YG is supported by a fellowship from IDB. IN is an Alon fellow and a faculty fellow of the Edmond J&#x202D;. &#x202C;Safra Center for Bioinformatics at Tel Aviv University&#x202D;.&#x202C; This research was supported by ISF grant 1499/10.

1. Creating a Scotch-tape Master5
<ol>
	<li>Draw or print out desired microchannel layout, to scale, on paper. In our case the design consists of two Y-shaped channels, each 3 mm wide (Figure 2).</li>
	<li>Cover a glass-slide with layers of scotch tape. The number of layers will determine the height of the channel (about 60 &mu;m per layer). We used 3 scotch tape layers.</li>
	<li>Place the layout design on a flat surface. Align the slide over the design pattern. Carefully cut the tape on the glass slide with a scalpel according to the layout.</li>
	<li>Remove the Scotch tape from all regions of the glass slide except those in the layout of the microchannel.</li>
	<li>Place the slide in a heating oven at 65 &deg;C for 2-3 min. Clean gently with ethanol.</li>
</ol>
2. Fabricating a PDMS Microfluidic Device
<ol>
	<li>Mix the base and curing components of PDMS as recommended by the manufacturer. We use a 10:1 ratio of base to curing agent. Pour about 30 ml of PDMS mixture into a Petri dish to a height of ~0.5 cm. Degas the PDMS in vacuum if needed. Submerge the glass slide in the PDMS with the patterned Scotch tape facing up. Placing the pattern after the PDMS prevents formation of air bubbles between the slide and dish bottom. Cure for 48 hr at 65 &deg;C, making sure the dish is horizontal using a bubble level.</li>
	<li>In a new 90 mm Petri dish pour 3 ml of PDMS mix, resulting in a layer about 0.5 mm deep. (If a spin coater is available, it can be used for this stage).</li>
	<li>Degas and cure as in 2.1.</li>
	<li>Gently cut out the microfluidic device to the desired size with a scalpel. This layer will be termed the "flow layer".</li>
	<li>Cut out a similarly sized piece of thin layer PDMS from the second Petri dish. This will be termed the "wells layer".</li>
	<li>Use an appropriately sized biopsy puncher (We use 1.2 mm ID) to punch holes for the inlets and outlets in the flow layer.</li>
	<li>Place the wells layer on a thick glass slide over the design layout and punch out wells, using 2 mm ID biopsy puncher, in alignment with the microchannels on the layout.</li>
	<li>Clean both PDMS layers as well as a 24 mm x 60 mm glass coverslip with ethanol. Air-dry and keep clean.</li>
	<li>Plasma treat both the glass coverslip and the wells layer PDMS using either standard plasma etcher (for non-reversible bonding) or a hand-held corona treater6 for reversible bonding. Carefully place the wells layer on top of the coverslip to cause adhesion (Figure 3). Gently rub out any air bubbles (if needed).</li>
</ol>
3. Cell Imaging Experiment
<ol>
	<li>Make sure you have the desired media ready in appropriate syringes connected to flexible plastic tubes with internal diameter of 0.02" (Tygon) in the syringe pump.</li>
	<li>Grow S. cerevisiae strains to the desired phase in liquid YPD. Vortex thoroughly and place 300 &mu;l of each strain into a microcentrifuge tube.</li>
	<li>Gently place 1 &mu;l of Concanavalin A (sigma) 2 mg/ml into each well. Wash out excess Concanavalin A from each well twice by gently pipetting water.</li>
	<li>While the Concanavalin A is drying, wash strains twice with 300 &mu;l of SC medium lacking glucose. Washing residual glucose from the cell walls helps proper adherence to the Concanavalin A.</li>
	<li>Vortex cells thoroughly. Pipette 0.75 &mu;l or less of each cell suspension into its own well (Figure 3). Gently wash out residual cells with rich medium. The next two steps need to be performed quickly in order to prevent cells from drying up.</li>
	<li>Plasma treat the chip and the wells thoroughly using the hand-held corona treater, being careful not to hit the wells directly. Carefully place the chip on the wells, paying close attention to the alignment between the two (this step can be done under a stereoscope). Gently press to adhere the two layers of PDMS. Slowly fill the device completely with about 50 &mu;l rich medium, making sure no air bubbles are left inside the channel.</li>
	<li>Connect the device, inserting the tubes into the appropriate inlets and start media flow. Take care that no air bubbles are introduced into the device as these can be tricky to remove; this can be accomplished by disconnecting an inlet or outlet and gently tapping on the device where the bubbles are, being careful not to break the glass coverslip.</li>
	<li>Place under microscope and find appropriate imaging points in each well.</li>
	<li>Keep the cells under rich medium flow for 1 hr or longer to allow recovery if needed.</li>
	<li>Start the experiment: we use constant flow from each of the two syringe pumps to prevent backflow of the media, changing relative flow rates as desired. Setting pump A to 0.8 ml/hr and pump B to 0.2 ml/hr results in medium A flowing over 80% of the width of the channel (Figure 4). This can be dynamically changed by changing the two flow rates. The new conditions stabilize within seconds along the full length of the channel2.</li>
</ol>

<ol>
	<li>Dertinger, S. K., Jiang, X., Li, Z., Murthy, V. N., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradients+of+substrate-bound+laminin+orient+axonal+specification+of+neurons.">Gradients of substrate-bound laminin orient axonal specification of neurons.</a> Proceedings of the National Academy of Sciences of the United States of America. 99, 12542-12547 (2002).</li><li>Hersen, P., McClean, M. N., Mahadevan, L., Ramanathan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+processing+by+the+HOG+MAP+kinase+pathway.">Signal processing by the HOG MAP kinase pathway.</a> Proc. Natl. Acad. Sci. U.S.A. 105, 7165-7170 (2008).</li><li>Paliwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAPK-mediated+bimodal+gene+expression+and+adaptive+gradient+sensing+in+yeast.">MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast.</a> Nature. 446, 46-51 (2007).</li><li>Bennett, M. R., Hasty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+for+measuring+gene+network+dynamics+in+single+cells.">Microfluidic devices for measuring gene network dynamics in single cells.</a> Nat. Rev. Genet. 10, 628-638 (2009).</li><li>Shrirao, A. B., Perez-Castillejos, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+Fabrication+of+Microfluidic+Devices+by+Replicating+Scotch-tape+Masters.">Simple Fabrication of Microfluidic Devices by Replicating Scotch-tape Masters.</a> Chips &#38; Tips. , (2010).</li><li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab Chip. 6, 1548-1549 (2006).</li><li>Gerber, D., Maerkl, S. J., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+microfluidic+approach+to+generating+protein-interaction+networks.">An in vitro microfluidic approach to generating protein-interaction networks.</a> Nat. Methods. 6, 71-74 (2009).</li></ol>

No conflicts of interest declared.

In this paper we present a simple benchtop method for creating a microfluidic device that allows following several yeast strains simultaneously under dynamic conditions. Following several strains in one channel provides a reliable tool for comparing dynamics of single cell responses in multiple strains. One advantage of our approach is the ability to fabricate the device with simple techniques without the need for a clean room. In fact, we have also carried out the protocol skipping the plasma treatment operations (in st...

Cells are constantly reacting to a dynamically changing environment, by changing their metabolism, transcriptional profile and cellular functions. To study these phenomena, methods that can quantify such changes are needed. One type of readout for such responses is the change in localization of stress related transcription factors in response to nutritional stress.
Microfluidic devices1 have been used to dynamically manipulate environmental conditions to cells2-4. They present several advantages for live cell imaging: the environment of the cells can be precisely controlled and dynamically changed; cells can be live-imaged at all times, including during manipulation of external conditions; and minimal reagent volumes are required. A very simple design for a microfluidic device, allowing alternation between two conditions, is a Y-shaped device2. We routinely use Y-shaped microfluidic devices that allow application of dynamic changes to the cells in the channel. We use this device to follow localization changes of fluorescently tagged stress related transcription factors in yeast cells. We follow these changes under changing nutritional conditions. For example, we can provide a pulse (or series of pulses) of glucose-containing medium within a period of no-glucose medium, at a very fine temporal resolution. Often in such experiments, one is interested in comparing the responses of different cell strains (for example, different mutants, or different wild isolates). The Y-shaped channel is limited to one strain in each channel - if more than one strain is used, there is no simple way to distinguish between the strains. To overcome this, different channels can be used. If we want to apply the same dynamics conditions to multiple strains, we would like to combine the Y-channel concept with multiple channels. There are two challenges in implementing this solution: It can be hard to apply the same conditions at the same time to all channels, and there is a geometric limitation to the number of Y-channels that can be fitted in one device. We were therefore looking for a way for viewing several strains in one channel under dynamic conditions.
Here we describe a two-layered microfluidic device intended for imaging multiple strains in one channel under the same dynamic conditions. The bottom layer consists of thin PDMS with a row of holes. This layer is attached to the glass coverslip creating wells into which we will place the different strains, adhering them with Concanavalin A to the glass. The second layer is a Y-shaped microfluidic device created using the scotch tape method5. After placing the strains in the wells the second layer is quickly aligned and attached to the first one, creating one channel with several wells that contain the different strains. This final device allows following several strains in one channel, where all strains are subjected to the same conditions at the exact same time. The whole design and production process can be done on the benchtop, using simple means, with no soft lithography.

<table border="1">
	<tbody>
 	<tr>
 	<td>Reagent/Equipment</td>
 <td>Company </td>
 <td>Catalogue number </td>
 </tr>
 <tr>
 	<td>PDMS- SYLGARD 184</td>
 <td>Dow Corning USA 	</td>
 <td></td>
 </tr>
 <tr>
 	<td>Vacuum desiccator</td>
 <td>Nalgene</td>
 <td>5310-0250</td>
 </tr>
 <tr>
 	<td>Biopsy punchers</td>
 <td>Ted Pella Inc.</td>
 <td>Harris Uni-Core 15076 (2 mm), 15074 (1.2 mm)</td>
 </tr>
 <tr>
 	<td>Syringe pumps</td>
 <td>Chemyx</td>
 <td>Fusion 200</td>
 </tr>
 <tr>
 	<td>Corona treater</td>
 <td>Electro-technic products</td>
 <td>BD-20</td>
 </tr>
 <tr>
 	<td>Tygon tubing</td>
 <td>Tygon</td>
 <td>S-54-HL</td>
 </tr>
 <tr>
 	<td>Concanavalin-A</td>
 <td>Sigma</td>
 <td>C7275</td>
 </tr>
 <tr>
 	<td>Scotch tape</td>
 <td>3M Scotch</td>
 <td>	Transparent Tape 1/2"</td>
 </tr>
 </tbody>
 </table>

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.

To demonstrate the separation between different strains we imaged two distinguishable yeast strains in alternate wells. Imaging the full wells shows no cell leakage between wells (Figure 5a,b). Both strains have the transcription factor MSN2 tagged with YFP. To test the simultaneous effect of dynamically changing conditions, we switched the flow rates in the two input channels, creating a step of no-glucose medium, which resulted in localization of Msn2-YFP to the nucleus (Figure 5c,d). ...

Watch this Scientific Journal Video about A Microfluidic Device for Studying Multiple Distinct Strains at JoVE.com

A Microfluidic Device for Studying Multiple Distinct 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 ...

a-microfluidic-device-for-studying-multiple-distinct-strains

Research

JoVE Journal

Bioengineering

8.7K Views.  Tel Aviv University. אנו מציגים שיטה פשוטה לייצר מכשירים המסוגלים החלת תנאים דינמיים דומים לזנים שונים מרובים microfluidic, ללא צורך בחדר נקי או ליתוגרפיה הרכה.

Video: A Microfluidic Device for Studying Multiple Distinct Strains

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

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 ...

Protocol for Biofilm Streamer Formation in a Microfluidic Device with Micro-pillars

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in porous media are relevant to several industrial and environmental processes such as wastewater treatment and CO2 sequestration. We used Pseudomonas fluorescens, a Gram-negative aerobic bacterium, to investigate biofilm formation in a microfluidic device that mimics porous media. The microfluidic device consists of an array of micro-posts, which were fabricated using soft-lithography. Subsequently, biofilm formation in these devices with flow was investigated and we demonstrate the formation of filamentous biofilms known as streamers in our device. The detailed protocols for fabrication and assembly of microfluidic device are provided here along with the bacterial culture protocols. Detailed procedures for experimentation with the microfluidic device are also presented along with representative results.

Protocols for the study of biofilm formation in a microfluidic device that mimics porous media are discussed. The microfluidic device consists of an array of micro-pillars and biofilm formation by Pseudomonas fluorescens in this device is investigated.

Several bacterial species possess the ability to attach to surfaces and colonize them in the form of thin films called biofilms. Biofilms that grow in ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Fully Automated Centrifugal Microfluidic Device for Ultrasensitive Protein Detection from Whole Blood

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform for reduced sample volume and assay time as well as full automation are required for potential use in point-of-care-diagnostics. Recently, we have demonstrated ultrasensitive detection of serum proteins, C-reactive protein (CRP) and cardiac troponin I (cTnI), utilizing a lab-on-a-disc composed of TiO2 nanofibrous (NF) mats. It showed a large dynamic range with femto molar (fM) detection sensitivity, from a small volume of whole blood in 30 min. The device consists of several components for blood separation, metering, mixing, and washing that are automated for improved sensitivity from low sample volumes. Here, in the video demonstration, we show the experimental protocols and know-how for the fabrication of NFs as well as the disc, their integration and the operation in the following order: processes for preparing TiO2 NF mat; transfer-printing of TiO2 NF mat onto the disc; surface modification for immune-reactions, disc assembly and operation; on-disc detection and representative results for immunoassay. Use of this device enables multiplexed analysis with minimal consumption of samples and reagents. Given the advantages, the device should find use in a wide variety of applications, and prove beneficial in facilitating the analysis of low abundant proteins.

This protocol demonstrates how to achieve femto molar detection sensitivity of proteins in 10 &#181;L of whole blood within 30 min. This can be achieved by using electrospun nanofibrous mats integrated in a lab-on-a-disc, which offers high surface area as well as effective mixing and washing for enhanced signal-to-noise ratio.

Enzyme-linked immunosorbent assay (ELISA) is a promising method to detect small amount of proteins in biological samples. The devices providing a platform ...

Co-culture of Living Microbiome with Microengineered Human Intestinal Villi in a Gut-on-a-Chip Microfluidic Device

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human gut-on-a-chip microphysiological device. We recapitulate the intestinal lumen-capillary tissue interface in a microfluidic device, where physiological mechanical deformations and fluid shear flow are constantly applied to mimic peristalsis. In the lumen microchannel, human intestinal epithelial Caco-2 cells are cultured to form a &#39;germ-free&#39; villus epithelium and regenerate small intestinal villi. Pre-cultured microbial cells are inoculated into the lumen side to establish a host-microbe ecosystem. After microbial cells adhere to the apical surface of the villi, fluid flow and mechanical deformations are resumed to produce a steady-state microenvironment in which fresh culture medium is constantly supplied and unbound bacteria (as well as bacterial wastes) are continuously removed. After extended co-culture from days to weeks, multiple microcolonies are found to be randomly located between the villi, and both microbial and epithelial cells remain viable and functional for at least one week in culture. Our co-culture protocol can be adapted to provide a versatile platform for other host-microbiome ecosystems that can be found in various human organs, which may facilitate in vitro study of the role of human microbiome in orchestrating health and disease.

We describe an in vitro protocol to co-culture gut microbiome and intestinal villi for an extended period using a human gut-on-a-chip microphysiological system.

Here, we describe a protocol to perform long-term co-culture of multi-species human gut microbiome with microengineered intestinal villi in a human ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

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 ...

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 ...

Setup of a Microfluidic Device for Combinatorial Plug  Production

Bilayer Microfluidic Device for Combinatorial Plug Production

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such systems have been used to encapsulate a variety of biochemical reactions - from incubation of single cells to implementation of PCR reactions, from genomics to chemical synthesis. Coupling the microfluidic channels with regulatory valves allows control over their opening and closing, thereby enabling the rapid production of large-scale combinatorial libraries consisting of a population of droplets with unique compositions. In this paper, protocols for the fabrication and operation of a pressure-driven, PDMS-based bilayer microfluidic device that can be utilized to generate combinatorial libraries of water-in-oil emulsions called plugs are presented. By incorporating software programs and microfluidic hardware, the flow of desired fluids in the device can be controlled and manipulated to generate combinatorial plug libraries and to control the composition and quantity of constituent plug populations. These protocols will expedite the process of generating combinatorial screens, particularly to study drug response in cells from cancer patient biopsies.

Author Spotlight: Integrating Computational and Experimental Approaches in Precision Oncology

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Droplet microfluidics is a versatile tool that allows the execution of a large number of reactions in chemically distinct nanoliter compartments. Such ...