We thank RainDance Technologies for the sample of PFPE-PEG surfactant utilized in this study, and we thank the BioMEMS Resource Center (Mehmet Toner, director) for the silicon wafer mold used to create PDMS channel replicas.

The protocols in this section describe the materials and equipment utilized specifically to obtain the experimental results presented. Note that alternative suppliers for chemicals and equipment may be utilized.
1. Device Fabrication and Soft Lithography
Standard soft lithography techniques,21 a number of which have been featured in previous JOVE articles,22 were used for creating polydimethylsiloxane (PDMS) microchannel networks bonded to glass substrates. Aside from master replica mold fabrication by SU-8 photolithography, the processes may be performed outside a clean room or clean hood; however, dust and particulates should still be minimized to achieve consistent results.
<ol>
 <li>Design a micro-channel pattern as shown in Figure 1 in AutoCAD (AutoDesk Inc.). Employ a third party manufacturer (Fineline Imaging Inc.) to print a high resolution (50,000 dpi) transparency mask on Mylar film or quartz where channels are transparent on a dark background.</li>
 <li>Create a silicon and SU-8 photoresist master for replica molding. Briefly, spin SU-8 2050 (MicroChem) negative photoresist with manufacturer's recommended rpm on a spin-coater to create a 52 &mu;m thick layer on a clean 7.5 cm or 10 cm silicon wafer. After soft bake, edge bead removal, UV exposure through a contact mask, post-exposure bake, development, and flood exposure, measure the actual thickness of the SU-8 layer using a Dektak profilometer (Veeco). Tape the master mold onto the bottom of a 4" or 5" Petri dish to prepare for PDMS replica molding.</li>
 <li>Mix PDMS elastomer base with elastomer curing agent (Dow Corning) in a 10:1 ratio w/w base to curing agent. Pour well-mixed PDMS precursor onto the silicon master to create a 2-3 mm final thickness layer. A mixture of 20 g elastomer base with 2 g curing agent is sufficient to cover a 4" diameter surface.</li>
 <li>Place the master mold and PDMS in vacuum desiccator (Jencons) to de-gas the uncured PDMS. Using a pressure regulator (Cole Parmer), slowly decrease the chamber gage pressure from 0" Hg to -27" Hg over 20 minutes to avoid excessive foaming. Leave device in vacuum chamber at -27" Hg for 30 minutes or until air bubbles disappear.</li>
 <li>Release vacuum and move master mold and PDMS to a 65 &deg;C oven (Thermo Scientific) for a minimum of four hours. The device may be left in the oven overnight to improve curing.</li>
 <li>Remove the device from oven and allow to cool. Carefully cut PDMS around circular wafer using a precision knife and peel out PDMS. Cut out device outline as shown in Figure 1 with a scalpel.</li>
 <li>Punch fluidic ports (three per device) in the three round regions shown in Figure 1 using a biopsy punch. For this device, use a 0.75 mm outer diameter punch (Harris).</li>
 <li>Adhere scotch tape to the patterned side of the PDMS and peel to remove any dust. As a cost-saving but viable alternative to conventional oxygen plasma apparatuses,21,22 plasma treat the patterned side of the PDMS and a clean 3" x 1" glass microscope slide using a hand-held laboratory corona treater (Electro-Technic Products Inc.).23 Note that this device should be used in a fume hood or well-ventilated area due to ozone discharge, and all watches and cell phones should be kept at least ten feet away. Adjust the corona discharge to attain a stable corona with minimal sparking. Slowly wave the electrode about 1/4" above each surface for around 20 seconds and then immediately bring the treated surfaces into contact to form a strong permanent bond before the PDMS surfaces return to their native state.</li>
 <li>Place the device on a metal plate, place in a cool oven, set the oven to 120 &deg;C, and bake overnight to complete bonding and to return the PDMS to its original hydrophobic state.24 During this high temperature baking, the glass surface of the channel will also be rendered hydrophobic due to deposition of a thin hydrophobic layer onto the glass. Alternatively, hydrophobic coatings such as Aquapel (PPG Industries) may be injected into the fluidic ports using a 1 mL syringe and a syringe needle.12 Carefully but firmly inject the Aquapel followed by purging air into the fluidic ports without breaking the PDMS to glass bond. Aggressively repeat the air purge on all inlet and outlet ports while wiping off any excess Aquapel in order to avoid any deposits that may clog the channels upon drying.</li>
</ol>
2. Sample Preparation
<ol>
 <li>Prepare a cell culture according to established procedures for your chosen cell type. For the particular device used in this study, 8-15 &mu;m particles or cells should adequately order for encapsulation. Smaller or larger cell types may require changing the dimensions of the focusing channel to achieve adequate Rep. For the method demonstration results shown in this paper, 9.9 &mu;m polystyrene microspheres (G1000, Thermo Scientific) are utilized as cell surrogates.</li>
 <li>Prepare the aqueous particle or cell suspension through gentle mixing. When using cells or polystyrene particles, concentration control is essential (see Figure 4) to achieve ideal ordered encapsulation. Using previous data12 as a guide, calculate the desired cell or particle concentration based on the ordered train spacing and micro-channel size as: one cell or particle per expected longitudinal train spacing times the focusing channel cross-sectional area. If the stock concentration (1% w/w) is inadequate, increase the concentration (here to 1.5% w/w) by gently centrifuging the stock sample, removing supernatant liquid, and re-suspending the particles by vortex mixing, or gentler mixing when using cells. Prepare an adequate volume to account for desired collection volume and for run time associated with flow tuning.</li>
 <li>Both cells and polystyrene particles have a specific gravity greater than one. Although not demonstrated in this protocol, for long-term experiments lasting on the order of many minutes to hours, buoyancy match the solution by adding a solute such as CaCl2 for particles or OptiPrep (Sigma-Aldrich) for cells.</li>
 <li>Prepare a 10 mL sample of the continuous fluorocarbon oil phase by mixing the fluorocarbon oil FC-40 (3M) and PFPE-PEG block copolymer surfactant25 (2.5% w/w) (RainDance Technologies) in a 15 mL centrifuge tube. Alternatively, light mineral oil (PTI Process Chemicals) can be utilized with ABIL-EM 90 surfactant (2.5% w/w) (Evonik Goldschmidt Corporation).</li>
</ol>
3. Experimental Setup
<ol>
 <li>Power on the inverted optical microscope (Axio Observer, Zeiss) and high speed camera (Phantom V310, Vision Research). Focus and inspect the channels for clogs and debris by either manually moving the device or by using a motorized microscope stage. Some small debris may be pushed out when liquid flows through. For large debris or obvious clogs, select another channel on the device as debris in the focusing channel can degrade ordering quality significantly. Note that clogs can often be removed under flow by pressing firmly on the PDMS surface above the affected region with blunt tweezers.</li>
 <li>Cut three lengths of PVC tubing (0.01" ID/0.03" OD, Tygon) for the aqueous inlet, oil inlet, and emulsion outlet. To minimize dead volume, cut just enough tubing to reach from the syringe pumps to the microscope stage. Cut tubing ends at a 45&deg; angle to facilitate insertion into fluidic ports.</li>
 <li>Use tweezers to press fit the tube ends into the fluidic ports punched in Step 1 and then press fit two 30 gauge blunt-tip stainless steel syringe needles (SmallParts) into the free ends of the respective aqueous and oil inlet tubes (no adhesive necessary). Place the outlet tubing into a waste reservoir. This tube will later be moved into a collection reservoir.</li>
 <li>Move the device and attached tubing to the microscope stage, align, and focus on the device nozzle using an available objective (20x was used for this experiment). Adjust for K hler illumination and other microscope settings as required for optimal recording.</li>
 <li>Fill a 1 mL syringe (BD) with the well-mixed aqueous phase and a 3 mL syringe (BD) with the oil phase solution prepared in Step 2. Note that any syringes of any volume may be used and should be carefully selected depending on the desired run times and minimization of any pulsatility. Tilt one syringe vertically and flick to move air bubbles to the syringe outlet. Slowly depress plunger enough to push the air to the syringe tip. Holding the syringe vertically, connect the syringes to the respective syringe needle already attached to the device in Step 3.3. Depress plunger to force the air through the syringe needle dead volume until fluid is pushed through the tubing almost to the device. Securely mount the syringe to a syringe pump (Nexus 3000, Chemyx) and engage the plunger block. Repeat connections for the second syringe and mount to a second syringe pump.</li>
 <li>Power on each syringe pump and program using the pump manufacturer's protocols. Set the initial flow rates to Qoil = 50 &mu;L/min and Qaq = 5 &mu;L/min for the oil phase and aqueous phase, respectively. Start the pumps.</li>
 <li>Wait for each fluid to enter the device and fill the channels, pushing out remaining dead air. This may take several minutes. If there is a large amount of air in the inlet tubing, temporarily increase each flow rate until the air is expelled. Do not increase the flow rates so high that large pressures occur in the channel, potentially leading to PDMS-to-glass bond failure.</li>
 <li>Using the initial flow rates, observe the formation of drops at the nozzle (results shown here: 20x magnification, frame rate 21005 fps, exposure 3 &mu;s). Reduce the camera field of view to only the nozzle to maximize the frame rate and reduce memory requirements if possible. Capture sample videos and confirm that the sampling rate is adequate to avoid aliasing.</li>
 <li>To avoid jetting (see Figure 2), start with low aqueous flow rates. Slowly increase the aqueous flow rate to observe ordering of particles in the long aqueous solution channel as the flow rate increases.</li>
 <li>If the particle concentration is too low to provide trains with relatively few "missing" particles and the sample was not buoyancy matched, physically tilt the syringe pump toward the syringe outlet to provide gradual settling of particles toward the syringe outlet. This method is demonstrated in the video protocol. Periodically rotating the syringe along its axis may also reduce undesired settling.</li>
 <li>Once adequate ordering occurs, adjust the oil flow rate to tune the generation frequency and size of drops. The mean drop volume may be calculated using the aqueous flow rate divided by the drop generation frequency as measured by video capture. Iteratively adjust both flow rates to achieve desired encapsulation rates and drop volumes.</li>
 <li>Once stable ordered encapsulation is confirmed, move the outlet tubing from the waste reservoir into a collection reservoir or feed it into another device for subsequent testing.</li>
 <li>Determine the collection time based on desired number of droplets and the calculated generation frequency.</li>
 <li>Record the fraction of drops containing 0, 1, 2,&hellip;, N particles to quantify efficiency using either drop generation video results or by pipetting a sample of collected emulsion for inspection.</li>
</ol>
4. Representative Results
Results are presented which achieve both controlled single-particle and controlled double-particle encapsulation (Figure 3). By cutting the FC-40 oil flow rate in half, single-particle encapsulation becomes two-particle encapsulation. Conversely, we could have increased the aqueous flow rate to deliver particles to the nozzle more quickly, but we also would have increased the risk of jetting of the aqueous stream. Histograms in Figure 3 present the fractional number of particles per drop for the two cases, along with comparisons to Poisson statistics. The occasional drops with zero particles are primarily due to "missing" particles in the ordered trains, while the cases where there are more encapsulated particles than desired result from local high particle concentrations and particles which sometimes migrate toward one of the two vertical focusing positions. Note that buoyancy matching as described in Section 2 was not utilized. Instead, the syringe pump was physically tilted to allow settling of particles toward the syringe outlet, leading to a high concentration of particles during the run.
An experimental run illustrating the need for proper particle and cell concentrations is shown in Figure 4. Without full ordering, localized groups of particles order and are encapsulated, but many drops are without particles. A histogram shows the decreased encapsulation efficiency for the desired two particle encapsulation.
<img alt="Figure 1" src="/files/ftp_upload/4096/4096fig1.jpg" /> 
Figure 1. Encapsulation device. a) Overall device with inlets, outlet, and long ordering channel. The device height is 52 &mu;m and the ordering channel width is 27 &mu;m. b) Both aqueous and oil inlets have large debris filters with gaps on the order of the ordering channel width for the enlarged view of the oil inlet. c)The enlarged nozzle view shows equal channel widths of 27 &mu;m for the aqueous and oil channels, followed by the nozzle contraction of 22 &mu;m and sudden expansion to a wider 61 &mu;m channel. Note that the dimensions of the device shown here have been verified using a profilometer after microfabrication and differ slightly from the nominal dimensions on the mask. A true image of the ordering channel and nozzle are available online as <a href="https://www.jove.com/files/ftp_upload/4096/4096fig1supplement.jpg" target="_blank">Supplemental Figure 1</a>. The <a href="https://www.jove.com/files/ftp_upload/4096/4096_mask.dwg" target="_blank">AutoCAD mask file</a> has also been included online as a supplement to this manuscript.
<img alt="Figure 2" src="/files/ftp_upload/4096/4096fig2.jpg" /> 
Figure 2. Hysteresis of a dripping to jetting transition using a wider device (80 &mu;m wide x 22 &mu;m high). a) At constant FC-40 flow rate (Qoil = 45 &mu;L/min), steady drop formation occurs at 10 kHz using an aqueous flow rate Qaq = 8 &mu;L/min. As the aqueous flow rate is slowly increased to 10 &mu;L/min, jetting of the aqueous fluid stream is triggered. b) When the flow rate is returned to 8 &mu;L/min jetting continues. Note that steady drop formation can be re-established by briefly pausing the aqueous flow pump (a 1 second pause is typical).
<img alt="Figure 3" src="/files/ftp_upload/4096/4096fig3.jpg" /> 
Figure 3. Single- and double- particle encapsulation. a) Drop formation with one cell per drop (Qoil = 60 &mu;L/min, Qaq = 9 &mu;L/min) with a drop generation rate of 6.1 kHz, average drop size of 24.4 pL, and a single-cell capture efficiencies Dk = 79.5% and Pk = 83.7% (&lambda; = 0.95) for a sample size of nd = 517 drops and np = 491 particles. b) Drop formation with two cells per drop is achieved simply by reducing the FC-40 flow rate Qoil to 30 &mu;L/min. The larger (39.8 pL) drops are formed at a rate of 3.8 kHz with a two-cell capture efficiency Dk = 71.5% and Pk = 79.5% (&lambda; = 1.80) for a sample size of nd = 383 drops and np = 689 particles. c-d) Two histograms compare the drop encapsulation particle efficiencies Dk of ordered single- and double- particle encapsulation with Poisson statistics (random encapsulation). Note that for both cases, particle spacing in the direction of flow is about 17-18 &mu;m for fully ordered, alternating particles. Supplemental videos showing both single- and double- particle encapsulation are available online. <a href="https://www.jove.com/files/ftp_upload/4096/4096movie3a.avi" target="_blank"> Click here to view Supplemental Movie 3a</a>. <a href="https://www.jove.com/files/ftp_upload/4096/4096movie3b.avi" target="_blank"> Click here to view Supplemental Movie 3b</a>.
<img alt="Figure 4" src="/files/ftp_upload/4096/4096fig4.jpg" /> 
Figure 4. Concentration greatly affects encapsulation efficiency. a) As the concentration decreases, full ordering does not occur, and thus "holes" in the trains emerge, leaving some drops with fewer than anticipated particles. b) The histogram shows the decreased efficiency (Dk = 55.9%, Pk = 70.9%) for two-particle encapsulation due to a lower value of &lambda; = 1.57 where there are nearly as many single-particle drops as there are double-particle drops. This figure results from Qoil = 30 &mu;L/min and Qaq = 9 &mu;L/min, the same flow conditions as for Figure 3b. A representative supplemental video is available online. <a href="https://www.jove.com/files/ftp_upload/4096/4096movie4.avi" target="_blank"> Click here to view Supplemental Movie 4</a>.

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Y., Abell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorosurfactants+for+microdroplets:+interfacial+tension+analysis.">Fluorosurfactants for microdroplets: interfacial tension analysis.</a> Journal of colloid and interface science. 350, 205-211 (2010).</li><li>Holt, D. J., Payne, R. J., Abell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+novel+fluorous+surfactants+for+microdroplet+stabilisation+in+fluorous+oil+streams.">Synthesis of novel fluorous surfactants for microdroplet stabilisation in fluorous oil streams.</a> Journal of Fluorine Chemistry. 131, 398-407 (2010).</li><li>Hatch, A. C., Fisher, J. S., Pentoney, S. L., Yang, D. L., Lee, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tunable+3D+droplet+self-assembly+for+ultra-high-density+digital+micro-reactor+arrays.">Tunable 3D droplet self-assembly for ultra-high-density digital micro-reactor arrays.</a> Lab on a chip. 11, 2509-2517 (2011).</li><li>Baret, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surfactants+in+droplet-based+microfluidics.">Surfactants in droplet-based microfluidics.</a> Lab on a chip. 12, 422-433 (2012).</li></ol>

JE is an inventor on a pending patent based on the technology utilized in this manuscript.

Despite relatively high degrees of ordering, not all drops will contain the proper number of particles or cells. Encapsulation efficiency may be calculated as the number of cells or particles that become encapsulated in drops with the desired occupancy divided by their total number. These raw data can be obtained either from an automated high speed video algorithm or from imaging a sample of collected emulsion. This can be compared to the fraction of particles Pk encapsulated in a drop containing k pa...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>AutoCAD</td>
 <td>AutoDesk</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Transparency Mask</td>
 <td>Fineline Imaging Inc.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>SU-8 Photoresist</td>
 <td>MicroChem</td>
 <td>2050</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dektak Profilometer</td>
 <td>Veeco</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Petri Dish</td>
 <td>BD Falcon</td>
 <td>351058</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PDMS Silicone Elastomer Kit</td>
 <td>Dow Corning Corp.</td>
 <td>Sylgard 184, Material Number (240)4019862</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vacuum Desiccator</td>
 <td>Jencons</td>
 <td>250-030</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vacuum Pump</td>
 <td>Alcatel Vacuum Technology</td>
 <td>2010 C2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vacuum Regulator</td>
 <td>Cole-Parmer</td>
 <td>EW-00910-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Oven</td>
 <td>Thermo Scientific</td>
 <td>Lindberg Blue M, OV800F</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Biopsy Punch, 0.75 mm</td>
 <td>Harris</td>
 <td>Uni-Core 15072</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Laboratory Corona 			Treater</td>
 <td>Electro-Technic 			Products Inc.</td>
 <td>BD-20AC, SKU 12051A</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glass Slides</td>
 <td>Gold Seal</td>
 <td>3010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Aquapel</td>
 <td>PPG Industries</td>
 <td>&nbsp;</td>
 <td>Alternative Strategy</td>
 </tr>
 <tr>
 <td>Polystyrene 			Microspheres, 9.9 &mu;m</td>
 <td>Thermo</td>
 <td>G1000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>OptiPrep</td>
 <td>Sigma-Aldrich</td>
 <td>D1556</td>
 <td>Not Demonstrated</td>
 </tr>
 <tr>
 <td>Luer-Lok Syringes</td>
 <td>BD</td>
 <td>1 			mL: 309628 
3 mL: 309585</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>FC-40 Fluorocarbon Oil</td>
 <td>3M Inc.</td>
 <td>Sigma Aldrich, F9755</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PFPE-PEG 			Fluorosurfactant</td>
 <td>RainDance Technologies</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Light Mineral Oil</td>
 <td>PTI Process Chemicals</td>
 <td>08042-47-5</td>
 <td>Alternative Strategy</td>
 </tr>
 <tr>
 <td>Mineral Oil Surfactant</td>
 <td>Evonik Goldschmidt 			Corporation</td>
 <td>ABIL EM 90</td>
 <td>Alternative Strategy</td>
 </tr>
 <tr>
 <td>Tygon PVC Tubing</td>
 <td>SmallParts</td>
 <td>TGY-010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>30 Gauge Luer-Lok 			Syringe Needle, 1/2"</td>
 <td>SmallParts</td>
 <td>NE-301PL-C</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Inverted Microscope</td>
 <td>Carl Zeiss Imaging</td>
 <td>Axio Observer.Z1 </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>High Speed Camera</td>
 <td>Vision Research</td>
 <td>Phantom V310</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Syringe Pumps (2)</td>
 <td>Chemyx Inc.</td>
 <td>Nexus 3000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Silicone Oil</td>
 <td>Dow Corning</td>
 <td>200 fluid, 10 cSt</td>
 <td>Optional for Emulsion 			Storage</td>
 </tr>
 </tbody>
</table>

high throughput single-cell and multiple-cell micro-encapsulation

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement from a bulk fluid environment with applications in high throughput screening, cytometry, and mass spectrometry. We describe a method to not only encapsulate single cells, but to repeatedly capture a set number of cells (here we demonstrate one- and two-cell encapsulation) to study both isolation and the interactions between cells in groups of controlled sizes. By combining drop generation techniques with cell and particle ordering, we demonstrate controlled encapsulation of cell-sized particles for efficient, continuous encapsulation. Using an aqueous particle suspension and immiscible fluorocarbon oil, we generate aqueous drops in oil with a flow focusing nozzle. The aqueous flow rate is sufficiently high to create ordering of particles which reach the nozzle at integer multiple frequencies of the drop generation frequency, encapsulating a controlled number of cells in each drop. For representative results, 9.9 &mu;m polystyrene particles are used as cell surrogates. This study shows a single-particle encapsulation efficiency Pk=1 of 83.7% and a double-particle encapsulation efficiency Pk=2 of 79.5% as compared to their respective Poisson efficiencies of 39.3% and 33.3%, respectively. The effect of consistent cell and particle concentration is demonstrated to be of major importance for efficient encapsulation, and dripping to jetting transitions are also addressed. 
Introduction 
Continuous media aqueous cell suspensions share a common fluid environment which allows cells to interact in parallel and also homogenizes the effects of specific cells in measurements from the media. High-throughput encapsulation of cells into picoliter-scale drops confines the samples to protect drops from cross-contamination, enable a measure of cellular diversity within samples, prevent dilution of reagents and expressed biomarkers, and amplify signals from bioreactor products. Drops also provide the ability to re-merge drops into larger aqueous samples or with other drops for intercellular signaling studies.1,2 The reduction in dilution implies stronger detection signals for higher accuracy measurements as well as the ability to reduce potentially costly sample and reagent volumes.3 Encapsulation of cells in drops has been utilized to improve detection of protein expression,4 antibodies,5,6 enzymes,7 and metabolic activity8 for high throughput screening, and could be used to improve high throughput cytometry.9 Additional studies present applications in bio-electrospraying of cell containing drops for mass spectrometry10 and targeted surface cell coatings.11 Some applications, however, have been limited by the lack of ability to control the number of cells encapsulated in drops. Here we present a method of ordered encapsulation12 which increases the demonstrated encapsulation efficiencies for one and two cells and may be extrapolated for encapsulation of a larger number of cells.
To achieve monodisperse drop generation, microfluidic "flow focusing" enables the creation of controllable-size drops of one fluid (an aqueous cell mixture) within another (a continuous oil phase) by using a nozzle at which the streams converge.13 For a given nozzle geometry, the drop generation frequency f and drop size can be altered by adjusting oil and aqueous flow rates Qoil and Qaq. As the flow rates increase, the flows may transition from drop generation to unstable jetting of aqueous fluid from the nozzle.14 
When the aqueous solution contains suspended particles, particles become encapsulated and isolated from one another at the nozzle. For drop generation using a randomly distributed aqueous cell suspension, the average fraction of drops Dk containing k cells is dictated by Poisson statistics, where Dk = &lambda;k exp(-&lambda;)/(k!) and &lambda; is the average number of cells per drop. The fraction of cells which end up in the "correctly" encapsulated drops is calculated using Pk = (k x Dk)/&Sigma;(k' x Dk'). The subtle difference between the two metrics is that Dk relates to the utilization of aqueous fluid and the amount of drop sorting that must be completed following encapsulation, and Pk relates to the utilization of the cell sample. As an example, one could use a dilute cell suspension (low &lambda;) to encapsulate drops where most drops containing cells would contain just one cell. While the efficiency metric Pk would be high, the majority of drops would be empty (low Dk), thus requiring a sorting mechanism to remove empty drops, also reducing throughput.15
Combining drop generation with inertial ordering provides the ability to encapsulate drops with more predictable numbers of cells per drop and higher throughputs than random encapsulation. Inertial focusing was first discovered by Segre and Silberberg16 and refers to the tendency of finite-sized particles to migrate to lateral equilibrium positions in channel flow. Inertial ordering refers to the tendency of the particles and cells to passively organize into equally spaced, staggered, constant velocity trains. Both focusing and ordering require sufficiently high flow rates (high Reynolds number) and particle sizes (high Particle Reynolds number).17,18 Here, the Reynolds number Re =uDh/&nu; and particle Reynolds number Rep =Re(a/Dh)2, where u is a characteristic flow velocity, Dh [=2wh/(w+h)] is the hydraulic diameter, &nu; is the kinematic viscosity, a is the particle diameter, w is the channel width, and h is the channel height. Empirically, the length required to achieve fully ordered trains decreases as Re and Rep increase. Note that the high Re and Rep requirements (for this study on the order of 5 and 0.5, respectively) may conflict with the need to keep aqueous flow rates low to avoid jetting at the drop generation nozzle. Additionally, high flow rates lead to higher shear stresses on cells, which are not addressed in this protocol. The previous ordered encapsulation study demonstrated that over 90% of singly encapsulated HL60 cells under similar flow conditions to those in this study maintained cell membrane integrity.12 However, the effect of the magnitude and time scales of shear stresses will need to be carefully considered when extrapolating to different cell types and flow parameters. The overlapping of the cell ordering, drop generation, and cell viability aqueous flow rate constraints provides an ideal operational regime for controlled encapsulation of single and multiple cells.
Because very few studies address inter-particle train spacing,19,20 determining the spacing is most easily done empirically and will depend on channel geometry, flow rate, particle size, and particle concentration. Nonetheless, the equal lateral spacing between trains implies that cells arrive at predictable, consistent time intervals. When drop generation occurs at the same rate at which ordered cells arrive at the nozzle, the cells become encapsulated within the drop in a controlled manner. This technique has been utilized to encapsulate single cells with throughputs on the order of 15 kHz,12 a significant improvement over previous studies reporting encapsulation rates on the order of 60-160 Hz.4,15 In the controlled encapsulation work, over 80% of drops contained one and only one cell, a significant efficiency improvement over Poisson (random) statistics, which predicts less than 40% efficiency on average.12
In previous controlled encapsulation work,12 the average number of particles per drop &lambda; was tuned to provide single-cell encapsulation. We hypothesize that through tuning of flow rates, we can efficiently encapsulate any number of cells per drop when &lambda; is equal or close to the number of desired cells per drop. While single-cell encapsulation is valuable in determining individual cell responses from stimuli, multiple-cell encapsulation provides information relating to the interaction of controlled numbers and types of cells. Here we present a protocol, representative results using polystyrene microspheres, and discussion for controlled encapsulation of multiple cells using a passive inertial ordering channel and drop generation nozzle.

Watch this Scientific Journal Video about High Throughput Single-cell and Multiple-cell Micro-encapsulation at JoVE.com

High Throughput Single-cell and Multiple-cell Micro-encapsulation

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates. We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

Microfluidic encapsulation methods have been previously utilized to capture cells in picoliter-scale aqueous, monodisperse drops, providing confinement ...

high-throughput-single-cell-and-multiple-cell-micro-encapsulation

Research

JoVE Journal

Bioengineering

18.6K Views.  Vanderbilt University. Combinando goccia generazione monodisperse con ordinamento inerziale di cellule e particelle, si descrive un metodo per incapsulare un numero desiderato di celle o particelle in una singola goccia a tassi kHz. Abbiamo dimostrato efficienze due volte superiori a quelli di incapsulamento non ordinate per raccogliere le gocce singola e doppia-particelle.

Video: High Throughput Single-cell and Multiple-cell Micro-encapsulation

Polymer Microarrays for High Throughput Discovery of Biomaterials

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a combinatorial library of materials to be screened in a parallel, high throughput format1. Herein is described the formation and characterization of a polymer microarray using an on-chip photopolymerization technique 2. This involves mixing monomers at varied ratios to produce a library of monomer solutions, transferring the solution to a glass slide format using a robotic printing device and curing with UV irradiation. This format is readily amenable to many biological assays, including stem cell attachment and proliferation, cell sorting and low bacterial adhesion, allowing the ready identification of 'hit' materials that fulfill a specific biological criterion3-5. Furthermore, the use of high throughput surface characterization (HTSC) allows the biological performance to be correlated with physio-chemical properties, hence elucidating the biological-material interaction6. HTSC makes use of water contact angle (WCA) measurements, atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). In particular, ToF-SIMS provides a chemically rich analysis of the sample that can be used to correlate the cell response with a molecular moiety. In some cases, the biological performance can be predicted from the ToF-SIMS spectra, demonstrating the chemical dependence of a biological-material interaction, and informing the development of hit materials5,3.

A description of the formation of a polymer microarray using an on-chip photopolymerization technique. The high throughput surface characterization using atomic force microscopy, water contact angle measurements, X-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry and a cell attachment assay is also described.

The discovery of novel biomaterials that are optimized for a specific biological application is readily achieved using polymer microarrays, which allows a ...

High-throughput Protein Expression Generator Using a Microfluidic Platform

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

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

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

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

High-throughput Synthesis of Carbohydrates and Functionalization of Polyanhydride Nanoparticles

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design efficacious vaccines carriers. Nanoparticle-based platforms can prolong the persistence of vaccine antigens, which could improve vaccine immunogenicity1. Several biodegradable polymers have been studied as vaccine delivery vehicles1; in particular, polyanhydride particles have demonstrated the ability to provide sustained release of stable protein antigens and to activate antigen presenting cells and modulate immune responses2-12.
The molecular design of these vaccine carriers needs to integrate the rational selection of polymer properties as well as the incorporation of appropriate targeting agents. High throughput automated fabrication of targeting ligands and functionalized particles is a powerful tool that will enhance the ability to study a wide range of properties and will lead to the design of reproducible vaccine delivery devices.
The addition of targeting ligands capable of being recognized by specific receptors on immune cells has been shown to modulate and tailor immune responses10,11,13 C-type lectin receptors (CLRs) are pattern recognition receptors (PRRs) that recognize carbohydrates present on the surface of pathogens. The stimulation of immune cells via CLRs allows for enhanced internalization of antigen and subsequent presentation for further T cell activation14,15. Therefore, carbohydrate molecules play an important role in the study of immune responses; however, the use of these biomolecules often suffers from the lack of availability of structurally well-defined and pure carbohydrates. An automation platform based on iterative solution-phase reactions can enable rapid and controlled synthesis of these synthetically challenging molecules using significantly lower building block quantities than traditional solid-phase methods16,17.
Herein we report a protocol for the automated solution-phase synthesis of oligosaccharides such as mannose-based targeting ligands with fluorous solid-phase extraction for intermediate purification. After development of automated methods to make the carbohydrate-based targeting agent, we describe methods for their attachment on the surface of polyanhydride nanoparticles employing an automated robotic set up operated by LabVIEW as previously described10. Surface functionalization with carbohydrates has shown efficacy in targeting CLRs10,11 and increasing the throughput of the fabrication method to unearth the complexities associated with a multi-parametric system will be of great value (Figure 1a).

In this article, a high throughput method is presented for the synthesis of oligosaccharides and their attachment to the surface of polyanhydride nanoparticles for further use in targeting specific receptors on antigen presenting cells.

Transdisciplinary approaches involving areas such as material design, nanotechnology, chemistry, and immunology have to be utilized to rationally design ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

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

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

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

Using Tomoauto: A Protocol for High-throughput Automated Cryo-electron Tomography

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native context at a molecular level. The technique involves initially preserving the sample in its native state by rapidly freezing the specimen in vitreous ice, then collecting a series of micrographs from different angles at high magnification, and finally computationally reconstructing a 3-D density map. The frozen-hydrated specimen is extremely sensitive to the electron beam and so micrographs are collected at very low electron doses to limit the radiation damage. As a result, the raw cryo-tomogram has a very low signal to noise ratio characterized by an intrinsically noisy image. To better visualize subjects of interest, conventional imaging analysis and sub-tomogram averaging in which sub-tomograms of the subject are extracted from the initial tomogram and aligned and averaged are utilized to improve both contrast and resolution. Large datasets of tilt-series are essential to understanding and resolving the complexes at different states, conditions, or mutations as well as obtaining a large enough collection of sub-tomograms for averaging and classification. Collecting and processing this data can be a major obstacle preventing further analysis. Here we describe a high-throughput cryo-ET protocol based on a computer-controlled 300kV cryo-electron microscope, a direct detection device (DDD) camera and a highly effective, semi-automated image-processing pipeline software wrapper library tomoauto developed in-house. This protocol has been effectively utilized to visualize the intact type III secretion system (T3SS) in Shigella flexneri minicells. It can be applicable to any project suitable for cryo-ET.

We present a protocol on how to utilize high-throughput cryo-electron tomography to determine high resolution in situ structures of molecular machines. The protocol permits large amounts of data to be processed, avoids common bottlenecks and reduces resource downtime, allowing the user to focus on important biological questions.

Cryo-electron tomography (Cryo-ET) is a powerful three-dimensional (3-D) imaging technique for visualizing macromolecular complexes in their native ...

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

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

High-throughput Identification of Bacteria Repellent Polymers for Medical Devices

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as contributing to antimicrobial resistance. One possible avenue for the reduction of device-associated infections is the identification of bacteria-repellent polymer coatings for these devices, which would prevent bacterial binding at the initial attachment step. A method for the identification of such repellent polymers, based on the parallel screening of hundreds of polymers using a microarray, is described here. This high-throughput method resulted in the identification of a range of promising polymers that resisted binding of various clinically relevant bacterial species individually and also as multi-species communities. One polymer, PA13 (poly(methylmethacrylate-co-dimethylacrylamide)), demonstrated significant reduction in attachment of a number of hospital isolates when coated onto two commercially available central venous catheters. The method described could be applied to identify polymers for a wide range of applications in which modification of bacterial attachment is important.

A high-throughput microarray method for the identification of polymers which reduce bacterial surface binding on medical devices is described.

Medical devices are often associated with hospital-acquired infections, which place enormous strain on patients and the healthcare system as well as ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...