This work was supported by NSF PHY-1659940.

1. Fabrication of microfluidic device
<ol>
	<li>Generate the desired microfluidic pattern using a layout design software (see Supplemental Materials).</li>
	<li>Fabricate the photomask. See Table of Materials for more details.
	<ol>
		<li>Utilizing a laser writer, write the pattern on a soda-lime glass plate coated with 100 nm of Cr and 500 nm of photoresist AZ1518.</li>
		<li>Develop the photoresist with developer AZ300MIF for 60 s.</li>
		<li>Etch away the chromium without protection from the photoresist using Cr-7 Chromium Etchant.</li>
		<li>Strip off residual photoresist using a photoresist stripper at 70 &#176;C for 45 min.</li>
	</ol>
	</li>
	<li>Pattern the photoresist. See Table of Materials for more details.
	<ol>
		<li>Spin coat HMDS on silicon wafer at 4000 rpm for 40 s.</li>
		<li>Spin coat photoresist AZ4330 on silicon wafer at 4000 rpm for 40 s.</li>
		<li>Soft bake the silicon wafer at 95 &#176;C for 60 s.</li>
		<li>Utilize the mask aligner to expose UV to the silicon wafer.</li>
		<li>Develop the photoresist with developer AZ300MIF for 4 min.</li>
	</ol>
	</li>
	<li>Perform DRIE etching. See Table of Materials for more details.
	<ol>
		<li>Dry-etch the wafer patterned with the photoresist using a Silicon Deep Reactive Ion Etching (DRIE) system for 100 &#181;m depth.</li>
		<li>Strip off the photoresist with acetone and plasma etch with a plasma etcher.</li>
	</ol>
	</li>
	<li>Wafer oxidation and silanization. See Table of Materials for more details.
	<ol>
		<li>Perform thermal oxidation on the etched silicon wafer in a furnace at 1100 &#176;C for 1 h.</li>
		<li>Wait for the silicon wafer to cool down, and then place the wafer into a desiccator with a few drops of trichloro-1H,1H,2H,2H-perfluorooctyl-silane (PFOTS) dripped in a small container near the wafer.</li>
		<li>Pump the pressure of the desiccator to 0.5 atm at room temperature for 60 min. The silicon wafer mold should become hydrophobic after proper silanization, which can be tested by adding several droplets of water onto the wafer.</li>
	</ol>
	</li>
	<li>Soft lithography. See Table of Materials for more details.
	<ol>
		<li>Mix pre-polymer and cross-linker (from polymethylsiloxane (PDMS) kit) at a 10:1 by weight ratio.</li>
		<li>Pour the mixed PDMS to create a 10 mm film in height onto the silanized silicon wafer mold.</li>
		<li>Degas the resulting PDMS-silicon wafer system in a desiccator for 30 min to 1 h.</li>
		<li>Incubate the PDMS-silicon wafer in a 70 &#176;C incubator overnight to cure the PDMS.</li>
		<li>Once the PDMS is cured, peel the PDMS film off the silicon wafer carefully.</li>
		<li>Using biopsy needles, punch through-holes at the inlet ports based on the location of the pattern on the PDMS and employ a circular punch to cut chips 27 mm in diameter.</li>
	</ol>
	</li>
	<li>PDMS layer bonding.
	<ol>
		<li>Heat cure two stacks of 27 mm PDMS cylinders (without patterning), which will become the reservoir layer and the capping layer of the device.</li>
		<li>Cut out two 7 mm circles around the inlets on the reservoir layer, and utilize a biopsy needle to punch through-holes at the inlet ports on the capping layer.</li>
		<li>Bond three stacks of PDMS (patterned stack, reservoir layer, capping layer) with oxygen plasma treatment.</li>
		<li>With the biopsy needle, punch through-holes at the outlets and the center port of the device.</li>
	</ol>
	</li>
</ol>
2. Media and cell line preparation
<ol>
	<li>Prepare media for culturing PC3 cell lines, including PC3-EMT and PC3-EPI: mix RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1x antibiotic-antimycotic. Generate cell lines as described previously14. 
	NOTE: PC3 cell lines are maintained in the above media in humidified incubators at 37 &#176;C with 5% CO2. Split cells and subculture every 3 days, before they reach 100% confluency.</li>
	<li>Transfect cell lines with cytoplasmic-labeled fluorescent markers for better visualization, as described previously12, such that PC3-EMT expresses cytoplasmic GFP and PC3-EPI expresses cytoplasmic mCherry. Note that the technology is also compatible with any fluorescent-labeled cells as well as brightfield imaging of unlabeled cells.</li>
</ol>
3. Experimental setup
<ol>
	<li>Fabrication of metal plate holder
	<ol>
		<li>Fabricate a plate holder that is sufficient to hold simultaneous gas-permeable culture dishes for parallel experiments by machining or 3D printing. The 3D CAD file (&#34;3-well plate.FCStd&#34;) can be found on GitHub as a reference (https://github.com/kechihl/3-well-plate).</li>
		<li>Unscrew the components of the holder (Figure 2) with a screwdriver. Disinfect the components via UV exposure for at least 1 h and leave in a sterile environment. 
		NOTE: The holder is designed to provide substantial conditions for thermal equilibrium and ideal gas compositions, with gas channel inlets that allow for airflow. More details can be found in our previous work12.</li>
		<li>Prepare up to three gas-permeable culture dishes, of which the cell culture membrane is relatively flexible.</li>
	</ol>
	</li>
	<li>Cell line seeding
	<ol>
		<li>24 hours before the start of the experiment, harvest PC3-EPI and PC3-EMT cells by trypsinization for 5 min. Add prewarmed culture media, then centrifuge at 150 x g for 3 min and discard the supernatant.</li>
		<li>Count cells of each PC3-EPI and PC3-EMT using a hemocytometer and isolate a total of 2.5 x 104 of each cell type for each gas-permeable culture dish.</li>
		<li>Mix and resuspend the two cell types in 2 mL of culture media and seed the cells into each of the gas-permeable culture dish.</li>
		<li>Leave the entire plate holder in the incubator overnight for cells to attach.</li>
		<li>Disinfect the PDMS devices via UV exposure for at least 1 h and leave in a sterile environment.</li>
	</ol>
	</li>
	<li>Setting up thermal control unit and gas supply system
	<ol>
		<li>As shown in Figure 3, set up a gas supply system that consists of both CO2 and O2 control units, a gas pump, a gas mixing chamber, a humidifier or bubbler, and three separate sets of gas valves and pressure gauges. More details can be found in our previous work12.</li>
		<li>Set up the CO2 and O2 control units such that they adjust the mixing rate of the gas from CO2 and N2 tanks and the O2 source. Alternatively, any gas supply system that provides gas under normoxia condition would work.</li>
		<li>Make sure the gas that is combined in the mixing chamber and humidified by a bubbler such that the relative humidity is increased up to 85% (as read out from a relative humidity monitor) and that the gas leads to three independent gas valves with pressure gauges in order to control and monitor gas flow rate in the plate holder.</li>
		<li>Place the entire plate holder in an on-stage incubation thermal control unit with separate heating subunits for the lid and the bottom plate, with all units set at 37 &#176;C. See Table of Materials for more details.</li>
	</ol>
	</li>
	<li>Installation of microfluidic device. See Table of Materials for more details.
	<ol>
		<li>Identify that the detailed components of the 3-well plate holder are in order, as in Figure 2. Each of the three wells are identical and independent, with a pair of gas channels for each well. Every well possesses a gas-permeable culture dish holder, a PDMS chip holder, a glass window holder, and a pair of 35 mm glass windows. These components can be assembled using the fitted screws. 
		NOTE: The glass windows ensure that there is a thermally-isolated space between the gas-permeable culture membrane and the well such that no water condensation occurs due to temperature differences at the interface. Note that the 3 wells are independent, and 3 experiments can be done separately.</li>
		<li>Pre-warm culture medium at 37 &#176;C and degas in a vacuum chamber for 20 min.</li>
		<li>Treat the PDMS chips using an oxygen plasma system for 30 s in order to maintain hydrophilicity.</li>
		<li>Set up the syringe system. Load two syringes slowly with growth media and other two syringes with desired reagent of interest (media, media with drug, etc.). Connect each individual syringe to a 50 cm tubing (0.020&#34; x 0.060&#34;OD) by a 23 G dispensing needle into one hollow steel pin. Insert a hollow steel pin into the other end of the tubing.</li>
		<li>Prime the tubing and insert the steel pin into each PDMS chip through the capping layer. Fill up the reservoir layer and wet the PDMS pattern layer pattern with media. The reservoir layer works as an on-chip bubble trap to prevent air bubbles from getting into the microfluidic pattern.</li>
		<li>Load a 1 mL syringe with culture media. Connect the syringe to a 5 cm tubing (0.020&#34; x 0.060&#34;OD) by a 23 G dispensing needle into one hollow steel pin. Insert a hollow steel pin into the other end of the tubing and prime the tubing.</li>
		<li>Insert the hollow steel pin into the center hole of the chip, where excessive media can be extracted out from the chip later during the chip sealing process.</li>
		<li>As each PDMS device requires four 10 mL syringes loaded onto a syringe pump, load two 10 mL syringes per chip in the forward deck. Place the two other syringes in the withdraw deck of the syringe system.</li>
		<li>Place chip directly on top of the gas-permeable culture membranes (with cells already adhered to the membranes). In order to avoid entrapping microbubbles in the microfluidic pattern, dispense 1 mL of prewarmed and degassed media into the 35 mm gas-permeable culture dish before assembly, and then make sure that the chip approaches the liquid surface with a 15-degree tilt angle.</li>
		<li>Clamp the gas-permeable culture dish and the well in the gas-permeable culture dish holder for each chip, with the PDMS chip holder pushing the PDMS device downward.</li>
		<li>Tape a sheet of a sealer on top of the PDMS device and clamp with the PDMS chip holder in order to prevent the chip from drying out.</li>
		<li>Set media flow rate around the array to be 20 &#181;L/h.</li>
		<li>Set the entire plate in the on-stage incubator on the motorized stage of an inverted microscope. Connect the gas supply system to the gas channels and pressurizing the gas-permeable culture membrane against the installed PDMS chip to ensure sealing of device. Maintain gauge pressure at 0.2 psi (1.4 x 104 Pa).</li>
		<li>Slowly extract excessive media in the chip using the 1-mL syringe from the center hole. Observe the chip under the microscope while extracting media and then stop extracting when the chip is sealed. Chip sealing should be obvious since a part of the cells would be crushed by the micro-structures.</li>
		<li>Connect the temperature sensing unit of the on-stage incubator to the 3-well plate and set to 37 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Single-cell time-lapse imaging
<ol>
	<li>Set up imaging software utilizing an inverted microscope to time-lapse image acquisition. Note that an inverted fluorescent microscope with fully motorized x-y stage, focus knob, shutter, and filter cubes is required for an EA experiment.</li>
	<li>Configure software to acquire images across two channels at 10x magnification for each chip with automatic image-stitching after one round of autofocus. Be aware that automatic correction systems like Perfect Focus do not guarantee satisfying image quality. It is more recommended to generate a customized focus surface for long-term image acquisition based on either autofocus or manual focus across the chip.</li>
	<li>Take images every hour and leave experiment running on the time scale of weeks. Monitor image quality on a daily basis and update the focus surface if necessary.</li>
	<li>Prepare the PDMS chip and gas-permeable culture dish for subsequent immunofluorescent analysis, as described previously13.</li>
</ol>
5. Image processing and analysis
<ol>
	<li>Post-experimental processing
	<ol>
		<li>Convert images into TIFF format for image processing and measurements utilizing Fiji/ImageJ.</li>
		<li>Compress TIFF files for ease of further image processing.</li>
	</ol>
	</li>
	<li>Fiji/ImageJ analysis
	<ol>
		<li>To identify cells, perform Background Subtraction and Particle Analysis to identify fluorescent bright-spots for cell location detection and automatic cell counting.</li>
		<li>Utilize plugins (Manual Tracking, Chemotaxis, Migration Tool, Trackmate) to analyze cell motility and migration.</li>
	</ol>
	</li>
</ol>

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No conflicts of interest declared.

Conventional cell culture was developed almost a century ago and remains the most frequently used preclinical model in biomedical research, despite its proven limited ability to predict clinical results in cancer17. Animal models offer the highest physiological relevance and reasonable genetic similarity to humans, but have long been acknowledged to have significant limitations in predicting human outcomes18. Among all the existing preclinical models, microfluidic cancer-on...

Cancer has been increasingly recognized as a complex ecosystem that depends not only on the continued dysregulation of mutated cell populations but also on vital interactions between cancer cells and the host microenvironment. In this sense, cancer evolves on an adaptive landscape manifested by a combination of factors, including a heterogeneous tumor microenvironment and crosstalk with a variety of host cells, all of which contribute selective pressures for further genetic or epigenetic changes1,2,3. In the context of solid tumors, uneven distribution of chemotherapeutics and other resource gradients contributes to their molecular heterogeneity and may play a role in the development of drug resistance, increased angiogenesis to particular tumor subpopulations, and even metastasis4,5,6. Conventional in vitro 2D cell culture studies, while possessing large-scale, convenient experimental capacity, provides mean-field, uniform, and fixed conditions, often lacking the precise spatial and temporal environmental control necessary to truly emulate in vivo tumor dynamics. Thus, there is a need for more representative ex vivo models to reproduce the tumor microenvironment prior to animal models in the drug development pipeline in order for a better prediction of cancer progression as well as responses to drugs within dynamical stress landscapes. Microfluidics have been proposed to bridge the gap between 2D cell culture studies and more complex in vivo animal studies that may not be able to support controllable quantitative studies7,8,9.
An ideal in vitro system to characterize cancer cell dynamics should possess the ability to generate a heterogeneous microenvironment to mimic the adaptive cellular responses that may take place in a tumor, as well as allow for the observation of these dynamics at a single-cell resolution. In this article, we describe a microfluidic cell culture platform, a PDMS-based device called the &#34;Evolution Accelerator&#34; (EA), that allows for parallel in vitro studies of cancer cell dynamics at cellular resolution with real-time data acquisition over the course of weeks, while stably maintaining gradients of stress across the culture landscape. The design of this platform is based on our previous work, in which the evolutionary dynamics of organisms in a metapopulation can be accelerated10,11. Specifically, in a group of spatially separated populations that interact at some level, when exposed to a heterogeneous stress landscape, the most fit species can dominate in a local population faster compared with that of a large uniform population. The advantageous species then migrate to neighboring microhabitats in search of resources and space, and eventually dominate the entire population. As shown in Figure 1, the pattern of the microfluidic EA chip is composed of (i) a pair of serpentine channels that provide fresh media circulation and construct fixed boundary conditions for chemical diffusion, and (ii) the hexagonal cell culture region which consists 109 interconnected hexagonal and 24 half-hexagonal chambers in the center, resembling a honeycomb structure. The chip is 100 &#181;m in depth. Media channels and cell culture region are connected with small slits (about 15 &#181;m wide), which prevent direct media flow and the resulting shear stress across the cell culture area, yet still allow chemicals to diffuse through small slits and exchange nutrients, metabolic waste, etc. The generation of chemical gradients is demonstrated in Figure 1B, where one media channel contains 0.1 mM of fluorescein while the other channel is free of fluorescein. Cells are cultured on a gas permeable membrane, encapsulated by the microstructures through the positive back pressure on the membrane against the chip. The components of the device holder are illustrated in Figure 2, and the experimental setup is illustrated in Figure 3, where the culture is maintained on an inverted microscope at 37 &#176;C, with above 85% relative humidity, and conditioned under normoxia gas composition.
This system provides detailed observation of localized cellular interactions via brightfield and fluorescent channels and allows for spatially-resolved downstream assays such as immunofluorescence, Western blot, or mass spectrometry. We have previously demonstrated as a proof-of-principle of this microfluidic cancer-on-chip model on the long-term co-culture of epithelial and mesenchymal PC3 prostate cancer cells12 as well as the emergence of drug-resistance polyploid giant cancer cells using the epithelial PC3 cell line13. While we present the application of this platform to understand the spatiotemporal dynamics of epithelial PC3 and mesenchymal prostate cancer cells under a stress gradient of docetaxel, the microfluidic system can be easily applied to any combination of cell lines and resource (i.e., drug, nutrient, oxygen) gradients.

<table><tbody><tr><td>10 mL BD Luer-Lok tip syringes</td><td>BD</td><td>14-823-16E</td><td></td></tr><tr><td>Antibiotic-Antimycotic</td><td>Sigma-Aldrich</td><td>A5955</td><td>1x anti-anti</td></tr><tr><td>AZ 300 MIF</td><td>Merck KGaA</td><td>18441123163</td><td>Photoresist developer</td></tr><tr><td>AZ1518</td><td>Merck KGaA</td><td>AZ1518</td><td>Photoresist</td></tr><tr><td>AZ4330</td><td>Merck KGaA</td><td>AZ4330</td><td>Photoresist</td></tr><tr><td>Cr Chromium Etchant</td><td>Sigma-Aldrich</td><td>651826</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Life Technologies Corporation</td><td>10437028</td><td></td></tr><tr><td>Heidelberg DWL 66+ laserwriter</td><td>Heidelberg Instruments</td><td>DWL66+</td><td>Writing photomask</td></tr><tr><td>Hexamethyldisilazane (HMDS)</td><td>Sigma-Aldrich</td><td>379212</td><td>For photoresist adhesion enhancement</td></tr><tr><td>Hollow steel pins</td><td>New England Small Tube</td><td>NE-1300-01</td><td>&amp;nbsp;.025 OD .017 ID x .500 long / type 304 WD fullhard</td></tr><tr><td>ibidi Heating System, Multi-Well Plates, K-Frame</td><td>ibidi</td><td>10929</td><td>On-stage incubator&amp;nbsp;</td></tr><tr><td>Luer-Lok 23 G dispensing needle</td><td>McMaster-Carr</td><td>75165A684</td><td>To connect syringes and tubings</td></tr><tr><td>Lumox dish 35</td><td>Sarstedt</td><td>94.6077.331</td><td>Gas-permeable cell culture dish</td></tr><tr><td>Microposit Remover 1165</td><td>Dow Electronic Materials</td><td>Microposit Remover 1165</td><td>Photoresist stripper</td></tr><tr><td>Microseal B Adhesive Sealer</td><td>Bio-Rad Laboratories</td><td>MSB1001</td><td>Adhesive sealer</td></tr><tr><td>O-Ring (for Lumox plate sealing)</td><td>McMaster-Carr</td><td>9452K114</td><td>Dash No. 27; 1-5/16&quot; ID x 1-7/16&quot; OD; Duro 70</td></tr><tr><td>O-Ring (for bottom glass window sealing)</td><td>McMaster-Carr</td><td>9452K74</td><td>Dash No. 20; 7/8&quot; ID x 1&quot; OD; Duro 70</td></tr><tr><td>Plasma-Preen Plasma Cleaning/Etching System</td><td>Plasmatic Systems, Inc</td><td>Plasma-Preen</td><td>Oxygen plasma system</td></tr><tr><td>RPMI 1640</td><td>Life Technologies Corporation</td><td>11875-093</td><td></td></tr><tr><td>Samco RIE800iPB DRIE</td><td>Samco</td><td>RIE800iPB</td><td>Deep reactive-ion etching system</td></tr><tr><td>Suss MA6 mask aligner</td><td>SUSS MicroTec</td><td>MA6</td><td>Mask aligner&amp;nbsp;</td></tr><tr><td>Sylgard 184 Silicone Elastomer</td><td>Fisher Scientific</td><td>NC9285739</td><td>PDMS elastomer</td></tr><tr><td>TePla M4L plasma etcher</td><td>PVA TePla</td><td>M4L</td><td>Plasma etcher</td></tr><tr><td>Trichloro-1H,1H,2H,2H-perfluorooctyl-silane (PFOTS)</td><td>Sigma-Aldrich</td><td>448931</td><td>For silicon wafer silanization</td></tr><tr><td>Tygon microbore tube (0.020&quot; x 0.060&quot;OD)</td><td>Cole-Parmer</td><td>EW-06419-01</td><td>Tubings for media delivery</td></tr></tbody></table>

generation of heterogeneous drug gradients across cancer populations on a microfluidic evolution accelerator for real-time observation

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. Microfluidic cancer-on-chip models have been proposed to bridge the gap between the oversimplified conventional 2D cultures and more complicated animal models, which have limited ability to produce reliable and reproducible quantitative results. Here, we present a microfluidic cancer-on-chip model that reproduces key components of a complex tumor microenvironment in a comprehensive manner, yet is simple enough to provide robust quantitative descriptions of cancer dynamics. This microfluidic cancer-on-chip model, the &#34;Evolution Accelerator,&#34; breaks down a large population of cancer cells into an interconnected array of tumor microenvironments while generating a heterogeneous chemotherapeutic stress landscape. The progression and the evolutionary dynamics of cancer in response to drug gradient can be monitored for weeks in real time, and numerous downstream experiments can be performed complementary to the time-lapse images taken through the course of the experiments.

Validation of optimum cell growth on chip 
A major goal of the experiment platform is to reproduce key components and interactions in a complex tumor microenvironment in a comprehensive manner, yet simple enough to provide quantitative, reliable and reproducible data. This goal can only be achieved if we have full control of the physical and biochemical environmental factors. We must either exclude the undesired factors or figure out a way to incorporate the uncontro...

Watch this Scientific Journal Video about Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation at JoVE.com

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

We present a microfluidic cancer-on-chip model, the &#34;Evolution Accelerator&#34; technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

Conventional cell culture remains the most frequently used preclinical model, despite its proven limited ability to predict clinical results in cancer. ...

generation-heterogeneous-drug-gradients-across-cancer-populations-on

Research

JoVE Journal

Bioengineering

6.3K Views.  Princeton University. We present a microfluidic cancer-on-chip model, the "Evolution Accelerator" technology, which provides a controllable platform for long-term real-time quantitative studies of cancer dynamics within well-defined environmental conditions at the single-cell level. This technology is expected to work as an in vitro model for fundamental research or pre-clinical drug development.

Video: Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

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 Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Biology

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Medicine

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor cells into neighboring tissue. Thus, invasion is a crucial step in metastasis, making the invasion process research and development of anti-metastatic drugs, highly significant. To address this demand, there is a need to develop 3D in vitro models which imitate the architecture of solid tumors and their microenvironment most closely to in vivo state on one hand, but at the same time be reproducible, robust and suitable for high yield and high content measurements. Currently, most invasion assays lean on sophisticated microfluidic technologies which are adequate for research but not for high volume drug screening. Other assays using plate-based devices with isolated individual spheroids in each well are material consuming and have low sample size per condition. The goal of the current protocol is to provide a simple and reproducible biomimetic 3D cell-based system for the analysis of invasion capacity in large populations of tumor spheroids. We developed a 3D model for invasion assay based on HMCA imaging plate for the research of tumor invasion and anti-metastatic drug discovery. This device enables the production of numerous uniform spheroids per well (high sample size per condition) surrounded by ECM components, while continuously and simultaneously observing and measuring the spheroids at single-element resolution for medium throughput screening of anti-metastatic drugs. This platform is presented here by the production of HeLa and MCF7 spheroids for exemplifying single cell and collective invasion. We compare the influence of the ECM component hyaluronic acid (HA) on the invasive capacity of collagen surrounding HeLa spheroids. Finally, we introduce Fisetin (invasion inhibitor) to HeLa spheroids and nitric oxide (NO) (invasion activator) to MCF7 spheroids. The results are analyzed by in-house software which enables semi-automatic, simple and fast analysis which facilitates multi-parameter examination.

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

A HMCA-based imaging plate is presented for invasion assay performance. This plate facilitates the formation of three-dimensional (3D) tumor spheroids and the measurement of cancer cell invasion into the extracellular matrix (ECM). The invasion assay quantification is achieved by semi-automatic analysis.

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

Cancer Research

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

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 ...

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

Generation of Heterogeneous Drug Gradients Across Cancer Populations on a Microfluidic Evolution Accelerator for Real-Time Observation

Summary

Explore More Videos

Chapters in this video

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

A Gradient-generating Microfluidic Device for Cell Biology

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

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

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments