Authors acknowledge the technical support from Zhifeng Cheng of Chansn Instrument (China) LTD. This work was supported by grants (National Natural Science Foundation of China,51927804).

1. Microfluidic chips design
<ol>
	<li>Design the microfluidic multiplexer consisting of 18 inlets, each of which is controlled by an individual valve and a peristaltic pump. To increase the liquid volume driven by per pumping cycle, have the peristaltic pump be composed of 3 control channels, which was purposely widened to 200 &#181;m, and 10 connected flow lines.</li>
	<li>Design the shear-free culture chamber. Replication of the 2-level culture unit is composed by a lower cell culture chamber (400 &#181;m x 400 &#18...

<ol>
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M. <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+microfluidic+devices+fabricated+in+layered+paper+and+tape.">Three-dimensional microfluidic devices fabricated in layered paper and tape.</a> Proceedings of the National Academy of Sciences. 105 (50), 19606-19611 (2008).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+analysis+on+a+droplet-in-oil+PCR+array.">DNA methylation analysis on a droplet-in-oil PCR array.</a> Lab on a Chip. 9 (8), 1059-1064 (2009).</li><li>Huang, N. T., Hwong, Y. J., Lai, R. 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+microfluidic+microwell+device+for+immunomagnetic+single-cell+trapping.">A microfluidic microwell device for immunomagnetic single-cell trapping.</a> Microfluidics and Nanofluidics. 22 (2), 16 (2018).</li><li>Galler, K., Br&#228;utigam, K., Gro&#223;e, C., Popp, J., Neugebauer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Making+a+big+thing+of+a+small+cell-recent+advances+in+single+cell+analysis.">Making a big thing of a small cell-recent advances in single cell analysis.</a> Analyst. 139 (6), 1237-1273 (2014).</li><li>Gr&#252;nberger, A., Wiechert, W., Kohlheyer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+microfluidics:+opportunity+for+bioprocess+development.">Single-cell microfluidics: opportunity for bioprocess development.</a> Current Opinion in Biotechnology. 29, 15-23 (2014).</li><li>Lin, H., Mei, N., Manjanatha, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+comet+assay+for+testing+genotoxicity+of+chemicals.">In vitro comet assay for testing genotoxicity of chemicals.</a> Optimization in Drug Discovery. , 517-536 (2014).</li><li>Bai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+water+collection+on+integrative+bioinspired+surfaces+with+star-shaped+wettability+patterns.">Efficient water collection on integrative bioinspired surfaces with star-shaped wettability patterns.</a> Advanced Materials. 26 (29), 5025-5030 (2014).</li><li>Zhao, J., Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+or+against+topographic+wettability+gradient:+movements+of+droplets+on+a+micropatterned+surface.">Following or against topographic wettability gradient: movements of droplets on a micropatterned surface.</a> Langmuir. 33 (21), 5328-5335 (2017).</li><li>Theberge, A. 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Various microfluidic devices have been developed to perform multiplexed and complex experiments17,18,19,20. For example, microwells made of an array of topological recesses can trap individual cells without the use of external force, showing advantageous characters including small sample size, parallelization, lower material cost, faster response, high sensitivity21...

High throughput techniques are crucial for biomedical and clinical studies. By parallelly conducting millions of chemical, genetic, or live cell and organoid tests, researchers can rapidly identify genes that modulate a bio-molecular pathway, and customize sequential drug input to one's specific needs. Robotics1 and microfluidic chips in combination with a device control program allow complex experimental procedures to be automated, covering cell/tissue manipulation, liquid handling, imaging, and data processing/control2,3. Therefore, hundreds and thousands of experimental conditions can be maintained on a single chip, according to the desired throughput4,5. 
In this protocol, we described the design and fabrication procedure of a microfluidic device, which consists of 1500 culture units, an array of enhanced peristaltic pumps and on-site mixing modulus. The 2-level cell culture chamber prevents unnecessary shear during medium exchange, which ensures an undisturbed culture environment for long-term live cell imaging. The studies demonstrate that the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery. Moreover, the advanced features of the microfluidic chip allow automated reconstitution of highly complex and dynamic microenvironmental conditions in vivo, like the everchanging cytokines and ligands compositions6,7, the completion of which takes months for conventional platforms like 96-well plate.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2713 Loker Avenue West</td>
			<td>Torrey pines scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AZ-50X</td>
			<td>AZ Electronic Materials, Luxembourg</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorotrimethylsilane(TMCS) 92360-25mL</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Incubator HP151</td>
			<td>Heal Force</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop Hole Puncher for PDMS chips WH-CF-14</td>
			<td>Suzhou Wenhao Microfluidic Technology Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM(L-glutamine, High Glucose, henol Red)</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electronic Balance UTP-313 Max:600g, e:0.1g, d:0.01g</td>
			<td>Shanghai Hochoice Apparatus Manufacturer Co.,LTD.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronection 0.25 mg/mL</td>
			<td>Millipore, Austria</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax 100x</td>
			<td>Gibco</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Incubator BGG-9240A</td>
			<td>Shanghai bluepard instruments Co.,Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Model Eclipse Ti2-E</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strep 10 Units/mL Penicillin 10 ug/mL Streptomycin</td>
			<td>Invitrogen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma cleaner PDC-002</td>
			<td>Harrick Plasma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polydimethylsiloxane(PDMS)</td>
			<td>Momentive</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>polylysine 0.01%</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater ARE-310</td>
			<td>Awatori Rentaro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater TDZ5-WS</td>
			<td>Cence</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin coater WH-SC-01</td>
			<td>Suzhou Wenhao Microfluidic Technology Co., Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3025</td>
			<td>MicroChem, Westborough, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3075</td>
			<td>MicroChem, Westborough, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The conventional on-chip peristaltic pump was firstly described by Stephen Quake in 2000, using which the peristalsis was actuated by the pattern 101, 100, 110, 010, 011, 001 8,10. The number 0 and 1 indicate &#34;open&#34; and &#34;close&#34; of the 3 horizontal control lines. Studies using more than 3 valves (e.g., five) have also been reported11. Even though the peristaltic pump composed by 3 control lines and 3 flow lines provides nano...

Watch this Scientific Journal Video about Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device at JoVE.com

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

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 ...

Microenvironment enable a highly complex and dynamic including ever changing cytokines, ligands concentration and composition. To create a similar culture environment is crucial for in vitro studies on complex life machinery such as stem cell differentiation, immune responses, and the development of organs on chip platforms. But to generate and deliver chemical signals of nanoliter volume and milliseconds accuracy is difficult using conventional biomedical approaches like pipetting.To meet the challenges, a culture platform which is capable of alternate image acquisition generation of dynamic combinatorial and sequential signal inputs is on high demand. In this protocol, the design and fabrication procedure of a microfluidic device are presented. The proposed microfluidic chip consists of 1500 culture units, array of enhanced peristaltic pumps and on-site mixing modulus.We demonstrate that platform is suitable for studies on single cells two dimensional cell populations and three dimensional neural spheres. The complex and dynamic environmental conditions on chip have great effects on neural stem cell differentiation and the self-renewal. The detail of design and fabrication procedures are as follows.Chip design was performed using AutoCAD software. To fit the size of the plate holder on microscope, the microfluidic chip is approximately seven by five centimeters in size And consists of 1, 500 culture chambers. One of important features is it has peristaltic pump which consists of eight flow channels and 200 micrometer wide control channels.The increase in the overlapping area between control and the flow channel increase 16 times as compared to the peristaltic pump, proposed by Stephen Quake in 2002, to about 15 nanoliters liquid and per pumping cycle and suffice the needs to maintain 1, 500 independent environmental conditions parallelly on chip. Another important part of the device is a two-layer culture chamber, which can maintain shear-free culture environment. The unwanted shear stress is prevented by directing it quick through the top layer.The cells, tissues and crucial conditional medium remain untouched at the bottom layer. Numerical simulation suggests that when the liquids are directed through culture chambers, left to right. the shear forces can be effectively prevented.Even at an input flow rate of 10 millimeter per second cell or micrometer-size tissue remain undisturbed at the bottom of the culture unit. We fabricated the replica molding or silicon wafer using UV lithography. The fluidic channels of 25 micrometers in height and 100 micrometers in weight are produced using SU-8 3025 negative photoresist.The culture chambers of 75 micrometers and 150 micrometers in height were fabricated using SU-8 3075 photoresist. AZ50X positive photoresist is used for the round-shaped well features which overlap with the control channels to ensure a good connection. To produce the microfludic chip, the patterned and blank silicon wafers were firstly treated with TMCS.Different quantities of PDMS 10 to 1 of monomer to catalyst ratio was thoroughly mixed and debubbled for one to two hours in the vacuum chamber at minus 0.85 megapascal. The control layer was obtained by spin coating PDMS at 2, 200 RPM. The silicon wafers were then transferred to incubator and incubated for 60 minutes at 80 centigrade.Different layers were aligned and bonded together using customized optical device and plasma etching machine. The inlet holes were then punched after another two hours thermal bonding at 80 centigrade. The chip was bonded to a PDMS-coated cover slip, which is the same size of 96-well plate and cured for at least 12 hours at 80 centrigrade before use.For operation, control channels were connected to miniature pneumatic solenoid valves through plastic tubing. The turning all of penumatic wells were controlled by a custom MATLAB program through the graphical user interface. Optimal closing pressures of push-up PDMS membrane valves were determined individually for each chip, which typically ranges from 25 to 30 PSI.By turning all of series of wells dynamically combinatorial and sequential inputs can be generated on chip. Cells were harvested at 80%confluency and resuspended with culture medium DMEM at a density about 10 to the power six per milliliter, which were then loaded into the chip by pressurizing the cell containing solution. For both adherent and the suspension culture of neuro stem cells, cells were collected and the spheres were directly loaded into the chip.For image acquisition, an inverted microscope with an automatic translational stage and the digital complementary metal-oxide semiconductor camera were used. The stage and image acquisition were controlled via the software shows the upper left corner and the lower right corner chambers as selecting points and move the translational stage to the points orderly to get the preliminary imaging with 4x objective lens. Change the 4x objective lens with the 20x one and adjust imaging parameters including light intensity, expose time, et cetera.Then locate the selecting points so the position of each chamber leaving 13 by 15 chamber matrix or to be defined by the program automatically. With determining the coordinates of the chambers, translational stage moves to each chamber where X, Y, Z focal plane can be fine tuned. Set the interval and the duration of imaging cycle.Save the path and then start imaging. This system is available for various target dynamic tracking, such as neural stem cells differentiation in brightfield and the fluorescence field of the process of neural stem cells sphere formation. Data analysis.Change the format in nd2 to Tif using the custom MATLAB program. And divide the data according to points. Select object to be analyzed is cell or tissue.Enter the threshold, object size, noise size to analysis. Or you can choose step-by-step analysis to optimal parameters. And then analyzing all the data.After save the result in MAT format, using the plot to draw the results or traces. In this paper, we described the protocol of the and culture of high-throughput microfluidic chip system based on the photolithography technology for the live cell microenvironment dynamic control. We also described some parameters that one should carefully control such as the closing pressure of PDMS membrane valves and the spin control of fluid in parallel of the chamber.In traditional cell in vitro culture the treatment of cell culture environment is performed manual, which is time consuming and laborious and fluid filled is not easy to standard control. Particularly minutes of solution. There are some problems such as generating inputs, immune drug concentration and large consumption.However, using our microfluidic system, it is possible to repeat it as stimulator at a fixed dose single fluid equal amount of uniform concentration, compare different cultures in different culture chambers at the same time. Different with some miniature temporal resolution back stimulus and with subsetting the resolution by regulating the fluid. In addition, our system can obtain a large number of comparative experimental results by simple operations.Our system is based on the principle of diffusion, creates minimal mechanical disturbance to the cellular microenvironment. For more stable cellular microenvironment chips you can increase the list of the buffer layer as the depth of the culture layer or reduce the input fluid of medium exchange, that is reduce the pressure, which will be here for the research on cell behavior. It is to be noticed that this study has examined only the culture of 3T3 and NSC cells.For other cells or cell lines, the culture conditions comparable with our parameters to adjust it.

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JoVE Journal

Bioengineering

3.8K ビュー.  Northwest University. 微小環境は、変化するサイトカイン、リガンド濃度、組成物など、非常に複雑で動的な環境を可能にします。同様の培養環境を作り出すためには、幹細胞の分化、免疫応答、チッププラットフォーム上の臓器の開発などの複雑な生命機械に関するインビトロ研究に不可欠です。しかし、ナノリットル体積とミリ秒の精度の化学信号を生成して提供することは、ピペット?...

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

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...

high-throughput protein expression generator using a microfluidic platform

High-throughput Protein Expression Generator Using a Microfluidic Platform

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental...

generating neuromuscular junctions using a microfluidic device

The video demonstrates the formation of neuromuscular junctions using a microfluidic device. Neural precursor cells and muscle progenitor cells are seeded into different wells within the device and left to mature into motor neurons and myotubes, respectively. The creation of a volume and chemical gradient leads to the development of active neuromuscular...

Generating Neuromuscular Junctions Using a Microfluidic Device

mechanostimulation of multicellular organisms through a high-throughput microfluidic compression system

Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

cell capture using a microfluidic device

Cell Capture Using a Microfluidic Device

Pause for a few seconds to allow the cell suspension to flow through the channel before adding the remaining half of the cell suspension in the lower well. Use a pen to mark the seeded side as NPC or equivalent for easy orientation of the device without a microscope and incubate for five minutes for cell attachment. Next, slowly top up the two NPC-seeded wells with an additional day 10 motor neuron medium to a total volume of 200 microliters per well. Using a 200-microliter pipette, remove DPBS...

production of sirna-loaded lipid nanoparticles using a microfluidic device

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named...

a microfluidic platform for high-throughput single-cell isolation and culture

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

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.

fabricating high-viscosity droplets using microfluidic capillary device with phase-inversion co-flow structure

Fabricating High-viscosity Droplets using Microfluidic Capillary Device with Phase-inversion Co-flow Structure

A phase-inversion co-flow device is demonstrated to generate monodisperse high-viscosity droplets above 1 Pas, which is difficult to realize in droplet...

a magnetic nanoparticle-based immunoassay using a microfluidic device

This video demonstrates a microfluidic system for a magnetic nanoparticle-based immunoassay. The detection system utilizes a microfluidic device incorporating a magnetic trap within its channels. Pre-formed immunocomplexes, comprised of antigen-coated magnetic nanoparticles bound to primary and peroxidase-conjugated secondary antibodies, are introduced into the channels. The immunocomplexes become captured in the porous magnetic trap upon applying an external magnetic field. Using a fluorogenic...

A Magnetic Nanoparticle-Based Immunoassay Using a Microfluidic Device

a microfluidic device for studying multiple distinct strains

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...

use of a high-throughput in vitro microfluidic system to develop oral multi-species biofilms

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

Prepare a suspension of iron microparticles of diameter 7.5 micrometers in 5% BSA. Incubate the chip and the microparticle suspension with the blocking solution for at least 1 hour at room temperature. Next, insert the microparticles into the chip with a syringe needle through the side channel outlet...

high-throughput measurement and classification of organic p in environmental samples

High-Throughput Measurement and Classification of Organic P in Environmental Samples

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P.  Up to 96 samples can be measured at one time in a standard...

double emulsion generation using a polydimethylsiloxane (pdms) co-axial flow focus device

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14...

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

high throughput microfluidic rapid and low cost prototyping packaging methods

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

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

tracking axonal transport of organelles in motor neurons using a microfluidic device

This video demonstrates a method for tracking axonal transport in motor neurons using a microfluidic chamber system. It involves culturing mouse embryonic spinal cord tissues in polymer-coated wells with nutrient medium to induce axonal growth. The movement of fluorescent dye-stained organelles in live imaging, confirms the bidirectional axonal...

Tracking Axonal Transport of Organelles in Motor Neurons Using a Microfluidic Device

fluorescence time-lapse imaging of the complete s. venezuelae life cycle using a microfluidic device

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Streptomyces are characterized by a complex life cycle that has been experimentally challenging to study by cell biological means. Here we present a protocol to perform fluorescence time-lapse microscopy of the complete life cycle by growing Streptomyces venezuelae in a microfluidic...

Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

visualization of biofilm formation in candida albicans using an automated microfluidic device

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological...

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

One day after plating, replace the SCEX medium in the proximal compartment, and add rich SCEX medium to the distal compartment, maintaining a volume gradient of at least 15 microliters per well between the distal and proximal wells. After four to six days, the axons should cross to the distal compartment and be ready for axonal transport...

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 ...

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

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 ...

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

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

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

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

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 ...

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

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 ...

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such as aspiration or coverslip compression have been used to mechanically stimulate whole embryos. These approaches limit experimental design as they are imprecise, require manual handling, and can process only a couple of embryos simultaneously. Microfluidic systems have great potential for automating such experimental tasks while increasing throughput and precision. This article describes a microfluidic system developed to precisely compress whole Drosophila melanogaster (fruit fly) embryos. This system features microchannels with pneumatically actuated deformable sidewalls and enables embryo alignment, immobilization, compression, and post-stimulation collection. By parallelizing these microchannels into seven lanes, steady or dynamic compression patterns can be applied to hundreds of Drosophila embryos simultaneously. Fabricating this system on a glass coverslip facilitates the simultaneous mechanical stimulation and imaging of samples with high-resolution microscopes. Moreover, the utilization of biocompatible materials, like PDMS, and the ability to flow fluid through the system make this device capable of long-term experiments with media-dependent samples. This approach also eliminates the requirement for manual mounting which mechanically stresses samples. Furthermore, the ability to quickly collect samples from the microchannels enables post-stimulation analyses, including -omics assays which require large sample numbers unattainable using traditional mechanical stimulation approaches. The geometry of this system is readily scalable to different biological systems, enabling numerous fields to benefit from the functional features described herein including high sample throughput, mechanical stimulation or immobilization, and automated alignment.

The present protocol describes the design, fabrication, and characterization of a microfluidic system capable of aligning, immobilizing, and precisely compressing hundreds of Drosophila melanogaster embryos with minimal user intervention. This system enables high-resolution imaging and recovery of samples for post-stimulation analysis and can be scaled to accommodate other multicellular biological systems.

During embryogenesis, coordinated cell movement generates mechanical forces that regulate gene expression and activity. To study this process, tools such ...