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.

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

Research

JoVE Journal

Bioengineering

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 ...

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. Biosensors relying on the principle of FRET have enabled real-time visualization of subcellular signaling events in live cells with high temporal and spatial resolution. Here, we describe the application of a genetically encoded Bile Acid Sensor (BAS) that consists of two fluorophores fused to the farnesoid X receptor ligand binding domain (FXR-LBD), thereby forming a bile acid sensor that can be activated by a large number of bile acids species and other (synthetic) FXR ligands. This sensor can be targeted to different cellular compartments including the nucleus (NucleoBAS) and cytosol (CytoBAS) to measure bile acid concentrations locally. It allows rapid and simple quantitation of cellular bile acid influx, efflux and subcellular distribution of endogenous bile acids without the need for labeling with fluorescent tags or radionuclei. Furthermore, the BAS FRET sensors can be useful for monitoring FXR ligand binding. Finally, we show that this FRET biosensor can be combined with imaging of other spectrally distinct fluorophores. This allows for combined analysis of intracellular bile acid dynamics and i) localization and/or abundance of proteins of interest, or ii) intracellular signaling in a single cell.

We provide a detailed protocol to study bile acid dynamics in living cells using a genetically encoded BAS FRET sensor. This Bile Acid Sensor represents a unique tool to study (regulation of) bile acid transport and FXR activation in a wide range of cell types.

F&#246;rster Resonance Energy Transfer (FRET) has become a powerful tool for monitoring protein folding, interaction and localization in single cells. ...

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 ...