Name: scalable fabrication of stretchable, dual channel, microfluidic organ chips
Uploaded: 2018-10-20T13:00:00
Description: Watch this Scientific Journal Video about Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips at JoVE.com

We thank M. Rousseau and S. Kroll for help with photography and videography and M. Ingram, J. Nguyen, D. Shea, and N. Wen for contributions to initial fabrication protocol development. This research was sponsored by the Wyss Institute for Biologically Inspired Engineering at Harvard University and the Defense Advanced Research Projects Agency under Cooperative Agreements #W911NF-12-2-0036 and #W911NF-16-C-0050, and FDA grant #HHSF223201310079C, NIH grants #R01-EB020004 and #UG3-HL141797-01, and Bill and Melinda Gates Foundation grants #OPP1163237 and #OPP1173198 to DEI. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the official policies, either expressed or implied, of the Defense Advanced Research Projects Agency, Food and Drug Administration, the National Institutes of Health, or the U.S. Government.

1. General Preparation
<ol>
	<li>To avoid debris, clean work area using packing tape and wipe down area with a cleanroom wipe and isopropyl alcohol.</li>
	<li>For all steps requiring PDMS, mix PDMS at a 10:1 ratio (10 g of cross linking agent, 100 g of elastomer base). Mix by hand or with a commercially available mixer. Use a planetary centrifugal mixer here: mixing for 2 minutes at 2000 rpm, then degassing the PDMS for 2 minutes at 2200 rpm.</li>
	<li>Clean all molds with air gun to blow out debris prior to use. ...

<ol>
	<li>Bhatia, S. N., 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=Microfluidic+organs-on-chips.">Microfluidic organs-on-chips.</a> Nature Biotechnology. 32 (8), 760-772 (2014).</li><li>Benam, K. H., 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=Engineered+In+vitro+Disease+Models.">Engineered In vitro Disease Models.</a> Annual Review of Pathology Mechanisms of Disease. 10 (1), 195-262 (2015).</li><li>Huh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Human+Disease+Model+of+Drug+Toxicity-Induced+Pulmonary+Edema+in+a+Lung-on-a-Chip+Microdevice.">A Human Disease Model of Drug Toxicity-Induced Pulmonary Edema in a Lung-on-a-Chip Microdevice.</a> Science Translational Medicine. 4 (159), 159ra147 (2012).</li><li>Huh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+Organ-Level+Lung+Functions+on+a+Chip.">Reconstituting Organ-Level Lung Functions on a Chip.</a> Science. 328 (5986), 1662-1668 (2010).</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 (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>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), E7-E15 (2016).</li><li>Musah, S., 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=Mature+induced-pluripotent-stem-cell-derived+human+podocytes+reconstitute+kidney+glomerular-capillary-wall+function+on+a+chip.">Mature induced-pluripotent-stem-cell-derived human podocytes reconstitute kidney glomerular-capillary-wall function on a chip.</a> Nature Biomedical Engineering. 1 (5), s41551-017-0069–017 (2017).</li><li>Somayaji, M. R., Das, D., Przekwas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Computational+approaches+for+modeling+and+analysis+of+human-on-chip+systems+for+drug+testing+and+characterization.">Computational approaches for modeling and analysis of human-on-chip systems for drug testing and characterization.</a> Drug Discovery Today. 21 (12), 1859-1862 (2016).</li><li>Prantil-Baun, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologically+Based+Pharmacokinetic+and+Pharmacodynamic+Analysis+Enabled+by+Microfluidically+Linked+Organs-on-Chips.">Physiologically Based Pharmacokinetic and Pharmacodynamic Analysis Enabled by Microfluidically Linked Organs-on-Chips.</a> Annual Review of Pharmacology and Toxicology. 58 (1), 37-64 (2018).</li><li>Mahler, G. J., Esch, M. B., Stokol, T., Hickman, J. J., Shuler, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Body-on-a-chip+systems+for+animal-free+toxicity+testing.">Body-on-a-chip systems for animal-free toxicity testing.</a> Alternatives to laboratory animals: ATLA. 44 (5), 469-478 (2016).</li><li>Miller, P. G., Shuler, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+demonstration+of+a+pumpless+14+compartment+microphysiological+system.">Design and demonstration of a pumpless 14 compartment microphysiological system.</a> Biotechnology and Bioengineering. 113 (10), 2213-2227 (2016).</li><li>Coppeta, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+portable+and+reconfigurable+multi-organ+platform+for+drug+development+with+onboard+microfluidic+flow+control.">A portable and reconfigurable multi-organ platform for drug development with onboard microfluidic flow control.</a> Lab Chip. 17 (1), 134-144 (2016).</li><li>Wikswo, J. 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=Scaling+and+systems+biology+for+integrating+multiple+organs-on-a-chip.">Scaling and systems biology for integrating multiple organs-on-a-chip.</a> Lab on a Chip. 13 (18), 3496-3511 (2013).</li><li>Sung, J. H., 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=Using+PBPK+guided+&amp;#34;Body-on-a-Chip&amp;#34;+Systems+to+Predict+Mammalian+Response+to+Drug+and+Chemical+Exposure.">Using PBPK guided &amp;#34;Body-on-a-Chip&amp;#34; Systems to Predict Mammalian Response to Drug and Chemical Exposure.</a> Experimental biology and medicine (Maywood, N.J.). 239 (9), 1225-1239 (2014).</li><li>Stokes, C., Cirit, M., Lauffenburger, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiome-on-a-Chip:+The+Challenge+of+&amp;#34;Scaling&amp;#34+in+Design,+Operation,+and+Translation+of+Microphysiological+Systems.">Physiome-on-a-Chip: The Challenge of &amp;#34;Scaling&amp;#34 in Design, Operation, and Translation of Microphysiological Systems.</a> CPT: Pharmacometrics &amp; Systems Pharmacology. 4 (10), 559-562 (2015).</li><li>Shirure, V. S., George, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+considerations+to+minimize+the+impact+of+drug+absorption+in+polymer-based+organ-on-a-chip+platforms.">Design considerations to minimize the impact of drug absorption in polymer-based organ-on-a-chip platforms.</a> Lab Chip. , (2017).</li><li>Huh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfabrication+of+human+organs-on-chips.">Microfabrication of human organs-on-chips.</a> Nature Protocols. 8 (11), 2135-2157 (2013).</li><li>Tran, T. T., 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=Exact+kinetic+analysis+of+passive+transport+across+a+polarized+confluent+MDCK+cell+monolayer+modeled+as+a+single+barrier.">Exact kinetic analysis of passive transport across a polarized confluent MDCK cell monolayer modeled as a single barrier.</a> Journal of Pharmaceutical Sciences. 93 (8), 2108-2123 (2004).</li><li>Henry, O. Y. F., 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=Organs-on-chips+with+integrated+electrodes+for+trans-epithelial+electrical+resistance+(TEER)+measurements+of+human+epithelial+barrier+function.">Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function.</a> Lab on a Chip. 17 (13), 2264-2271 (2017).</li><li>Maoz, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organs-on-Chips+with+combined+multi-electrode+array+and+transepithelial+electrical+resistance+measurement+capabilities.">Organs-on-Chips with combined multi-electrode array and transepithelial electrical resistance measurement capabilities.</a> Lab on a Chip. 17 (13), 2294-2302 (2017).</li><li>Benam, K. H., 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=Matched-Comparative+Modeling+of+Normal+and+Diseased+Human+Airway+Responses+Using+a+Microengineered+Breathing+Lung+Chip.">Matched-Comparative Modeling of Normal and Diseased Human Airway Responses Using a Microengineered Breathing Lung Chip.</a> Cell Systems. 3 (5), 456-466 (2016).</li></ol>

The fabrication process relies on high resolution 3D printed molds to pattern the PDMS top and bottom Organ Chip body components coupled with micromolded porous PDMS membranes. This critical approach was selected due to ease of prototyping combined with rapid transition into scaled up fabrication and replacement of tooling. The top component molds are designed to pattern ports in precise locations with defined vertical profiles during the casting step. This not only avoids the labor involved in manually punching access p...

An erratum was issued for: <a href="https://www.jove.com/video/58151/scalable-fabrication-stretchable-dual-channel-microfluidic-organ">Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips</a>.&#160; The Representative Results, Discussion, and References sections have been updated.
In the Representative Results section, the legend for Figure 5 has been updated&#160;from:
Figure 5: Permeability of inert tracer Cascade Blue through the microporous PDMS membrane. Cascade Blue hydrazide dye in medium was loaded into the top channel of the Organ Chip and perfused at 60 &#181;L/h to measure the flux of the dye across the membrane into the bottom channel containing medium. Empty chips were compared to Gut Chips with Caco2-BBe1 cells in the apical channel and human vascular endothelial cells (HUVEC) in the basal channel cultured for 6 days. Error bars indicate standard error of the mean.
to:
Figure 5: Permeability of inert tracer Cascade Blue through the microporous PDMS membrane. Cascade Blue hydrazide dye in medium was loaded into the top channel of the Organ Chip and perfused at 60 &#181;l/h to measure the flux of the dye across the membrane into the bottom channel containing medium. Empty chips were compared to Gut Chips with Caco2-BBe1 cells in the apical channel and human vascular endothelial cells (HUVEC) in the basal channel cultured for 6 days. The apparent permeability (Papp, cm/s) of the microporous PDMS membrane was determined using the dye concentration in the outlet channels. The gut chip cell layers provide a significantly increased barrier to permeability. Error bars indicate standard error of the mean.
In the Discussion section, the fourth paragraph has been updated from:
Troubleshooting the resulting Organ Chips takes place at two levels: during the fabrication process and during Organ Chip culture. We have developed a visual method for quality assurance (QA) of through-hole formation in the cast membranes that greatly accelerates the production process while improving the quality and reliability of assembled Organ Chips. This QA method allows for process troubleshooting, and we recommend keeping a record of process conditions to enable tracking fabrication problems that may occur during cell culture. During Organ Chip culture, inert tracer dyes are the simplest method of measuring barrier function to troubleshoot the fabrication process and cell culture steps. Lucifer Yellow has been used historically due to its small molecular mass and innate fluorescence, but Cascade Blue offers similar properties with a narrower emission spectrum that is less likely to interfere with downstream assays. Larger molecules, such as poly-ethyleneglycol (PEG)- or dextran-conjugated fluorophores are larger and consequently result in lower permeability overall and lower sensitivity. The apparent permeability (Papp, cm/s) of tracer dyes can be used to determine barrier function properties of organs or tissues (Figure 4). The following equation can be used to calculate Papp between the dosing channel and receiving channel and is derived from equations used primarily for Transwell studies19,20 and corrects for tracer dye loss caused by absorption into PDMS by comparing the two output flows and not relying on mass balance assumptions at the outflow.
<img alt="Equation 1" src="/files/ftp_upload/58151/58151eq1.jpg" />
to:
Troubleshooting the resulting Organ Chips takes place at two levels: during the fabrication process and during Organ Chip culture. We have developed a visual method for quality assurance (QA) of through-hole formation in the cast membranes that greatly accelerates the production process while improving the quality and reliability of assembled Organ Chips. This QA method allows for process troubleshooting, and we recommend keeping a record of process conditions to enable tracking fabrication problems that may occur during cell culture. During Organ Chip culture, inert tracer dyes are the simplest method of measuring barrier function to troubleshoot the fabrication process and cell culture steps. Lucifer Yellow has been used historically due to its small molecular mass and innate fluorescence, but Cascade Blue offers similar properties with a narrower emission spectrum that is less likely to interfere with downstream assays. Larger molecules, such as poly-ethyleneglycol (PEG)- or dextran-conjugated fluorophores are larger and consequently result in lower permeability overall and lower sensitivity. The apparent permeability (Papp, cm/s) of tracer dyes can be used to determine barrier function properties of organs or tissues (Figure 5). The following equation derived by Tran, et al.19 can be used to calculate Papp between the dosing channel and receiving channel, which partially corrects for tracer dye loss caused by absorption into PDMS by averaging the two output flows and not relying on mass balance assumptions at the outflow.
<img alt="Equation 1" src="/files/ftp_upload/58151/58151eq1v2.jpg" />
The References section has been updated&#160;from:
<ol start="19">
	<li>Blaser, D.W. Determination of drug absorption parameters in Caco-2 cell monolayers with a mathematical model encompassing passive diffusion, carrier-mediated efflux, non-specific binding and phase II metabolism. at &#60;http://edoc.unibas.ch/655/1/DissB_7998.pdf&#62; (2007).</li>
	<li>Hubatsch, I., Ragnarsson, E.G.E., Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nature Protocols. 2 (9), 2111-2119 (2007).</li>
	<li>Henry, O.Y.F. et al. Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function. Lab on a Chip. 17 (13), 2264-2271 (2017).</li>
	<li>Maoz, B.M. et al. Organs-on-Chips with combined multi-electrode array and transepithelial electrical resistance measurement capabilities. Lab on a Chip. 17 (13), 2294-2302 (2017).</li>
	<li>Benam, K.H. et al. Matched-Comparative Modeling of Normal and Diseased Human Airway Responses Using a Microengineered Breathing Lung Chip. Cell Systems. 3 (5), 456-466.e4 (2016).</li>
</ol>
to:
<ol start="19">
	<li>Tran, T.T. et al. Exact kinetic analysis of passive transport across a polarized confluent MDCK cell monolayer modeled as a single barrier. Journal of Pharmaceutical Sciences. 93 (8), 2108&#8211;2123 (2004).</li>
	<li>Henry, O.Y.F. et al. Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function. Lab on a Chip. 17 (13), 2264-2271 (2017).</li>
	<li>Maoz, B.M. et al. Organs-on-Chips with combined multi-electrode array and transepithelial electrical resistance measurement capabilities. Lab on a Chip. 17 (13), 2294-2302 (2017).</li>
	<li>Benam, K.H. et al. Matched-Comparative Modeling of Normal and Diseased Human Airway Responses Using a Microengineered Breathing Lung Chip. Cell Systems. 3 (5), 456-466.e4 (2016).</li>
</ol>

An erratum was issued for: <a href="https://www.jove.com/video/58151/scalable-fabrication-stretchable-dual-channel-microfluidic-organ">Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips</a>.&#160; The Representative Results, Discussion, and References sections have been updated.

Erratum: Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

Here, we describe the fabrication of dual channel, vascularized Organ-on-a-Chip (Organ Chip) microfluidic culture devices using a scalable protocol amenable for use by research groups lacking access to cleanrooms and traditional soft lithography tools. These devices have been developed to recapitulate human organ-level functions for understanding normal and disease physiology, as well as drug responses in vitro1,2. Critical to engineering this functionality are two perfused microfluidic channels separated by a semi-permeable membrane (Figure 1). This design enables recreation of tissue-tissue interfaces between at least two types of tissues, typically organ parenchymal cells on one side of the porous membrane and vascular endothelium on the other, as well as their exposure to fluid flow. In addition, because the elastomeric polymer, poly (dimethyl siloxane) (PDMS), is used to fabricate the Organ Chip body and membrane components, cyclic mechanical strain can be applied to the entire engineered tissue-tissue interface via the elastic membrane to mimic the natural physical microenvironment of living organs, such as breathing motions in the lung and peristalsis in the intestine.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58151/58151fig1.jpg" /> 
Figure 1: Organ Chip cross section. Organ Chips consist of two channels separated by a porous, elastic membrane that can be seeded with cells on both sides. Top channel cross sections are 1 mm wide x 1 mm high, bottom channel cross sections are 1 mm wide x 0.2 mm high, and vacuum channels in both and bottom parts are 0.3 mm wide, 0.5 mm high, and spaced 0.3 mm from the fluidic channels.&#160;<a href="https://www.jove.com/files/ftp_upload/58151/58151fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
These stretchable, dual channel, Organ Chips have been used for demonstrating the impact of breathing motion on nanoparticle absorption in the lung and drug-induced pulmonary edema3,4; effects of peristaltic motion on differentiation5 and bacterial overgrowth in the intestine5,6,7; and influence of cyclic deformations due to the pulsation of the heart on differentiation and maturation of glomerular podocytes in the kidney8. Additionally, these two-lumen devices that contain an endothelium-lined vascular channel separated by an extracellular matrix (ECM)-coated membrane from parenchymal cells within a separately accessible channel are well suited for characterization of drug PK parameters and new target discovery, which has been limited in single perfusion channel systems. Moreover, multiple Organ Chips may be linked together via their vascular channels to effectively create a human body-on-chips, which could offer an attractive human in vitro platform for therapeutics development9,10. Unlike most micro-physiological systems (MPS)11,12,13, the Organ Chips contain two microfluidic channels separated by a porous membrane that facilitates vascular-parenchymal interactions to recapitulate in vivo organ function. This not only simplifies linking of different organs together by perfusing a common medium through the vascular channels, but the compartmentalization of tissues and fluids mimics in vivo functions and supports pharmacokinetic experimentation and modeling as well as in vitro-in vivo extrapolation9,10 that is difficult or impossible in single channel MPS14,15,16. The popularity of PDMS in microfluidic devices has led to the development of tools to overcome the material&#39;s inherent ability to absorb small molecules10,17. However, the large numbers of chips required to support biological studies where the use of microbial agents and PDMS-absorbing compounds make reuse of Organ Chips difficult necessitates a scalable manufacturing process even for small research groups. The protocol described here presents a method for the device fabrication suitable for the use in academic laboratories, including those lacking access to cleanrooms and soft lithography. This protocol aims to broaden access to Organ Chips by a broad range of researchers seeking to use the stretchable, dual-channel devices for exploring basic biological processes as well as translational therapeutic development.
Leveraging best practices from micromanufacturing fields coupled with design for manufacturing, a robust approach was developed for fabricating Organ Chip devices in large quantities with high reproducibility and yield. The fabrication protocol described here provides a scalable method for Organ Chip production. We describe the use of an optional Mold-in-Place Jig (MiP; design details in Supplemental Materials) coupled with polyurethane gasket strips to enable scaling up of casting PDMS components. The glossy side of polyurethane strips produce optically smooth PDMS parts while the textured side facilitates demolding. We also describe the use of an optional Automated Membrane Fabricator (AMF) that provides uniform compression of membrane wafer molds during curing for fabricating up to 24 membranes per batch. The design is broadly applicable for studies of organs that are composed of tissues that experience mechanical strain and perfusion, and these chips can be produced with low chip-to-chip variability in quantities required to meet the needs of small and large research groups alike. The workflow is amenable to a batch or assembly line format, and readily compatible with quality assessment protocols for control of production processes, personnel training, and responsive troubleshooting. We hope that this protocol will expand access to the capabilities of dual channel, stretchable, Organ Chips for basic and translational research.

<table border="0" cellpadding="0" cellspacing="0" style="width:1212px;" width="1211">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:501px;">Personal Protective Equipment</td>
			<td style="width:179px;"></td>
			<td style="width:133px;"></td>
			<td style="width:399px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hairnet</td>
			<td>VWR</td>
			<td>89107-770</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tyvek lab coat</td>
			<td>VWR</td>
			<td>13450-506</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Extended cuff gloves</td>
			<td>VWR</td>
			<td>89521-898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cutting mat</td>
			<td>VWR</td>
			<td>102096-430</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tile cutter</td>
			<td>McMaster-Carr</td>
			<td>26765A31</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mold-in-place (MIP) top molds</td>
			<td>Protolabs, Inc.</td>
			<td>custom</td>
			<td>printed in Prototherm 12120</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mold-in-place (MIP) bottom molds</td>
			<td>Protolabs, Inc.</td>
			<td>custom</td>
			<td>printed in Prototherm 12121</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Duckbill curved forceps</td>
			<td>VWR</td>
			<td>63041-864</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sharp tipped forceps</td>
			<td>Electron Microscopy Sciences</td>
			<td>72700-D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Metal spatula</td>
			<td>VWR</td>
			<td>&#160;82027-528</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Deep reactive ion etch (DRIE)&#160; pillar array wafers</td>
			<td>Sensera, Inc.</td>
			<td>custom</td>
			<td>Four 50 x 50 mm pillar arrays per wafer; pillars 7 um wide, 50 um tall, spaced hexagonally 40 um apart</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Textured polycarbonate .01&#8221; thick</td>
			<td>McMaster-Carr</td>
			<td>85585K33</td>
			<td>cut to 45 mm square</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PDMS blocks (40 x 40 x 5 mm)</td>
			<td>n/a</td>
			<td>custom</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminar flow hood</td>
			<td>Germfree</td>
			<td>BVBI</td>
			<td>cast in-house</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Air gun</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">60&#176;C level oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vacuum desiccator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mass balance</td>
			<td></td>
			<td></td>
			<td>accuracy to 0.1 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma machine</td>
			<td>Diener</td>
			<td>Nano</td>
			<td>oxygen plasma capability is critical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sylgard 184 poly (dimethylsiloxane) (PDMS) base/curing agent kit</td>
			<td>Ellsworth Adhesives&#160;</td>
			<td>4019862</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mixing cup</td>
			<td></td>
			<td></td>
			<td>Ensure adequate ventilation when handling prepolymer due to low levels of ethylbenzene</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 mL syringe</td>
			<td>VWR</td>
			<td>10099-395</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cleanroom wipes</td>
			<td>VWR</td>
			<td>TWTX1080</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">25 x 75 mm glass microscope slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Packing tape</td>
			<td>VWR</td>
			<td>500043-724</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scotch tape</td>
			<td>VWR</td>
			<td>500026-873</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Die-cut Polyurethane (PU) strips</td>
			<td>Atlantic Gasket, Inc.</td>
			<td>custom: AGWI2X3</td>
			<td>&#160;1/8&#8221; thick; 60 Durometer Black Polyurethane; 2&#8221; x 3&#8221;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polycarbonate film .005&#8221; thick</td>
			<td>McMaster-Carr</td>
			<td>85585K102</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 x 100 x 15 mm square gridded petri dishes</td>
			<td>VWR</td>
			<td>60872-480</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Aluminum foil</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optional Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thinky PDMS Mixer</td>
			<td>Thinky</td>
			<td>ARE-310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mold-in place (MIP) jig</td>
			<td>in-house</td>
			<td></td>
			<td>screw clamp compression jig</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Automated membrane fabricator (AMF)</td>
			<td>in-house</td>
			<td></td>
			<td>pneumatic compression piston array with programmable heater</td>
		</tr>
	</tbody>
</table>

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.

The protocol presented here describes the scalable fabrication of PDMS Organ Chips. These devices enable culture of two distinct perfused tissue types on an elastic porous membrane (Figure 1). The PDMS channels are cast using 3D printed molds, which accelerates prototyping of new designs (Figure&#160;2A and 2B). Top channels are cast in molds under compression against a compliant polyurethane gasket to produce co...

Watch this Scientific Journal Video about Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips at JoVE.com

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

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 ...

The overall goal of this protocol is to describe the fabrication of organ chipped microfluidic devices for recapitulating organ level functionality in vitro. This protocol describes a way to fabricate organ chipped devices recapitulating organ level function in vitro. The func, the devices, such as these, are actually fabricated using 3D printed molds out of a soft silicon rubber.This rubber enables us to actually imbue these devices with mechanical cues that enables us to stretch the tissue as you would get in let's say a lung or a gut. We also add profusion, which mimics blood flow and the flow of other bodily fluids within organ systems. Now taken together, these devices enable us to actually recreate and try to understand the complex physiology that happens in vivo.But do this in vitro. So essentially we're experimenting on humans, but not on people. And so it's a very effective way of bridging the divide between animal studies, that are always done in pre-clinical development of therapeutics, and then before human studies, that are called clinical trials, where there is a much greater risk to the human, to human safety.And so these devices help bridge the divide and actually enable us to develop new therapeutics and understand the basic biology that actually happens in complex systems such as human. Top channel preparation. Wipe down the glossy side of each polyurethane piece with ethanol and clean room wipes.Place glossy side of the polyurethane over the open side of the mold in place mold. Place the mold and polyurethane assemblies into the jig, textured side against the end of the jig. Continue to do this until all the molds have been placed within the jig.Tighten the jig by turning its handle using a wrench, until the jig spacing is 25 millimeters in width. Make a boat of aluminum foil surrounding the mold in place jig. Pour PDMS into each mold's well until full.Once the entire jig is filled, place the jig into the vacuum desiccator. After one hour, remove from desiccator. And place into 60 degree Centigrade oven for at least four hours.Disassemble the jig using a wrench. Remove the polyurethane strips from each mold. And discard.Carefully demold PDMS parts from their molds and lay them feature side up. Line up the blade of the tile scraper at the end tab notch. And cut away each end to cingulate the top components.Store finished parts in square Petri dishes. Store parts in positive pressure cabinets at room temperature. Bottom channel preparation.Pour 10.5 grams of PDMS into the mold. The air gun can be used very gently to move the PDMS over the space. Place molds into vacuum desiccator.After one hour, move the molds to a level 60 degree Centigrade oven. Place mold on table in a laminar flow hood. Loosen the PDMS from edge of the mold.Grip one corner and gently peel back the PDMS from the mold's surface. When fully removed, invert and lay on work surface so that channel features are face up. Cut along outside edges with tile cutter.With flat paddle forceps, lay parts feature side up on tape to remove any debris. Remove part from packing tape. Drag the loose end of the part across the slide.The loose end will laminate with the glass. Cover features with Scotch tape. Store parts in positive pressure cabinets at room temperature.PDMS membrane preparation. Check that the wafers are free of PDMS on the back. Place each membrane wafer in the designated slots.Use the one mL syringe to place 09 mL of PDMS onto the center of each membrane wafer post array. Let the PDMS sit for a minimum of five minutes. Allow PDMS to spread throughout the post of the membrane wafer.Do not proceed to the next step until at least 75%of the post array is covered in PDMS. Plasma treat the polycarbonate strip using the conditions described in the protocol. Remove polycarbonate sheet from the plasma machine and use scissors to cut the polycarbonate sheets into 45 millimeter by 45 millimeter squares.Lay the plasma treated side of the polycarbonate squares onto the PDMS, centered on the membrane wafer. Place PDMS spacer on the center of the polycarbonate square. Then place precut textured polycarbonate.Insert trays so that tray three is in the back, tray two is in the middle, and tray one is in the front. Tray one has a notch for alignment. Open the output pressure valve, and very slowly open the input pressure valve.This is so that the four kilograms of force is gradually applied to each membrane wafer, as opposed to instantly which may break the wafers. Flip the AMF switch to on to begin the curing cycle. Then close the input pressure valve and open the output pressure valves to release the pressure from the air cylinders.Remove the trays and bring them to the laminar flow hood. Carefully peel off the textured polycarbonate. Carefully remove the PDMS spacer.Inspect the PDMS membrane for areas with through holes. Use a marker to trace the outline of the through hole area. Also mark any holes or defects on the membranes.This is an example of an unmarked and marked membrane. Using wafer handling tweezers, loosen wafers from the tray. Remove each membrane from the wafer and place on Petri dish.The PDMS membrane will demold from the wafer and will be bonded to the polycarbonate backing. Store parts in positive pressure cabinets at room temperature. Top assembly and preparation.Using matte finished Scotch tape, clean the PDMS membranes as well as the insides of the Petri dish to remove debris. Thoroughly tape the feature side of each tall channel top to remove debris. Place tops in Petri dish with PDMS membrane.Be aware that some membranes may take one or two top parts. The main channels of each top part should fit within the marked area within the membrane. Load the Petri dishes into the plasma.Plasma treat membrane and top according to the written protocol. Once the bonding cycle has finished, remove the dishes and lay the activated top parts onto the membrane. Place in 60 degree Centigrade oven for at least two hours.Using a scalpel, trace around the perimeter of the bonded top to separate it from the polycarbonate carrier. Once the part is traced, peel the top from the polycarbonate. The PDMS membrane that has bonded to the top should peel from the carrier as well.Using tweezers remove the membrane from the ports that access the bottom channel. Do not leave any parts of the membrane covering the access port. Additionally remove any debris or dust with tweezers.Chip assembly. Feature side up, plasma treat top assemblies with bottoms. Under an inverted microscope, align the top assembly with the bottom half.Place in 60 degree Centigrade oven for at least two hours.Results. The protocol presented here describes the scalable fabrication of PDMS organ chips. These devices enable culture of two distinct profuse tissue types on an elastic porous membrane.The PDMS channels are cast using 3D printed molds, which accelerates prototyping of new designs. Top channels are cast in molds under compression against a compliant polyurethane gasket to produce components with molded ports. While bottom channel components are cast in trays and handled on microscope slide backing.This fabrication approach combines multi scale patterning of the parts into a single step, which saves time, improves reproducibility and traceability, and reduces debris generated by port punching and multiple cutting steps. The porous membranes are critical to the function of the organ chip, and the fabrication approach based on casting against patterned silicon wafers results in membranes of consistent thickness and surface finish. Handling via polycarbonate carriers allows for larger batch production and storage.The assembled organ chip consists of two profusion channels in an optically transparent package. In the overlapping region, a porous PDMS membrane enables tissue-tissue interaction of metabolites, proteins, therapeutics, pathogens, and cells to recapitulate organ chip function while two parallel channels on either side are used to provide mechanical strain, using cyclic vacuum actuation. The porosity of the PDMS membrane bio-medically supports the flux of metabolites, growth factors, and even cells between the vasculature and organ parenchyma.The apparent permeability of the membrane was determined using the di-concentration in the outlet channels with and without Caco-2 gut cells. The gut chip cell layers provide a significantly increased barrier to permeability. The organ chip can be actuated using the parallel vacuum channels to quantitatively and reproducibly apply cyclic strain loading to the membrane, and therefore the cultured tissues.This cyclic strain combined with media profusion supports cellular differentiation to better mimic in vivo organ physiology, such as formation of villi in the gut chip. Using the protocol described here, you should now be able to fabricate a stretchable PDMS organ chip.

Research

JoVE Journal

Bioengineering

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