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

Not: Bizim mikroakışkan cihaz imalatı için photomask tasarımları bilgisayar destekli tasarım (CAD) yazılımı kullanılarak çizildi. tasarımlar daha sonra ticari bir hizmeti kullanarak krom, fotoğraf maskeleri imal etmek yararlanılmıştır. PDMS cihazlar yumuşak litografi teknikleri kullanılarak yapılmıştır. 11 Litografi tarafından Usta Kalıpları 1. Fabrikasyon <ol><li> Fotolitografi işlem 12 önce, bir alt-tabaka 4-inç çaplı silisyum gofret kullanımı ve 10 dakika boyunca 120 ° C de, geleneksel bir fırında gofret dihidrat. </li><li> Bir plazma temizleyici 30 saniye boyunca 100 watt oksijen plazma tedavisi ile susuz silikon gofret temizleyin. </li><li> Aşağıdaki pişirme işlemi için, sırası ile 65 ° C &#39;de iki ocak ve 95 ° C, ön ısıtma. </li><li> spin kaplayıcı ile temizlenir silikon gofret üzerinde olumsuz fotorezist (PR) Coat 5 g; 30 sn 1,200 rpm (SU-8 50) spin mikrokanal tabakası üretmek. </li><li> PR yerleştirin12 dakika boyunca 65 ° C&#39;de önceden ısıtılmış bir sıcak plaka üzerine gofret kaplanmış ve yumuşak bir fırında işlemini gerçekleştirmek için (100 mikron kalınlığında desenler) 33 dakika boyunca 95 ° C&#39;de bir ısıtılmış sıcak plaka aktarın. </li><li> Pişirme sonrasında, bir yarı otomatik maske hizalama sahibinin PR kaplı silikon gofret yerleştirin ve bir 25400 dpi çözünürlük şeffaflık photomask onu hizaya getirin. </li><li> Silikon gofret PR desen oluşturmak için 500 mJ / cm2 dozunda UV ışığı (365 nm) PR kaplı silikon gofret Açığa. </li><li> hizalama gelen gofret çıkarın ve 12 dakika süreyle 95 ° C&#39;de post-Pişirme için bir ocak üzerine yerleştirin. </li><li> hizalama işaretlerini ortaya çıkarmak için 12 dakika ve azot gazı ile hafifçe kuru için çapraz bağlanmamış PR yıkayın SU-8 geliştirici (propilen glikol monometil eter asetat, PGMEA) çözeltide gofret bekletin. </li><li> Yine, spin kaplayıcı tarafından gofret üzerinde olumsuz fotorezist ceket 5 g; 300 ve 30 sn için 30 saniye ve 1200 rpm&#39;de (SU-8 10) 700 rpm&#39;de (SU-8 100) spin# 181; m kalınlığında desen ve 27 mikron kalınlığında desen sırasıyla mikro katmanı yapmak. </li><li> (27 um, derin çekme çukurlu katmanı), 4 dakika ve 8 dakika boyunca 95 ° C &#39;de 65 ° C&#39; de bir sıcak plaka üzerinde PR kaplanmış gofret yerleştirin; 40 dakika boyunca 65 ° C &#39;de ve (300 um derin bir kültür çukurlu tabakası için), 110 dakika boyunca 95 ° C&#39; de. </li><li> Soğuduktan sonra, UV ışığı ile donatılmış maske hizalama PR kaplı silikon gofret yerleştirin. </li><li> 250 mJ / cm 2 (27 mikron kalınlığında deseni) ve 700 mJ / dozunda cm 2 (300 mikron kalınlığında pattern) UV ışığı (365 nm) PR kaplı silikon gofret Açığa. </li><li> sırasıyla 5 dakika boyunca (27 mikron kalınlığında deseni) ve 30 dakika (300 mikron kalınlığında kalıp) 95 ° C &#39;de gofret pişirilir. </li><li> sırasıyla 6 dakika (27 mikron kalınlığında deseni) ve 25 dakika (300 mikron kalınlığında desen), için PGMEA tarafından çapraz bağlanmamış PR yıkayın ve sonra azot gazı ile kurulayın. </li><li> desen özelliklerini yüksekliğini ölçmekBir tarayıcı lazer Profilometrenin xy-sahnede gofret koyarak tarama lazer profilometreyle gofret. <ol><li> açıkça desen 20X objektif mercek altında &quot;kamera görüntüsü&quot; gözlem modu kullanarak gofret özelliklerini göstermek için odak düzlemi ayarlayın. </li><li> &quot;Lazer görünüm&quot; &quot;kamera görüntüsü&quot; dan gözlem moduna geçmek ve özellikleri üst ve alt pozisyonları ayarlayın. </li><li> sırasıyla ölçüm modu, alanı, kalite ve saydam (üstte) kadar z-zift, 1 satır (1.024 x 1), yüksek doğruluk ve 0.5 mikron, ayarlayın ve ardından ölçüme başlamak için başlangıç ​​altını basın. </li></ol></li></ol> Tek hücreli İzolasyon PDMS Cihazlarının 2. Hazırlık <ol><li> PDMS döküm önce, daha kolay ana kalıpları PDMS kopyaları kalkmasına yapar hidrofobik bir yüzey yaratmak için trichlorosilane ile ana kalıpları Silanize. <ol><li> usta kalıpları ve içeren bir ağırlık tekne yerleştirin200 kurutucuda trichlorosilane ul ve 15 dakika boyunca vakum (-85 kPa) uygulanır. </li><li> vakumu durdurmak ve daha sonra en az 1 saat oda sıcaklığında ana kalıpları Silanize için desikatörde ana kalıpları bırakın. </li><li> desikatöre master kalıpları çıkarın ve 10 cm Petri kabındaki her ana kalıp yerleştirin. </li></ol></li><li> bir taban ve 10 arasında bir oranda bir sertleştirme ajanı ihtiva eden 17.6 g PDMS polimer kitinin bir toplam tutarını karıştırın: 1, ve daha sonra Petri kabındaki ana kalıp PDMS dökün. </li><li> desikatörde Petri kabı yerleştirin ve PDMS hava kabarcıklarını çıkarmak için 1 saat vakum (-85 kPa) uygulanır. </li><li> desikatör Petri çanağı çıkarın ve 3 65 ° C sıcaklıkta, geleneksel bir fırında yer - PDMS tedavisi için 6 saat. </li><li> ana kalıpları tedavi PDMS kopyaları çıkarın ve 0 iç-çapına sahip bir puncher&#39;ait yakalama-kuyu dizi PDMS çoğaltma Mikrokanallı iki ucunda iki bir giriş olarak delikleri ve bir çıkış yumruk.Akışkan kanalının (Şekil 2A) için 75 mm. </li><li> PDMS kopyaları yüzeyini temizlemek ve sonra kısa bir oksijen plazma tedavisi (14 sn için 100 watt) için bir plazma temizleyici PDMS kopyaları yerleştirmek için bant kullanın. </li><li> oksijen plazma makineden PDMS kopyaları çıkartın. </li><li> Bir stereomikroskop altında elle bir üst (yakalama mikro içeren) ve (kültür mikro içeren) bir alt PDMS çoğaltma hizalayın ve temas getirmek. </li><li> Nihai cihaz oluşturmak PDMS yinelemeler arasında sürekli bir bağlantı sağlamak için 24 saat boyunca 65 ° C sıcaklıkta bir fırın içinde hizalanmış PDMS yinelemeler yerleştirin. </li><li> deiyonize (Di) -su doldurulmuş kapta PDMS cihaz ıslatın ve PDMS cihazın mikrokanaldan havayı çıkarmak için 15 dakika boyunca vakum altında (-85 kPa) bir desikatör içinde kap yerleştirmek. </li><li> Bir doku kültürü kaputu DI su dolu PDMS cihaz yerleştirin ve UV ışığı (ışığın dalga boyu: 254 nm) kullanmak 30 mil için cihazın sterilize etmekn. </li><li> 1X PBS çözeltisi içinde% 5 sığır serum albumin (BSA) ile PDMS cihazında distile su yerine ve PDMS yüzeye yapışmasını engellemek için hücrelerin 30 dakika boyunca 37 ° C&#39;de inkübe edin. BSA kaplama izole hücre transferi verimini artırmak için kritik yakalama oyuklu kültür oyuklu: edin. </li><li> sterilize 1x PBS çözeltisi ile PDMS cihazın% 5 BSA çözüm değiştirin. </li></ol> Tek hücreli Süspansiyon 3. hazırlanması <ol><li> Kültür Nöral hücre KT98 ve insan karsinoma hücre A549 ve MDA-MB-435, biz kök PDMS cihaz hücre deneyi için hücreler hazırlamak için geleneksel bir hücre kültürü inkübatöründe (37 ° C,% 5 CO2 ve% 95 nem) Petri kabındaki . </li><li> Çıkarın ve geçirilerek kültür ortamı 70 yetiştirilen hücreler kültür kabı (% 10 fetal bovin serumu ve% 1 antibiyotik ile birlikte DMEM bazal ortamı) iptal -% 80 birleşir. </li><li> Yavaşça sterilize PBS üç kez hücreleri yıkayın. &lt;/ Li&gt; <li> (Ayrıntılı bilgi için Malzeme Listesi bakınız) çıkarın ve PBS atmak ve proteolitik, kolajenolitik ve DNaz faaliyetleri ile rekombinant enzim karışımı 2 ml ekleyin. </li><li> 5 dakika boyunca oda sıcaklığında kültürü çanak inkübe ve daha sonra hücre ayrılması için kültür çanağı dokunun. </li><li> hücrelerin dağıtılması için steril PBS 4 ml ve sonra 15 ml konik bir tüp hücre süspansiyonu aktarın. </li><li> 3 dakika boyunca 300 xg&#39;de tüp Santrifüj ve süpernatant kaldırmak. </li><li> Yavaşça PBS sterilize 1 ml hücre pelletini ve standart Tripan Mavi dışlama yöntemi 13 kullanarak canlı hücre sayısını. Not: Bu yeniden süspansiyon haline adımı tek hücre izolasyon verimini artırmak için iyi ayrışmış tek hücre süspansiyonu hazırlamak için kritik önem taşır. </li></ol> 4. Tek hücreli İzolasyon ve klonal Kültür <ol><li> 2.5 x10 - 2.2 arasında bir konsantrasyonda Yük hücresi süspansiyonu 50 ul <subir el pipet ile cihazın çıkış deliğinden PDMS cihazın Mikrokanallı içine p&gt; 6 hücre / ml. Not: Hücre süspansiyonu yükleme pipet Mikrokanallı içine hava kabarcıkları almaktan kaçınması durdu ve ilk durağı konumunda düzenlenecek gerekmektedir. </li><li> eşit hücreleri ile tüm Mikrokanallı doldurmak için giriş delikten hücre süspansiyonu başka bir 50 ul yükleyin. </li><li> giriş ve çıkış delikleri damlacıklar gelen hidrostatik basınç ile uyarılan akışını önlemek için bir tıkaç (3 mm uzunluğunda, 1 mm çapında kesim naylon misina) ile çıkış deliği mühür. Not: Fişler kullanmadan önce bir doku kültürü kaput içinde sterilize UV ​​idi. </li><li> , Kültür ortamı ile 1 ml steril şırınga doldurun ondan hava kabarcıklarını çıkarmak ve bir şırınga pompası kurmak. </li><li> Bir 23 G, kör bir iğne ve bir poli-tetrafluoroethene (PTFE) boru ile PDMS cihazın giriş deliğine orta yüklü şırınga bağlayın. </li><li> çıkış deliği ve tüm prizden çıkarınow 2 dk zaman aralığı hücreleri yerçekimi kuvveti tarafından tek hücreli yakalama-kuyu içine yerleşmek için izin vermek. </li><li> şırınga pompası tarafından tahrik edilen 600 | il / dk&#39;lık bir akış oranında kültür ortamında 300 ul uncaptured hücreleri yıkamak. </li><li> Cihazı stabilize etmek 2 dakika bekleyin ve kapalı bir kültür sistemi oluşturmak için fişler ile giriş ve çıkış delikleri kapatın. </li><li> kültür mikro yakalanan tek hücreleri aktarmak için el cihazı çevirin. </li><li> 100 mm doku kültürü çanağı içinde PDMS cihaz yerleştirin ve mikrokanaldan kültür ortamı buharlaşmayı önlemek için cihaz etrafında steril 10 ml PBS ilave edin. </li><li> Tek hücrelerinin klonal kültürü için geleneksel bir hücre kültürü inkübatöründe (37 ° C,% 5 CO2 ve% 95 nem) kültür çanağı hareket. </li></ol> 5. Kültür Orta Yenileme <ol><li> kültür 1 gün sonra, hücre pr geliştirmek için taze ortam PDMS cihazında bir kültür ortamı yerineoliferation. </li><li> Bir sonraki adımı yaparken Mikrokanallı içine hava kabarcıkları almaktan kaçınması giriş ve çıkış delikleri alanların yakınında PDMS cihazın üstündeki kültür ortamının iki damlacıkları yerleştirin. </li><li> Bir giriş olarak, mikrokanalın iki ucunun yakınındaki PDMS cihazın üst ve mikrokanal için bir çıkışı iki delikler. Not: PDMS cihazın tam iki kat üzerinden yumruk etmeyin; Bunun yerine, sadece PDMS üst katmanı sayesinde yumruk. </li><li> Bir 23 G, kör bir iğne ve PTFE tüp vasıtasıyla girişe taze ortam ihtiva eden bir 1 ml&#39;lik plastik bir şırınga bağlayın. </li><li> eski orta yerine 5 dakika süreyle cihaza 120 ul taze orta akış. </li><li> giriş mühür ve delikler çıkış ve hücre kültürü inkübatör cihazı geri dönmek için iki fişleri takın. </li><li> 5.5 adımları 5.4 tekrarlayarak ardından sızdırmazlık tapaları, kaldırarak kültür ortamının kültürünün döneminde her 2 günde bir yenileyin. </li></ol>

<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>

The authors declare that they have no competing financial interests.

Mikro-tabanlı bir aygıt sistemleri 6,14 örneğin 6 yakalama geniş çaplı tek bir hücre tek kök hücre çoğalması 15 gibi tek hücre manipülasyonu ve analiz için kullanılmıştır. De boyut, sayı ve şekil olsa da, özel uygulamalar için ayarlanabilmektedir kuyunun boyutu artar olduğunda tek hücre izolasyon verimi, her zaman ele almaktadır. 9,15 Mikro boyutu hücre yayılması ve büyümesi için izin vermek için büyütülür ise ...

Şu anda bir kültür alanı ayrı ayrı tek hücre yerleştirilmesi genellikle sınırlayıcı seyreltme veya floresan-aktive edilmiş hücre sıralama (FACS) kullanılarak elde edilir. sadece kolayca elde edilebilir Bir pipet ve doku kültür plakaları, gereğine göre bir çok laboratuarlar için, sınırlayıcı seyreltme, uygun bir yöntemdir. Bu durumda, bir hücre süspansiyonu seri uygun bir hücre yoğunluğuna kadar seyreltilir ve daha sonra bir el ile bir pipet kullanarak kültür kuyu içine yerleştirilir. Bu bölmeli tek hücreler daha sonra, 1 ve koloni oluşumu 2 tarama genetik heterojenliğinin olarak, hücre analizi için kullanılmıştır. Sınırlayıcı seyreltme yöntemi Poisson dağılımı doğa% 37 3 maksimum olasılık tek hücreli olayları kısıtlar çünkü Ancak, bu yöntem yardım için bir robot kolu kullanarak olmadan düşük verimlilik ve emek-yoğun, olduğunu. entegre robot kol ile FACS makineleri doğru Plac tarafından Poisson dağılımının sınırlama üstesinden gelebilirBir anda 4 de hiç bir kültürde tek hücreli ing. Ancak, yüksek mekanik kayma gerilmesi 5 (böylece, hücre canlılığını indirdi) ve makine alım ve işletme maliyetleri çok laboratuvarda kullanımını sınırlıdır. Çok verimli bir mikro 6 içine tek hücreler yüklemek için yukarıdaki sınırlamaları aşmak için, mikro cihazlar geliştirilmiştir. Ancak, mikro-yüklü hücreler nedeniyle tek hücreli yükleme olasılığını maksimize etmek için tek bir hücre olduğu yakın microwell her boyutunu yapma ihtiyacı, çoğalmaya için yeterli alan vermeyin. Kültür deneyleri daha büyük mikro-birçok hücre bazlı uygulamalar (örneğin, klonojenik analiz 7), gerekli olduğu için (90 - 650 um çapında ya da yan uzunluğunda), aynı zamanda uzun bir hücre kültürleri için izin vermek için kullanılmıştır. Ancak, sınırlayıcı seyreltme yöntemi gibi, onlar da 10 arasında değişen, düşük tek hücre yükleme verimliliği sahip -.% 30 89 Daha önce, 10 cihazı poli-dimetilsiloksan (PDMS) ile yapıldı. Ayrı ayrı mikro tek hücrelerin izole edilmesi ve izole edilmiş hücrelerin klonojenik deneyde uygulanmasını göstermek için yüksek verimli mikroakışkan platformu geliştirilmiş ve mikro dizilerinin iki grup ihtiva eder adres büyük ölçüde olan boyutu kuyucuklarda tek bir hücreyi yüklenirken verimliliğini artırabilir farklı mikro boyutlarda olan hücrenin önemli ölçüde daha büyüktür. Özellikle, bu &quot;çift kuyu&quot; kavramı kültür alanının büyüklüğü esnek o basit farklı hücre tiplerini ve uygulamalarına uygun cihazın tasarımını ayarlamak için yapım, tek hücreli yakalama verimliliği etkilemeden ayarlanmasına imkan verir. Bu yüksek verimli bir yöntem, hücre heterojenite çalışmaları ve monoklonal hücre çizgisi oluşturulması için uzun vadeli bir hücre kültür deneyleri için yararlı olacaktır.

<table border="0" cellpadding="0" cellspacing="0" width="1323">
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			<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>
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		<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>
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		<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>
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		<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>
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		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Thermo Hyclone</td>
			<td>SH30071.03HI</td>
			<td></td>
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			<td height="20" style="height:20px;">Antibiotics</td>
			<td>Biowest</td>
			<td>L0014-100</td>
			<td>Glutamine-Penicillin-Streptomycin</td>
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			<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>
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		<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>
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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.

Tek hücre izolasyonu ve kültürü için mikroakışkan platformu mikro dizileri iki set (Şekil 2A) ile bir mikrokanal (yükseklik 200 mm) içerir. Mikro dizilerin iki set yakalama oyuk (çapı 25 mm ve derinliği 27 mm), sırasıyla, tek hücreli izolasyonu ve kültürü için, kültür oyuk (çapı 285 mm ve derinlik 300 mm), ve her olarak adlandırılır üst görünümü (Şekil 2B) görüldüğü zaman yakalama-kuyu kültür-kuyu merkezinde konuml...

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.

Yüksek verim Tek hücreli İzolasyonu ve Kültür mikroakışkan Platformu

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 ...

a-microfluidic-platform-for-high-throughput-single-cell-isolation

Research

JoVE Journal

Bioengineering

11.2K Görüntüleme.  National Health Research Institutes, Taiwan. 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. 

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

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.

Bir mikroakışkan Platform kullanarak Yüksek throughput Protein Ekspresyonu Jeneratör

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

Biyomühendislik

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.

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.

Yüksek Verim Micro-encapsulation Tek hücreli ve Çoklu-hücre

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

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip: İnsan Microarrayed hücrelerde DNA hasarına ölçülmesi için bir Yüksek verim 96 Oyuklu Platformu

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

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 ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Sağlam, mikroakışkan Hücre içi Delivery Platform olarak hücre Sıkma

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

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Parçacıkların Mekansal Takip için Çoklanmış Elektronik Algılama ile mikroakışkan Platformu

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

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Hücre farklılaşması ve Çekiş Kuvvetleri Korelatif Analizi için Yüksek verim Hücre Mikroarray Platformu

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

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

Boyuna Imaging'de Caenorhabditis elegans için bir mikrosıvısal Platform

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

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.

Tek hücreli genom sıralama için bir üretilen iş Ultrahigh mikrosıvısal Platform

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

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Dinamik Sıkıştırma ile Chondrocytes Uyarıcı için Bir Mikroakışkan Platform

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