We sincerely thank Dr. Javier Rodr&#237;guez, Dr. Mar&#237;a Luisa L&#243;pez, Carlos Matell&#225;n, and Juan Francisco Rodr&#237;guez for very helpful suggestions, discussions, and/or preliminary data. We also kindly thank the contributions of Sergio F&#233;rnandez, Pedro Herreros, and Lara Stolzenburg to this project. Special thanks go to Dr. Marta Garc&#237;a for GFP-labelled hFBs and hKCs. Finally, we recognize the excellent technical assistance of Guillermo Vizca&#237;no and Ang&#233;lica Corral. This work was supported by the &#34;Programa de Actividades de I+D entre Grupos de Investigaci&#243;n de la Comunidad de Madrid&#34;, Project S2018/BAA-4480, Biopieltec-CM. This work was also supported by the &#34;Programa de excelencia&#34;, Project EPUC3M03, CAM. CONSEJER&#205;A DE EDUCACI&#211;N E INVESTIGACI&#211;N.

1. Chip design and micromachining parameters
<ol>
	<li>Design the microfluidic chip layers with FreeCAD open-source design software; refer to Table 1 for the dimensions of the channels. Include four 2.54 mm diameter holes in the design to use a custom-made aligner for a correct layer superposition.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Length (&#956;m)</td>
			<td>Width (&#956;m)</td>
		</tr>
		<tr>
			<td>Lower chamber</td>
			<td>28,400</td>
			<td>800</td>
		</tr>
		<tr>
			<td>Upper chamber</td>
			<td>31,000</td>
			<td>800</td>
		</tr>
	</tbody>
</table>
Table 1: Dimensions of the upper and lower channels of the device.
<ol start="2">
	<li>Cut 95 &#181;m-thick, adhesive, transparent vinyl sheets into 30 cm x 30 cm squares to fit in the plotter properly.</li>
	<li>Use Brother CanvasWorkspace software to create a 30 cm x 30 cm workspace, and fill it with the designed patterns for the different layers of the chip (Figure 1A). Store it in a .svg file.</li>
	<li>Cut the 30 cm x 30 cm vinyl sheets with the edge plotter (Figure 1B-D).
	<ol>
		<li>Stick the vinyl sheet to a low tack adhesive mat, and eliminate all the air bubbles if necessary.</li>
		<li>Upload the .svg file to the plotter, and set the cutting parameters: cutting blade: level 3; cutting pressure: level 0; cutting speed: level 1. Place the adhesive mat with the vinyl into the plotter, and start the cutting process.</li>
	</ol>
	</li>
	<li>Cut the top channel pattern on 12 &#181;m-thick double-sided tape vinyl by following the previous steps.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62353/62353fig1v2.jpg" /> 
Figure 1: Chip design and micromachining process. (A) Software layout showing the working space filled with both the top and bottom patterns designed for the chip. (B) Edge plotter during cutting process; cutting blade, whole vinyl sheet, and adhesive mat are shown. (C) Patterned vinyl being detached from the cut sheet. (D) Sample of an adhesive vinyl layer patterned with the top channel design. <a href="https://www.jove.com/files/ftp_upload/62353/62353fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. PDMS layer fabrication
<ol>
	<li>Mix the PDMS and curing agent in a ratio of 10:1 (v/v), and place the mixture under vacuum for 15 min to remove air bubbles. Pour 55 mL of the mixture into a 55 cm2 square culture dish to obtain a 2 mm-thick layer. Remove bubbles with a needle.</li>
	<li>Cure the mixture (step 2.1) in an oven for 1 h at 80 &#176;C. Unmold the PDMS and cut it into rectangles with the chip dimensions. Make holes for the tubing with an 18 G syringe needle.</li>
</ol>
3. Chip assembly
NOTE: For better understanding, see Figure 2.
<ol>
	<li>Assemble the whole device using an aligner to adjust channels, inlets, and outlets properly. Pile up four vinyl layers (with the corresponding bottom micropattern) for assembling the lower channel, keeping the cover tape of the bottom layer to avoid sticking to the aligner.</li>
	<li>Cut and place the polycarbonate (PC) porous membrane on top of the lower channel to separate it from the upper one. Be careful not to cover the inlets of the lower channel.</li>
	<li>Add ten vinyl layers with the upper chamber design. Stick a double-sided tape vinyl layer with the top-channel pattern on top. Remove the chip from the aligner and stick it on the glass slide.</li>
	<li>Place a 2-mm-thick PDMS sheet on top of the double-sided tape vinyl layer to provide appropriate anchoring for the tubing and to avoid leakage. Leave a weight on top of the chip overnight to ensure the chip is completely watertight. Sterilize the chip by flushing 70% v/v ethanol for 5 min, and then wash with distilled H2O.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62353/62353fig02.jpg" /> 
Figure 2: Microfluidic chip assembly. (A) General scheme of the assembly of the device. Lower and upper chambers are composed of four and eleven superimposed vinyl sheets, respectively. (B) Top and lateral views of the microfluidic chip. Top and bottom channels are represented in pink and blue, respectively. (C) Image of the chip assembly using a custom-made aligner. (D) Chip image after complete assembly. <a href="https://www.jove.com/files/ftp_upload/62353/62353fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Pump connections
NOTE: The graphical representation of pumps connections is shown in Figure 3.
<ol>
	<li>Connect Pump 1 to the Upper Chamber Inlet 1 (UCi1).</li>
	<li>Connect Pump 2 to the Upper Chamber Inlet 2 (UCi2).</li>
	<li>Connect Pump 3 to the Lower Chamber Inlet (LCi).</li>
	<li>Connect Upper Chamber Outlet (UCo) and Lower Chamber Outlet (LCo) to a waste tube.</li>
	<li>Connect the syringes to each inlet using polytetrafluoroethylene (PTFE) tubes and 18 G stainless steel connectors.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62353/62353fig03.jpg" /> 
Figure 3: Pump connections and inlets/outlets location. (A) Diagram showing the connection of the three different pumps to their respective inlets. Outlets connect to a waste container. (B) Chip image with labeled inlets and outlets. Abbreviations: LCi = lower chamber inlet; LCo = lower chamber outlet; UCi1 =&#160;upper chamber inlet 1; UCi2 = upper chamber inlet 2; UCo = upper chamber outlet. <a href="https://www.jove.com/files/ftp_upload/62353/62353fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Cell culture
NOTE: The HaCaT cell line has a commercial origin. Human primary fibroblasts come from healthy donors and were obtained from the collection of biological samples of human origin registered in &#34;Registro Nacional de Biobancos para Investigaci&#243;n Biom&#233;dica del Instituto de Salud Carlos III&#34;.
<ol>
	<li>Work in a cell culture hood, previously sterilized under ultraviolet light and wiped with ethanol.</li>
	<li>Thaw H2B-GFP-HaCaT cells (human immortalized skin keratinocytes, hKCs) and GFP-human primary fibroblasts (hFBs) at 37 &#176;C, add 2 mL of culture medium, and centrifuge for 7 min at 20 &#176;C at 250 &#215; g. 
	NOTE: H2B-GFP-HaCaT cells are human immortalized keratinocytes modified to express a hybrid histone H2B-green fluorescent protein (GFP), providing their nuclei with green fluorescence. GFP-hFBs are human primary fibroblasts transformed with the vector pLZRS-IRES-EGFP to express cytoplasmic green fluorescence. These cells were modified following previously published protocols30,31</li>
	<li>Culture both hKCs and hFBs in 1x DMEM supplemented with 10% fetal bovine serum and 1% of antibiotic/antimycotic solution. Pre-warm the culture medium at 37 &#176;C before use.</li>
	<li>Detach the cells by washing them with 1x phosphate-buffered saline (PBS), adding 2 mL of trypsin/ethylenediamine tetraacetic acid (EDTA) and incubating them for 10 min at 37 &#176;C.</li>
	<li>Inactivate trypsin adding 4 mL of culture medium. Resuspend the cells, and transfer them to a 15 mL tube. Remove 10 &#181;L to count cells on a Neubauer chamber and determine the appropriate concentration.</li>
	<li>Centrifuge the 15 mL tube for 7 min at 20 &#176;C at 250 &#215; g. Remove the supernatant, and resuspend the pellets at the desired concentration: hFBs at 50,000 cells/mL and hKCs at 5&#183;&#215; 106 cells/mL.</li>
</ol>
6. Fibrinogen pre-gel preparation
<ol>
	<li>Activate thrombin by adding 1mL of CaCl2 (1% w/v in NaCl) to the vial.</li>
	<li>Add the following components to obtain 1 mL of a fibrin hydrogel at a final concentration of fibrin of 3.5 mg/mL: 59 &#181;L of activated thrombin (10 NIH units/mL), 59 &#181;L of tranexamic acid (Table of Materials, 100 mg/mL), 764 &#181;L of culture medium containing 50,000 hFBs/mL, 118 &#181;L of fibrinogen (20 mg/mL in NaCl (0.9% w/v)). 
	&#8203;NOTE: Fibrinogen must be added at the last moment.</li>
</ol>
7. Parallel flow protocol
<ol>
	<li>Pump 1x PBS with pump 3 through the LCi at 50 &#181;L/min during the whole process.</li>
	<li>Pump sacrificial fluid (1x PBS) with pump 2 through the UCi2 at 100 &#181;L/min.</li>
	<li>Load the syringe with the pre-gel, rapidly place it into pump 1, and run it at 200 &#181;L/min (Figure 4A).</li>
	<li>Stop pumps 1 and 2 once the pre-gel exits the UCo.</li>
	<li>Leave the chip without removing the tubing at 37 &#176;C for at least 10 min to allow gelation.</li>
	<li>Pump&#160;culture medium at 50 &#181;L/h with pump 3 through UCi2 overnight.</li>
	<li>Block UCi1 with a cap.</li>
</ol>
8. hKCs monolayer seeding
<ol>
	<li>Check under the microscope that hFBs are spread 24 h after the generation of the dermal compartment.</li>
	<li>Introduce the hKCs with pump 2 through UCi2 at 5 &#215;&#183;106 cells/mL at 40 &#181;L/min for 1 min (Figure 4B).</li>
	<li>Leave the chip overnight at 37 &#176;C in a humidity-saturated incubator for cell attachment.</li>
	<li>Pump fresh culture medium with pump 3 only through LCi at 50 &#181;L/min (Figure 4C).</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62353/62353fig04.jpg" /> 
Figure 4: Microfluidic protocol for the generation of the dermo-epidermal construct. (A) Transverse cross-section showing the parallel flow process to generate the dermal compartment. (B) Keratinocyte monolayer seeding 24 h after dermal compartment generation. (C) Cell culture maintenance inside the microfluidic device. (D) Cross-sectional recreation of the skin inside the chip. <a href="https://www.jove.com/files/ftp_upload/62353/62353fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
9. Cell viability assay
NOTE: Live/Dead kit stains cells with green or red fluorescence depending on their live or dead state. For proper viability differentiation, non-fluorescent hKCs and hFBs must be used in this step. All the steps in the procedure are carried out through UCi2 with pump 2.
<ol>
	<li>Wash the top channel with 1x PBS for 5 min at 50 &#181;L/min to remove culture medium.</li>
	<li>Pump air to remove the 1x PBS at 50 &#181;L/min.</li>
	<li>Prepare Calcein AM/Ethidium homodimer-1 Kit (Live/Dead) solution by following the manufacturer&#39;s instructions.</li>
	<li>Pump Live/Dead solution at 50 &#181;L/min for 2 min.</li>
	<li>Incubate 30 min at 37 &#176;C in the dark.</li>
	<li>Wash top channel by pumping 1x PBS at 50 &#181;L/min for 2 min to remove any remaining reagent.</li>
	<li>Observe the sample under the confocal microscope. Use an excitation wavelength of 495/590 nm and an emission wavelength of 519/617 nm for live and dead cells, respectively.</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+bioprinting+of+functional+human+skin:+production+and+in+vivo+analysis.">3D bioprinting of functional human skin: production and in vivo analysis.</a> Biofabrication. 9 (1), 015006 (2016).</li><li>Mori, N., Morimoto, Y., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+integrated+with+perfusable+vascular+channels+on+a+chip.">Skin integrated with perfusable vascular channels on a chip.</a> Biomaterials. 116, 48-56 (2017).</li><li>Kim, H. J., Li, H., Collins, J. J., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+microbiome+and+mechanical+deformation+to+intestinal+bacterial+overgrowth+and+inflammation+in+a+human+gut-on-a-chip.">Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (1), 7-15 (2016).</li><li>Shah, 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=A+microfluidics-based+in+vitro+model+of+the+gastrointestinal+human-microbe+interface.">A microfluidics-based in vitro model of the gastrointestinal human-microbe interface.</a> Nature Communications. 7, 11535 (2016).</li><li>Marx, U., 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-on-a-chip'+developments:+A+translational+cuttingedge+alternative+to+systemic+safety+assessment+and+efficiency+evaluation+of+substances+in+laboratory+animals+and+man.">Human-on-a-chip' developments: A translational cuttingedge alternative to systemic safety assessment and efficiency evaluation of substances in laboratory animals and man.</a> Alternatives to Laboratory Animals. 40 (5), 235-257 (2012).</li><li>Bein, 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=Microfluidic+organ-on-a-chip+models+of+human+intestine.">Microfluidic organ-on-a-chip models of human intestine.</a> Cellular and Molecular Gastroenterology and Hepatology. 5 (4), 659-668 (2018).</li><li>Bennet, D., Estlack, Z., Reid, T., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microengineered+human+corneal+epithelium-on-a-chip+for+eye+drops+mass+transport+evaluation.">A microengineered human corneal epithelium-on-a-chip for eye drops mass transport evaluation.</a> Lab on a Chip. 18, 1539-1551 (2018).</li><li>Kim, H. J., Huh, D., Hamilton, G., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+gut-on-a-chip+inhabited+by+microbial+flora+that+experiences+intestinal+peristalsis-like+motions+and+flow.">Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow.</a> Lab on a chip. 12, 2165-2174 (2012).</li><li>Kim, H. J., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut-on-a-chip+microenvironment+induces+human+intestinal+cells+to+undergo+villus+differentiation.">Gut-on-a-chip microenvironment induces human intestinal cells to undergo villus differentiation.</a> Integrative Biology. 5 (9), 1130-1140 (2013).</li><li>O'Neill, A. T., Monteiro-Riviere, N. A., Walker, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+microfluidic+human+epidermal+keratinocyte+culture.">Characterization of microfluidic human epidermal keratinocyte culture.</a> Cytotechnology. 56 (3), 197-207 (2008).</li><li>Ren, K., Chen, Y., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+materials+for+microfluidics+in+biology.">New materials for microfluidics in biology.</a> Current Opinion in Biotechnology. 25, 78-85 (2014).</li></ol>

The authors declare that they have no competing financial interests.

The motivation to develop this method was the desire to model skin diseases and study the effects of new and innovative therapies in a high-throughput platform. To date, this laboratory produces these dermo-epidermal equivalents by casting-either manually or with the help of the 3D bioprinting technology-the fibrin gel with fibroblasts into a cell culture insert plate and seeding the keratinocytes on top of it. Once the keratinocytes reach confluence, the 3D culture is exposed to the air-liquid interface, which induces k...

Recently, advances have been made toward the development and production of in vitro human skin models for the analysis of the toxicity of cosmetic and pharmaceutical products1. Researchers in pharmaceutical and skin care industries have been using animals, mice being the most common, to test their products2,3,4,5. However, testing products on animals is not always predictive of the response in humans, which frequently leads to drug failure or adverse effects in humans and consequently to economic losses5,6. The UK was the first country that prohibited the use of animals for cosmetic testing in 1998. Later, in 2013, the EU banned the testing and approbation of cosmetics in animals (EU Cosmetics Regulation No. 1223/2009)7.
This prohibition is also being considered by other countries such as in &#39;The Humane Cosmetics Act&#39; in the USA8. In addition to ethical concerns, the anatomical differences between animal and human skin make animal testing time-consuming, expensive, and often ineffective. Furthermore, the global in vitro toxicology testing market size is expected to reach USD 26.98 billion by 20259. For these reasons, there is a need to develop new methods and alternatives for those in vitro studies, such as bioengineered human skin models, that enable testing for safety and toxic effects of cosmetics and drugs without the use of animals.
There are two different kinds of commercially available, in vitro, human skin models. The first type consists of stratified epidermal equivalents containing multiple layers of differentiating keratinocytes that are seeded on different materials. Some of them have been approved by the Organization for Economic Co-operation and Development (OECD) and validated by the (European Centre for the Validation of Alternative Methods (ECVAM) for skin corrosion and irritation testing, such as EpiDerm or SkinEthic10,11,12. The second type are full-skin equivalents with a layer of differentiating human keratinocytes seeded on a three-dimensional (3D) scaffold that contains fibroblasts, such as T-Skin and EpiDerm-FT. However, these models are cultured under static conditions, which makes them unable to accurately represent human physiological conditions.
Recent interest has focused on generating in vitro 3D skin models in cell culture-insert (CCI) formats with dynamic perfusion13,14,15,16,17,18,19. However, these systems cannot be considered stricto sensu as microfluidic skin-on-chips as per their classical definition in the field. Ingber&#39;s definition for organs-on-a-chip states that the organ must be placed inside the microfluidic channels, which is a condition that only a few devices fulfil20,21. Skin-on-chips have so far modelled mostly simple epithelia as single-cell layers and/or dermal cell layers separated by a porous membrane22,23. Although there have been some advances modeling skin in microfluidic systems16,24, there is currently no literature showing an organ-on-a-chip system that fits Ingber&#39;s definition, capable of producing a multilayered skin in situ and including both epithelial and stromal components.
In this work, a new, cost-effective, robust, vinyl-based microfluidic platform for skin-on-a-chip applications is presented. This platform was produced by micro-machining, which provides more simplicity in the fabrication process, as well as increased flexibility and versatility in the layout of the device, overcoming some of the limitations of PDMS25. A way to introduce a simplified skin construct through a parallel flow controlled with syringe pumps was also designed. Parallel flow allows two fluids with very different viscosities (a buffer and fibrin pre-gel in this case) to be perfused through a channel without mixing with each other. As a proof of concept, a dermo-epidermal construct containing fibroblasts embedded in a fibrin matrix mimicking the dermis was introduced in the device, on top of which a monolayer of keratinocytes was loaded to emulate the undifferentiated epidermis. The dermal compartment height can be modulated by modifying the flow rates. The main novelty of this work, compared to previously described models22,26,27,28,29, is the development of a 3D construct inside a microchamber by means of microfluidics. Although this article presents a simplified undifferentiated skin, the long-term goal is to generate and characterize a fully differentiated skin construct to demonstrate its viability and functionality for drug and cosmetic testing purposes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amchafibrin</td>
			<td>Rottafarm</td>
			<td></td>
			<td>Tranexamic acid</td>
		</tr>
		<tr>
			<td>Antibiotic/antimycotic</td>
			<td>Thermo Scientific HyClone</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Culture plates</td>
			<td>Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen Life Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Double-sided tape vynil</td>
			<td>ATP Adhesive Systems</td>
			<td></td>
			<td>GM 107CC, 12 &#181;m thick</td>
		</tr>
		<tr>
			<td>Edge plotter</td>
			<td>Brother</td>
			<td></td>
			<td>Scanncut CM900</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Scientific HyClone</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>Extracted from human plasma</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-Human dermal fibroblasts</td>
			<td>-</td>
			<td></td>
			<td>Primary. Gift from Dr. Marta Garc&#237;a</td>
		</tr>
		<tr>
			<td>H2B-GFP-HaCaT cell line</td>
			<td>ATCC</td>
			<td></td>
			<td>Immortalized keratinocytes. Gift from Dr. Marta Garc&#237;a</td>
		</tr>
		<tr>
			<td>Live/dead kit</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membrane</td>
			<td>Merk TM</td>
			<td></td>
			<td>5 &#181;m pore size</td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane</td>
			<td>Dow Corning</td>
			<td></td>
			<td>Sylgard 184</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td>Terumo</td>
			<td></td>
			<td>5 mL</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td>10 NIH/vial</td>
		</tr>
		<tr>
			<td>Transparent adhesive vinyl</td>
			<td>Mactac</td>
			<td></td>
			<td>JT 8500 CG-RT, 95 &#181;m thick</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA</td>
			<td>Sigma Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td>IDEX</td>
			<td></td>
			<td>Teflon, 1/16&#8221; OD, 0.020&#8221; ID</td>
		</tr>
	</tbody>
</table>

generation of a simplified three-dimensional skin-on-a-chip model in a micromachined microfluidic platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

The designed chip is composed of two fluidic chambers separated by a 5 &#181;m pore size PC membrane that allows the growth of the cell by allowing the passage of growth-promoting molecules from the lower chamber. The upper chamber holds the tissue construct, in this case, a monolayer of hKCs on a fibrin hydrogel containing hFBs.
The height of the channels is determined by the number of adhesive sheets added to each channel. The lower chamber is composed of 4 layers (380 &#181;m) and the upper...

Watch this Scientific Journal Video about Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform at JoVE.com

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

generation-simplified-three-dimensional-skin-on-chip-model

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Bioengineering

3.9K Views.  Universidad Carlos III de Madrid (UC3M). Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps. 

Video: Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

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. ...

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 ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...