Name: a microfluidic model of biomimetically breathing pulmonary acinar airways
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This work was supported in part by the European Commission (FP7 Program) through a Career Integration Grant (PCIG09-GA-2011-293604), the Israel Science Foundation (Grant nr. 990/12) and the Technion Center of Excellence in Environmental Health and Exposure Science (TCEEH). Microfabrication of microfluidic chips was conducted at the Micro-Nano Fabrication Unit (MNFU) of the Technion and supported by a seed grant from the Russel Berrie Institute of Nanotechnology (RBNI) at Technion. The authors thank Avshalom Shai for assistance during deep reactive ion etching (DRIE) and Molly Mulligan and Philipp Hofemeier for helpful discussions.

1. Master Fabrication
<ol>
	<li>Use deep reactive ion etching (DRIE) of a silicon on insulator (SOI) wafer to fabricate a master silicon wafer as described in former works32, 33. 
	NOTE: DRIE is preferred to standard SU-8 micromachining due to the high aspect ratio features (40 &#181;m wide and 90 &#181;m deep trenches).</li>
</ol>
2. Casting and Sealing of the Microfluidic Device
<ol>
	<li>Mix PDMS and curing agent at a 10:1 weight ratio inside a clean small contain...

<ol>
	<li>Kleinstreuer, C., Zhang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airflow+and+Particle+Transport+in+the+Human+Respiratory+System.">Airflow and Particle Transport in the Human Respiratory System.</a> Annu. Rev. Fluid Mech. 42 (1), 301-334 (2010).</li><li>Sznitman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Respiratory+microflows+in+the+pulmonary+acinus.">Respiratory microflows in the pulmonary acinus.</a> J. Biomech. 46 (2), 284-298 (2013).</li><li>Tsuda, A., Henry, F. S., Butler, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gas+and+aerosol+mixing+in+the+acinus.">Gas and aerosol mixing in the acinus.</a> Respir. Physiol. Neurobiol. 163 (1-3), 139-149 (2008).</li><li>Kleinstreuer, C., Zhang, Z., Donohue, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Drug-Aerosol+Delivery+in+the+Human+Respiratory+System.">Targeted Drug-Aerosol Delivery in the Human Respiratory System.</a> Annu. Rev. Biomed. Eng. 10 (1), 195-220 (2008).</li><li>Semmler-Behnke, M., Kreyling, W. G., Schulz, H., Takenaka, S., Butler, J. P., Henry, F. S., Tsuda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+delivery+in+infant+lungs.">Nanoparticle delivery in infant lungs.</a> Proc. Natl. Acad. Sci. 109 (13), 5092-5097 (2012).</li><li>Sznitman, J., Heimsch, F., Heimsch, T., Rusch, D., Rosgen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Convective+Alveolar+Flow+Induced+by+Rhythmic+Breathing+Motion+of+the+Pulmonary+Acinus.">Three-Dimensional Convective Alveolar Flow Induced by Rhythmic Breathing Motion of the Pulmonary Acinus.</a> J. Biomech. Eng. 129 (5), 658-665 (2007).</li><li>Tsuda, A., Henry, F. S., Butler, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chaotic+mixing+of+alveolated+duct+flow+in+rhythmically+expanding+pulmonary+acinus.">Chaotic mixing of alveolated duct flow in rhythmically expanding pulmonary acinus.</a> J. Appl. Physiol. 79 (3), 1055-1063 (1995).</li><li>Henry, F. S., Butler, J. P., Tsuda, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinematically+irreversible+acinar+flow:+a+departure+from+classical+dispersive+aerosol+transport+theories.">Kinematically irreversible acinar flow: a departure from classical dispersive aerosol transport theories.</a> J. Appl. Physiol. 92 (2), 835-845 (2002).</li><li>Kumar, H., Tawhai, M. H., Hoffman, E. A., Lin, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+geometry+on+airflow+in+the+acinar+region+of+the+human+lung.">The effects of geometry on airflow in the acinar region of the human lung.</a> J. Biomech. 42 (11), 1635-1642 (2009).</li><li>Lee, D. Y., Lee, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+of+particle+transport+in+an+expanding+or+contracting+alveolated+tube.">Characteristics of particle transport in an expanding or contracting alveolated tube.</a> J. Aerosol Sci. 34 (9), 1193-1215 (2003).</li><li>Tsuda, A., Butler, J. P., Fredberg, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+alveolated+duct+structure+on+aerosol+kinetics.+I.+Diffusional+deposition+in+the+absence+of+gravity.">Effects of alveolated duct structure on aerosol kinetics. I. Diffusional deposition in the absence of gravity.</a> J. Appl. Physiol. 76 (6), 2497-2509 (1994).</li><li>Tsuda, A., Butler, J. P., Fredberg, J. 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D., Korbel, K., Kline, T. L., Jorgensen, S. M., Eaker, D. R., Bohle, R. M., Ritman, E. L., Langheinrich, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchrotron-Based+Micro-CT+Imaging+of+the+Human+Lung+Acinus.">Synchrotron-Based Micro-CT Imaging of the Human Lung Acinus.</a> Anat. Rec. Adv. Integr. Anat. Evol. Biol. 293 (9), 1607-1614 (2010).</li><li>Tsuda, A., Filipovic, N., Haberth&#252;r, D., Dickie, R., Matsui, Y., Stampanoni, M., Schittny, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Finite+element+3D+reconstruction+of+the+pulmonary+acinus+imaged+by+synchrotron+X-ray+tomography.">Finite element 3D reconstruction of the pulmonary acinus imaged by synchrotron X-ray tomography.</a> J. Appl. Physiol. 105 (3), 964-976 (2008).</li><li>Berg, E. J., Weisman, J. L., Oldham, M. 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A critical feature of the microfluidic acinar platform presented here is its ability to reproduce physiologically-realistic breathing motions that give rise to physiological flow profiles and velocities within acinar ducts and within alveoli. Since the microfluidic channels are produced with a relatively low aspect ratio (i.e., wd/h &#8776; 3.9, where wd is the duct width and h is the duct height), the measured flows show more plug-like ...

A detailed quantification of respiratory flow dynamics in the distal, alveolated regions of the lungs is paramount towards understanding airflow mixing in the pulmonary acinus and predicting the fate of inhaled aerosols in the deepest airways1&#8211;3. This latter aspect is of particular concern when addressing on the one hand the hazards of inhaled pollutant particles or conversely in seeking novel strategies for improved and targeted drug delivery of inhaled therapeutics to localized lung sites4, 5&#160;as well as for systemic delivery.
To date, respiratory flows in the deep pulmonary acinar regions have been typically investigated in silico using computational fluid dynamics (CFD) or alternatively in vitro with scaled-up experimental models following hydrodynamic similarity matching. In the past few decades, CFD methods have been increasingly applied to study acinar flow phenomena, from single alveolar models6, 7&#160;and alveolated ducts8&#8211;12&#160;to more elaborate in silico models that capture anatomically-realistic acinar tree structures with multiple generations of alveolated ducts and up to several hundreds of individual alveoli13&#8211;15.
Together, numerical efforts have been pivotal in shedding light on the role and influence of wall motion during breathing movements on ensuing acinar airflow patterns. In the absence of breathing motion, static alveoli feature recirculating flows within their cavities that exhibit no convective exchange of air between the acinar duct and the alveolus6, 7; in other words, alveolar flows would be entirely isolated from flows within the acinar trees and exchange of air would result uniquely from diffusive mechanisms. With the existence of cyclic expansions of the alveolar domain, however, alveolar flow topologies are drastically modified and the resulting flow patterns inside alveoli are intimately tied to the location of an alveolus along the acinar tree (e.g., proximal vs. distal generations).
In particular, it has been hypothesized in simulations that alveolar flow patterns are strongly influenced by the ratio of alveolar to ductal flow rates such that proximal generations of the pulmonary acinar tree, where this ratio is relatively large following mass conservation across a tree structure, feature complex recirculating flows inside the alveolar cavities with irreversible fluid pathlines. With each deeper acinar generation, the ratio of alveolar to ductal flow rates gradually decreases such that distal acinar generations exhibit more radial-like streamlines that are reminiscent of simple inflations and deflations of a balloon. With advances in modern imaging modalities, lung imaging data16, 17&#160;of rodents, including rat and mouse, have given rise to some of the first CFD simulations of anatomically-reconstructed acinar flows in reconstructed alveoli. Despite such promising progress, these recent studies are still limited to addressing airflow phenomena in terminal alveolar sacs only18, 19&#160;or a few alveoli surrounding a single duct20. As a result, state-of-the-art investigations of respiratory flow phenomena in the acinus remain dominated by studies focusing on generic anatomically-inspired geometries of the acinar environment2.
On the experimental side, various setups featuring an airway with one or several alveoli have been developed over the years21&#8211;24. Yet, there exists no experimental models of bifurcating alveolated airways that are capable of mimicking physiological respiration by expanding and contracting in a breathing-like fashion. Given a lack of attractive experimental platforms at hand, the study of acinar transport phenomena remains limited with regards to validating computational studies and critically, there remains a dearth of experimental data available. In recent years, Ma et al. (2009) have constructed a scaled-up rigid-wall model of an acinus consisting of three acinar generations; however, the lack of wall motion in this model limited its capability to capture realistic alveolar flow patterns under breathing conditions.
Other scaled-up experiments including a moving wall model based on anatomical data from cast replica were recently introduced25; however, since the model only captured the last two acinar generations (i.e., terminal sacs), it failed to capture the complex recirculating flows that characterize more proximal acinar generations. These latter examples of scaled-up experiments further underline the ongoing limitations with such approaches. Specifically, no existing experiment has thus far demonstrated the hypothesized transition from recirculating to radial flows along the acinus and thereby confirm numerical predictions of flow topologies hypothesized to exist in real pulmonary acinar trees7, 15. Perhaps most critically, scaled-up experiments are extremely limited in investigating inhaled particle transport and deposition dynamics26&#160;due to difficulties in matching all relevant non-dimensional parameters (e.g., particle diffusion, a critical transport mechanism for sub-micron particles, is completely neglected).
With ongoing experimental challenges, new experimental platforms that permit investigations of respiratory air flows and particle dynamics in complex moving walls acinar networks are sought. Here, an anatomically-inspired in vitro acinar model is introduced. This microfluidic platform mimics pulmonary acinar flows directly at the representative acinar scale, and broadens the growing range of pulmonary microfluidic models27, including bronchial liquid plug-flows28&#8211;30&#160;and the alveolar-capillary barrier31.
Namely, the present design features a simplified five generation alveolated airway tree with cyclically expanding and contracting walls, where cyclic motions are achieved by controlling pressure inside a water chamber which surrounds the thin PDMS lateral walls and where the top wall is deformed by an additional water chamber sitting directly above the acinar structure. Unlike common multilayer microfluidic devices, this chamber is simply formed by embedding the barrel section of a syringe inside the PDMS Device, and does not require preparation of an additional PDMS mold.
The miniaturized approach presented here offers a simple and versatile means for reproducing complicated acinar structures with moving walls as compared to scaled-up models while capturing the underlying characteristics of the acinar flow environment. This platform can be used for flow visualization using fluid-suspended particles inside the airways (see Representative Results below). In the near future, the model will be used with airborne particles for studying inhaled acinar particle dynamics.

<table>
	<tbody>
		<tr>
			<td>Polydimethylsiloxane (PDMS) and curing agent</td>
			<td>Dow Corning</td>
			<td>(240)4019862</td>
			<td>SYLGARD&#174; 184 SILICONE ELASTOMER KIT</td>
		</tr>
		<tr>
			<td>Plastipak 2 ml syringe</td>
			<td>BD</td>
			<td>300185</td>
			<td></td>
		</tr>
		<tr>
			<td>Norm-Ject Luer slip 1 ml syringe</td>
			<td>Henke Sass Wolf </td>
			<td>4010-200V0</td>
			<td></td>
		</tr>
		<tr>
			<td>1mm Biopsy punch</td>
			<td>Kai Medical</td>
			<td>BP-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Corona Treater</td>
			<td>Electro-Technic Products</td>
			<td>BD-20AC</td>
			<td></td>
		</tr>
		<tr>
			<td>PHD Ultra Syringe pump</td>
			<td>Harvard apparatus</td>
			<td>703006</td>
			<td></td>
		</tr>
		<tr>
			<td>Dyed red rqueous fluorescent particles</td>
			<td >Thermo-Scientific</td>
			<td ></td>
			<td >Uncatalloged 0.86 &#181;m beads were used</td>
		</tr>
		<tr>
			<td>Glycerin AR</td>
			<td>Gadot</td>
			<td>830131320</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowMaster MITAS micro-particle image velocimetry (&#181;PIV) system </td>
			<td>LaVision</td>
			<td>1108630</td>
			<td ></td>
		</tr>
	</tbody>
</table>

a microfluidic model of biomimetically breathing pulmonary acinar airways

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards optimizing drug inhalation techniques as well as predicting deposition patterns of potentially toxic airborne particles in the pulmonary alveoli. Here, soft-lithography techniques are used to fabricate complex acinar-like airway structures at the truthful anatomical length-scales that reproduce physiological acinar flow phenomena in an optically accessible system. The microfluidic device features 5 generations of bifurcating alveolated ducts with periodically expanding and contracting walls. Wall actuation is achieved by altering the pressure inside water-filled chambers surrounding the thin PDMS acinar channel walls both from the sides and the top of the device. In contrast to common multilayer microfluidic devices, where the stacking of several PDMS molds is required, a simple method is presented to fabricate the top chamber by embedding the barrel section of a syringe into the PDMS mold. This novel microfluidic setup delivers physiological breathing motions which in turn give rise to characteristic acinar air-flows. In the current study, micro particle image velocimetry (&#181;PIV) with liquid suspended particles was used to quantify such air flows based on hydrodynamic similarity matching. The good agreement between &#181;PIV results and expected acinar flow phenomena suggest that the microfluidic platform may serve in the near future as an attractive in vitro tool to investigate directly airborne representative particle transport and deposition in the acinar regions of the lungs.

Computer-aided design (CAD) and microscope images of the in vitro acinar platform are presented in Fig. 1. The biomimetic acinar model features five generations of branching rectangular channels lined with alveolar-like cylindrical cavities (Fig. 1). Here, the model generations are numbered from generation 1 (for the most proximal generation) to generation 5 (for the most distal generation). Note that only the channel inlet leading to generation 1 is open to the outer environmen...

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A Microfluidic Model of Biomimetically Breathing Pulmonary Acinar Airways

Soft-lithography was utilized to produce a representative true-scale model of pulmonary alveolated airways that expand and contract periodically, mimicking physiological breathing motion. This platform recreates respiratory acinar flows on a chip, and is anticipated to facilitate experimental investigation of inhaled aerosol dynamics and deposition in the pulmonary acinus.

Quantifying respiratory flow characteristics in the pulmonary acinar depths and how they influence inhaled aerosol transport is critical towards ...

The overall goal of this model system, is to mimic the geometry and breathing motion of the Pulmonary Acinus for studying Acinar airflow patterns and airborne micro-particle trajectories. This method can help answer key questions in the field of Acinar Transport Phenomena. Such as, the effects of gravity, drug, and diffusion on particle deposition outcomes.The main advantage of this technique is that experiments are done inside a one-to-one scale model rather than a scaled up model. This allows for accurate observation of particle Brownian motion. The implications of this technique extend towards inhalation therapy, because a better understanding of aerosolized drug deposition can be achieved.To begin, use deep reactive ion etching of a silicon on insulator wafer to fabricate a master silicon wafer as described previously. Next, mix the PDMS and curing agent at a ten-to-one weight ratio inside a clean small container. Degas the mixture in a desiccator under vacuum until all of the air bubbles are removed.Place the master wafer into a petri dish, and pour the degassed PDMS mixture to a height of approximately one millimeter above the master wafer. Degas the poured PDMS once again for at least 40 minutes, until all air bubbles are removed above the wafer, and bubbles below the wafer are minimized. Make sure that the wafer is as close as possible to the bottom of the plate.If necessary, press the wafer gently to the bottom using two stirring sticks and degas once again. Then, bake the PDMS at 65 degrees Celsius for 20 minutes in a natural convection oven. While the PDMS is baking, file the barrel section of a plastic two milliliter syringe, using a fine grit sand paper to improve its adherence to PDMS.In addition, use the sand paper to flatten the base of the syringe barrel by placing the sand paper on a flat surface and sliding the base of the syringe barrel on top of it. Clean off any debris from the syringe using pressurized air. Then place the barrel section of the syringe on top of the first PDMS layer with the large opening facing the surface of the PDMS.Pour a second layer of PDMS on top of the first one, to a height of about five millimeters, while ensuring that the PDMS does not enter the barrel of the syringe. Then, degas the PDMS once again. Bake the entire set-up at 65 degrees Celsius for at least two hours in a natural convection oven to harden the PDMS.Cut through the PDMS mold around the patterned region of the master wafer using a scalpel. While cutting, the scalpel should weakly touch the surface of the wafer. Then, gently insert a thin tool, such as wafer forceps, in the notch created by the scalpel, and peel off the PDMS cast from the master wafer.Place the cast on a soft surface covered with aluminum foil, so that the patterned side faces up and punch a hole in the PDMS at the chamber inlet and channel inlet, using a one millimeter biopsy punch. Next, add some of the PDMS with curing agent to a clean glass slide and spin coat a thin layer at 3, 000 RPM's for 30 seconds. Then, bake the slide at 65 degrees Celsius for at least one hour.Clean the slide and PDMS cast using scotch tape. Treat the surfaces of the coated glass slide and the molded PDMS with oxygen plasma using a corona treater. Note that each surface should be treated for at least one minute.Then, gently press the surfaces together and bake them overnight at 65 degrees Celsius. Mix water suspended fluorescent polystyrene particles with water and glycerol in a glass vile to obtain a 64-to-36 volume to volume ratio of glycerol to water containing 0.25%particles by weight. Next, place a drop of the glycerol solution on top of the channel inlet and a drop of DI water on the chamber inlet.Then, set the apparatus inside a desiccator and pull a vacuum for about five minutes. Before releasing the vacuum, wait for the bubbles that form in the drops of glycerol solution and DI water to pop. Upon vacuum release, the liquids are drawn into the voids inside the device.If residual air remains inside the channels, eliminate it by applying external pressure on the fluids using a syringe and allowing the air to diffuse into the PDMS. Next, inject about two millimeters of deionized water into the top chamber until it is fully filled with water. Then, cover the chamber with a 19 gauge blunt syringe tip.Cut the tip of another blunt 19 gauge syringe tip and insert this tip into the side chamber inlet. Connect both syringe tips to a one milliliter syringe using thin, Teflon tubing and a t-shaped connector. Make sure that the one milliliter syringe, Teflon tubing, t-shaped connector, and top chamber are all filled with water, without any bubbles.Then, connect the one milliliter syringe to a syringe pump and program it to mimic a quiet title breathing cycle as described in the accompanying text protocol. In order to obtain micro-particle vilo symmetry images, place the device on to the stage of an inverted microscope. Then, turn on the double pulsed Nd:YAG laser and load the image analysis software on the computer.While the device is being actuated, obtain a series of nine-to-twelve phase locked double frame images of the particle seeded flow, using a micro-particle image vilo symmetry system in accordance with the manufacturers'specifications. To achieve phase locked double frame images, acquire a double frame series at 10 hertz. Then, reorganize the data so that all framed pairs that are separated by a full cycle time form a new time series.Use the sum of correlation algorithm to compute phase locked velocity vector map so the resulting flow field from the image series. Repeat this process several times with varying lag times between the first and second frames of each frame pair for resolving different flow regions inside the alveolar cavity. Next, use a data analysis program to stitch together the individual flow maps into a complete and high detailed map of flow patterns by averaging overlapping data points.A critical feature of the micro-fluidic acinar platform presented here, is its ability to reproduce physiologically realistic breathing motions that give rise to physiological flow profiles and velocities within acinar ducts and within alveoli. Flow velocity profiles across the width of the channels show a steady drop of flow rates towards deeper acinar generations. Flow profiles near and within alveolar cavities show that flow magnitudes drop steeply along the opening of alveoli.Resulting in flow velocity that are two to three orders of magnitudes slower inside alveoli compared to the ducts. In addition, flow patterns change considerably with increasing acinar generation. While generation one features a re-circulation zone, which roughly coincides with the center of the alveolas, generation three's characterized by a re-circulation zone which is shifted toward the proximal side of alveolas with a more open stream line pattern.Finally, radial stream lines with no re-circulation zone are observed in device generation five. Once mastered, this technique can be done in a few hours if it is performed properly. Following this procedure, which enables flow visualization in acinar geometries, the platform can be used to track single airborne particles in order to explore the dynamics and the position of inhaled particles.

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Research

JoVE Journal

Bioengineering

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

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 ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

A Microfluidic Chip for ICPMS Sample Introduction

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). The chip produces monodisperse aqueous sample droplets in perfluorohexane (PFH). Size and frequency of the aqueous droplets can be varied in the range of 40 to 60 &#181;m and from 90 to 7,000&#160;Hz, respectively. The droplets are ejected from the chip with a second flow of PFH and remain intact during the ejection. A custom-built desolvation system removes the PFH and transports the droplets into the ICPMS. Here, very stable signals with a narrow intensity distribution can be measured, showing the monodispersity of the droplets. We show that the introduction system can be used to quantitatively determine iron in single bovine red blood cells. In the future, the capabilities of the introduction device can easily be extended by the integration of additional microfluidic modules.

We present a discrete droplet sample introduction system for inductively coupled plasma mass spectrometry (ICPMS). It is based on a cheap and disposable microfluidic chip that generates highly monodisperse droplets in a size range of 40&#8722;60 &#181;m at frequencies from 90 to 7,000 Hz.

This protocol discusses the fabrication and usage of a disposable low cost microfluidic chip as sample introduction system for inductively coupled plasma ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

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 ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the spheroid cultures, a perfusable vascular network in the spheroids remains a critical challenge for long-term culture required to maintain and develop their functions, such as protein expressions and morphogenesis. The protocol presents a novel method to integrate a perfusable vascular network within the spheroid in a microfluidic device. To induce a perfusable vascular network in the spheroid, angiogenic sprouts connected to microchannels were guided to the spheroid by utilizing angiogenic factors from human lung fibroblasts cultured in the spheroid. The angiogenic sprouts reached the spheroid, merged with the endothelial cells co-cultured in the spheroid, and formed a continuous vascular network. The vascular network could perfuse the interior of the spheroid without any leakage. The constructed vascular network may be further used as a route for supply of nutrients and removal of waste products, mimicking blood circulation in vivo. The method provides a new platform in spheroid culture toward better recapitulation of living tissues.

The protocol describes how to engineer a perfusable vascular network in a spheroid. The spheroid's surrounding microenvironment is devised to induce angiogenesis and connect the spheroid to the microchannels in a microfluidic device. The method allows the perfusion of the spheroid, which is a long-awaited technique in three-dimensional cultures.

A spheroid (a multicellular aggregate) is regarded as a good model of living tissues in the human body. Despite the significant advancement in the ...

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

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

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

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