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. 
	CAUTION: Do not use metal forceps to remove debris as it will damage the surface of the molds.</li>
</ol>
2. Top Channel Preparation
<ol>
	<li>Wipe down the glossy side of each polyurethane piece with ethanol and cleanroom wipes. Make sure all residual ethanol is dried from the polyurethane surface.</li>
	<li>Place the glossy side of the polyurethane over the open side of the MiP mold to create a seal on the open side of the mold, leaving only a well-like opening at the top of the mold for pouring PDMS. 
	NOTE: Check that every mold is covered securely by polyurethane piece or the PDMS will leak from molds during pouring.</li>
	<li>Place the mold and polyurethane assemblies into a MiP jig, with the textured side against the end of the MiP jig. Continue to do this until all molds have been placed in the jig.</li>
	<li>Tighten the MiP jig by turning the handle using a wrench until the jig spacing is 25 mm in width.</li>
	<li>Make a boat of aluminum foil surrounding the MiP jig to prevent excess PDMS from leaking onto surfaces.</li>
	<li>Pour PDMS into each of the molds well until full. 
	NOTE: Each chip top component requires about 3 mL of PDMS.</li>
	<li>Once the entire jig is filled, place the jig into the vacuum desiccator. Pull vacuum at -80 kPa for 1 h to degas PDMS.</li>
	<li>After 1 h, remove the MiP jig from the desiccator and place in 60 &#176;C oven for at least 4 h to cure PDMS.</li>
	<li>Disassemble the MiP jig using a wrench, loosen the jig by turning the handle counter clockwise. Once molds are free from compression, remove molds from jig.</li>
	<li>Remove the polyurethane strips from each mold and discard.</li>
	<li>Carefully de-mold PDMS parts from their molds and lay them feature-side up.</li>
	<li>Line up the blade of tile scraper at end tab notch and cut away each end to singulate the top components.</li>
	<li>Check parts for any of the following failure modes and discard any unsatisfactory parts: scratches in the main channel, large debris above the channel area, large bubbles, deformed vacuum channels.</li>
	<li>Store finished parts in square Petri dishes within pressure positive cabinets at room temperature.</li>
</ol>
3. Bottom Channel Preparation
<ol>
	<li>Pour approximately 10.5 g of PDMS into molds until the PDMS reaches the top of the cavity.
	<ol>
		<li>Inspect the bottom channel mold for PDMS cured to the bottom of mold.</li>
		<li>If dirty, scrape old PDMS from the bottom of the mold since an uneven surface on the bottom of the mold can cause uneven thickness of the final parts. 
		NOTE: For small &#60;2 cm2 areas that are uncovered, the air gun can be used very gently to move PDMS over the space.</li>
	</ol>
	</li>
	<li>Place molds into vacuum desiccator for 1 h.</li>
	<li>After 1 h, move the molds into a level 60 &#176;C oven for &#62;4 h.</li>
	<li>Place mold on table in laminar flow hood. Loosen PDMS from one edge of the mold.</li>
	<li>Grip one corner and gently peel back the PDMS from the mold surface.</li>
	<li>When fully removed, lay on work surface, so that channel features are face up.</li>
	<li>Cut parts along outside edges with tile cutters, placing tile cutter blade in notched PDMS as in step 2.12.</li>
	<li>Lay parts feature side up on tape to remove any debris.</li>
	<li>Remove part from packing tape. Drag the loose end of the part across the slide. The loose end will laminate with the glass. 
	NOTE: It is critical to avoid stretching the part while laying it down. If bubble is trapped between part and glass, gently lift the part with the forceps and re-lay.</li>
	<li>Perform quality control inspection of parts. Check parts for any failure modes and discard any unsatisfactory parts, including ones that contain scratches in the main channel, large debris, large bubbles, or deformed vacuum channels.</li>
	<li>Cover features with tape.</li>
	<li>Store parts in a positive pressure cabinet at room temperature.</li>
</ol>
4. PDMS Membrane Preparation
<ol>
	<li>Check that the wafers are free of PDMS on the back.</li>
	<li>Place each membrane wafer in the designated slots in the AMF trays.</li>
	<li>Use the 1 mL syringe to place 0.09 mL of PDMS onto the center of each membrane wafer post array. Let PDMS sit for a minimum of 5 min to allow PDMS to spread throughout the posts of the membrane wafer. 
	NOTE: Do not proceed to next step until at least 75% of the post array is covered in PDMS. The quality of membranes improves the longer the PDMS is allowed to wick into post region so longer wait times in this step are preferred.</li>
	<li>Plasma treat the polycarbonate strip at 20 W for 45 s, O2 gas at 0.80 mbar in a plasma machine.</li>
	<li>Remove the polycarbonate sheet from the plasma machine and use scissors to cut the polycarbonate sheets into 45 mm x 45 mm squares. 
	NOTE: Minimize contact with the plasma treated surface to prevent dust from sticking to the polycarbonate.</li>
	<li>Gently lay the plasma treated side of the polycarbonate squares onto the liquid PDMS centered on the membrane wafer. Ensure that the polycarbonate and the PDMS are in contact. 
	NOTE: Avoid air pockets between the polycarbonate and the PDMS.</li>
	<li>Place the pre-cut PDMS spacer on the center of the polycarbonate square.</li>
	<li>Place the pre-cut textured polycarbonate sheet on the PDMS block to keep the assembly from bonding to the compression plate.</li>
	<li>Insert tray so that Tray 3 is in the back, Tray 2 is in the middle, and Tray 1 is in the front. Tray 1 has a notch for alignment.</li>
	<li>Open the output pressure valve and very slowly open the input pressure valve. Only then close the output pressure valve. 
	NOTE: This is so that the output 4 kg of force is gradually applied to each membrane wafer as opposed to instantly, which may break the wafers.</li>
	<li>Flip the AMF switch to ON to begin the curing cycle. Cure wafer under 4 kg (16 kPa) of compression and a ramping temperature cycle listed in Table 1.</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0" style="width:323px;" width="322">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="39">
			<td height="39" style="height:39px;width:91px;">Step</td>
			<td style="width:131px;">Temperature (&#176;C)</td>
			<td style="width:101px;">Duration (min)</td>
		</tr>
		<tr height="20">
			<td align="right" height="20" style="height:20px;">1</td>
			<td align="right">20</td>
			<td align="right">20</td>
		</tr>
		<tr height="20">
			<td align="right" height="20" style="height:20px;">2</td>
			<td align="right">35</td>
			<td align="right">10</td>
		</tr>
		<tr height="20">
			<td align="right" height="20" style="height:20px;">3</td>
			<td align="right">45</td>
			<td align="right">10</td>
		</tr>
		<tr height="20">
			<td align="right" height="20" style="height:20px;">4</td>
			<td align="right">50</td>
			<td align="right">60</td>
		</tr>
		<tr height="20">
			<td align="right" height="20" style="height:20px;">5</td>
			<td align="right">60</td>
			<td align="right">120</td>
		</tr>
		<tr height="18">
			<td align="right" height="18" style="height:19px;">6</td>
			<td align="right">20</td>
			<td>Hold</td>
		</tr>
	</tbody>
</table>
Table 1 - Membrane curing conditions
<ol start="12">
	<li>Close the input pressure valve and open the output pressure valve to release pressure from the air cylinders.</li>
	<li>Remove the trays and bring them to the laminar flow hood.</li>
	<li>Carefully peel off the textured polycarbonate and carefully remove the PDMS spacer. 
	NOTE: Watch the PDMS spacer to ensure it does not also peel off the polycarbonate carrier, if this occurs start peeling from a different corner.
	<ol>
		<li>Inspect the PDMS membrane through the polycarbonate carrier for areas with through-holes and use a marker to trace the outline of the through-hole area and mark any holes or defects in the membranes.</li>
		<li>Using wafer handling forceps, loosen wafers from the tray.</li>
		<li>Remove each membrane from the wafer and place in Petri dish. 
		NOTE: The PDMS membrane will de-mold from the membrane wafer and will be adhered to the polycarbonate backing. If PDMS starts detaching from the polycarbonate carrier, peel from a different region.</li>
		<li>Store membranes and wafers in Petri dishes in a positive pressure cabinet at room temperature.</li>
	</ol>
	</li>
</ol>
5. Top Assembly and Preparation
<ol>
	<li>Using matte tape, clean the PDMS membranes as well as the insides of the Petri dish to remove debris.</li>
	<li>Thoroughly tape the feature side of each top component to remove debris.</li>
	<li>Place top channel part (&#34;top&#34;) feature side up in Petri dish with PDMS membrane. 
	NOTE: Be aware that some membranes may be used for one or two top parts depending on size of the usable area. The main channels of each top part should fit within the marked area of the membrane.</li>
	<li>Load the Petri dishes into the plasma machine.</li>
	<li>Plasma treat membrane and top at 20 W for 45 s, O2 gas at 0.80 mbar.</li>
	<li>Once the bonding cycles has finished, remove the dishes and lay the activated parts feature side down on top of the membrane and ensure part is fully laminated with membrane with no bubbles.</li>
	<li>Place parts into 60 &#176;C oven for at least 2 h to anneal.</li>
	<li>Using a scalpel, trace around the perimeter of the bonded top to separate top-membrane assembly from the polycarbonate carrier. 
	NOTE: Do not cut the carrier.</li>
	<li>Once the part is traced, peel the assembly from the polycarbonate. The PDMS membrane that is bonded to the top should peel from the carrier.</li>
	<li>Using sharp tipped forceps, remove the membrane from the ports that access the bottom channel, and remove any debris or dust with forceps under a stereoscope. 
	NOTE: Do not leave any part of the membrane covering the access port.</li>
</ol>
6. Chip Assembly
<ol>
	<li>Feature side up, plasma treat assemblies with bottom components using the conditions in step 5.5.</li>
	<li>Under an inverted microscope, align the top assembly with microscope slide to the bottom half.</li>
	<li>Place in 60 &#176;C oven for at least 2 h.</li>
	<li>Chip Quality Control Inspection 
	NOTE: Pay close attention to the main ports and channel of chip. Check for failure modes by eye and also under the microscope.
	<ol>
		<li>To check that the chip is bonded fully, tug lightly on each corner of the chip to check for delaminating parts.</li>
		<li>Look at the channel of the chip to check for a wrinkled or sagging membrane, which will appear as a wavy pattern or a light deflection in the channel.</li>
		<li>Perform a microscope inspection to inspect for debris in the main channel. 
		NOTE: Debris in non-critical areas, such as the vacuum channels is acceptable.</li>
		<li>With the chip still on the inverted microscope, inspect main channel and vacuum channels for delamination. 
		NOTE: Delamination in non-critical areas (e.g., the edge of the chip) is acceptable.</li>
		<li>Check that the main channels are aligned to within 50-60 &#181;m (1-2 membrane pores). 
		NOTE: It is crucial that the channels are not overlapping with vacuum channels.</li>
		<li>Check that the membrane between the main channels and the inlet and outlet channels is intact without any apparent holes. 
		NOTE: Any hole in the membrane can lead to a leaky chip or cell growth outside the channels.</li>
	</ol>
	</li>
	<li>Store chips in Petri dishes in a positive pressure cabinet at room temperature.</li>
</ol>

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D.E.I. is a founder and holds equity in Emulate, Inc., and chairs its scientific advisory board. J.P. is presently an employee of Emulate, Inc. R.N., Y.C., J.P., and D.E.I. are inventors on intellectual property that has been licensed to Emulate, Inc.

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 ports but also reduces debris in the workplace, enables reproducible port alignment to interface manifolds or instrumentation, and produces parts with control over the fit and sealing of inserted tubing or pins for fluidic and pneumatic connections. The molds are stacked on top of each other in a compression jig, separated by compliant polyurethane sheets to facilitate through-hole casting of ports. By stacking multiple parts in a single jig, a single user can cast large quantities of components complete with ports in a single step. Material selection and manufacturing method for the molds are critical to provide the necessary feature resolution, low surface roughness, and high degree of flatness for device assembly and subsequent imaging applications. Stereolithography can meet these requirements, although materials with high deflection temperatures (&#62; 80 &#176;C) and compatibility with PDMS curing reduce the available polymer range. Various commercially available resins, including glass-filled resins, meet these criteria.
The elastic porous PDMS membrane is arguably the most unique and critical component of an Organ Chip while being the most complex to fabricate. A deep reactive ion etches (DRIE) process outsourced to a vendor is used to microfabricate 50 x 50 mm hexagonal arrays of pillars (7 &#181;m diameter, 40 &#181;m apart, 50 &#181;m tall, C4F8 coated) that are used to pattern pores in the PDMS membrane. The quality of the pillar arrays is critical to achieving robust membrane casting. In particular, pillars must be etched to tight tolerances with smooth vertical profiles to avoid undercuts or excessive sidewall roughness that can lead to mold failure. Care should be taken to avoid &#34;grassing&#34; at the bottom of the etched region, which can affect membrane demolding and cell attachment. Membrane fabrication with successful through-hole patterning and device integration is the single most complex section of the protocol. Critically, placing 0.09 mL of PDMS on each wafer and allowing adequate time for it to spread is essential to avoid incomplete through-hole molding. Properly plasma treating the polycarbonate backing is required to achieving robust backing of the membrane for demolding and bonding steps without wrinkling or stretching. The backing provides a robust means of demolding the cast membrane from the fragile silicon wafer.
The compressive load applied to each wafer is also essential for uniform through-hole fabrication. Earlier efforts using weights hindered membrane production and resulted in poor yields due to non-uniform force distribution. To overcome the production bottleneck, we optimized the previously published membrane fabrication protocol18 and built an Automated Membrane Fabricator (AMF) to parallelize the process. The AMF consists of 24 pneumatic pistons supported over a programmable hot plate to provide controlled compressive force throughout a programmed PDMS curing process. A polycarbonate backing film is placed on the uncured PDMS and then uniformly compressed using pneumatic pistons of the AMF while being heated to polymerize the PDMS. Critically, the gradual curing process described in the protocol results in higher quality membranes than a single step to the maximum temperature, where feathering patterns resulting from bubble development during the curing process were observed. While optional, the AMF increases throughput significantly beyond what is possible using weights in an oven.
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" />
Vr is the volume in mL of receiving channel effluent after time t; Vd is the volume in mL of the dosing channel effluent after time t; A is the area of membrane through-hole region in cm2 (0.167 cm2 for this device); t is the time of effluent collection in seconds; Cr is the measured change in concentration of the tracer dye in the receiving channel effluent; Cd is the measured concentration of the tracer dye in the dosing channel effluent. Key assumptions for this equation to be valid include: 1) steady tracer dye dosing concentration over time t, 2) the concentration of Cr is small compared to Cd, and 3) the permeability of the system is uniformly distributed across the culture region. Although this equation can be used for static systems, care must be taken to check that the assumptions hold true. Electrical methods, including trans-epithelial electrical resistance (TEER) are commonly implemented in Transwell studies and recently have been incorporated into PDMS Organ Chips for instant and continuous barrier function measurements as well20,21.

Limitations of this protocol include the elasticity of PDMS as well as the manual casting and assembly process that limits production rates. PDMS is a versatile polymer that is well-suited for Organ Chips requiring mechanical strain actuation, but its elasticity can hinder production. Parts can be difficult to handle without deformation and membranes require backing films for manipulation. As a result, automation of Organ Chip production can be limited. The casting process, unlike hot embossing or injection molding used for thermoplastic polymers, is batch-based and therefore also limits throughput.
Organ Chips enable in vitro studies of human organ- and body-level functions in vivo by perfusing a common medium through the vascular channels. By reconstituting physiological tissue-tissue interfaces, flux of molecules between the vascular and parenchymal compartments, mechanical cues, and fluidic shear and transport, these devices promote histodifferentiation and are capable of recapitulating in vivo-like functions of both normal and diseased organs. The compartmentalization of tissues and fluids in two compartments mimics their in vivo functions, and Organ Chip studies are amenable to time-resolved 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 microchannel structures can be leveraged for other applications, including investigating the impact of dynamic tobacco smoke exposure with bidirectional breathing in human small airway epithelium to develop novel biomarkers of lung damage22. The defined positions of the planar membranes and high optical clarity of the devices make them uniquely suited for image-based analyses and integration of embedded sensors. The mechanical stimulation enabled by integrated vacuum channels and elastomeric materials provides functionalities not possible in Transwell systems. We have demonstrated that mechanical strain is essential for recapitulation of certain in vivo physiological functions, including nanoparticle absorption in the lung4, pulmonary edema3 and differentiation of mature iPS-derived glomerular podocytes8.
Future applications of this protocol may include integration of various sensing modalities that can be used to provide real-time readouts of Organ Chip response to stimuli such as drugs, toxins, or radiation. The protocol presented here could be extended to non-PDMS materials with different optical, mechanical, and chemical properties, including biodegradable materials. The Organ Chip protocol presented here should enable researchers to fabricate devices that offer a high degree of control over the microenvironment of healthy and pathophysiologic tissues and organs, which can be leveraged for therapeutic development, including target discovery, toxicity and pharmacokinetic assessments, as well as for personalized medicine.

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><tbody><tr><td>&lt;em&gt;Personal Protective Equipment&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Hairnet</td><td>VWR</td><td>89107-770</td><td></td></tr><tr><td>Tyvek lab coat</td><td>VWR</td><td>13450-506</td><td></td></tr><tr><td>Extended cuff gloves</td><td>VWR</td><td>89521-898</td><td></td></tr><tr><td>&lt;em&gt;Equipment&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Cutting mat</td><td>VWR</td><td>102096-430</td><td></td></tr><tr><td>Tile cutter</td><td>McMaster-Carr</td><td>26765A31</td><td></td></tr><tr><td>Mold-in-place (MIP) top molds</td><td>Protolabs, Inc.</td><td>custom</td><td>printed in Prototherm 12120</td></tr><tr><td>Mold-in-place (MIP) bottom molds</td><td>Protolabs, Inc.</td><td>custom</td><td>printed in Prototherm 12121</td></tr><tr><td>Duckbill curved forceps</td><td>VWR</td><td>63041-864</td><td></td></tr><tr><td>Sharp tipped forceps</td><td>Electron Microscopy Sciences</td><td>72700-D</td><td></td></tr><tr><td>Metal spatula</td><td>VWR</td><td>&amp;nbsp;82027-528</td><td></td></tr><tr><td>Deep reactive ion etch (DRIE)&amp;nbsp; 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><td>Textured polycarbonate .01&amp;rdquo; thick</td><td>McMaster-Carr</td><td>85585K33</td><td>cut to 45 mm square</td></tr><tr><td>PDMS blocks (40 x 40 x 5 mm)</td><td>n/a</td><td>custom</td><td></td></tr><tr><td>Laminar flow hood</td><td>Germfree</td><td>BVBI</td><td>cast in-house</td></tr><tr><td>Air gun</td><td></td><td></td><td></td></tr><tr><td>60&amp;deg;C level oven</td><td></td><td></td><td></td></tr><tr><td>Vacuum desiccator</td><td></td><td></td><td></td></tr><tr><td>Mass balance</td><td></td><td></td><td>accuracy to 0.1 g</td></tr><tr><td>Plasma machine</td><td>Diener</td><td>Nano</td><td>oxygen plasma capability is critical</td></tr><tr><td>&lt;em&gt;Supplies&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Sylgard 184 poly (dimethylsiloxane) (PDMS) base/curing agent kit</td><td>Ellsworth Adhesives&amp;nbsp;</td><td>4019862</td><td></td></tr><tr><td>Mixing cup</td><td></td><td></td><td>Ensure adequate ventilation when handling prepolymer due to low levels of ethylbenzene</td></tr><tr><td>1 mL syringe</td><td>VWR</td><td>10099-395</td><td></td></tr><tr><td>Cleanroom wipes</td><td>VWR</td><td>TWTX1080</td><td></td></tr><tr><td>25 x 75 mm glass microscope slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Packing tape</td><td>VWR</td><td>500043-724</td><td></td></tr><tr><td>Scotch tape</td><td>VWR</td><td>500026-873</td><td></td></tr><tr><td>Die-cut Polyurethane (PU) strips</td><td>Atlantic Gasket, Inc.</td><td>custom: AGWI2X3</td><td>&amp;nbsp;1/8&amp;rdquo; thick; 60 Durometer Black Polyurethane; 2&amp;rdquo; x 3&amp;rdquo;</td></tr><tr><td>Polycarbonate film .005&amp;rdquo; thick</td><td>McMaster-Carr</td><td>85585K102</td><td></td></tr><tr><td>100 x 100 x 15 mm square gridded petri dishes</td><td>VWR</td><td>60872-480</td><td></td></tr><tr><td>&amp;nbsp;Aluminum foil</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Optional Equipment&lt;/em&gt;</td><td></td><td></td><td></td></tr><tr><td>Thinky PDMS Mixer</td><td>Thinky</td><td>ARE-310</td><td></td></tr><tr><td>Mold-in place (MIP) jig</td><td>in-house</td><td></td><td>screw clamp compression jig</td></tr><tr><td>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 components with molded ports (Figure 2C) while bottom channel components are cast in trays and handled on microscope slide backing (Figure 2D). 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 (Figure 3). Handling via polycarbonate carriers allows for larger batch production and storage.
The assembled Organ Chip (Figure 4) consists of two perfusion 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 biomimetically supports the flux of metabolites, growth factors, and even cells between the vasculature and organ parenchyma (Figure 5). The apparent permeability (Papp, cm/s) of the membrane was determined using the dye concentration in the outlet channels with and without Caco2 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 (Figure 6). This cyclic strain combined with media perfusion supports cellular differentiation to better mimic in vivo organ physiology, such as formation of villi in the Gut Chip.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58151/58151fig2.jpg" /> 
Figure 2: Channel fabrication with 3D printed molds. Organ chip parts are cast against high resolution 3D printed molds (A and B), which allows for greater design versatility and prototyping than traditional soft lithography. Top channel parts (C) are cured under compression eliminating the need for punching ports in the finished parts. Each triplicate casting is singulated with a single cut. Bottom channel parts (D) are placed on glass slides to facilitate ease of use and imaging. Scale bars are approximately 1 cm in all images. <a href="https://www.jove.com/files/ftp_upload/58151/58151fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58151/58151fig3.jpg" /> 
Figure 3: The porous PDMS membrane is cast using DRIE patterned silicon wafers. (A) Rendering of&#160;7 &#181;m diameter, 50 &#181;m tall micropillars etched using DRIE into a silicon wafer. (B) PDMS is cured on this array under 4 kg of compression (16 kPa) to create a 50 &#181;m thick membrane with an array of 7 &#181;m diameter though holes spaced hexagonally 40 &#181;m apart. <a href="https://www.jove.com/files/ftp_upload/58151/58151fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58151/58151fig4.jpg" /> 
Figure 4: Photograph of an assembled PDMS Organ Chip. Red dye fills the larger apical channel used for parenchymal cells while the blue dye highlights the basal channel typically used for vascular endothelium. <a href="https://www.jove.com/files/ftp_upload/58151/58151fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58151/58151fig5.jpg" /> 
Figure 5: Permeability of inert tracer Cascade Blue through the microporous PDMS membrane.&#160;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. <a href="https://www.jove.com/files/ftp_upload/58151/58151fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58151/58151fig6.jpg" /> 
Figure 6: Application of membrane strain using vacuum side channels. Plot indicates linear strain modulation of membrane in response to an applied vacuum pressure. Cyclic uniaxial strain is applied uniformly to the culture region of the Organ Chip using applied vacuum to parallel side channels. The Strain correlates linearly with decreasing vacuum pressure at approximately 1% strain for every -10 kPa change in vacuum pressure (R2 = 0.992). Error bars indicate standard deviation of the mean.&#160;<a href="https://www.jove.com/files/ftp_upload/58151/58151fig5large.jpg" style="font-size: 14px;" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Materials:&#160;<a href="https://www.jove.com/files/ftp_upload/58151/MIP_Jig.7z" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Universidad Científica del Sur

Research

JoVE Journal

Bioengineering

26.6K Views.  Harvard University. 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.

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

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.

The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.

Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.

The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.

We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays.  ...

Biology

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging techniques.
The one-time use packaging technique employs UV-based and temperature curing epoxies to connect microtubes to access holes, wire-bonding for integrated circuit connections, and silver epoxy for electrical connections. This method is based on a robust assembly technique that can support relatively high pressure close to 1 psi and does not need any support to strengthen the microfluidic architecture.
Reusable packaging techniques consist of PDMS-based microtube interconnectors and anisotropic adhesive films for electrical connections. These devices are more sensitive and fragile. Consequently, Plexiglas support is added to the microfluidic structure to improve the electrical contact when anisotropic adhesive films are used, and also to strengthen the microfluidic architecture. In addition, a micromanipulator is needed to maintain tubes while using a thin PDMS layer to connect them to the access holes. Different PDMS layer thicknesses, ranging from 0.45-3 mm, are tested to compare the best adherence versus injection rates. Applied injection rates are varied from 50-300 &#956;l/hr for 0.45-3 mm PDMS layers, respectively. These techniques are mainly applicable for low-pressure applications. However, they can be extended for high-pressure ones through plasma-oxygen process to permanently seal the PDMS to glass substrates. The main advantage of this technique, besides the fact that it is reusable, consists of keeping the device observable when the microchannel length is very short (in the range of 3 mm or lower).

In this article we describe different techniques for microfluidic rapid prototyping platforms. The proposed techniques are based on ultraviolet (UV) sensitive and temperature curing epoxies, polydimethylsiloxane (PDMS) based tubing, wire-bonding, and anisotropic adhesive films. The assembling procedures presented are developed for both one-time use devices as well as reusable microfluidic systems.

In this work, 3 different packaging and assembly techniques are presented. They can be classified into two categories: one-time use and reusable packaging ...

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

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

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

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

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

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

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

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

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

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

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

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

Developmental Biology

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

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

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

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

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