cell reconstructs: To make basic medium (500 mL), mix the following reagents: 490 mL keratinocyte serum-free medium, 1.8 mL bovine pituitary extract, 10 mL dialyzed fetal bovine serum, 500 &mu;l of 10 &mu;g/mL SCF, 562.5 &mu;l of 4 &mu;g/mL bFGF and 500 &mu;l of 264 &mu;g/mL ET-3. Medium I: Add 10 &mu;l EGF of 100 &mu;g/mL to 100 mL basic medium. Medium II: Add 2 &mu;l EGF to 100 mL basic medium. Medium III: Add 720 &mu;l CaCl2 of 1 M to 300 mL basic medium.</li>
 <li>For melanoma skin reconstruct: To make Medium I (100mL), mix the following reagents: 72.5 mL of DMEM, 24 mL of F12 (HAM's), 2 mL L-glutamine of 200 mM, 200 &mu;l hydrocortisone of 269 &mu;g/mL, 200 &mu;l ITES of 500X, 200 &mu;l O-phosphorylethanolamine of 0.05 M, 200 &mu;l adenine of 90 mM, 200 &mu;l Progesterone of 2 nM, 240 &mu;l CaCl2 of 1 M, 200 &mu;l Triiodothyronine of 10 nM and 100 &mu;l of chelexed newborn calf serum (dissolve 3 g of Chelex 100 in 100 mL of serum, stir 1.5 hours at 4 &deg;C, filter sterilize). To make Medium II (100mL), mix the following reagents: 72.5 mL of DMEM, 24 mL of F12 (HAM's), 2 mL L-glutamine of 200 mM, 200 &mu;l hydrocortisone of 269 &mu;g/mL, 200 &mu;l ITES of 500X, 200 &mu;l O-phosphorylethanolamine of 0.05 M, 200 &mu;l adenine of 90 mM, 200 &mu;l Progesterone of 2 nM, 240 &mu;l CaCl2 of 1 M, 200 &mu;l Triiodothyronine of 10 nM and 100 &mu;l newborn calf serum. To make Medium III (300mL), mix the following reagents: 142.5 mL of DMEM, 142.5 mL of F12 (HAM's), 6 mL of L-glutamine of 200 mM, 600 &mu;l hydrocortisone of 269 &mu;g/mL, 600 &mu;l ITES of 500X, 600 &mu;l O-phosphorylethanolamine of 0.05 M, 600 &mu;l adenine of 90 mM, 600 &mu;l Progesterone of 2 nM, 720 &mu;l CaCl2 of 1 M, 600 &mu;l Triiodothyronine of 10 nM and 6 mL newborn calf serum.</li>
 </ol>
 </li>
 <li>Trypsinize human keratinocytes from culture flasks with 0.05% trypsin/EDTA, neutralize trypsin with soy bean trypsin inhibitor (250 mg/L in 1x phosphate buffered saline), spin down, resuspend a cell pellet at 4.17 x106 cells/mL in skin reconstruct medium I.</li>
 <li>Trypsinize human melanocytes or melanoma cells from culture flasks with 0.05% trypsin/EDTA, neutralize trypsin with soy bean trypsin inhibitor, spin down, resuspend a cell pellet at 0.83 x106 cells/mL in skin reconstruct medium I.</li>
 <li>Remove washing medium from both inside and outside of each insert.</li>
 <li>Add skin reconstruct medium I (1.5 mL to the inside and 10 mL to the outside of each insert).</li>
 <li>Take 600 &mu;l cell suspension of keratinocytes and 600 &mu;l cell suspension of melanocytes or melanoma cells, mix well. Dispense 200 &mu;l mixed cell suspension drop by drop to the inside of each insert. For dermal stem cell reconstruct, use 100 &mu;l of keratinocyte suspension only. Incubate for 2 days at 37 &deg;C.</li>
 <li>Aspirate skin reconstruct medium I from both the inside and the outside of each insert. Add skin reconstruct medium II (2 mL to the inside and 10 mL to the outside). Incubate for another 2 days at 37 &deg;C.</li>
 <li>Aspirate skin reconstruct medium II from the inside and the outside of each insert, add 7.5 mL skin reconstruct medium III to only the outside. From this point, the surface of reconstructs starts being exposed to the air. Change medium III every other day until day 18.</li>
 <li>Harvest skin reconstruct at day 18:
 <ol>
 <li>Aspirate media from the inside and the outside of inserts.</li>
 <li>Remove inserts from tray with forceps.</li>
 <li>Cut out the reconstruct (including the polycarbonate filter) by tracing a circle close to the edge with a scalpel blade.</li>
 <li>Cut the reconstruct and the filter in half on a hard surface.</li>
 </ol>
 </li>
 <li>For paraffin sections: place a half of the reconstruct in a histology cassette between 2 black TBS biopsy papers and soak the whole cassette in 10% formalin for more than 4 hours. Then place the cassette in 70% ethanol and store it at 4 &deg;C until you are ready to process the reconstruct for paraffin embedding.</li>
 <li>For frozen sections:
 <ol>
 <li>Place the other half of reconstruct in 50% sucrose at 4 &deg;C for 1-2 hours, then change the sucrose to 2M and store it at 4 &deg;C for another 1-2 hours.</li>
 <li>Dispense optimal cutting temperature freezing media (OCT) into a disposable base mold so that it fills about 50% of the boat. Avoid any bubbles in the OCT.</li>
 <li>Grab the edge of the reconstruct with forceps and remove the reconstruct from the sucrose. Place it on Kimwipes until the Kimwipes absorb the sucrose.</li>
 <li>Transfer the reconstruct into the base mold on top of the OCT, using forceps and a spatula. Cover the top of the reconstruct with more OCT until the base mold is completely full. Make sure you do not have any bubbles in the OCT which will make cutting difficult.</li>
 <li>Place the base mold evenly on crushed dry ice, and allow the OCT to freeze completely.</li>
 <li>Wrap the base mold in tin foil and store it at -70 &deg;C until you are ready to cut it with a cryostat.</li>
 </ol>
 </li>
 <li>Graft a skin reconstruct on a mouse:
 <ol>
 <li>Prepare a 50 mL falcon tube with 5 mL of DMEM (for freezing and defrosting mouse skin).</li>
 <li>Use a female SCID hairless outbred mouse around 6 weeks.</li>
 <li>The mouse is anesthetized with isoflurane (EZ anesthesia system): initial anesthesia is performed in the induction chamber with 4% isoflurane (before mouse is moved to the surgical bed) and maintain anesthesia through a nosecone breather during procedure (isoflurane: 1.5-2%). Heat support by a heated pad to prevent hypothermia and sterile ophthalmic lubricant applied to prevent ocular injury during anesthesia.</li>
 <li>Put 600 mL water in a beaker and leave on top of a hot plate to keep water temperature between 40 - 60 &deg;C.</li>
 <li>Clean the mouse skin with compound benzoin tincture and alcohol prep swabs.</li>
 <li>Mark a line on top of the mouse back to keep the same position for suturing later.</li>
 <li>Cut a round wound bed about 1.2 cm in diameter on the mouse upper back using Iris scissors and forceps. Cover the wound bed with sterile gauze sponges. Put the round mouse skin in 5 mL of DMEM, then leave in liquid nitrogen container until it totally frozen. Defrost the tube in hot water on top of a hot plate until totally thawed. Repeat 3 times.</li>
 <li>Detach the skin reconstruct from the transwell using surgical blade and remove the membrane very carefully, transfer the skin reconstruct (epidermis side up) on top of the wound bed.</li>
 <li>Place the mouse skin (epidermis side up) on top of the skin reconstruct.</li>
 <li>Make the first suture on the marker previously labeled, the second on the bottom, then two more sutures on sides to fix the mouse skin.</li>
 <li>Clean mouse skin after grafting. Remove nosecone and keep mouse on the heated pad until awake and ambulatory.</li>
 <li>Put the mouse into a new cage.</li>
 <li>Post operative analgesia: Meloxicam (1 mg/kg) by subcutaneous injection immediately after surgery, 24 hours and 48 hours after surgery.</li>
 </ol>
 </li>
</ol>
2. Representative Results:
Skin reconstructs with normal melanocytes and dermal stem cells 
The skin reconstructs are cultured in a special 6-well tray with inserts. The dermal compartment is cultured in DMEM with 10% FBS for the first four days (Fig. 1A). The skin reconstruct medium I is used when keratinocytes are seeded with melanocytes or melanoma cells (Fig. 1B). The epidermis is exposed to the air at day 9 and this allows keratinocytes to differentiate (Fig. 1C). At day 18, the epidermis of skin reconstructs is composed of stratified keratinocyte layers. The undifferentiated basal layer and sequentially differentiated layers are vertically oriented (Fig. 1D). Staining the section of reconstructs with the melanocytic marker S100 shows that melanocytes are aligned in the basal layer of the epidermis and communicate with multiple keratinocytes through dendrite extensions (Fig. 1E). The dermal compartment contains fibroblasts embedded in a collagen type I matrix. Deposited collagen IV indicates the basement membrane, which separates the epidermis from the dermis (Fig. 1F). When dermal stem cells (labeled with GFP lentiviral vector) are embedded with fibroblasts in a collagen I matrix, they migrate to the epidermis and differentiate into melanocytes (1), (Fig. 2). Multiple layers of keratinocytes in the epidermis are developed.
Skin reconstructs of melanoma tumors 
Clinicopathological studies indicate that melanomas progress in a stepwise manner: common acquired nevi, dysplastic nevi, RGP (radial growth phase) melanomas, VGP (vertical growth phase) melanomas and metastatic melanomas (7). Different stages of melanoma cell lines are morphologically similar to each other in 2-D culture (Fig. 3A-D) but when they are incorporated in skin reconstructs, the behavior of the cells reflects their in vivo characteristics. The location and the growth rate of normal melanocytes are tightly controlled in skin reconstructs (Fig. 3E). RGP primary melanomas WM35 proliferate predominantly in the epidermis (Fig. 3F), whereas VGP melanomas WM793 grow invasively into the dermis (Fig. 3G). Metastatic melanomas 1205Lu aggressively invade deep into the dermis (Fig. 3H).
<img alt="Figure 1" src="/files/ftp_upload/2937/2937fig1.jpg" /> 
Figure 1. Skin reconstructs with normal melanocytes. The gross appearance of the skin reconstructs is shown in A-C. A. Fibroblasts mixed with collagen are grown in DMEM containing 10% FBS and form dermal compartment. B. Keratinocytes and melanocytes are seeded on top of the dermis and grow in skin reconstruct medium. C. The epidermis is exposed to air at day 9. D. H&amp;E-stained skin reconstruct presents the epidermis comprised vertically, oriented basal layer, and sequentially differentiated stratified cell layers. The dermis contains fibroblasts embedded in a collagen type I matrix. E. S100-positive melanocytes (black arrows) are aligned at the basement membrane and communicate with multiple keratinocytes. F. Collagen IV-staining indicates the basement membrane, which separates the epidermis from the dermis. All the stainings in D-F were performed on formalin-fixed, paraffin-embedded sections.
<img alt="Figure 2" src="/files/ftp_upload/2937/2937fig2.jpg" /> 
Figure 2. Dermal stem cells in skin reconstructs migrate to the epidermis and differentiate into melanocytes. At day 5 after seeding keratinocytes, single GFP-positive cells (green) start migrating out from spheres. The epidermis is still composed of a single layer. At day 8, a few cells reach the epidermis-dermis interface. By day 10, GFP-positive cells are tightly aligned at the basement membrane position. The migrated GFP-positive cells in the epidermis express melanocytic marker HMB45 (red, as indicated by white arrows). Nuclei are stained with DAPI (blue). The epidermis is developed as multiple layers. The basement membrane is indicated with white dotted lines.
<img alt="Figure 3" src="/files/ftp_upload/2937/2937fig3.jpg" /> 
Figure 3. Skin reconstructs of different stages of melanomas: A-D. Normal melanocytes and melanoma cells grown in 2D cultures. A. Melanocytes from foreskin. B. RGP WM35 cells. C. VGP WM793 cells. D. Metastatic melanoma 1205Lu cells. E. Normal melanocytes are located at the basement membrane. F. RGP melanoma WM35 cells grow as cell clusters in the epidermis. G. VGP melanoma WM793 cells invade into the dermis through basement membrane. H. Metastatic melanoma 1205Lu cells aggressively invade deep into dermis.
 
Troubleshooting
<table border="1">
 <tbody>
 <tr>
 <td>Problem</td>
 <td>Troubleshooting</td>
 </tr>
 <tr>
 <td>Collagen mixture does not solidify</td>
 <td>Collagen mixture color should be straw-yellow to pink, otherwise pH is wrong and collagen may not gel. If the color is bright yellow, more sodium bicarbonate should be added drop by drop.</td>
 </tr>
 <tr>
 <td>Collagen prematurely precipitates in the mixuture</td>
 <td>Keep all components on ice until the collagen mixture is placed onto the insert</td>
 </tr>
 <tr>
 <td>Contracted collagen is not even (one side is thicker than the other side)</td>
 <td>Calibrate the shelf of incubator</td>
 </tr>
 <tr>
 <td>The epidermis is formed with less than three keratinocyte layers.</td>
 <td>Use undifferentiated keratinocytes at lower passages</td>
 </tr>
 </tbody>
</table>

<ol>
	<li>Sun, T., Jackson, S., Haycock, J. W., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+of+skin+cells+in+3D+rather+than+2D+improves+their+ability+to+survive+exposure+to+cytotoxic+agents.">Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents.</a> J Biotechnol. 122, 372-381 (2006).</li><li>Smalley, K. S., Lioni, M., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+isn't+flat:+taking+cancer+biology+to+the+next+dimension.">Life isn't flat: taking cancer biology to the next dimension.</a> In Vitro Cell Dev Biol Anim. 42, 242-247 (2006).</li><li>Sun, T., Norton, D., McKean, R. J., Haycock, J. W., Ryan, A. J., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+3D+cell+culture+system+for+investigating+cell+interactions+with+electrospun+fibers.">Development of a 3D cell culture system for investigating cell interactions with electrospun fibers.</a> Biotechnol Bioeng. 97, 1318-1328 (2007).</li><li>Kremer, M., Lang, E., Berger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypical+engineering+of+differentiated+composite-skin+equivalents+of+human+keratinocytes+in+a+collagen-GAG+matrix+(INTEGRA+Artificial+Skin)+in+a+perfusion+culture+system.">Organotypical engineering of differentiated composite-skin equivalents of human keratinocytes in a collagen-GAG matrix (INTEGRA Artificial Skin) in a perfusion culture system.</a> Langenbecks Arch Surg. 386, 357-363 (2001).</li><li>Esc&#225;mez, M. J., Garc&#237;, M., Larcher, F., Meana, A., Mu&#241;oz, E., Jorcano, J. L., Del R&#237;o, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+model+of+wound+healing+in+genetically+modified+skin-humanized+mice.">An in vivo model of wound healing in genetically modified skin-humanized mice.</a> J Invest Dermatol. 123, 1182-1191 (2004).</li><li>Li, L., Fukunaga-Kalabis, M., Yu, H., Xu, X., Kong, J., Lee, J. T., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+dermal+stem+cells+differentiate+into+functional+epidermal+melanocytes.">Human dermal stem cells differentiate into functional epidermal melanocytes.</a> J Cell Sci. 123, 853-860 (2010).</li><li>Meier, F., Nesbit, M., Hsu, M. Y., Martin, B., Van Belle, P., Elder, D. E., Schaumburg-Lever, G., Garbe, C., Walz, T. M., Donatien, P., Crombleholme, T. M., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanoma+progression+in+skin+reconstructs:+biological+significance+of+bFGF.">Human melanoma progression in skin reconstructs: biological significance of bFGF.</a> Am J Pathol. 156, 193-200 (2000).</li><li>Yu, H., McDaid, R., Lee, J., Li, L., Kumar, S. M., Elder, D. E., Van Belle, P., Gimotty, P., Guerra, M., Hammond, R., Nathanson, K. L., Dalla Palma, M., Herlyn, M., Xu, X. <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+BRAF+mutation+and+p53+inactivation+during+transformation+of+a+subpopulation+of+primary+human+melanocytes.">The role of BRAF mutation and p53 inactivation during transformation of a subpopulation of primary human melanocytes.</a> Am J Pathol. 174, 2367-2377 (2009).</li><li>Lee, J. T., Li, L., Brafford, P. A., van den Eijnden, M., Halloran, M. B., Sproesser, K., Haass, N. K., Smalley, K. S., Tsai, J., Bollag, G., Herlyn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PLX4032,+a+potent+inhibitor+of+the+B-Raf+V600E+oncogene,+selectively+inhibits+V600E-positive+melanomas.">PLX4032, a potent inhibitor of the B-Raf V600E oncogene, selectively inhibits V600E-positive melanomas.</a> Pigment Cell Melanoma Res. 23, 820-827 (2010).</li><li>Tsai, J., Lee, J. T., Wang, W., Zhang, J., Cho, H., Mamo, S., Bremer, R., Gillette, S., Kong, J., Haass, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+selective+inhibitor+of+oncogenic+B-Raf+kinase+with+potent+antimelanoma+activity.">Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity.</a> Proc Natl Acad Sci U S A. 105, 3041-3046 (2008).</li></ol>

No conflicts of interest declared.

We have described generating 3D skin reconstructs with normal human melanocytes, dermal stem cells and melanoma cells. When grown in monolayer culture, melanocytic cells present a similar morphology (flattened, spindle or more dendritic-shaped) regardless of their origin of clinical stage. In contrast, 3D skin reconstructs recapitulate stage-specific properties of melanoma cells. Normal human melanocytes reside at the basement membranes between the epidermal and the dermal layers as single cells. RGP primary melanoma cells grow as small clusters along the basement membrane whereas more aggressive VGP melanoma cells grow as large clusters breaking through the basement membrane into the dermis. Metastatic melanoma cells grow in all directions and invade deep into the dermis as single cells or clusters. Quantitative analyses can be performed on this model by measuring the depth of invasion and the extent of proliferation. In addition to characterization of normal and malignant melanocytic cells, using the model we can directly induce the differentiation of dermal stem cells into matured melanocytes, which home to the basal layer of epidermis and establish bona fide communication with keratinocytes through upregulation of E-cadherin (6). 
Using viral vectors, we are able to either activate or inactivate gene function for a better understanding of the dynamics and functional significance of genes expressed by each cell type in skin reconstructs, which promises to be an efficient model to study not only transformation mechanisms of melanocytes, but also progression of melanoma (8). Long-term observation of cell phenotypes has become possible through grafting of skin reconstructs to immunodeficient animals. Such a human skin-mouse chimera is an excellent research tool to study melanomagenesis and melanoma metastasis.
Skin reconstructs can also be a useful platform for drug assessment. Many drugs which eradicate cancer cells in 2D culture conditions often have little effect in experimental and clinical applications. As seen in the in vivo tumor, melanoma cells in 3D cultures are often resistant to the drugs which cells in 2D cultures respond to, suggesting that the microenvironment modulates signaling pathways in melanoma. For successful drug discovery, the 3D skin reconstruct model is an ideal preclinical tool to predict the effects of compounds in vivo (9,10). 
In summary, skin reconstruct models bridge the gap between in vitro and in vivo studies. They will lead to a better understanding of which genes are involved in transformation and how stem cells contribute to that transformation.

<table border="1">
	<tbody>
	
		<tr>
			<th>Name of the reagent</th>
			<th>Company</th>
			<th>Catalogue number</th>
		</tr>
		<tr>
			<td>10x EMEM</td>
			<td>Bio Whittaker</td>
			<td>12-684F</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Gibco</td>
			<td>25030-081</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH-30071.02</td>
		</tr>
		<tr>
			<td>Bovine Tendon Acid-Extracted Collagen</td>
			<td>Organogenesis</td>
			<td>200/50</td>
		</tr>
		<tr>
			<td>Tissue Culture 6-well Trays with Inserts</td>
			<td>Organogenesis</td>
			<td>9285</td>
		</tr>
		<tr>
			<td>Keratinocyte Serum Free Medium</td>
			<td>Gibco</td>
			<td>17005042</td>
		</tr>
		<tr>
			<td>Dialyzed Fetal Calf Serum</td>
			<td>Hyclone</td>
			<td>SH-30079.03</td>
		</tr>
		<tr>
			<td>recombinant Human Stem Cell Factor </td>
			<td>Fitzgerald Industries </td>
			<td>RDI-307-255X</td>
		</tr>
		<tr>
			<td>Basic FGF </td>
			<td>Fitzgerald Industries </td>
			<td>30R-AF015</td>
		</tr>
		<tr>
			<td>Endothelin-3, human</td>
			<td>American Peptide Co.</td>
			<td>88-5-10</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma</td>
			<td>C-7902</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>JRH biosciences</td>
			<td>56430-10L</td>
		</tr>
		<tr>
			<td>F12 (HAM's)</td>
			<td>Gibco</td>
			<td>11765-54</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma</td>
			<td>H-0888</td>
		</tr>
		<tr>
			<td>Insulin, Transferrin, Ethanolamine, Selenium (ITES)</td>
			<td>BioWhittaker</td>
			<td>17839Z</td>
		</tr>
		<tr>
			<td>O-Phosphorylethanolamine</td>
			<td>Sigma</td>
			<td>P0503</td>
		</tr>
		<tr>
			<td>Adenine</td>
			<td>Sigma</td>
			<td>A9795</td>
		</tr>
		<tr>
			<td>Progesterone</td>
			<td>Sigma</td>
			<td>P-8783</td>
		</tr>
		<tr>
			<td>Triiodothyronine</td>
			<td>Sigma</td>
			<td>T-5516</td>
		</tr>
		<tr>
			<td>Newborn calf serum</td>
			<td>Hyclone</td>
			<td>SH3011802</td>
		</tr>
		<tr>
			<td>10% buffer formalin </td>
			<td>Surgipath</td>
			<td>00600</td>
		</tr>
		<tr>
			<td>Optimal cutting temperature freezing media (OCT) </td>
			<td>Sakura</td>
			<td>4583</td>
		</tr>
		<tr>
			<td>Multi cassettes </td>
			<td>Surgipath </td>
			<td>02293-BX</td>
		</tr>
		<tr>
			<td>TBS biopsy papers </td>
			<td>Triangle Biomedical Sciences </td>
			<td>BP-B</td>
		</tr>
		<tr>
			<td>TBS biopsy papers </td>
			<td>Triangle Biomedical Sciences </td>
			<td>BP-B</td>
		</tr>
		<tr>
			<td>Disposable Base Molds</td>
			<td>Fisher</td>
			<td>15-182-501C</td>
		</tr>
		<tr>
			<td>Compound benzoin tincture</td>
			<td>Professional Disposables, Inc,</td>
			<td>S42450</td>
		</tr>
		<tr>
			<td>Alcohol Prep Swabs</td>
			<td>CVS pharmacy</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Silk Black Braided 5-0</td>
			<td>Ethicon</td>
			<td>682G</td>
		</tr>
		<tr>
			<td>Gauze sponges (sterile)</td>
			<td>CVS pharmacy</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz</td>
			<td>RS5070</td>
		</tr>
		<tr>
			<td>Iris scissors</td>
			<td>Roboz</td>
			<td>RS5913</td>
		</tr>
		<tr>
			<td>Surgical blades</td>
			<td>Feather</td>
			<td>2976</td>
		</tr>
		<tr>
			<td>EZ anesthesia</td>
			<td>Euthanex Corp.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SCID hairless outbred mouse</td>
			<td>Charles river</td>
			<td>&nbsp;</td>
		</tr>		
	</tbody>
</table>

the three-dimensional human skin reconstruct model: a tool to study normal  skin and melanoma progression

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

Watch this Scientific Journal Video about The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression at JoVE.com

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

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

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

the-three-dimensional-human-skin-reconstruct-model-tool-to-study

Research

JoVE Journal

Bioengineering

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

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

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

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

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

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

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

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

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

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

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

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 ...

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

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

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

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

Generation of Self-assembled Vascularized Human Skin Equivalents

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

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

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