Name: high throughput single-cell and multiple-cell micro-encapsulation
Uploaded: 2012-06-15T12:00:00
Description: Watch this Scientific Journal Video about High Throughput Single-cell and Multiple-cell Micro-encapsulation at JoVE.com

Wij danken Raindance Technologies voor de steekproef van PFPE-PEG oppervlakte-actieve stof gebruikt in deze studie, en we danken de BioMEMS Resource Center (Mehmet Toner, directeur) voor de silicium wafer mal gebruikt om PDMS kanaal replica&#39;s te creëren.

De protocollen in dit hoofdstuk beschrijven de materialen en uitrusting die specifiek gebruikt om de gepresenteerde experimentele resultaten te verkrijgen. Merk op dat alternatieve leveranciers van chemicaliën en apparatuur kan worden gebruikt. 1. Apparaat Fabricage en zachte lithografie Standaard zachte lithografie technieken, 21 waarvan een aantal zijn gekenmerkt in voorgaande Jove artikelen, werden 22 gebruikt voor het maken polydimethylsi...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+dispersions+using+"flow+focusing"+in+microchannels.">Formation of dispersions using "flow focusing" in microchannels.</a> Applied Physics Letters. 82, 364 (2003).</li><li>Utada, A., Fernandez-Nieves, A., Stone, H., Weitz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dripping+to+Jetting+Transitions+in+Coflowing+Liquid+Streams.">Dripping to Jetting Transitions in Coflowing Liquid Streams.</a> Physical Review Letters. 99, (2007).</li><li>Chabert, M., Viovy, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+high-throughput+encapsulation+and+hydrodynamic+self-sorting+of+single+cells.">Microfluidic high-throughput encapsulation and hydrodynamic self-sorting of single cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 105, 3191-3196 (2008).</li><li>Segr&#237;, G., Silberberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radial+Particle+Displacements+in+Poiseuille+Flow+of+Suspensions.">Radial Particle Displacements in Poiseuille Flow of Suspensions.</a> Nature. 189, 209-210 (1961).</li><li>Carlo, D. D. i. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inertial+microfluidics.">Inertial microfluidics.</a> Lab on a chip. 9, 3038-3046 (2009).</li><li>Carlo, D. D. i., Edd, J., Humphry, K., Stone, H., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+Segregation+and+Dynamics+in+Confined+Flows.">Particle Segregation and Dynamics in Confined Flows.</a> Physical Review Letters. 102, (2009).</li><li>Humphry, K. J., Kulkarni, P. M., Weitz, D. A., Morris, J. F., Stone, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axial+and+lateral+particle+ordering+in+finite+Reynolds+number+channel+flows.">Axial and lateral particle ordering in finite Reynolds number channel flows.</a> Physics of Fluids. 22, 081703 (2010).</li><li>Lee, W., Amini, H., Stone, H. A., Carlo, D. D. i. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+self-assembly+and+control+of+microfluidic+particle+crystals.">Dynamic self-assembly and control of microfluidic particle crystals.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 22413 (2010).</li><li>Duffy, D. C., McDonald, J. C., Schueller, O. J. A., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+prototyping+of+microfluidic+systems+in+poly(dimethylsiloxane.">Rapid prototyping of microfluidic systems in poly(dimethylsiloxane.</a> Anal. Chem. 70, 4974-4984 (1998).</li><li>Kotz, K., Cheng, X., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+Device+Fabrication+and+Surface+Modification.">PDMS Device Fabrication and Surface Modification.</a> J. Vis. Exp. (8), e319 (2007).</li><li>Haubert, K., Drier, T., Beebe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PDMS+bonding+by+means+of+a+portable,+low-cost+corona+system.">PDMS bonding by means of a portable, low-cost corona system.</a> Lab on a chip. 6, 1548-1549 (2006).</li><li>Hatch, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=1-Million+droplet+array+with+wide-field+fluorescence+imaging+for+digital+PCR.">1-Million droplet array with wide-field fluorescence imaging for digital PCR.</a> Lab on a chip. , 3838-3845 (2011).</li><li>Holtze, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocompatible+surfactants+for+water-in-fluorocarbon+emulsions.">Biocompatible surfactants for water-in-fluorocarbon emulsions.</a> Lab on a chip. 8, 1632-1639 (2008).</li><li>Garstecki, P., Stone, H., Whitesides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+for+Flow-Rate+Controlled+Breakup+in+Confined+Geometries:+A+Route+to+Monodisperse+Emulsions.">Mechanism for Flow-Rate Controlled Breakup in Confined Geometries: A Route to Monodisperse Emulsions.</a> Physical Review Letters. 94, (2005).</li><li>Garstecki, P., Fuerstman, M. J., Stone, H. A., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+droplets+and+bubbles+in+a+microfluidic+T-junction-scaling+and+mechanism+of+break-up.">Formation of droplets and bubbles in a microfluidic T-junction-scaling and mechanism of break-up.</a> Lab on a chip. 6, 437-446 (2006).</li><li>Nie, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emulsification+in+a+microfluidic+flow-focusing+device:+effect+of+the+viscosities+of+the+liquids.">Emulsification in a microfluidic flow-focusing device: effect of the viscosities of the liquids.</a> Microfluidics and Nanofluidics. , (2008).</li><li>Holt, D. J., Payne, R. J., Chow, W. 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>

Ondanks de relatief hoge mate van bestellen, zullen niet alle druppels bevatten het juiste aantal deeltjes of cellen. Inkapselefficiëntie kan worden berekend als het aantal cellen of deeltjes die raken ingekapseld in druppels met de gewenste bezetting gedeeld door hun aantal. Deze ruwe data kunnen worden verkregen bij een geautomatiseerde high speed video-algoritme of van beeldvorming een monster van de verzamelde emulsie. Dit kan vergeleken worden met de fractie deeltjes Pk ingekapseld in een drupp...

<table border="1"><tbody><tr><td> Naam van het serum </td><td> Vennootschap </td><td> Catalogusnummer </td><td> Reacties </td></tr><tr><td> AutoCAD </td><td> AutoDesk </td><td></td><td></td></tr><tr><td> Transparantie Mask </td><td> Fineline Imaging Inc </td><td></td><td></td></tr><tr><td> SU-8 fotolak </td><td> Microchem </td><td> 2050 </td><td></td></tr><tr><td> Dektak profilometer </td><td> Veeco </td><td></td><td></td></tr><tr><td> Petrischaal </td><td> BD Falcon </td><td> 351058 </td><td></td></tr><tr><td> PDMS Siliconen Elastomeer Kit </td><td> Dow Corning Corp </td><td> Sylgard 184, Materiaal nummer (240) 4019862 </td><td></td></tr><tr><td> Vacuümexsiccator </td><td> Jencons </td><td> 250-030 </td><td></td></tr><tr><td> Vacuümpomp </td><td> Alcatel Vacuum Technology </td><td> 2010 C2 </td><td></td></tr><tr><td> Vacuümregelaar </td><td> Cole Parmer- </td><td> EW-00910-10 </td><td></td></tr><tr><td> Oven </td><td> Thermo Scientific </td><td> Lindberg Blue M, OV800F </td><td></td></tr><tr><td> Biopsie Punch, 0,75 mm </td><td> Harris </td><td> Uni-Core 15072 </td><td></td></tr><tr><td> Laboratorium Corona Treater </td><td> Electro-Technic Products Inc </td><td> BD-20AC, SKU 12051A </td><td></td></tr><tr><td> Objectglaasjes </td><td> Gold Seal </td><td> 3010 </td><td></td></tr><tr><td> Aquapel </td><td> PPG Industries </td><td></td><td> Alternatieve strategie </td></tr><tr><td> Polystyreen Microspheres, 9,9 micrometer </td><td> Thermo </td><td> G1000 </td><td></td></tr> &lt;tr&gt; <td> OptiPrep </td><td> Sigma-Aldrich </td><td> D1556 </td><td> Niet aangetoond </td></tr><tr><td> Luer-Lok spuiten </td><td> BD </td><td> 1 ml: 309628 3 ml: 309585 </td><td></td></tr><tr><td> FC-40 Fluorocarbon Oil </td><td> 3M Inc </td><td> Sigma Aldrich, F9755 </td><td></td></tr><tr><td> PFPE-PEG fluor </td><td> Raindance Technologies </td><td></td><td></td></tr><tr><td> Lichte minerale olie </td><td> PTI Proceschemicaliën </td><td> 08042-47-5 </td><td> Alternatieve strategie </td></tr><tr><td> Minerale olie oppervlakteactieve </td><td> Evonik Goldschmidt Corporation </td><td> ABIL EM 90 </td><td> Alternatieve strategie </td></tr><tr><td> Tygon PVC-buis </td><td> SmallParts </td><td> TGY-010 </td><td></td></tr><tr><td> 30 Gauge Luer-Lok naald, 1/2 &quot; </td><td> SmallParts </td><td> NE-301PL-C </td><td></td></tr><tr><td> Geïnverteerde microscoop </td><td> Carl Zeiss Imaging </td><td> Axio Observer.Z1 </td><td></td></tr><tr><td> High Speed ​​Camera </td><td> Vision Research </td><td> Phantom V310 </td><td></td></tr><tr><td> Spuit Pompen (2) </td><td> Chemyx Inc </td><td> Nexus 3000 </td><td></td></tr><tr><td> Siliconenolie </td><td> Dow Corning </td><td> 200 vloeistof 10 cSt </td><td> Optioneel voor Emulsion opslag </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

De combinatie van monodisperse druppel generatie met traagheid ordening van cellen en deeltjes, beschrijven we een methode om een ​​gewenst aantal cellen of deeltjes in te kapselen in een enkele druppel op kHz tarieven. We laten zien efficiëntie twee maal groter dan die van niet-geordende inkapseling voor enkel-en dubbel-deeltje druppels.

High Throughput Single-cel en Multiple-cel Micro-encapsulatie

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

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

Combinatoriële Synthese van en high-throughput eiwit Op de markt van Polymer Film en Nanodeeltje bibliotheken

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

High-throughput synthese van koolhydraten en functionalisering van polyanhydride Nanodeeltjes

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

High Throughput Microfluidic Rapid en Low Cost Prototyping Verpakking

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

Met behulp van Tomoauto: Een protocol voor 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 ...

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

Een microfluïdische platform voor High-throughput Single-cell Isolatie en Cultuur

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

High-throughput Identificatie van bacteriën 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 ...

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

3D Microtissues voor injecteerbare regeneratieve therapie en 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

Een met-throughput Microfluidic Platform voor eencellige genoom Sequencing

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