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.

scalable-fabrication-stretchable-dual-channel-microfluidic-organ

Research

JoVE Journal

Bioengineering

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

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 ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Image-guided, Laser-based Fabrication of Vascular-derived Microfluidic Networks

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic networks embedded in PEGDA hydrogels. Here, we describe the creation of virtual masks that allow for image-guided laser control; the photopolymerization of a micromolded PEGDA hydrogel, suitable for microfluidic network fabrication and pressure head-driven flow; the setup and use of a commercially available laser scanning confocal microscope paired with a femtosecond pulsed Ti:S laser to induce hydrogel degradation; and the imaging of fabricated microfluidic networks using fluorescent species and confocal microscopy. Much of the protocol is focused on the proper setup and implementation of the microscope software and microscope macro, as these are crucial steps in using a commercial microscope for microfluidic fabrication purposes that contain a number of intricacies. The image-guided component of this technique allows for the implementation of 3D image stacks or user-generated 3D models, thereby allowing for creative microfluidic design and for the fabrication of complex microfluidic systems of virtually any configuration. With an expected impact in tissue engineering, the methods outlined in this protocol could aid in the fabrication of advanced biomimetic microtissue constructs for organ- and human-on-a-chip devices. By mimicking the complex architecture, tortuosity, size, and density of in vivo vasculature, essential biological transport processes can be replicated in these constructs, leading to more accurate in vitro modeling of drug pharmacokinetics and disease.

This protocol outlines the implementation of image-guided, laser-based hydrogel degradation to fabricate vascular-derived, biomimetic microfluidic networks embedded in poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These biomimetic microfluidic systems may be useful for tissue engineering applications, generation of in vitro disease models, and fabrication of advanced "on-a-chip" devices.

This detailed protocol outlines the implementation of image-guided, laser-based hydrogel degradation for the fabrication of vascular-derived microfluidic ...

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

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

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

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

A Simple and Scalable Fabrication Method for Organic Electronic Devices on Textiles

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices cannot be worn under everyday conditions. Therefore, textiles are commonly considered the best substrate to accommodate electronic devices in wearable use. In this paper, we describe how to selectively pattern organic electroactive materials on textiles from a solution in an easy and scalable manner. This versatile deposition technique enables the fabrication of wearable organic electronic devices on clothes.

In this paper, we present a protocol to selectively deposit organic materials on textiles, which allows for the direct integration of organic electronic devices with wearables. The fabricated devices can be fully integrated in textiles, respecting their mechanical appearance and enabling sensing capabilities.

Today, wearable electronics devices combine a large variety of functional, stretchable, and flexible technologies. However, in many cases, these devices ...

Reconfigurable Microfluidic Channel with Pin-discretized Sidewalls

Microfluidic components need to have various shapes to realize different key microfluidic functions such as mixing, separation, particle trapping, or reactions. A microfluidic channel that deforms even after fabrication while retaining the channel shape enables high spatiotemporal reconfigurability. This reconfigurability is required in such key microfluidic functions that are difficult to achieve in existing "reconfigurable" or "integrated" microfluidic systems. We describe a method for the fabrication of a microfluidic channel with a deformable sidewall consisting of a laterally aligned array of the ends of rectangular pins. Actuating the pins in their longitudinal directions changes the pins' end positions, and thus, the shape of discretized channel sidewalls.Pin gaps can cause unwanted leakage or adhesion to adjacent pins caused by meniscus forces. To close the pin gaps, we have introduced hydrocarbon-fluoropolymer suspension-based gap filler accompanied by an elastomeric barrier. This reconfigurable microfluidic device can generate strong temporal in-channel displacement flow, or can stop the flow in any region of the channel. This feature will facilitate, on demand, the handling of cells, viscous liquids, gas bubbles, and non-fluids, even if their existence or behavior is unknown at the time of fabrication.

A microfluidic channel with deformable sidewalls offers flow control, particle handling, channel dimension customization and other reconfigurations while in use. We describe a method for fabricating a microfluidic channel with sidewalls made of an array of pins that allows their shape to change.

Microfluidic components need to have various shapes to realize different key microfluidic functions such as mixing, separation, particle trapping, or ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.