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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;.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&#160;</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&#34; ID x 1-7/16&#34; 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&#34; ID x 1&#34; 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&#160;</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&#34; x 0.060&#34;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. ...

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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

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

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 ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

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 ...