Bu çalışma, Ulusal Bilim Vakfı (CBET 0.846.453) ve Ulusal Sağlık Enstitüleri (1R01GM089999) tarafından kısmen desteklenmiştir.

1. Standart SU-8 fotolitografi 1 (Bu video görünmez) kullanılarak silikon ustaların Fabrikasyon. <ol><li> SU-8 &quot;efendileri&quot; (SÜ-8 2050, MicroChem, Newton, MA) bir temiz oda farklı bileşenleri (farklı kalınlıktaki PDMS membranlar, ko-kültür odası) imalatı için. Oluşturmak için standart SU-8 fotolitografi yöntemleri kullanın. Böyle bir ana kalıp herhangi bir mikroimalat tesisi (örneğin, Stanford Mikroakiskan <a href="http://thebigone.stanford.edu/foundry/">Döküm</a> http://thebigone.stanford.edu/foundry/ imal edilebilir) . </li><li> PDMS çoğaltma önce ana PDMS kolay bırakma kolaylaştırmak için bir vakum kaynağına bağlı bir desikatöre fluorosilane buharı SU-8 ustaları maruz bir kağıt havlu fluorosilane bir damla ekleyin ve bir dakika süreyle vakum uygulamak. Vakum çıkarın ve fluorosilane birikimi için 30 dakika izin verin. SU-8 ana ileride kullanmak üzere kapalı bir kapta tutun. </li></ol> 2. SU-8 master 2 PDMS Replica kalıplama <ol><li> Akışkan katman ve kontrol katmanı SU-8 ustadan PDMS çoğaltma kalıplama yapılır. </li><li> 10:01 ağırlıkça PDMS pre-polimer ve çapraz bağlayıcı karıştırın. 1 saat (ya da hava kabarcıkları PDMS karışımından kaldırılana kadar) için bir desikatöre oranı ve gazını. </li><li> Petri SU-8 ana yerleştirin ve dikkatle SU-8 ustanın üstüne PDMS karışımı dökün. Tekrar Degas PDMS. </li><li> Spin kalıp kalıp etrafında 100μm yeknesak bir PDMS kalınlığı elde etmek için bir dakika için 1200 rpm. </li><li> PDMS tedavi için bir saat PDMS ve 80 SU-8 ana kalıp ° C içeren petri ısıtın. </li><li> Kaldır tedavi PDMS kaplı ocak SU-8 ana. </li><li> SU-8 master PDMS kalıp soyun. </li><li> Iki bakteri girişler, bir çıkış ve bir pnömatik bağlantı noktası için 20 gauge künt uç iğne kullanarak PDMS kalıp dört delik delin. </li></ol> 3. Çok katmanlı PDMS cihazları Bağlar: Bu cihaz üç katmandan ana kanal, orta pnömatik tabakası, ve bakteriler için kanalları içeren bir alt bakteriyel katman içeren bir üst tabaka ile monte edilir. Üç kat aşağıda açıklandığı şekilde monte edilir. <ol><li> Metanol ile PDMS membranlar ıslak ve temiz bir forseps kullanarak SU-8 ana kalıp dikkatle onları soymak. Bir teflon levha üzerine yerleştirin membran. </li><li> Bir oksijen plazma odası pnömatik membran ve akışkan katman yerleştirin. 40 sn (oksijen basıncı 10 sccm, RF güç 100W) için plazma açın. </li><li> Plazma maruz kaldıktan sonra, 10 dakika içinde alt PDMS kanal membran forseps kullanarak hizalayın. Membran ve PDMS katmanları ekleme metanol katmanları serbest dolaşımına yardımcı olur ve hizalamayı kolaylaştırır. Bu durumda, cihaz, metanol buharlar kaçmasına izin için bir gazlı bez parçası üzerinde yer almaktadır. </li><li> 8 saat 80 ° C&#39;de ocak Cure hizalı PDMS katmanları </li><li> Bağ üst kanal birleştirilmiş kompozit PDMS aygıtına 3.4 - pnömatik membran alt tabakası üzerine yapıştırma sonra, adımları 3.2 tekrarlayın. </li></ol> 4. PDMS cihaz 3,4 birleştirmek <ol><li> Ökaryotik hücre ve bakteri ekim için kullanılan giriş ve çıkış delikleri delikler delin. </li><li> Limanlarına Tygon boru (0,01 inç OD) bağlayın ve 20ml bir şırınga ile çekerek pnömatik katmanı işletmek. </li><li> PDMS model ve bir oksijen plazma odasına önceden temizlenmiş cam slayt yerleştirin. Plazma açın vakum uygulamadan 20 sn (oksijen basıncı 20 sccm, RF güç 300W). </li><li> Adanın duvar cam slayt (PDMS) yapıştırma arasındaki temasını önlemek için, hemen bir şırınga cihaza bağlayın ve şırınga çekerek ve bir yerde tutarak vakum uygulamak. </li><li> Camına PDMS modeli (2 dakika içinde) yavaşça basın. </li><li> Cure PDMS cihazı 80 ° C&#39;de 10 dakika. </li></ol> 5. Fibronektin ile cam yüzey Tedavisi <ol><li> UV altında 15 dakika Tygon boru her bağlantı noktasına bağladıktan sonra cihaz sterilize edin. </li><li> Dolgu ve kanal kanal herhangi bir enkaz kaldırmak için steril PBS akışı. Hava kabarcıklarını çıkarmak için, 10 dakika boyunca akışı uygulayın. Hava kabarcıkları gaz geçirgenliği sayesinde kolayca PDMS kaçabilir. </li><li> Pnömatik kanal kanal içinde hava kabarcıklarının oluşmasını önlemek için içine bir renk boya çözüm doldurun. </li><li> Fibronektin 1 mcg / ml cam ökaryotik hücre tohumlama kolaylaştırmak için, cihazın içine tanıtmak ve 37 en az 40 dakika boyunca inkübe ° C </li><li> 1 ml büyüme medya ile aşırı fibronektin yıkayın. </li></ol> 6. Ortakçı E. Oluşumu ve ölçüm coli biyofilm INadalar <ol><li> OD 600 M9 minimal medya commensal bakterilerin (örneğin, E. coli BW25113/pCM18) sulandırınız ~ 1 kapalı odaları / adalarda komensal biyofilm oluşturan. </li><li> Pozitif sıvı basıncı uygulayarak adanın duvar kapatın. Ev yapımı bir hava-sıvı basıncı dönüştürücü kanalda hava kabarcığı oluşumunu önlemek için kullanılır. </li><li> Komensal E. tanıtın coli valf ile adalar içine kapalı ve bakteriler 32 az 1 saat takmak için izin ° C. </li><li> Unattached bakterilerin yıkayın ve 12 saat 0.25mL/hr seyreltik bakteriyel bir süspansiyon (OD 600 ~ 0.05) serpmek. </li><li> Gevşek bağlı bakteri kaldırmak ve ökaryotik hücre eki için hazırlamak için taze DMEM büyüme medya ile biyofilm durulayın. </li><li> Resim Leica TCS SP5 Konfokal Lazer Tarama Mikroskobu (Bannockburn, IL) ve biyofilm mimarisi IMARIS 5 yazılımı kullanarak görselleştirmek kullanarak biyofilm. </li></ol> 7. Bakteriyel adaları 6-8 çevresinde ökaryotik hücrelerin Seeding <ol><li> 5 x 10 5 hücre / mL ekim yoğunluğu için DMEM medya doku kültürü şişesi ve tekrar süspansiyon HeLa hücrelerinde Trypsinize. </li><li> 2 ml / dk Picoplus şırınga pompası (Harvard Apparattus, MA) kullanarak bir akış hızında cihazın içine HeLa hücrelerinde tanıtın. Adanın duvar böylece bakteriyel biyofilm HeLa hücre bölgeden ayrı düşürdü olduğunu emin olun. Bu adım, tohum tekdüzelik tespit edilebilir ve böylece ışık mikroskobu aşamasında yapılmalıdır. </li><li> % 5 CO 2 inkübatör kanal ve 37 6 saat inkübe ° C hücre süspansiyon hareketi azaltmak için hücre çıkışı sıkıştırın. </li><li> Unattached hücreleri çıkarmak için 0.5 ml / saat besi cihazı serpmek. </li><li> Yenile HeLa ve ortakçı E. DMEM medya coli, her 6 saatte bir. </li></ol> 8. Ada ve ökaryotik hücrelerde maruz patojen bakterilerin Giriş <ol><li> E. hazırlayın coli O157: H7 (EHEC) (moi) 100:1 200:1 (EHEC HeLa hücre başına sayısı) bir enfeksiyon çokluğu HeLa hücreleri enfekte. </li><li> Durulayın ve gevşek bağlı komensal bakteri yıkayın. </li><li> Adaları içine seyreltik EHEC süspansiyon (OD 600 ~ 0.1) tanıtmak. </li><li> 37 ° C akışı olmadan 6 saat süreyle% 5 CO2 inkübatör komensal biyofilm sinyallere maruz kalacağı EHEC izin cihaz inkübe edin. </li><li> 6 saat HeLa hücrelerinde komensal adalar EHEC maruz PDMS duvar kaldırın. </li><li> PBS ile kanal durulayın ve Live / Dead kiti (L3224, Invitrogen, CA) kullanılarak canlı ve ölü hücreleri için leke. </li><li> Zeiss Axiovert 200M floresan mikroskop ve görüntü birden çok yerde, canlı ve ölü hücreleri sayısı tahmin ediyoruz. </li></ol> 9 - Temsilcisi Sonuçlar 9 GI yolu enfeksiyonları olaylar dizisi HeLa hücrelerinde bir tek tabaka ve ortakçı E. gelişmekte tarafından taklit edildi coli biyofilm önce komensal biyofilm EHEC HeLa hücreleri enfeksiyona maruz. Mikroakışkan ko-kültür modeli imalatı dahil adımları Şekil 1&#39;de gösterilmiştir. Şekil 2A, ko-kültür cihaz 3 boyutlu render gösterir . Co-kültür cihaz ökaryotik hücre kültür odası ve bakteriyel adanın iki bölge farklı bir renk boyalar (bakteriyel ada için ökaryotik bölgeler ve yeşil mor) Şekil 2B gösterilmiştir. Şekil 2C renk boyalar kullanarak bakteriyel kültür bölgeleri çevreleyen ökaryotik bölgeden izole fizibilite gösterilen Şekil 3 epitel hücreleri ve bakteriler ko-kültür temsilcisi sonuçlarını gösterir. Şekil 3A komensal biyofilm (yeşil) ada kolonizasyonu EHEC (kırmızı). Şekil 3B, epitel hücreleri ve ortakçı E. yan yana gösterir bakteriyel adada EHEC coli. EHEC ve GFP ifade komensal bakteriler ifade RFP epitel hücreleri, bakteri ada izole fizibilite gösteren, PDMS duvar indirdi adada lokalizedir. <img src="/files/ftp_upload/1749/1749fig1.jpg" alt="Şekil 1" /> Şekil 1: Hücre ekim düzeni ko-kültür modeli. (1) bir ada PDMS duvar oluşturmak için indirilir, ada haline komensal bakteriler tanıtıldı ve fibronektin adanın etrafında aktı. (2) HeLa hücrelerinde adanın çevresindeki bölgelerde numaralı seribaşı. (3) HeLa hücrelerinde izdiham ulaşmak ve komensal biyofilm geliştirmiştir sonra, EHEC adanın içine tanıtıldı. (4) PDMS duvar kaldırdıEHEC için adanın çevresindeki HeLa hücrelerinde açığa kadar. Vanasının Ankastre detayları gösterir. <img src="/files/ftp_upload/1749/1749fig2a.jpg" alt="Şekil 2a" /> <img src="/files/ftp_upload/1749/1749fig2bc.jpg" alt="Şekil 2BC" /> Şekil 2: epitel hücreleri ve bakteriler ko-kültür için mikroakışkan bir model . Epitel hücreleri arasında bakteriyel adalar oluşturulması için pnömatik tahrikli yakalama bölgeleri gösteren ko-kültür cihazın (A) Üç boyutlu çevrimiyle. Her bakteriyel ada (1200 mikron çap ve 1000 mm arayla), büyüme ortamı sağlanması ve adanın atık kaldırarak için ayrı bir giriş ve çıkış vardır. (B) renk boya ile farklı bölgeler (epitel hücre bölgesine, bakteriyel adalar) gösteren ko-kültür cihaz Mikrografik. (C), pnömatik yakalama sistem sadakat, pnömatik-aktif bir kanal (mavi) kullanarak PDMS duvar (sol panel) düşürücü, kapalı ada mor boya tanıtan ve 48 saat için etrafında sarı boya akan gösterilir PDMS duvar (sağ panel) ortaya çıktığında, adanın bölgenin çevresindeki sarı boya maruz kalmaktadır. Ölçek çubuğu 500 mikron temsil eder. <img src="/files/ftp_upload/1749/1749fig3.jpg" alt="Şekil 3" /> Şekil 3: Co-HeLa hücre ve bakteri kültürü. (A) RFP ifade EHEC ve GFP ifade E. Floresan görüntü coli BW25113 ada. (B) iletilen Yerleşimi, yeşil ve kırmızı floresan görüntüler cihaz. Ölçek çubuğu 200 mikron temsil eder.

<ol>
	<li>McDonald, 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=Prototyping+of+microfluidic+devices+in+poly(dimethylsiloxane)+using+solid-object+printing.">Prototyping of microfluidic devices in poly(dimethylsiloxane) using solid-object printing.</a> Anal Chem. 74, 1537-1545 (2002).</li><li>Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+fabrication+of+integrated+passive+valves+and+pumps+for+flexible+polymer+3-dimensional+microfluidic+systems.">Design and fabrication of integrated passive valves and pumps for flexible polymer 3-dimensional microfluidic systems.</a> Biomed Microdevices. 4, 117-121 (2002).</li><li>Baek, J. Y., Park, J. Y., Ju, J. I., Lee, T. S., Lee, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pneumatically+controllable+flexible+and+polymeric+microfluidic+valve+fabricated+via+in+situ+development.">A pneumatically controllable flexible and polymeric microfluidic valve fabricated via in situ development.</a> J Micromech Microeng. 15, 1015-1020 (2005).</li><li>Grover, W. H., Ivester, R. H., Jensen, E. C., Mathies, R. A., A, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+multiplexed+control+of+latching+pneumatic+valves+using+microfluidic+logical+structures.">Development and multiplexed control of latching pneumatic valves using microfluidic logical structures.</a> Lab Chip. 6, 623-631 (2006).</li><li>Lee, J., Jayaraman, A., Wood, T. K., K, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indole+is+an+inter-species+biofilm+signal+mediated+by+SdiA.">Indole is an inter-species biofilm signal mediated by SdiA.</a> BMC Microbiol. 7, 42-42 (2007).</li><li>Hsu, C. H., 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=Microfluidic+devices+with+tunable+topographies.">Microfluidic devices with tunable topographies.</a> Appl Phys Lett. 86, 023508-023508 (2005).</li><li>Hui, E. E., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromechanical+control+of+cell-cell+interactions.">Micromechanical control of cell-cell interactions.</a> Proc Natl Acad Sci USA. 104, 5722-5726 (2007).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+sensing+hydrogel+microstructures+into+micropatterned+hepatocellular+cocultures.">Integrating sensing hydrogel microstructures into micropatterned hepatocellular cocultures.</a> Langmuir. 25, 3880-3886 (2009).</li><li>Kim, J., Hegde, M., Jayaraman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-culture+of+epithelial+cells+and+bacteria+for+investigating+host-pathogen+interactions.">Co-culture of epithelial cells and bacteria for investigating host-pathogen interactions.</a> Lab-on-Chip. , (2009).</li></ol>

Patojen eki ve kolonizasyon için Konvansiyonel Testler patojenlerin eklenir içine ökaryotik hücrelerin doku kültürü tabak tek tabaka kullanmaktadır. Bu modeller ökaryotik hücreleri üzerinde geliştirilen bir sofrada yemek yiyen bakteri biyofilm dahil değil gibi fizyolojik ilgili değildir. Basit ek ökaryotik hücrelere ön yetişen bir bakteri kültürü biyofilm zamanla gelişecek yüksek yapılar organize olarak bu konformasyon yol açması olası değildir ve neredeyse imkansız değilse kültür ökaryo...

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<td>SU-8 2050</td>
<td>&nbsp;</td>
<td>Microchem Corp, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>high-resolution (16,256 dpi) photolithography mask</td>
<td>&nbsp;</td>
<td>Fineline-Imaging Inc, CO</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Trichloro(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)silane</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>77279</td>
<td>&nbsp;</td>
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<td>PDMS</td>
<td>&nbsp;</td>
<td>Dow Corning, WI</td>
<td>184 SIL ELAST KIT 0.5KG</td>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH30002.02</td>
<td>&nbsp;</td>
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<td>Programmable spin coater</td>
<td>&nbsp;</td>
<td>Laurell Tech Corp</td>
<td>WS0650S</td>
<td>&nbsp;</td>
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<td>Mask aligner</td>
<td>&nbsp;</td>
<td>Neutronix-Quintel, PA</td>
<td>Q4000</td>
<td>&nbsp;</td>
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<td>Oxygen plasma etcher</td>
<td>&nbsp;</td>
<td>March Plasma System, CA</td>
<td>CS-1701</td>
<td>&nbsp;</td>
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<td>Syringe pump</td>
<td>&nbsp;</td>
<td>Harvard Apparatus, MA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Live/Dead Viability/Cytotoxicity Kit</td>
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<td>Invitrogen</td>
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microfluidic co-culture of epithelial cells and bacteria for investigating soluble signal-mediated interactions

Insan gastrointestinal (Gİ) sistem bağırsak epitel hücreleri ve patojen olmayan (ortakçı) bakteri bir arada olduğu benzersiz bir ortam. Patojen komensal katmanında karşılaştığı mikroçevresinin kolonizasyon ölçüde belirlenmesinde önemli olduğu ileri sürülmektedir. Patojen kolonizasyon araştırmak için geçerli kültür yöntemleri de gastrointestinal sistem mikroçevresinin taklit bir şekilde bakteri ve epitel hücreleri birlikte kültür etkinleştirmek gibi bu hipotezi araştırmak için uygun değildir. Burada ökaryotik hücre ve bakteri bağımsız kültür sağlayan bir mikroakışkan ko-kültür modeli tanımlamak ve patojen kolonizasyon komensal mikroçevresinin etkisi test. Komensal gelişmekte olan ko-kültür modeli göstermiştir.<em&gt; Escherichia coli</em&gt; Enterohemorajik tanıtımı tarafından takip HeLa hücreleri arasındaki biyofilm<em&gt; E. coli</em&gt; Komensal ada (EHEC), gastrointestinal sistem enfeksiyonu olaylar dizisi taklit eden bir dizi.

Watch this Scientific Journal Video about Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions at JoVE.com

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

Bu protokol, eşzamanlı ve lokalize epitel hücreleri ve bakteri kültürü için bir mikroakışkan ortak kültür modeli açıklamaktadır. Bu model, varsayılan probiyotik bakteri suşlarının etkinliğini patogenezinde farklı çözünür moleküler sinyallerin rolünü araştıran gibi ekran için kullanılabilir.

Çözünür Sinyal aracılı Etkileşimleri Araştırılması için Epitel Hücreleri ve Bakteriler mikroakışkan Co-kültür

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

microfluidic-co-culture-epithelial-cells-bacteria-for-investigating

Research

JoVE Journal

Biology

14.0K Görüntüleme.  Texas A&M University. Bu protokol, eşzamanlı ve lokalize epitel hücreleri ve bakteri kültürü için bir mikroakışkan ortak kültür modeli açıklamaktadır. Bu model, varsayılan probiyotik bakteri suşlarının etkinliğini patogenezinde farklı çözünür moleküler sinyallerin rolünü araştıran gibi ekran için kullanılabilir.

Video: Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

Nörotoksisite Yüksek İçerik Analizi Nöronal ve Astrosit Hücre Co-Kültür Testi

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biyoloji

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

Çok bölmesi MSS Nöron glia Co-kültür mikroakışkan Platformu

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Üç boyutlu Karışık-hücre Sfero Co-kültürden Meme Epitel Hücrelerinin İzolasyonu

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Biyoparlaklık Rezonans Enerji Transferi kullanma Canlı Hücrelerde Protein-protein etkileşimleri araştıran

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

Sitometrisi ile Böbrek Hücrelerine sitozolik kalsiyum Ölçümleri

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Fare Kolon Mukozanın Mikrodisseksiyon için Cryosectioning Yöntemi

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Mikroakışkan Aygıtlar Kullanma Tek Budding Maya Hücreleri ömrü ve Hücresel fenotipleri Ölçmek için

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

Bakteri, Maya ve İnsan Hücrelerinde Geçici Olarak Oluşan Moleküler Şaperon Meclislerinin Yerinde İzlenmesinde

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

Tehlike Değerlendirmesi İçin Epitel Hücreleri ve Primer İmmün Hücrelerden Oluşan Çok Hücreli İnsan Alveoler Modeli

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...