Name: a microfluidic platform for high-throughput single-cell isolation and culture
Uploaded: 2016-06-16T15:00:00
Description: Watch this Scientific Journal Video about A Microfluidic Platform for High-throughput Single-cell Isolation and Culture at JoVE.com

This work was supported by a grant from the National Health Research Institutes (03-A1 BNMP11-014).

주 : 우리의 미세 유체 소자의 제작을위한 포토 마스크 설계는 컴퓨터 이용 설계 (CAD) 소프트웨어를 사용하여 그려졌다. 디자인이어서, 상용 서비스를 이용하여 크롬 포토 마스크를 제조하는데 사용 하였다. PDMS의 장치는 소프트 리소그래피 기술을 사용하여 제조 하였다. 11 리소그래피에 의해 마스터 금형 1. 제작 <ol><li> 포토 리소그래피 공정 (12) 전에, 기판...

<ol>
	<li>Meacham, C. E., Morrison, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+heterogeneity+and+cancer+cell+plasticity.">Tumour heterogeneity and cancer cell plasticity.</a> Nature. 501 (7467), 328-337 (2013).</li><li>Vermeulen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+cloning+of+colon+cancer+stem+cells+reveals+a+multi-lineage+differentiation+capacity.">Single-cell cloning of colon cancer stem cells reveals a multi-lineage differentiation capacity.</a> P Natl Acad Sci USA. 105 (36), 13427-13432 (2008).</li><li>Shapiro, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Practical flow cytometry. , (2005).</li><li>Leong, K. G., Wang, B. E., Johnson, L., Gao, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+prostate+from+a+single+adult+stem+cell.">Generation of a prostate from a single adult stem cell.</a> Nature. 456 (7223), 804-808 (2008).</li><li>Shapiro, E., Biezuner, T., Linnarsson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+sequencing-based+technologies+will+revolutionize+whole-organism+science.">Single-cell sequencing-based technologies will revolutionize whole-organism science.</a> Nat Rev Genet. 14 (9), 618-630 (2013).</li><li>Rettig, J. R., Folch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+single-cell+trapping+and+imaging+using+microwell+arrays.">Large-scale single-cell trapping and imaging using microwell arrays.</a> Anal. Chem. 77 (17), 5628-5634 (2005).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+fibrin+gels+promote+selection+and+growth+of+tumorigenic+cells.">Soft fibrin gels promote selection and growth of tumorigenic cells.</a> Nat Mater. 11 (8), 734-741 (2012).</li><li>Charnley, M., Textor, M., Khademhosseini, A., Lutolf, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+column:+microwell+arrays+for+mammalian+cell+culture.">Integration column: microwell arrays for mammalian cell culture.</a> Integr. Biol. 1 (11-12), 11-12 (2009).</li><li>Lindstrom, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-density+microwell+chip+for+culture+and+analysis+of+stem+cells.">High-density microwell chip for culture and analysis of stem cells.</a> PloS one. 4 (9), e6997 (2009).</li><li>Lin, C. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+dual-well+device+for+high-throughput+single-cell+capture+and+culture.">A microfluidic dual-well device for high-throughput single-cell capture and culture.</a> Lab Chip. 15 (14), 2928-2938 (2015).</li><li>Xia, Y. N., 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=Soft+lithography.">Soft lithography.</a> Angew Chem Int Edit. 37 (5), 550-575 (1998).</li><li>Shin, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+assay+for+simultaneous+culture+of+multiple+cell+types+on+surfaces+or+within+hydrogels.">Microfluidic assay for simultaneous culture of multiple cell types on surfaces or within hydrogels.</a> Nat Protoc. 7 (7), 1247-1259 (2012).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypan+blue+exclusion+test+of+cell+viability.">Trypan blue exclusion test of cell viability.</a> Curr. Protoc. Immunol. Appendix 3 (Appendix 3B), (2001).</li><li>Lindstrom, S., Andersson-Svahn, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Miniaturization+of+biological+assays+-+Overview+on+microwell+devices+for+single-cell+analyses.">Miniaturization of biological assays - Overview on microwell devices for single-cell analyses.</a> Bba-Gen Subjects. 1810 (3), 308-316 (2011).</li><li>Lecault, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+analysis+of+single+hematopoietic+stem+cell+proliferation+in+microfluidic+cell+culture+arrays.">High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays.</a> Nat Methods. 8 (7), 581-593 (2011).</li><li>Park, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+trapping+in+larger+microwells+capable+of+supporting+cell+spreading+and+proliferation.">Single cell trapping in larger microwells capable of supporting cell spreading and proliferation.</a> Microfluid Nanofluid. 8 (2), 263-268 (2010).</li><li>Tirino, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells+in+solid+tumors:+an+overview+and+new+approaches+for+their+isolation+and+characterization.">Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization.</a> FASEB J. 27 (1), 13-24 (2013).</li><li>Chen, P. C., Huang, Y. Y., Juang, 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=MEMS+microwell+and+microcolumn+arrays:+novel+methods+for+high-throughput+cell-based+assays.">MEMS microwell and microcolumn arrays: novel methods for high-throughput cell-based assays.</a> Lab Chip. 11 (21), 3619-3625 (2011).</li><li>Liang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+Screening+Using+a+Library+of+Human+Induced+Pluripotent+Stem+Cell-Derived+Cardiomyocytes+Reveals+Disease-Specific+Patterns+of+Cardiotoxicity.">Drug Screening Using a Library of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Reveals Disease-Specific Patterns of Cardiotoxicity.</a> Circulation. 127 (16), 1677-1691 (2013).</li></ol>

마이크로 웰 기반 장치 시스템은 6,14 그러한 6 트래핑 대규모 단일 셀 및 단일 조혈 줄기 세포 증식 15 단일 세포 조작 및 분석을 위해 사용되어왔다. 물론 크기, 개수, 형상 비록 특정 애플리케이션을 위해 조절 될 수 있으며, 웰의 크기가 증가 될 때 단일 세포 분리 효율은 항상 노출되어있다. 9,15 마이크로 웰의 크기가 세포의 확산과 성장을 ?...

현재 배양 공간에 개별적으로 하나의 셀을 배치는 일반적으로 제한 희석 또는 형광 - 활성화 된 세포 분류 (FACS)를 사용하여 달성된다. 그것은 단지 쉽게 사용할 수 있습니다 피펫 및 조직 배양 플레이트를 필요로 많은 실험실의 경우, 희석을 제한하는 편리한 방법입니다. 이러한 경우에, 세포 현탁액을 연속적으로 적당한 세포 밀도로 희석 한 다음 수동 피펫을 이용하여 배양 웰에 두었다. 이러한 복실 단셀 그런 다음 1 콜로니 형성이 스크리닝 이질성 유전자로서 세포 분석을 위해 사용된다. 한계 희석법의 푸 아송 분포의 특성은 37 % (3)의 최대 확률 단세포 이벤트를 제한하기 때문에,이 방법을 지원하기위한 로봇 팔을 이용하지 않고 낮은 처리량과 노동력이있다. 통합 된 로보트 팔 FACS 기계 정확하게 PLAC으로 푸 아송 분포의 한계를 극복 할 수있다한 번에 4에서 잘 문화에서 하나의 셀을 보내고. 그러나, 높은 기계적 전단 응력은 5 (따라서, 세포 생존 능력을 저하) 및 기계 구입 및 운영 비용은 많은 실험실에서의 사용을 제한하고있다. 고효율 마이크로 웰 (6)에 하나의 셀을로드에 위의 한계를 극복하기 위해, 마이크로 장치가 개발되었다. 그러나, 마이크로 웰은로드 셀 인해 단일 ​​셀 로딩 가능성을 최대화하기 위해 하나의 셀에 가까운 마이크로 웰 각의 크기의 제조가 필요로 증식 할 충분한 공간을 제공하지 않는다. 배양 분석이 더 많은 마이크로 웰 세포 - 기반 애플리케이션 (예를 들면, 클론 원성 분석 7)에 요구되는 바와 같이 (90 - 650 ㎛의 직경 또는 측면 길이) 또한 확장 세포 배양을 허용하기 위해 이용되어왔다. 그러나, 한계 희석법 같이, 또한 (10)에 이르기까지, 낮은 단일 셀 로딩 효율을 갖는다 -. 30 % 89 이전에, 우리는 (10) 장치는 폴리 디메틸 실록산 (PDMS)으로 제작 하였다. 각각의 마이크로 웰에서 단일 세포를 분리하고, 분리 된 세포의 클론 원성 분석에서의 응용을 증명하기 위해 높은 처리량 미세 플랫폼을 개발하고, 마이크로 웰 어레이들의 두 세트를 포함하는 한 대체로 크기가 마이크로 웰의 단일 셀 로딩의 효율을 향상시킬 수있는 다른 마이크로 웰 크기와 세포보다 상당히 크다. 특히,이 &quot;이중 - 웰&quot;개념 배양 영역의 크기는 유동적이 간단 다른 세포 유형 및 애플리케이션에 적합하도록 장치의 설계를 조정할 수있어 단일 세포 포집 효율에 영향을주지 않고 조절 될 수있다. 이 고효율 방법은 세포 이질성 연구 및 모노클로 날 세포주 확립 장기간 세포 배양 실험에 유용 할 것이다.

<table border="0" cellpadding="0" cellspacing="0" width="1323">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">AutoCAD software</td>
			<td style="width:225px;">Autodesk</td>
			<td style="width:235px;">AutoCAD LT 2011</td>
			<td style="width:463px;">Part No. 057C1-74A111-1001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Silicon wafer&#160;</td>
			<td>Eltech corperation</td>
			<td style="width:235px;">SPE0039</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conventional oven</td>
			<td>YEONG-SHIN company</td>
			<td style="width:235px;">ovp45</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plasma cleaner</td>
			<td>Nordson</td>
			<td style="width:235px;">AP-300</td>
			<td>Bench-Top Plasma Treatment System</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 50 negative photoresist</td>
			<td>MicroChem</td>
			<td>Y131269</td>
			<td style="width:463px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 100 negative photoresist</td>
			<td>MicroChem</td>
			<td>Y131273</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spin coater</td>
			<td>Synrex Co., Ltd.</td>
			<td>SC-HMI 2&#34; ~ 6&#34;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hotplate</td>
			<td>YOTEC company</td>
			<td>YS-300S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Msak aligner</td>
			<td>Deya Optronic CO.</td>
			<td>A1K-5-MDA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 developer</td>
			<td>Grand Chemical Companies</td>
			<td>GP5002-000000-72GC</td>
			<td>Propylene glycol monomethyl ether acetate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scanning laser profilometer</td>
			<td>KEYENCE</td>
			<td>VK-X 100</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trichlorosilane</td>
			<td>Gelest, Inc</td>
			<td>SIT8174.0</td>
			<td style="width:463px;">TRIDECAFLUORO-1,1,2,2-TETRAHYDROOCTYL.&#160; Hazardous. Corrosive to the respiratory tract., reacts violently with water.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Desiccator</td>
			<td>Bel-Art Products&#160;</td>
			<td>F42020-0000</td>
			<td>SPACE SAVER VACUUM DESICCATOR 190MM WHITE BASE</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polydimethylsiloxane (PDMS) kit</td>
			<td>Dow corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Harris Uni-Core puncher</td>
			<td>Ted Pella Inc.</td>
			<td>15072</td>
			<td>with 0.75 mm inner-diameter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Removable tape</td>
			<td>3M Company</td>
			<td>Scotch Removable Tape 811</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereomicroscope</td>
			<td>Leica Microsystems</td>
			<td>Leica E24</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bovine serum albumin (BSA)</td>
			<td>Bersing Technology</td>
			<td>ALB001.500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM basal medium</td>
			<td>Gibco</td>
			<td style="width:235px;">12800-017</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Thermo Hyclone</td>
			<td>SH30071.03HI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Antibiotics</td>
			<td>Biowest</td>
			<td>L0014-100</td>
			<td>Glutamine-Penicillin-Streptomycin</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recombinant enzyme mixture</td>
			<td>Innovative cell technology</td>
			<td style="width:235px;">AM-105</td>
			<td>Accumax</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DiIC12(3) cell membrane dye</td>
			<td>BD Biosciences</td>
			<td style="width:235px;">354218</td>
			<td>Used as a cell tracker</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td style="width:235px;">703007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plastic syringe (1 mL)</td>
			<td>BD Biosciences</td>
			<td style="width:235px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">23 gauge blunt needles</td>
			<td>Ever Sharp Technology, Inc.</td>
			<td style="width:235px;">TD21</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Poly-tetrafluoroethene (PTFE) tubing</td>
			<td>Ever Sharp Technology, Inc.</td>
			<td style="width:235px;">TFT-23T</td>
			<td>&#160;inner diameter, 0.51 mm; outer diameter, 0.82 mm</td>
		</tr>
	</tbody>
</table>

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.

단일 세포 분리 및 배양 용 마이크로 유체 플랫폼은 마이크로 웰 어레이의 두 세트 (도 2A)와 마이크로 채널 (200 ㎛의 높이)를 포함한다. 마이크로 웰 어레이의 두 세트 캡처 웰 (직경 25 μm의 깊이에 27 μm의) 각각 단일 세포의 분리 및 문화에 대한 문화 웰 (직경 285 μm의 깊이에서 300 μm의), 각 되나된다 탑 뷰 (도 2B)에서 볼 때 캡처 - 웰 배양 웰의 중?...

Watch this Scientific Journal Video about A Microfluidic Platform for High-throughput Single-cell Isolation and Culture at JoVE.com

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

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.

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 ...

Research

JoVE Journal

Bioengineering

High-throughput Protein Expression Generator Using a Microfluidic Platform

마이크로 유체 플랫폼을 사용하여 높은 처리량 단백질 표현 생성기

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

연구

생체공학

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

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 ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

CometChip : Microarrayed 인간의 세포에서 DNA 손상을 측정하기위한 높은 처리량 96 글쎄 플랫폼

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

High Throughput Microfluidic Rapid and Low Cost Prototyping Packaging Methods

높은 처리량 마이크로 유체 신속하고 저렴한 비용으로 프로토 타입 포장 방법

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

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

강력한, 미세 유체 세포 내 전달 플랫폼으로 휴대 뒤쪽에서

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

입자의 공간 추적을위한 멀티 플렉스 전자 감지와 미세 유체 플랫폼

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

세포 분화 및 견인 힘의 상관 관계 분석을위한 높은 처리량 세포 마이크로 어레이 플랫폼

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

꼬마 선 충 에 경도 이미징을 위한 미세 플랫폼

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

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

단일 셀 게놈 시퀀싱 초고 처리량 미세 플랫폼

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

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

동적 압축으로 연골 세포 자극을 위한 미세 유체 플랫폼

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...