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>
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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. 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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...

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>
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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 ...

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Bioengineering

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