Name: perfusable vascular network with a tissue model in a microfluidic device
Uploaded: 2018-04-04T14:00:00
Description: Watch this Scientific Journal Video about Perfusable Vascular Network with a Tissue Model in a Microfluidic Device at JoVE.com

This work was supported by CREST JST (grant number JPMJCR14W4), Society for the Promotion of Science (JSPS) KAKENHI (grant number 25600060, 16K16386), The Center of Innovation Program from MEXT and JST, Project Focused on Developing Key Evaluation Technology from Japan Agency for Medical Research and Development, AMED, Mizuho Foundation for the Promotion of Sciences. Microfabrication was supported by Kyoto University Nano Technology Hub.

1. Fabrication of the Microfluidic Device Mold
<ol>
	<li>Design the pattern of the microfluidic device using commercially available software (Clewin5 or AutoCAD 2016, etc.). For the function of the Clewin5, see the user manual (http://manualzz.com/doc/7159150/clewin-user-s-manual). 
	NOTE: The design file is available in Supplementary File 1.</li>
	<li>Transfer the design file to a micro pattern generator and load the tool with a chromium mask coated with the positive photoresist.</li>
	<...

<ol>
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A., Gibot, L., Lacroix, D., Yarmush, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Annual Review of Biomedical Engineering. 15, 177-200 (2013).</li><li>Inamori, M., Mizumoto, H., Kajiwara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Approach+for+Formation+of+Vascularized+Liver+Tissue+by+Endothelial+Cell-Covered+Hepatocyte+Spheroid+Integration.">An Approach for Formation of Vascularized Liver Tissue by Endothelial Cell-Covered Hepatocyte Spheroid Integration.</a> Tissue Engineering Part A. 15 (8), 2029-2037 (2009).</li><li>Kunz-Schughart, L. 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Previous reports show that hLFs secrete a cocktail of multiple angiogenic factors, such as angiopoietin-1, angiogenin, hepatocyte growth factor, transforming growth factor-&#945;, tumor necrosis factor and some extracellular matrix proteins29,30. This assay relies on the angiogenic secretion from hLFs in a coculture spheroid, which is the limitation of the technique. Therefore, it is critical for a stable vascular formation to set a coculture spheroid at the bott...

Shifting from a monolayer (two-dimensional) culture to a three-dimensional culture is motivated by the need to work with culture models that mimic the cellular functions of living tissues1,2,3. Flat and hard plastic substrates commonly used in cell culture do not resemble most of the extracellular environments in the human body. In fact, many studies demonstrate that three-dimensional culture recreate tissue-specific architecture, mechanical and biochemical cues, and cell-cell communication, which have not been observed in conventional two-dimensional culture4,5,6,7,8.
A multicellular aggregate or spheroid, is one of the most promising techniques to realize this three-dimensional culture9,10. Cells secrete the extracellular matrix (ECM) and can interact with others in the spheroid. Although some other bioengineering approaches11,12,13,14, such as cell stacking, successfully replicate spatial complexity of the human body, these approaches have only two or three kinds of cells for the ease of analysis and focused on only one function of target organs. In contrast, cells in spheroids are exposed to different culture environments depending on their positions in the spheroid due to the heterogeneous supply of nutrients, oxygen, and paracrine and autocrine signaling molecules in the spheroid. This feature of spheroids partially mimics in vivo culture condition and enable the cells in spheroids to create much more complex, organized tissue structure in vitro than those cultured in stacking tissue9,15,16. Note that if a spheroid is comprised of a single kind of cells, the function of the cells in the spheroid is not uniform due to the heterogeneous environment in the spheroid. In the past few years, spheroid cultures allowed embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) or tissue-resident stem cells to mimic in vivo developmental sequences and recreate mini-organs such as the brain17, liver18and kidney19,20.
Despite significant progress in spheroid culture techniques, culturing large spheroids for a long time is still problematic. In a three-dimensional tissue, cells need to be located within 150-200 &#181;m of a blood vessel because of the limited supply of oxygen and nutrients21. Vascular networks within the spheroid are necessary to recapitulate exchanging substances between blood and tissues in vivo. To achieve this, other groups have co-cultured endothelial cells with target cells22,23,24 or induced the differentiation of pluripotent cells into CD31-positive cells20. Nevertheless, the reported vessel-like structures do not have the open ends of the lumina to supply oxygen and nutrients to the center of the spheroid. To mimic the vascular role to nourish cells in the three-dimensional culture, open-ended and perfusable vascular network must be developed in the spheroid.
During the past few years, some research groups in the microengineering field reported methods to construct a perfusable vascular network, spontaneously formed in a microfluidic device by utilizing angiogenic factors from cocultured fibroblast cells25,26. These vascular networks have a similar morphology to their in vivo counterparts and can be remodeled by environmental factors, making them suitable for mimicking vascular functions in a spheroid culture. The purpose of this protocol is to construct a perfusable vascular network in a spheroid using a microfluidic platform27. The microfluidic device is modified from the previously reported device25 so that a spheroid can be incorporated. By directing the angiogenic secretion from fibroblast cells in a spheroid to endothelial cells in microchannels, angiogenic sprouts from the microchannels anastomosed with the spheroid and formed a perfusable vascular network. This method allows a direct delivery of a wide range of substances, such as fluorescent molecules and micrometer-scale beads into the interior of a spheroid, which provides the framework for a long-term tissue culture with vascular networks.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AutoCAD 2017</td>
			<td>Autodesk</td>
			<td>AutoCAD 2017</td>
			<td></td>
		</tr>
		<tr>
			<td>A chromium mask coated with AZP 1350.</td>
			<td>CLEAN SURFACE TECHNOLOGY</td>
			<td>CBL2506Bu-AZP</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pattern generator</td>
			<td>Heidelberg</td>
			<td>uPG101</td>
			<td></td>
		</tr>
		<tr>
			<td>MF CD-26 developer</td>
			<td>Rohm and haas electronic materials</td>
			<td>-</td>
			<td>Developer in protocol 1.4</td>
		</tr>
		<tr>
			<td>S-Clean</td>
			<td>Sasaki Chemical</td>
			<td>S-24</td>
			<td>Chromium etchant in protocol 1.5</td>
		</tr>
		<tr>
			<td>Aceton</td>
			<td>Wako</td>
			<td>012-00343</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon Wafer</td>
			<td>Canosis</td>
			<td>SiJ-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>MIKASA</td>
			<td>1H-D7</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane (HMDS)</td>
			<td>Tokyo Ohka Kogyo</td>
			<td>H0089</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3050</td>
			<td>MicroChem</td>
			<td>-</td>
			<td>Negative photoresist in protocol 1.9</td>
		</tr>
		<tr>
			<td>UV Exposure</td>
			<td>Nanometric Technology Inc</td>
			<td>LA310s</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>MicroChem</td>
			<td>Y020100</td>
			<td>Developer for the negative photoresist in protocol 1.13</td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Wako</td>
			<td>163-04841</td>
			<td></td>
		</tr>
		<tr>
			<td>Surhace profiler</td>
			<td>Vecco</td>
			<td>Veeco Dektak XT-S</td>
			<td></td>
		</tr>
		<tr>
			<td>(Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane</td>
			<td>Sigma</td>
			<td>448931</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning Toray</td>
			<td>184W/C</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch (1.0mm)&#160;</td>
			<td>Kai Industries</td>
			<td>BP-10F</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy Punch (2.0mm)&#160;</td>
			<td>Kai Industries</td>
			<td>BP-20F</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma System</td>
			<td>Femto Science</td>
			<td>COVANCE</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>MATSUNAMI GLASS</td>
			<td>C024241</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Dishes</td>
			<td>Iwaki</td>
			<td>1000-035</td>
			<td></td>
		</tr>
		<tr>
			<td>RFP Expressing Human Umbilical Vein Endothelial Cell</td>
			<td>Angio Proteomie</td>
			<td>cAP-0001RFP</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Human Lung Fibroblasts</td>
			<td>Lonza</td>
			<td>CC-2512</td>
			<td></td>
		</tr>
		<tr>
			<td>Endothelial Cell Growth Medium</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast Growth Media Kits</td>
			<td>Lonza</td>
			<td>CC-3132</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Fisher Scientific</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Wako</td>
			<td>168-23191</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05w/v% Trypsin-0.53mmol/l EDTA&#8226; 4Na Solution with Phenol Red</td>
			<td>Wako</td>
			<td>204-16935</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (Phosphate Buffered Salts)</td>
			<td>Takara bio</td>
			<td>T900</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Sumitomo bakelite</td>
			<td>631-21031</td>
			<td></td>
		</tr>
		<tr>
			<td>1000ul Chip</td>
			<td>NIPPON Genetics</td>
			<td>FG-402</td>
			<td></td>
		</tr>
		<tr>
			<td>200ul&#160; Chip</td>
			<td>NIPPON Genetics</td>
			<td>FG-301</td>
			<td></td>
		</tr>
		<tr>
			<td>10ul Chip</td>
			<td>NIPPON Genetics</td>
			<td>37650</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td>Model 370</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP Expressing Human Umbilical Vein Endothelial Cell</td>
			<td>Angio Proteomie</td>
			<td>cAP-0001GFP</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibrinogen from bovine plasma</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>Aprotinin from bovine lung</td>
			<td>Sigma</td>
			<td>A6279</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Thrombin from bovine plasma</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H21492</td>
			<td>Fluorescent dye to stain nuclei in protocol 5.5</td>
		</tr>
		<tr>
			<td>Paraformaldehyde Solution</td>
			<td>Wako</td>
			<td>163-25983</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>OLYMPUS</td>
			<td>IX71</td>
			<td></td>
		</tr>
		<tr>
			<td>Degital CCD Camera</td>
			<td>OLYMPUS</td>
			<td>ORCA-R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Laser Scanning Biological Microscope</td>
			<td>OLYMPUS</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>OLYMPUS</td>
			<td>IX-83</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate-dextran</td>
			<td>Sigma</td>
			<td>FD70S</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 shows a design and photo of the microfluidic device. It has three parallel channels, in which channel 2 contains the spheroid well. Channels 1 and 3 are used for the HUVEC culture and channel 2 is for the spheroid. Each channel is separated by trapezoidal microposts designed to pattern PDMS. The microposts prevent the hydrogel in channel 2 from leaking into channels 1 and 3 by surface tension and allow exchanging substances between the spheroid and H...

Watch this Scientific Journal Video about Perfusable Vascular Network with a Tissue Model in a Microfluidic Device at JoVE.com

Perfusable Vascular Network with a Tissue Model in a Microfluidic Device

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 ...

The overall goal of this procedure is to use a microfluidic platform to construct a perfusable vascular network in a multicellular aggregate, or spheroid. This method can help address key questions in the alternate shape of regenerative medicine fields, such as how is a vascular network important in vivo and in vitro tissue models. The main advantage of this technique is that the route of drug and supplement administration can be simulated in vitro via delivery of regions of interest directly into the spheroids.Generally, individuals new to this method will struggle because of the difficulty in introducing the cells into the target area in the microfluidic device. After casting the PDMS prepolymer, degas the material in a vacuum chamber for two hours, followed by overnight curing at 80 degrees Celsius in an air-vented oven. The next morning, peel the PDMS from the silicon wafer, and use a two-millimeter-diameter hole punch to create holes in the material at the indicated positions.Use a one-millimeter-diameter punch to create the spheroid well, and use adhesive tape to clean the PDMS slab and a 24-by-24 millimeter glass cover slip. Treat the clean slab with air plasma for 40 seconds, and bond the PDMS slab onto the cover slip. Then, cure the PDMS at 80 degrees Celsius for at least 12 hours.Two to three days before seeding the microfluidic device, add three times 10 to the fifth freshly thawed hLF, RFP-HUVECs, and GFP-HUVECs to 10 milliliters of fresh endothelial medium in a 10-millimeter dish. When the hLFs and RFP-HUVECs reach sub-confluency, suspend the hLFs and RFP-HUVECs in the endothelial medium to final concentrations at 1.0 times 10 to the fifth and 2.5 times 10 to the fourth cells per millimeter, respectively. After two days in suspension culture, in a biosafety cabinet, add a 99-microliter droplet of freshly prepared master mix solution into the center of a 35-millimeter Petri dish on ice.To load the microfluidic device, use a modified 100-microliter micropipette tip to transfer a spheroid and 100 microliters of medium into a second, room-temperature Petri dish. Then, aspirate the spheroid in a minimum amount of medium, and turn the pipette upright so that the spheroid moves to the bottom of the pipette tip by gravity. Touch the pipette tip to the meniscus of the master mix droplet to eject the spheroid into the solution without depressing the pipette plunger.Next, quickly add one microliter of freshly prepared thrombin to the droplet, and gently mix the thrombin into the solution. With the pipette set to seven microliters, slowly transfer the spheroid into the spheroid well of the PDMS slab. The most critical step of the procedure is injecting the spheroid with enough gel to allow the spheroid to settle at the bottom of the device.After loading, remove the pipette tip gently to prevent leakage between the microposts. Incubate the spheroid at 37 degrees Celsius for 15 minutes. When the fibrin has solidified, slowly inject 20 to 30 microliters of endothelial medium into holes one A and three A to load channels one and three, respectively.Then, transfer the device onto a wet lab wipe in a 100-millimeter dish, and incubate the device at 37 degrees Celsius and 5%CO2 for 24 hours to facilitate the removal of any bubbles at the interface between the medium and the fibrin. The next day, add two milliliters of 0.05%trypsin-EDTA to the sub-confluent GFP-HUVECs, and stop the trypsin reaction with four milliliters of complete medium when the cells have completely detached. Collect the endothelial cells by centrifugation and resuspend the pellet in fresh endothelial medium at a five times 10 to the sixth cells per milliliter concentration.Next, inject 20 microliters of HUVECs into hole one B to load channel one, and place the device at 37 degrees Celsius for 30 minutes, tilted at a 90 degree angle to ensure that the HUVECs adhere to the fibrin in channel two. After loading channel three in the same manner, place the device in a new 100-millimeter dish containing a damp lab wipe in the cell culture incubator for seven to 14 days, replacing half of the medium in channels one and three daily. These representative images taken on day zero of HUVEC loading, show fibrin gel loaded into channel two only, without any leakage into channels one or three and with the HUVECs successfully attached to the sidewall of the fibrin gel.The spheroid can also be observed to be properly settled at the bottom of the device. Angiogenic sprouts are observed beginning on day one, with the longest sprout reaching the spheroid on day three in this experiment, and with most of the angiogenic sprouts reaching the spheroid by day seven. Optimally, after four days in the device, flow can be observed through the vascular lumens with the vascular angle defined as the direction of the vascular tip and the center of the spheroid from the vascular root.The vascular angles decrease in a time-dependent manner indicating migration of the angiogenic sprouts toward the spheroid. RFP and GFP-HUVECs can coordinately form a single vascular lumen, clearly indicating how angiogenic sprouts from channels one and three anastomose to RFP-HUVECs in the spheroid to form a continuous vascular network. Further, FITC-dextran injected into channel one flows into the constructed vascular network and interior of the spheroid, eventually reaching channel three, implying that the integrated vascular network could supply nutrients to and remove waste products from the spheroid.After its development, this technique paved the way for researchers in the organic electric field to explore the efficacy of drugs or supplements of interest in in vitro tissue models.

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Research

JoVE Journal

Bioengineering

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 ...

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 ...

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 ...

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 ...

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 ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

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 ...

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 ...

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells&#39; behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

This report describes a microfluidic chip-based method to set up a single cell culture experiment in which high-efficiency pairing and microscopic analysis of multiple single cells can be achieved.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases ...