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

우리는이 연구에서 널리 사용 PFPE-PEG 계면 활성제의 샘플 레인 댄스 기술 감사, 우리는 PDMS 채널 복제본을 만드는 데 사용되는 실리콘 웨이퍼 금형을위한 BioMEMS 자원 센터 (Mehmet 토너, 감독)을 감사드립니다.

이 섹션의 프로토콜 제시 실험 결과를 얻기 위해 특별히 활용 자재 및 장비를 설명합니다. 화학 물질 및 장비의 대체 공급 업체가 활용할 수 있습니다. 1. 장치 제조 및 소프트 리소그래피 표준 소프트 리소그래피 기법, 21 이전 주피터 문서에 등장되어있는 다수의, 22는 유리 기판에 보세 polydimethylsiloxane (PDMS) microchannel 네트워크를 만드는 ?...

<ol>
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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. 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주문 상대적으로 높은 학위에도 불구하고, 모든 물방울 입자 또는 세포의 적절한 숫자를 포함합니다. 봉지 효율은 세포 또는 그 총 개수로 나눈 원하는 인과 드랍스에 캡슐된다 입자의 개수로 계산됩니다. 이러한 원시 데이터가 수집 유제의 견본 자동화된 고속의 비디오 알고리즘이나 이미지에서 어느 얻을 수있다. 이것은 K 입자와 K 입자를 포함하는 것입니다 드랍스 D K의 분수를 ...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> 의 AutoCAD </td><td> 오토 데스크 </td><td></td><td></td></tr><tr><td> 투명 마스크 </td><td> Fineline 이미징 주식 회사 </td><td></td><td></td></tr><tr><td> SU-8 레지스트 </td><td> MicroChem </td><td> 2050 </td><td></td></tr><tr><td> Dektak의 Profilometer </td><td> Veeco </td><td></td><td></td></tr><tr><td> 페트리 요리 </td><td> BD 팔콘 </td><td> 351,058 </td><td></td></tr><tr><td> PDMS 실리콘 엘라스토머 키트 </td><td> 다우 코닝 주식 회사 </td><td> Sylgard 184, 자재 번호 (241) 4019862 </td><td></td></tr><tr><td> 진공 건조기 </td><td> Jencons </td><td> 250-030 </td><td></td></tr><tr><td> 배기기 </td><td> 알카텔 진공 기술 </td><td> 2010 C2 </td><td></td></tr><tr><td> 진공 레귤레이터 </td><td> 콜 - Parmer </td><td> EW-00910-10 </td><td></td></tr><tr><td> 오븐 </td><td> 써모 과학 </td><td> 린드버그 블루 M, OV800F </td><td></td></tr><tr><td> 생검 펀치, 0.75 mm </td><td> 해리스 </td><td> 단일 코어 15,072 </td><td></td></tr><tr><td> 실험실 코로나 Treater </td><td> 전기 제품 기술 주식 회사 </td><td> BD-20AC, SKU 12051A </td><td></td></tr><tr><td> 유리 슬라이드 </td><td> 골드 인감 </td><td> 3010 </td><td></td></tr><tr><td> Aquapel </td><td> PPG 산업 </td><td></td><td> 대체 전략 </td></tr><tr><td> 폴리스티렌 Microspheres, 9.9 μm의 </td><td> 써모 </td><td> G1000 </td><td></td></tr> &lt;TR&gt; <td> OptiPrep </td><td> 시그마 - 올드 리치 </td><td> D1556 </td><td> 입증되지 않음 </td></tr><tr><td> Luer-Lok의 주사기 </td><td> BD </td><td> 한 ML : 309,628 3 ML : 309,585 </td><td></td></tr><tr><td> FC-40 플루오로 카본 오일 </td><td> 3M 주식 회사 </td><td> 시그마 알드리치, F9755 </td><td></td></tr><tr><td> PFPE-PEG의 불화 계면 활성제 </td><td> 레인 댄스 기술 </td><td></td><td></td></tr><tr><td> 가벼운 미네랄 오일 </td><td> PTI 프로세스 케미칼 </td><td> 08042-47-5 </td><td> 대체 전략 </td></tr><tr><td> 미네랄 오일 계면 활성제 </td><td> Evonik Goldschmidt 주식 회사 </td><td> 아빌 EM 90 </td><td> 대체 전략 </td></tr><tr><td> Tygon PVC 튜빙 </td><td> SmallParts </td><td> TGY-010 </td><td></td></tr><tr><td> 30 게이지 Luer-Lok 주사기 바늘, 1 / 2 &quot; </td><td> SmallParts </td><td> NE-301PL-C </td><td></td></tr><tr><td> 거꾸로 현미경 </td><td> 칼 자이스 혈구 이미징 </td><td> Axio Observer.Z1 </td><td></td></tr><tr><td> 고속 카메라 </td><td> 비전 연구 </td><td> 팬텀 V310 </td><td></td></tr><tr><td> 주사기 펌프 (2) </td><td> Chemyx 주식 회사 </td><td> 넥서스 3000 </td><td></td></tr><tr><td> 실리콘 오일 </td><td> 다우 코닝 </td><td> 200 유체, 10 CST </td><td> 에멀젼 저장을위한 옵션 </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

세포 및 입자의 관성 주문과 monodisperse 드롭 생성을 결합하여, 우리는 kHz에서 속도에서 한 방울의 세포 또는 입자의 원하는 숫자를 캡슐하는 방법을 설명합니다. 우리는 두 번 단일 및 이중 입자 술을 정렬되지 않은 캡슐 분들을 초과 효율성을 보여줍니다.

높은 처리량 마이크로 캡슐 단일 셀 및 다중 셀

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

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JoVE Journal

Bioengineering

Polymer Microarrays for High Throughput Discovery of Biomaterials

Biomaterials 높은 처리량 발견을위한 폴리머 Microarrays

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

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생체공학

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

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

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높은 처리량 마이크로 유체 신속하고 저렴한 비용으로 프로토 타입 포장 방법

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

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높은 처리량 단일 세포의 분리 및 문화를위한 미세 유체 플랫폼

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 ...

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의료 기기에 대한 박테리아의 높은 처리량 식별 구충제 폴리머

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

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

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