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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<li>Cubo, N., Garcia, M., del Cañizo, J. F., Velasco, D., Jorcano, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((3D+bioprinting+of+functional+human+skin%3A+production+and+in+vivo+analysis%5BTitle%5D)+AND+%22Biofabrication%22%5BJournal%5D)">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/pubmed?term=((Skin+integrated+with+perfusable+vascular+channels+on+a+chip%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Contributions+of+microbiome+and+mechanical+deformation+to+intestinal+bacterial+overgrowth+and+inflammation+in+a+human+gut-on-a-chip%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((A+microfluidics-based+in+vitro+model+of+the+gastrointestinal+human-microbe+interface%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Human-on-a-chip%27+developments%3A+A+translational+cuttingedge+alternative+to+systemic+safety+assessment+and+efficiency+evaluation+of+substances+in+laboratory+animals+and+man%5BTitle%5D)+AND+%22Alternatives+to+Laboratory+Animals%22%5BJournal%5D)">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/pubmed?term=((Microfluidic+organ-on-a-chip+models+of+human+intestine%5BTitle%5D)+AND+%22Cellular+and+Molecular+Gastroenterology+and+Hepatology%22%5BJournal%5D)">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/pubmed?term=((A+microengineered+human+corneal+epithelium-on-a-chip+for+eye+drops+mass+transport+evaluation%5BTitle%5D)+AND+%22Lab+on+a+Chip%22%5BJournal%5D)">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/pubmed?term=((Human+gut-on-a-chip+inhabited+by+microbial+flora+that+experiences+intestinal+peristalsis-like+motions+and+flow%5BTitle%5D)+AND+%22Lab+on+a+chip%22%5BJournal%5D)">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/pubmed?term=((Gut-on-a-chip+microenvironment+induces+human+intestinal+cells+to+undergo+villus+differentiation%5BTitle%5D)+AND+%22Integrative+Biology%22%5BJournal%5D)">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/pubmed?term=((Characterization+of+microfluidic+human+epidermal+keratinocyte+culture%5BTitle%5D)+AND+%22Cytotechnology%22%5BJournal%5D)">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/pubmed?term=((New+materials+for+microfluidics+in+biology%5BTitle%5D)+AND+%22Current+Opinion+in+Biotechnology%22%5BJournal%5D)">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><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 &amp;micro;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&amp;iacute;a</td></tr><tr><td>H2B-GFP-HaCaT cell line</td><td>ATCC</td><td></td><td>Immortalized keratinocytes. Gift from Dr. Marta Garc&amp;iacute;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 &amp;micro;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 &amp;micro;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&amp;rdquo; OD, 0.020&amp;rdquo; 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

Research

JoVE Journal

Bioengineering

4.1K Views.  Universidad Carlos III de Madrid (UC3M). Dit is een nieuw, lithografievrij protocol voor het modelleren van een 3D-skin-on-chip met behulp van microgemachineerde, zelfklevende vinyllagen en een parallelstroommethodologie voor het genereren van dermale compartimenten. Micromachined vinyl biedt meer eenvoud in het fabricageproces en veelzijdigheid in de lay-out van het apparaat, waarbij enkele van de beperkingen van PDMS en traditionele lithografiebenaderingen worden overwonnen. Deze meerlagige skin-on-a-chip kan worden gebruikt voor ...

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

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Biology

Procedure for the Development of Multi-depth Circular Cross-sectional Endothelialized Microchannels-on-a-chip

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, and observation and quantification are very challenging. However, conventional in vitro microvessel assays have limitations when representing in vivo microvessels with respect to three-dimensional (3D) geometry and providing continuous fluid flow. Using a combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics, we have developed a multi-depth circular cross-sectional endothelialized microchannels-on-a-chip, which mimics the 3D geometry of in vivo microvessels and runs under controlled continuous perfusion flow. A positive reflowable photoresist was used to fabricate a master mold with a semicircular cross-sectional microchannel network. By the alignment and bonding of the two polydimethylsiloxane (PDMS) microchannels replicated from the master mold, a cylindrical microchannel network was created. The diameters of the microchannels can be well controlled. In addition, primary human umbilical vein endothelial cells (HUVECs) seeded inside the chip showed that the cells lined the inner surface of the microchannels under controlled perfusion lasting for a time period between 4 days to 2 weeks.

A microchannels-on-a-chip platform was developed by the combination of photolithographic reflowable photoresist technique, soft lithography, and microfluidics. The endothelialized microchannels platform mimics the three-dimensional (3D) geometry of in vivo microvessels, runs under controlled continuous perfusion flow, allows for high-quality and real-time imaging&#160;and can be applied for microvascular research.

Efforts have been focused on developing in vitro assays for the study of microvessels because in vivo animal studies are more time-consuming, expensive, ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Developmental Biology

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart muscle, the myocardium, is notoriously difficult to accomplish in vitro. Mainly, obstacles lie in the need for human cardiomyocytes (CMs) that are either adult or exhibit adult-like phenotypes and can successfully replicate the myocardium&#39;s cellular complexity and intricate 3D architecture. Unfortunately, due to ethical concerns and lack of available primary patient-derived human cardiac tissue, combined with the minimal proliferation of CMs, the sourcing of viable human CMs has been a limiting step for cardiac tissue engineering. To this end, most research has transitioned toward cardiac differentiation of human induced pluripotent stem cells (hiPSCs) as the primary source of human CMs, resulting in the wide incorporation of hiPSC-CMs within in vitro assays for cardiac tissue modeling.
Here in this work, we demonstrate a protocol for developing a 3D mature stem cell-derived human cardiac tissue within a microfluidic device. We specifically explain and visually demonstrate the production of a 3D in vitro anisotropic cardiac tissue-on-a-chip model from hiPSC-derived CMs. We primarily describe a purification protocol to select for CMs, the co-culture of cells with a defined ratio via mixing CMs with human CFs (hCFs), and suspension of this co-culture within the collagen-based hydrogel. We further demonstrate the injection of the cell-laden hydrogel within our well-defined microfluidic device, embedded with staggered elliptical microposts that serve as surface topography to induce a high degree of alignment of the surrounding cells and the hydrogel matrix, mimicking the architecture of the native myocardium. We envision that the proposed 3D anisotropic cardiac tissue-on-chip model is suitable for fundamental biology studies, disease modeling, and, through its use as a screening tool, pharmaceutical testing.

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

The leading cause of death worldwide persists as cardiovascular disease (CVD). However, modeling the physiological and biological complexity of the heart ...

A Microphysiological System to Study Leukocyte-Endothelial Cell Interaction during Inflammation

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of activated leukocytes across the vascular endothelium into key organs can lead to organ failure. A physiologically relevant biomimetic microfluidic assay (bMFA) has been developed and validated using several experimental and computational techniques, which can reproduce the entire leukocyte rolling/adhesion/migration cascade to study leukocyte-endothelial cell interactions. Microvascular networks obtained from in vivo images in rodents were digitized using a Geographic Information System (GIS) approach and microfabricated with polydimethylsiloxane (PDMS) on a microscope slide. To study the effect of shear rate and vascular topology on leukocyte-endothelial cell interactions, a Computational Fluid Dynamics (CFD) model was developed to generate a corresponding map of shear rates and velocities throughout the network. The bMFA enables the quantification of leukocyte-endothelial cells interactions, including rolling velocity, number of adhered leukocytes in response to different shear rates, number of migrated leukocytes, endothelial cell permeability, adhesion molecule expression and other important variables. Furthermore, by using human-related samples, such as human endothelial cells and leukocytes, bMFA provides a tool for rapid screening of potential therapeutics to increase their clinical translatability.

In this protocol, a biomimetic microfluid assay, which can reproduce a physiologically relevant microvascular environment and reproduce the entire leukocyte adhesion/migration cascade, is employed to study leukocyte-endothelial cell interactions in inflammatory disease.

Leukocyte-endothelial cell interactions play an important role in inflammatory diseases such as sepsis. During inflammation, excessive migration of ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

Immunology and Infection

A Reconfigurable Barrier Tissue Platform with Open-Well and Microfluidic Modes

Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. However, these platforms have not gained widespread adoption in bioscience laboratories where open-well, membrane-based approaches serve as the gold standard for mimicking tissue barriers, despite lacking fluid flow capabilities. This issue can be primarily attributed to the incompatibility of existing microphysiological systems with standard protocols and tools developed for open-well systems.
Here, we present a protocol for creating a reconfigurable membrane-based platform with an open-well structure, flow enhancement capability, and compatibility with conventional protocols. This system utilizes a magnetic assembly approach that enables reversible switching between open-well and microfluidic modes. With this approach, users have the flexibility to begin an experiment in the open-well format using standard protocols and add or remove flow capabilities as needed. To demonstrate the practical usage of this system and its compatibility with standard techniques, an endothelial cell monolayer was established in an open-well format. The system was reconfigured to introduce fluid flow and then switched to the open-well format to conduct immunostaining and RNA extraction. Due to its compatibility with conventional open-well protocols and flow enhancement capability, this reconfigurable design is expected to be adopted by both engineering and bioscience laboratories.

Author Spotlight: Developing a Unique Modular Microphysiological System to Mimic Human Barrier Tissue

This protocol describes a reconfigurable membrane-based cell culture platform that integrates the open-well format with fluid flow capabilities. This platform is compatible with standard protocols and allows for reversible transitions between open-well and microfluidic culture modes, accommodating the needs of both engineering and bioscience laboratories.

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. ...

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

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