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...

<ol>
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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+heterogeneous+environment+and+docetaxel+gradient+in+the+emergence+of+polyploid,+mesenchymal+and+resistant+prostate+cancer+cells.">The role of heterogeneous environment and docetaxel gradient in the emergence of polyploid, mesenchymal and resistant prostate cancer cells.</a> Clinical &amp; Experimental Metastasis. 36 (2), 97-108 (2019).</li><li>Roca, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factors+OVOL1+and+OVOL2+induce+the+mesenchymal+to+epithelial+transition+in+human+cancer.">Transcription factors OVOL1 and OVOL2 induce the mesenchymal to epithelial transition in human cancer.</a> PloS One. 8 (10), e76773 (2013).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>Tinevez, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trackmate:+An+open+and+extensible+platform+for+single-particle+tracking.">Trackmate: An open and extensible platform for single-particle tracking.</a> Methods. 115, 80-90 (2017).</li><li>Caballero, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-on-chip+models+of+cancer+metastasis+for+future+personalized+medicine:+From+chip+to+the+patient.">Organ-on-chip models of cancer metastasis for future personalized medicine: From chip to the patient.</a> Biomaterials. 149, 98-115 (2017).</li><li>Akhtar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+flaws+and+human+harms+of+animal+experimentation.">The flaws and human harms of animal experimentation.</a> Cambridge Quarterly of Healthcare Ethics. 24 (4), 407-419 (2015).</li><li>Sontheimer-Phelps, A., Hassell, B. A., Ingber, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+cancer+in+microfluidic+human+organs-on-chips.">Modelling cancer in microfluidic human organs-on-chips.</a> Nature Reviews Cancer. 19 (2), 65-81 (2019).</li><li>Bogorad, M. I., DeStefano, J., Karlsson, J., Wong, A. D., Gerecht, S., Searson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+in+vitro+microvessel+models.">Review: in vitro microvessel models.</a> Lab on a Chip. 15, 4242-4255 (2015).</li><li>Za&#241;udo, J. G. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+control+of+cellular+decision-making+networks+in+the+epithelial-to-mesenchymal+transition.">Towards control of cellular decision-making networks in the epithelial-to-mesenchymal transition.</a> Physical Biology. 16 (3), 031002 (2019).</li><li>Morris, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+population+solitary+waves+can+defeat+rings+of+funnels.">Bacterial population solitary waves can defeat rings of funnels.</a> New Journal of Physics. 19 (3), 035002 (2017).</li></ol>

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. ...

This microfluidic cancer on-chip model which we call the evolution accelerator, provides a controllable platform for long term, real time quantitative studies of cancer dynamics within well defined environmental conditions at a single cell level. The technology allows us to probe the evolutionary dynamics of cancer under a tumor-like stress landscape. Providing information including morphology, population, motility, and migration over time on the cellular level.With the ability to control and monitor the behavior of mixed tumor populations under well controlled stress gradients our technology may present a more physiologically relevant model for preclinical drug development. The presented pressure sealing packaging method can also be implemented in other microfluidic systems that requires sophisticated adjustment of ambient gas composition as well as the downstream experiment capacity. The keys to successfully perform a long term experiment is to secure a physically and biochemically stable environment.Factors including temperature, humidity, and gas composition should be monitored at all times. To begin, fabricate a plate holder that is sufficient to hold simultaneous gas permeable culture dishes on a 3D printing machine. Unscrew the components of the customized three well sample plate with screw driver.Disinfect the components via UV exposure for at least one hour and leave in a sterile environment. To start cell line seeding add five milliliters of trypsin into each PC3-Epi or PC3-EMT cell culture in a 10 centimeter culture dish and run the trypsinization for five minutes in an incubator at 37 degrees celsius. Transfer the cells from the culture dishes into 15 milliliter centrifuge tubes and then centrifuge at 150 times g for three minutes.Discard the supernatant. Re-suspend each of the cell pellets in the 15 milliliter centrifuge tubes and count the cells of each PC3-MP or PC3-EMT using a hemocytometer. Isolate a total of 2.5 times 10 to the four of each cell type.Mix the two isolated cell types and add in two milliliters of culture media. Seed two milliliters of the cell culture into each of the three gas permeable culture dishes. Leave the gas permeable in the incubator at 37 degrees celsius over night for the cells to attach.Disinfect the PDMS devices via UV exposure for at least one hour and leave in a sterile environment. Now set up a gas supply system that consists of both carbon dioxide and oxygen control units, a gas pump, a gas mixing chamber, a humidifier or bubbler, and three separate sets of gas valves, and pressure gauges. Alternatively use a gas supply system that provides normoxia condition.Make sure the relative humidity in the mixing chamber is increased to above 85%Set up an onstage incubation thermal control unit at 37 degrees celsius with separate heating sub-units for the lid and the bottom plate. The next day take out the gas permeable culture dishes from the incubator and identify that the detailed components are in order, Every well possesses a gas permeable culture dish holder, a PDMS chip holder, a glass window holder, and a pair of 35 milliliter wide glass windows. Use the fitted screws to assemble these components and clamp the gas permeable culture dish.Then transfer the pre-warmed culture medium to a vacuum chamber to de-gas for 20 minutes. Treat the PDMS chip in an oxygen plasma system for 30 seconds in order to maintain hydrophilicity. Next load two syringes slowly with 10 milliliters of de-gassed growth media and the other two syringes with 10 milliliters of de-gassed desired reagent of interest.For example media and media with drug. Connect each individual syringe to a 50 centimeter tubing by a 23 gauge dispensing needle into one hollow steel pin. Insert a hollow steel pin into the other end of the tubing.Push the media into the tubing to 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 with media. The reservoir layer works as on-chip bubble trap to prevent air bubbles from getting into the microfluidic pattern.Then load a one milliliter syringe with culture media. Connect the syringe to a five centimeter tubing by a 23 gauge dispensing needle into one hollow steel pin. Insert a hollow steel pin into the other end of the tubing and prime the tubing.Insert the hollow steel pin into the center hole of the chip for excessive media to be extracted later. Load two 10 milliliter syringes per chip in the forward deck and place the other two syringes in the withdrawal deck of the syringe system. In order to avoid entrapping microbubbles in the microfluidic pattern, dispense one milliliter of the pre-warmed and de-gassed media into the 35 milliliter deep gas permeable culture dish.Position the chips directly on top of the gas permeable culture membranes, where the chip approaches the liquid surface with a 15 degree tilt angle. Clamp the PDMS chip with the PDMS chip holder, pushing the PDMS device downward. Tape a sheet of a sealer on top of the PDMS device and clamp with PDMS chip holder, in order to prevent the chip from drying out.Set media flow rate around the array to be 20 microliters per hour. Position the entire plate in the onstage incubator on the motorized stage of an inverted microscope. Connect the gas supply system to the gas channels through gas tubing and maintain the gauge pressure at 0.2 PSI.This pressurizes the gas permeable culture membrane against the installed PDMS chip to ensure sealing of the device. Slowly extract excessive media in the chip using the one milliliter syringe from the center hole. Observe the chip under the microscope while extracting media and then stop extracting when the chip is sealed.Connect the temperature sensing unit of the onstage incubator to the three well plate and set to 37 degrees celsius. In this study PC3 cultured in an evolution accelerator without the presence of external stress had growth curves while aligned. Suggesting the identicality of the cell proliferation profile as a function of position across the chip.The initial slope of the curves reveals the proliferation rate. The doubling time for cells in the device is around 24 hours. GFP expressing PC3-EMT and mCherry expressing PC3-Epi cell lined were co-cultured in the evolution accelerator.Starting from 40%confluence the coculture grew 40%confluent within three days. While the growth curve of total confluence is in the form of logistic growth model. The population of PC3-EMT continued to expand and PC3-Epi was suppressed in the asymptotic phase.PC3-Epi and PC3-EMT cells were densely seated in the center of the device and were allowed to freely migrate toward the empty region. The normalized histogram of the speed of the speed of both phenotypes shows the velocity of PC3-EMT was significantly higher than PC3-Epi by a factor of 1.8 times. It is very important to avoid creating microbubbles at all cost while the culture media should be pre-warmed and de-gassed.The PDMS chip should be wetted properly and handled gently. Due to the soft re-sealable nature of our pressure sealing method many downstream experiments can be preformed. Such as subclinical population extraction, local metabolite analysis, and spatially resolved immunofluorescence and sequencing.Our technology demonstrates a way to reproduce key components and interactions in a complex super-micro environment in a comprehensive manner. Yet simple enough to provide quantitative, reliable, and reproducible data.

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Research

JoVE Journal

Bioengineering

6.3K vistas.  Princeton University. Este modelo de cáncer microfluídico en chip que llamamos acelerador de evolución, proporciona una plataforma controlable para estudios cuantitativos a largo plazo y en tiempo real de la dinámica del cáncer dentro de condiciones ambientales bien definidas a un nivel de célula única. La tecnología nos permite sondear la dinámica evolutiva del cáncer bajo un paisaje de estrés similar a un tumor. Proporcionar información que incluye morfología, población, motilidad y migración a lo...