Bu araştırma Sağlık hibe numaraları R01NS050452 ve R01EB009429 Ulusal Enstitüsü tarafından finanse edildi. Konsol cips imalatı Cornell Üniversitesi&#39;nde bulunan Nanofabrikasyona Tesisinde işbirlikçileri tarafından dışarıdan gerçekleştirildi. Konsol üretim sürecinde kullanılan tüm ekipmanlar bu tesiste yer oldu. Konsol mikro-üretim ile yardım için Mandy Esch ve Jean-Matthieu Prot Special thanks. Konsol işlevsellik video animasyon UCF de Sentetik Gerçeklik Lab Charles Hughes, Alex Zelenin ve Eric Imperiale tarafından oluşturulmuştur.

1. Konsol Chip Fabrikasyon Açıklanan imalat adımlarının gösterilen ayrıntıları Şekil 1 &#39;de verilmiştir. <ol><li> Bir fırın içinde silikon üzerinde yalıtkan (SOI) gofret yerleştirin ve bunları kurutmak için 20 dakika boyunca 125 ° C&#39;de pişirilir. </li><li> Bir plazma kullanılarak susuz SOI gofretin sap tabakası üzerine silikon oksit, 1.5 mikron kalınlığında bir tabaka yatırın kimyasal buhar çöktürme (PECVD) aracı artırıldı. </li><li> Cihaz katman yukarı bakacak şekilde spin lak mandrenin gofret yerleştirin. , Gofret merkezli olduğundan emin olun 1,000 rpm / saniye bir hızlanma 60 saniye boyunca 3000 rpm&#39;de gofret merkezinde ve spin P20 astar 2 ml dağıtmak. P20 astar cihaz fotorezist tabakaya yapışmasını teşvik etmektedir. </li><li> Gofret spin lak mandrenin görünse de, o ile 1000 rpm / saniye oranında hızı 60 saniye boyunca 3000 rpm&#39;de S1818 gofretin ortasına ışığa ve spin 2 ml dağıtmakFotorezist 1.5 mikron kalınlığında bir tabaka btain. </li><li> Sıcak bir plaka üzerinde gofret yerleştirin ve 1 dakika boyunca 115 ° C&#39;de bir fırında yumuşak gerçekleştirin. </li><li> Cihaz üzerinde bir tabaka ışığa için konsol maskeden desen transfer etmek için bir iletişim fotolitografi maske hizalama kullanılarak sert kontakt UV ışınlarına maruz kalma gerçekleştirin. 125 mj / S1818 fotodirenç için cm 2 poz enerji değerine göre pozlama süresini hesaplayın. </li><li> Hafifçe ileri ve geri gofretin çalkalayarak, 1 dakika boyunca 726 MIF fotorezist geliştirici gofret daldırarak fotorezist geliştirir. </li><li> De-iyonize (DI) su ile durulayın gofret ve tercihen bir sıkma durulama kurutma makinesi (SRD) aracını kullanarak, kurutun. </li><li> Aseton batırılmış bir pamuk bez kullanılarak (kenardan 5 mm kadar) gofretin kenarından fotorezist çıkarın. </li><li> Fotorezist tarafı yukarı bakacak şekilde, bir Derin Reaktif İyon Asitli (DRIE) aracında gofret yükleyin ve devi ile desenli silikon tabakasını etch için bir reçete çalıştırınce katmanı. Bir aşındırma durak olarak gömülü oksit katmanı işlevleri, yani tam bir iz sağlamak için gerekli olması beklenmektedir% 100&#39;den daha fazla döngü için% 50 ile bir iz gerçekleştirin. </li><li> Fotorezist şerit 20 dakika boyunca sıcak bir fotorezist Banyo çözeltisi içindeki gofret yerleştirin. DI su ile gofret durulayın hızlı-dökümü-rinser (QDR) gofret aktarın. Fotorezist şerit ardından, durulama ve gofretin kuruması için SRD aracında gofret yükleyin. Beklendiği gibi konsol desen görünür sağlamak için bir mikroskop altında gofret kontrol edin. </li><li> Bir PECVD alet gofretin cihaz katmanda un katkılı bir silikon oksit 1 mikron kalınlığında bir tabaka bırakın. Daha fazla işlem kademesi sırasında konsollar için koruyucu tabaka olarak bu oksit tabakası çalışır. </li><li> Gofretin arka tarafının başlatmak üzere yukarı bakacak şekilde sap tabakası ile dönel kaplayıcı aynası üzerine, gofret yerleştirin. Olun gofret 3000 rpm&#39;de P20 gofretin ortasına astar ve spin 2 ml dağıtılması, ortalanır1000 rpm / saniye oranında hızı 60 sn. </li><li> Gofret spin lak mandrenin görünse de, bir 6.5 mikron kalınlığında bir tabaka elde edilmesi için 1000 rpm / saniye oranında hızı 45 saniye boyunca 2000 rpm&#39;de gofretin merkezi ve spin üzerine SPR220-4.5 fotodirenç 2 ml dağıtmak Fotorezist. </li><li> 2 dakika boyunca 115 ° C&#39;de bir fırında yumuşak gerçekleştirmek için bir yakınlık sıcak bir plaka üzerinde gofret yerleştirin. </li><li> Ön / arka uyum yeteneğine sahip bir fotolitografya temas hizalama kullanarak, ön yan konsol desen arka pencere maske hizalayın ve kolu katmanda fotodirenç için arka pencere maskeden desen transfer etmek için bir sert kontakt UV maruziyetini gerçekleştirin. 480 mj / SPR220-4.5 fotodirenç için cm 2 poz enerji değerine göre hesaplanan bir pozlama süresi kullanın. </li><li> UV&#39;ye maruz kalmadan sonra, çok ince bisküviler bir sonraki işleme aşamasından önce 30 dakika süre ile karanlık bir yerde kalmasını sağlar. </li><li> Gofretin daldırarak fotorezist geliştirilmesi2 dakika süre ile, 726 MIF fotorezist geliştirici hafifçe ileri ve geri gofreti çalkalandı. </li><li> De-iyonize (DI) su ile durulayın gofret ve tercihen bir sıkma durulama kurutma makinesi (SRD) aracını kullanarak, kurutun. </li><li> 12 saat boyunca 90 ° C&#39;de bir fırında gofret yerleştirin. </li><li> Florlu gazlar ile RIE sistemi ve oksit gravür için bir tarifi kullanarak gofret ters silikon oksit tabakası etch. Gofretin ters desenli oksit daha sonraki işlemler için bir aşındırma maske olarak görev yapar. Maruz kalan penceresinde silikon oksit tamamen kazınmış olduğundan emin olmak için bir mikroskop altında gofret kontrol edin. </li><li> Aseton batırılmış bir bez kullanılarak temiz oda (kenardan 5 mm kadar) gofretin kenarında fotorezist çıkarın. </li><li> Bir DRIE aracında gofret yüklemek ve bir aşındırma durak olarak gömülü oksit tabakası işleyişi ile 500 um&#39;lik bir derinliğe desenli tabaka sap oyulması için bir tarifi çalıştırın. Aşırı ısınmasını önlemek için birden çok çalışır içine bu etch bölünmüşsilikon tutarsız aşındırma neden gofret. Bu aşındırma adımı tamamen dirseklerin altında silikon kaldırır. </li><li> Dirseklerin altında gömülü oksit tabakası ve dirseklerin üstüne koruyucu oksit katman şerit,% 25 sulandırılmış HF pürüzlendirici kullanan bir ıslak aşındırma adımı gerçekleştirir. Not: Bu adım silikon konsolun alt yüzeyi serbest bırakır ve aynı zamanda lazer ile dirsekli araştırmak için bir erişim sağlamak için altına bir pencere açar. </li><li> DI su banyoları dizi ve azot ile dikkatlice kuru kullanarak gofret durulayın. Konsollar kendi üslerinde yalnızca desteklenen yana, doğrudan dirseklerin üzerine DI su veya inert gaz güçlü spreyler kullanmayın. </li><li> Sap tabakası aşındırma adımı esnasında üretilen klevajın çizgileri boyunca gofret bireysel fiş bölmektedir. </li></ol> 2. Hücre Kültürü <ol><li> Daha önce yayınlanmış yöntemlerden 17&#39;ye göre 13F lamelleri hazırlayın. NOT: Eğer 13F koyrslips mevcut değildir, 95 ° C&#39;nin üzerindeki bir temas açısına sahip herhangi bir hidrofobik yüzey kullanılabilir. </li><li> Konsol cips ve% 70 etanol çözeltisi içinde 13F lamelleri sterilize ve bir akış kaputu kurumaya bırakın. </li><li> Bir standart 12 plaka içindeki 13F lamelleri üstüne bireysel konsol cips yerleştirin. </li><li> Kaplayın hücre tipi için optimize biyopolimer veya yüzey modifikasyonu ile dirsekleri standart hücre kültür protokollere göre kullanılır. </li><li> Istenen konsantrasyona kendi özel büyüme ortamında hücrelerin yeniden askıya. NOT: Bu protokol konsol penceresinden düşen ve istenilen konsol yüzeyine yapışan olmayan hücrelerin önemli bir sayıda neden olur. Hücre preparasyonlar, bu sebepten, standart preparatları için bir örtücü cam, daha konsantre 3-4x yapılmalıdır. Örneğin, sıçan iskelet kas uydu hücreleri, tipik olarak tohumlama 500-700 hücre / mm kare 15,16 ilk önce tohum ekme yoğunluğu kullanılarak yürütülürVe / meydan aa 2000 hücre olacak şekilde konsol alt tabakalar kullanılmaktadır. Optimizasyon deneyler, uygun bir tohum yoğunluğu belirlemek için yeni bir hücre kaynakları ile yapılmalıdır. </li><li> Orta kabarcık tamamen konsol pencere kapsayan sağlanması konsol çip yüzeyi üzerine, hücre süspansiyonu 200 ul pipetle. Orta pencereden fitiller ve dirseklerin üstüne bir statik kabarcık oluşturmak vermezse 13F lamel değiştirmek ve kaplama reattempt. </li><li> Bir kuluçka yongası içeren plaka aktarın ve hücreler, en az 1 saat (tercihen 2-3 saat) boyunca yapışmaya izin verir. </li><li> Bu kaplama tamamlandıktan sonra, büyüme ortamı, 1 ml kültür, bir 13F lamel olmaksızın temiz bir çukuruna çipi transferi, ve en fazla steril forseps kullanır. </li><li> Inkübatör plaka dönün. </li><li> Lamelleri in vitro bakım için standart bir protokole uygun hücreleri korumak. Iskelet kası hücreleri için,Hücreler birleştiklerinde, gerekli olacaktır hale kez ortam bileşiminin bir anahtar miyotüp oluşumunu tetiklemek için karıştırılabilir. </li></ol> Konsol Saptırma Analiz için Donanım ve Yazılım 3.Kurulum <ol><li> Dik elektrofizyoloji mikroskop aşamasında içine ısıtılmış kültür çanak yerleştirin. </li><li> Isıtılmış mikroskop kademesine ile henüz (+ 10 mM HEPES) içinde süspanse edilmektedir besleme ortamının 3 ml ilave edilir. </li><li> 15 mm&#39;lik bir ayırma mesafesi ile ısıtılmış kültür çanak iç paslanmaz çelik elektrotlar monte ve bir darbe üreteci arasında değişen yoğunlukta, frekans ve dalga biçimi alan uyarım darbelerini üretebilen bağlamak, sistem alan uyarımı üretmek için izin Uygun zaman hücrelerin. </li><li> , Mikroskop tablonun alt tarafına, X ve Y platformlarının üzerine monte edilen bir helyum-neon lazer cıvata ve lazer ışını, bir 30 ° &#39;lik açı relativ de ısıtılmış kültür çanak tabanı boyunca yönlendirilecek şekilde ayarlayınkonsolun düzlemine e. </li><li> , Mikroskop sahne alt etmek, XY öteleme aşamalarında üzerine monte edilmiş bir kadran fotoğraf-dedektör modülü cıvata ve 4 kadranın merkezinde yansıyan lazer ışını topraklar. Şekil 2, böylece konumunu ayarlamak kurmak donanımın bir bakış sağlar Açıklanan protokol uygulanması için gerekli. </li><li> Cantilevers üzerinden tarama doğrusal aktüatörler kontrol etmek için bir yazılım program yazın. Şekil 3&#39;te sunulan akış şemasına değini ile yazılım programı yazmak. Bu sistem ile kullanılmak üzere programlanmış bir grafik arayüz Şekil 4&#39;te sunulmuştur. </li></ol> 4. Kaydı Konsol Sapma Veri <ol><li> Konsol analiz donanımı ve ilgili yazılımlar açın. </li><li> Ortama ısıtılmış sahne termistoru yerleştirin ve 37 ° C okumak için sabırsızlanıyorum. </li><li> Cantileve ile aşamaya konsol çipi takınSahnenin sağ tarafında yönelmiştir rs. </li><li> Mikroskop ışık kaynağı açın. </li><li> Konsol varsayarsak sahnenin sağında, konsol 1 yöneliktir: görünüm içine konsolun kenarlarını getirmek ve konsol 1. NOT ucunda lazer ışını konumlandırmak için lazer / fotoğraf dedektör kontrol yazılımı kullanmak için Odak mikroskop Dizinin sol üst konumlandırılmış biridir ve sayılar sol alt aşağı 16 çalıştırın. Konsol 17 sağ üst pozisyonda ve sağ alt (Şekil 5A) içinde 32 hareket eder. </li><li> Basın kayıt yazılımı üzerinde &quot;oynamak&quot;. </li><li> Sinyal foto-dedektörü kontrol kademeli motora ayarlayarak, x ve y çerçeveleri hem de &quot;0&quot; okur, böylece foto-dedektörü yerleştirin. </li><li> Lazer / fotoğraf-detektör kontrol yazılımı Set konsol 1 pozisyon (Şekil 4). </li><li> . Adımı 4.7 tekrarlayın, konsolun 16 ucuna lazer taşıyın ve cantilev seter lazer / fotoğraf-detektör kontrol yazılımı 16 pozisyon. </li><li> Lazer / fotoğraf-detektör kontrol yazılımı adımı 4.7. Ve set konsol 32 pozisyonunu tekrar, konsolun 32 ucuna lazer taşıyın. </li><li> Lazer / fotoğraf-detektör kontrol yazılımı adımı 4.7. Ve set konsol 17 pozisyonunu tekrar, konsolun 17 ucuna lazer taşıyın. </li><li> Mikroskop ışık kaynağı ve laboratuarda havai ışığı kapatın. </li><li> Kayıt yazılımı basın &quot;rekor&quot;. </li><li> 1 Hz frekansta 40 msn&#39;de, 5 V bakliyat için nabız jeneratörü donanımı ayarlayın ve makineyi açın. NOT: İsteğe bağlı olarak, bu noktada belirli bir hücre kaynakları için optimize edilmiş stimülasyon protokolleri istihdam, belirtilen ayarları insan ve kemirgen hücre kaynaklarının 12,13,15,16 kullanılarak toplanan verilere dayalı kurallar vardır. </li><li> Lazer / fotoğraf dedektör kontrol yazılımı kullanarak, her on 5 saniye durdurma, 32 konsol dizi boyunca tarama donanımı ayarlayınör. </li><li> 32 konsolun Tarama işlemi tamamlandığında, daha sonra kayıt yazılımı ve veri dosyasını getirmek, uyarıcı kapatın. </li><li> Kontraktil aktivite kanıt her bir dirsek gelen kaydedilen iz inceleyin. Olumlu tepkiler her konsolun bir kenara not edin. Saptırma taban üzerinde en az 0,1 V ise, bir daralma, bir tepe noktası olarak tanımlanır. </li><li> Lazer / fotoğraf dedektör kontrol yazılımı üzerinde tarama protokolü herhangi bir sigara duyarlı konsol çıkarın. </li><li> Aktif konsollar daha sonra hücrenin spontan kontraktil aktivitenin bir okuma almak için uyarı olmaksızın rescanned edilebilir. </li><li> Kültürlenen hücrelerin fonksiyonel çıktıya üzerindeki etkisini gözlemlemek için ortama terapötik bir bileşik ilave edildikten sonra, ya da geniş alan elektriksel uyarımı olmaksızın taramaları çalıştırın. </li><li> Elektriksel Uzun süre ve Contr tarama seviyeleri için hücreleri uyararak yorgunluk değerlendirmeler yürütmek<html> actility belirli bir eşiğin altına düşmesi pik gücü için ne kadar sürdüğünü ölçmek için. </li><li> Motor nöronlar, kas ile ko-kültür içinde muhafaza edilir deneylerde, nöromüsküler nöronal bir uyarıcı motonöron-miyotüp konsol ko-kültürler işlemi ile bağlantı oluşumu (glutamat gibi) veya sinaptik inhibitör ölçün (örneğin, D-tubokurarin) ve tarama spontan aktivitede artış ve azalışlar sırasıyla 16. </li><li> Planlanan deneyler ihtiyaçlarına göre belirlenecektir gibi özel taramalar yapar. </li></ol> Konsol Sapma Verilerin 5. Analiz <ol><li> Kullanım Stoney denklemleri 15 hücre tabakası veya miyotüp (Pascal), ve (nano-Newton) hücresel kasılma gücünün doğrudan ölçümü stres bir okuma içine (Volt) ham konsol sapma verilerini dönüştürmek için (aşağıda ayrıntılı) güncellendi: reklam / 51866 / 51866eq1.jpg &quot;/&gt; Denklem 1 <img alt="Denklem 2" fo:content-width="2in" width="300" src="/files/ftp_upload/51866/51866eq2.jpg" /> Denklem 2 </li><li> Δ = konsol ucu sapma ve stres miyotüp tarafından üretilen yerlerde, σ c üniforma kalın filmi konsol, C dedektörün tam genişliğini varsayarak miyotüp tarafından üretilen = stres, = fotoğrafın üstüne lazer konumuna gerilimi ilişkin sistemine özgü katsayısı -detector, θ = konsolun düzlemine lazer ve detektör açısının E Si silikon elastik modüle = t Si konsolun kalınlığı = t = f miyotüp kalınlığı v = Si oranı arasında Zehir silikon, L = konsol uzunluğu, dedektör konsol ucundan lazer P = yolu uzunluğu, w <sub&gt; Si konsolun genişliği =. Bu denklemlerde kullanılan terimleri açıklayan şematik Şekil 6&#39;da verilmiştir. </li><li> Düzgün bir film miyotüp olduğunu varsayarak, miyotüp yapan kuvvet Stoney uygulama için kullanılan stresten kuvvet hesaplama ve varsayılan hücre bir kesit alana eşitleyerek, 3. Denklem yol filmde kuvvete eşittir denklem. <img alt="Denklem 3" fo:content-width="1.5in" src="files/ftp_upload/51866/51866eq3.jpg" /> Denklem 3 </li><li> Fonksiyonel Veri toplama, imünositokimyasal analizler için konsol fiş düzeltmek veya standart teknikler kullanılarak protein veya DNA analizi için hücreler kullanır. </li><li> İsteğe bağlı olarak, daha sonraki bir zaman noktasında, işlevsel performansı yeniden değerlendirme amacıyla kuluçka yerine moleküler analiz için hazırlanırken hücreleri döner. </li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+muscle+fiber+contractile+properties+in+adults+with+muscular+dystrophy+treated+with+MYO-029.">Single muscle fiber contractile properties in adults with muscular dystrophy treated with MYO-029.</a> Muscle Nerve. 39, 3-9 (2009).</li><li>Sung, J. 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=Microfabricated+mammalian+organ+systems+and+their+integration+into+models+of+whole+animals+and+humans.">Microfabricated mammalian organ systems and their integration into models of whole animals and humans.</a> Lab on a chip. 13, 1201-1212 (2013).</li><li>Wilson, K., Molnar, P., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+functional+myotubes+with+a+Bio-MEMS+device+for+non-invasive+interrogation.">Integration of functional myotubes with a Bio-MEMS device for non-invasive interrogation.</a> Lab on a chip. 7, 920-922 (2007).</li><li>Wilson, K., Das, M., Wahl, K. J., Colton, R. J., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+contractile+stress+generated+by+cultured+rat+muscle+on+silicon+cantilevers+for+toxin+detection+and+muscle+performance+enhancement.">Measurement of contractile stress generated by cultured rat muscle on silicon cantilevers for toxin detection and muscle performance enhancement.</a> PLoS ONE. 5, (2010).</li><li>Binnig, G., Quate, C. F., Gerber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atomic+Force+Microscope.">Atomic Force Microscope.</a> Physical Review Letters. 56, 930-933 (1986).</li><li>Pirozzi, K. L., Long, C. J., McAleer, C. W., Smith, A. S., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+of+embryonic+skeletal+muscle+myotube+physical+characteristics+with+contractile+force+generation+on+an+atomic+force+microscope-based+bio-microelectromechanical+systems+device.">Correlation of embryonic skeletal muscle myotube physical characteristics with contractile force generation on an atomic force microscope-based bio-microelectromechanical systems device.</a> Applied physics letters. 103, 83108 (2013).</li><li>Smith, A., Long, C., Pirozzi, K., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+system+for+high-content+screening+of+neuromuscular+junctions+in+vitro.">A functional system for high-content screening of neuromuscular junctions in vitro.</a> Technology. 1, 37-48 (2013).</li><li>Stenger, D. A., 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=Coplanar+molecular+assemblies+of+amino-+and+perfluorinated+alkylsilanes:+characterization+and+geometric+definition+of+mammalian+cell+adhesion+and+growth.">Coplanar molecular assemblies of amino- and perfluorinated alkylsilanes: characterization and geometric definition of mammalian cell adhesion and growth.</a> Journal of the American Chemical Society. 114, 8435-8442 (1992).</li><li>Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+defined+long-term+in+vitro+tissue+engineered+model+of+neuromuscular+junctions.">A defined long-term in vitro tissue engineered model of neuromuscular junctions.</a> Biomaterials. 31, 4880-4888 (2010).</li><li>Rumsey, J. W., Das, M., Bhalkikar, A., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+the+mechanosensory+circuit+of+the+stretch+reflex+arc:+Sensory+neuron+innervation+of+intrafusal+muscle+fibers.">Tissue engineering the mechanosensory circuit of the stretch reflex arc: Sensory neuron innervation of intrafusal muscle fibers.</a> Biomaterials. 31, 8218-8227 (2010).</li><li>Das, M., Rumsey, J. W., Bhargava, N., Stancescu, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+tissue+engineering:+A+maturation+model+promoting+long-term+survival+of+myotubes,+structural+development+of+the+excitation-contraction+coupling+apparatus+and+neonatal+myosin+heavy+chain+expression.">Skeletal muscle tissue engineering: A maturation model promoting long-term survival of myotubes, structural development of the excitation-contraction coupling apparatus and neonatal myosin heavy chain expression.</a> Biomaterials. 30, 5392-5402 (2009).</li><li>Das, M., 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=Developing+a+novel+serum-free+cell+culture+model+of+skeletal+muscle+differentiation+by+systematically+studying+the+role+of+different+growth+factors+in+myotube+formation.">Developing a novel serum-free cell culture model of skeletal muscle differentiation by systematically studying the role of different growth factors in myotube formation.</a> In Vitro Cell Dev Biol Anim. 45, 378-387 (2009).</li><li>Stenger, D. A., Pike, C. J., Hickman, J. J., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+determinants+of+neuronal+survival+and+growth+on+self-assembled+monolayers+in+culture.">Surface determinants of neuronal survival and growth on self-assembled monolayers in culture.</a> Brain research. 630, 136-147 (1993).</li><li>Hickman, J. 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=Rational+pattern+design+for+in+vitro+cellular+networks+using+surface+photochemistry.">Rational pattern design for in vitro cellular networks using surface photochemistry.</a> Journal of Vacuum Scienc., &amp; Technology A: Vacuum, Surfaces, and Films. 12, 607-616 (1994).</li><li>Das, M., Molnar, P., Devaraj, H., Poeta, M., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+and+morphological+characterization+of+rat+embryonic+motor+neurons+in+a+defined+system.">Electrophysiological and morphological characterization of rat embryonic motor neurons in a defined system.</a> Biotechnology progress. 19, 1756-1761 (2003).</li><li>Rumsey, J. W., 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=Node+of+Ranvier+formation+on+motor+neurons+in+vitro.">Node of Ranvier formation on motor neurons in vitro.</a> Biomaterials. 30, 3567-3572 (2009).</li><li>Murugan, R., Molnar, P., Rao, K. P., Hickman, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterial+Surface+patterning+of+self+assembled+monolayers+for+controlling+neuronal+cell+behavior.">Biomaterial Surface patterning of self assembled monolayers for controlling neuronal cell behavior.</a> International journal of biomedical engineering and technology. 2, 104-134 (2009).</li><li>Guo, X., Johe, K., Molnar, P., Davis, H., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+human+fetal+spinal+cord+stem+cell+line,+NSI-566RSC,+and+its+induction+to+functional+motoneurons.">Characterization of a human fetal spinal cord stem cell line, NSI-566RSC, and its induction to functional motoneurons.</a> Journal of Tissue Engineering and Regenerative Medicine. 4, 181-193 (2010).</li><li>Varghese, K., 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+new+target+for+amyloid+beta+toxicity+validated+by+standard+and+high-throughput+electrophysiology.">A new target for amyloid beta toxicity validated by standard and high-throughput electrophysiology.</a> PLoS ONE. 5, e8643 (2010).</li><li>Natarajan, A., 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=Patterned+cardiomyocytes+on+microelectrode+arrays+as+a+functional,+high+information+content+drug+screening+platform.">Patterned cardiomyocytes on microelectrode arrays as a functional, high information content drug screening platform.</a> Biomaterials. 32, 4267-4274 (2011).</li><li>Davis, 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=Rat+Cortical+Oligodendrocyte-Embryonic+Motoneuron+Co-Culture:+An+Axon-Oligodendrocyte+Interaction+Model.">Rat Cortical Oligodendrocyte-Embryonic Motoneuron Co-Culture: An Axon-Oligodendrocyte Interaction Model.</a> Journal of biomaterials and tissue. 2, 206-214 (2012).</li><li>Natarajan, A., DeMarse, T., Molnar, P., Hickman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+In+Vitro+Feed-Forward+Networks.">Engineered In Vitro Feed-Forward Networks.</a> J Biotechnol Biomater. 3, 2 (2013).</li></ol>

The authors have nothing to disclose.

Hücresel daralma kanıt için mikro konsol analiz kritik adımlar mikroskop sahne içindeki konsol çip yerleştirme ve dizideki köşe konsolun ucuyla lazer ve foto-dedektör sonraki hizalama vardır. Bu doğru yapılmazsa, o zaman yazılım potansiyel veri toplama sırasında yanlış negatif birikimine neden, dizideki kalan konsolun pozisyonlarını hesaplamak için mümkün olacaktır. Operatörler konsol çip lazer pozisyonları kalibre önce kültür çanak alt ile aynı hizada yalan emin olmak için dikkatli olm...

The in vitro culture of muscle cells from both human and rodent sources has been possible for decades1,2. However, while standard coverslip preparations are useful for biochemical assessment, they do not facilitate analysis of the cell&#8217;s primary functional output (contractility), and therefore are of somewhat limited value as a means to assess cellular maturation and performance. In order to maximize the amount of data obtainable from such in vitro cultures, it is necessary to advance the development of systems capable of housing such cells in configurations that permit the real-time assessment of their functional performance. The establishment of a multitude of three dimensional muscle models has made some progress toward fulfilling this need, and such systems have been used in a number of publications as a means to assess the contractile capacity of cultured muscle cells in vitro3-5. While such systems are invaluable for tissue modeling and reconstruction studies, they are limited in their applicability for studies of single cell responses. In such cases where single fiber studies are necessary, complex and labor intensive ex vivo methodologies remain the only option6-10. Furthermore, current movement toward the development of complex, multi-organ platforms for drug development and screening protocols requires the establishment of systems which are non-invasive, easily scalable and which integrate readily with supporting cells and tissue models11. 
Microscale cantilevers offer a simple method for assessing the functional contractile capacity of single cells/small populations of cells12,13. The technique is based on modified Atomic Force Microscopy (AFM) technology14, and uses a laser and photo-detector system to measure microscale cantilever deflection in response to cultured myotube contractile activity. Modified Stoney&#8217;s equations are then used to calculate stress in the myotube, and the force exerted by the myotube in order to generate the observed substrate deflection15. A scanning program has been written which enables simultaneous assessment of multiplexed cantilever arrays, offering potential moderate to high through-put applications for drug toxicity/efficacy studies15,16. Such technology may prove invaluable in the development of functional, pre-clinical assays for predicting drug efficacy in vivo. Furthermore, fabrication of cantilever chips in silicon does not impede post analysis processing of cells for standard biomolecular assays such as immunostaining, western blotting and PCR. 
This manuscript provides detailed instructions on the fabrication and preparation of microscale silicon cantilevers, the hardware and software set-up, and the operating guidelines for assessing the functionality of contractile cells cultured on these chips. Standard cell culture techniques can be implemented for plating and maintenance of cells on these surfaces, hence any contractile cell type for which reliable culture parameters exist should be able to integrate with this device with ease. The relatively simple 2D culture parameters utilized in this system makes integration of other cell models or addition of cell types that can interact with muscle (such as innervating neurons) straight-forward, greatly increasing the applicability of this model in the development of more complex functional in vitro assays and multi-organ models of mammalian systems.

<table border="0" cellpadding="0" cellspacing="0" width="690">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:299px;">Name of material/ equipment</td>
			<td style="width:128px;">Company</td>
			<td style="width:108px;">Catalog number</td>
			<td style="width:156px;">Comments/ Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle growth medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurobasal medium</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">21103-049&#160;</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">B27 (50x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17504044</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glutamax (100x)</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">35050061</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">G5 supplement</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">17503-012&#160;</td>
			<td dir="LTR" style="width:156px;">1x</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Glial-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRG400B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Brain-Derived Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRB600B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Ciliary Neurotrophic Factor</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC400A</td>
			<td dir="LTR" style="width:156px;">40 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-3</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN500B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Neurotrophin-4</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRN501B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Acidic Fibroblast Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">13241-013&#160;</td>
			<td dir="LTR" style="width:156px;">25 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vascular Endothelial Growth Factor</td>
			<td dir="LTR" style="width:128px;">Life Technologies</td>
			<td dir="LTR" style="width:108px;">P2654</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Cardiotrophin-1</td>
			<td dir="LTR" style="width:128px;">Cell sciences</td>
			<td dir="LTR" style="width:108px;">CRC700B</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Heparin Sulphate</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">D9809&#160;</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Leukemia Inhibitory Factor</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">L5158&#160;</td>
			<td dir="LTR" style="width:156px;">20 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">Vitronectin</td>
			<td dir="LTR" style="width:128px;">Sigma</td>
			<td dir="LTR" style="width:108px;">V0132</td>
			<td dir="LTR" style="width:156px;">100 ng/ ml</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Primary rat muscle differentiation medium</td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;">NB Activ 4</td>
			<td dir="LTR" style="width:128px;">Brain Bits LLC</td>
			<td dir="LTR" style="width:108px;">NB4-500</td>
			<td dir="LTR" style="width:156px;">N/A</td>
		</tr>
		<tr height="17">
			<td dir="LTR" height="17" style="height:17px;width:299px;"></td>
			<td dir="LTR" style="width:128px;"></td>
			<td dir="LTR" style="width:108px;"></td>
			<td dir="LTR" style="width:156px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 red diode laser</td>
			<td>Newport</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Photo-detector</td>
			<td>Noah Industries</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Model 2100 Pulse stimulator</td>
			<td>A-M systems</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Multiclamp 700B Digitizer</td>
			<td>Axon Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Patch clamp microscope and stage</td>
			<td>Olympus</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Delta T4 culture dish controller</td>
			<td>Bioptechs</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Axoscope software</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">LabVIEW software</td>
			<td>National Instruments</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">37oC, 5% CO2 incubator</td>
			<td>NAPCO</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class 2 microbiological flow hood</td>
			<td>Labconco</td>
			<td>N/A</td>
			<td></td>
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			<td height="17" style="height:17px;">Pipettes and tips</td>
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utilization of microscale silicon cantilevers to assess cellular contractile function in vitro

Daha fazla ön ve biyolojik olarak uygun in vitro tahlillerin gelişimi tohumlanmış hücrelerin fonksiyonel değerlendirilmesini sağlamak çok yönlü hücre kültür sistemlerinin ilerlemesi dayanmaktadır. Bu amaçla, mikro konsol bükme teknolojisi daralma kaynaklı alt tabakanın değerlendirilmesi yoluyla, iskelet, kalp ve düz kas hücreleri dahil hücre tipleri, bir dizi kasılma işlevselliğini ölçmek için hangi ile bir platform sunuyor. Birden fazla mesaj göndermiş konsol dizilerin uygulanması ilaç yeterlilik ve toksisitesini, hastalığın ilerlemesini fenotip ve yanı sıra, kas ve diğer hücre-hücre etkileşimleri değerlendirmek için yüksek verimli protokollere orta geliştirmek için bir araç sağlar. Bu yazıda, bu amaç için güvenilir bir konsol dizileri imal edilmesi için ayrıntıları ve bu yüzeyler üzerinde başarıyla kültürü hücreleri için gerekli olan bir yöntem sağlar. Ayrıca açıklama fonksiyonel anal gerçekleştirmek için gerekli adımlar sağlanırkontraktil hücre tipleri Ysis yeni lazer ve foto-dedektör sistemi kullanarak bu tür diziler üzerinde muhafaza. Vurgular hassasiyet ve bu sistem ile mümkün kontraktil fonksiyonun tekrar analiz tekrarlanabilir niteliği verilen temsili veriler, hem de çalışma geniş olan bu tür önlemlerin hale gelebilir. Bu sistemin başarılı yaygınlaşmasının araçlarla araştırmacılar doku performansı, hastalık gelişimi ve yeni terapötik tedaviye yanıtın daha doğru tahminlere yol açan, in vitro hızlı, düşük maliyetli fonksiyonel çalışmalar yapmak sağlayabilir.

Dirseklerin kontraktil hücrelerinin başarılı kültürü, standart hücre kültürü teknikleri (Şekil 5) kullanılarak, nispeten basit bir işlemdir. Yakalanma hücreleri destekleyici dirseklerin yüzdesi, hücre tipine bağlı olarak değişir incelenmiş ve belirli bir kültür tekniği kullanılmış olmasıdır. Sıçan arka bacak türetilen birincil embriyonik hücreleri kullanarak, kontraktil aktivite incelendi konsolun tespit% 12 (n = 4). Açıklanan lazer ve foto-dedektör sistemi kullanıl...

Watch this Scientific Journal Video about Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro at JoVE.com

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

Bu protokol, in vitro kas hücrelerinin kasılma ölçmek için esnek kültür yüzeyleri olarak mikro silikon konsolun kullanımını tarif etmektedir. Hücresel kasılması, ölçülen kaydedilen ve in vitro kontraktil fonksiyon ölçmek için non-invazif ve ölçeklenebilir sisteminin temin edilmesiyle, kuvvet çıktılan dönüştürülebilir dirsekli bükme neden olur.

Mikro Silikon konsolun Kullanımı Hücresel kasılma fonksiyonunu değerlendirmek için<em&gt; İn Vitro</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

utilization-microscale-silicon-cantilevers-to-assess-cellular

Research

JoVE Journal

Bioengineering

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

10.1K Görüntüleme.  University of Central Florida.  Bu protokol, in vitro kas hücrelerinin kasılma ölçmek için esnek kültür yüzeyleri olarak mikro silikon konsolun kullanımını tarif etmektedir. Hücresel kasılması, ölçülen kaydedilen ve in vitro kontraktil fonksiyon ölçmek için non-invazif ve ölçeklenebilir sisteminin temin edilmesiyle, kuvvet çıktılan dönüştürülebilir dirsekli bükme neden olur. 

Video: Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

Utilization of Plasmonic and Photonic Crystal Nanostructures for Enhanced Micro- and Nanoparticle Manipulation

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological research. Perhaps the most widely used physical force to achieve noninvasive manipulation of small particles has been dielectrophoresis(DEP).1 However, DEP on its own lacks the versatility and precision that are desired when manipulating cells since it is traditionally done with stationary electrodes. Optical tweezers, which utilize a three dimensional electromagnetic field gradient to exert forces on small particles, achieve this desired versatility and precision.2 However, a major drawback of this approach is the high radiation intensity required to achieve the necessary force to trap a particle which can damage biological samples.3 A solution that allows trapping and sorting with lower optical intensities are optoelectronic tweezers (OET) but OET's have limitations with fine manipulation of small particles; being DEP-based technology also puts constraint on the property of the solution.4,5
This video article will describe two methods that decrease the intensity of the radiation needed for optical manipulation of living cells and also describe a method for orientation control. The first method is plasmonic tweezers which use a random gold nanoparticle (AuNP) array as a substrate for the sample as shown in Figure 1. The AuNP array converts the incident photons into localized surface plasmons (LSP) which consist of resonant dipole moments that radiate and generate a patterned radiation field with a large gradient in the cell solution. Initial work on surface plasmon enhanced trapping by Righini et al and our own modeling have shown the fields generated by the plasmonic substrate reduce the initial intensity required by enhancing the gradient field that traps the particle.6,7,8 The plasmonic approach allows for fine orientation control of ellipsoidal particles and cells with low optical intensities because of more efficient optical energy conversion into mechanical energy and a dipole-dependent radiation field. These fields are shown in figure 2 and the low trapping intensities are detailed in figures 4 and 5. The main problems with plasmonic tweezers are that the LSP's generate a considerable amount of heat and the trapping is only two dimensional. This heat generates convective flows and thermophoresis which can be powerful enough to expel submicron particles from the trap.9,10 The second approach that we will describe is utilizing periodic dielectric nanostructures to scatter incident light very efficiently into diffraction modes, as shown in figure 6.11 Ideally, one would make this structure out of a dielectric material to avoid the same heating problems experienced with the plasmonic tweezers but in our approach an aluminum-coated diffraction grating is used as a one-dimensional periodic dielectric nanostructure. Although it is not a semiconductor, it did not experience significant heating and effectively trapped small particles with low trapping intensities, as shown in figure 7. Alignment of particles with the grating substrate conceptually validates the proposition that a 2-D photonic crystal could allow precise rotation of non-spherical micron sized particles.10 The efficiencies of these optical traps are increased due to the enhanced fields produced by the nanostructures described in this paper.

Plasmonic tweezers and photonic crystal nanostructures are shown to produce useful enhancements in the efficiency and orientation control of optically trapping micro- and nano-particles.

Gelişmiş mikro ve Nanopartikül Manipülasyon Plasmonic ve Fotonik Kristal Nanoyapıların Kullanımı

A method to manipulate the position and orientation of submicron particles nondestructively would be an incredibly useful tool for basic biological ...

Biyomühendislik

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Yarı yapay Moleküler Makine ve Kullanımı DNA Hasar Tespiti için in vitro Meclisi

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Parametrized Diyastolik Dolum Biçimcilik yoluyla Transmitral Akışının Modellenmesi Kinematik tabanlı Analizi ile Küresel Diyastolik Fonksiyonu Kantitasyonu

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

Mikro Schlieren Tekniği ile bir mikroakışkan Aygıt karıştırma homojen Analizi

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

Yeniden İnşa İnsan Kornea gibi Epitelyal (RHCE) Doku Modeli kullanılarak Göz tahriş Kimyasalların Tehlike Tanımlaması Göz tahrişi Testi (EIT)

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

Techniques for the Evolution of Robust Pentose-fermenting Yeast for Bioconversion of Lignocellulose to Ethanol

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme saccharification processes release sugars that can be fermented by yeast. Traditional industrial yeasts do not ferment xylose (comprising up to 40% of plant sugars) and are not able to function in concentrated hydrolyzates. Concentrated hydrolyzates are needed to support economical ethanol recovery, but they are laden with toxic byproducts generated during pretreatment. While detoxification methods can render hydrolyzates fermentable, they are costly and generate waste disposal liabilities. Here, adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of the native Scheffersomyces stipitis strain NRRL Y-7124 that are able to efficiently convert hydrolyzates to economically recoverable ethanol despite adverse culture conditions. Improved individuals are enriched in an evolving population using multiple selection pressures reliant on natural genetic diversity of the S. stipitis population and mutations induced by exposures to two diverse hydrolyzates, ethanol or UV radiation. Final evolution cultures are dilution plated to harvest predominant isolates, while intermediate populations, frozen in glycerol at various stages of evolution, are enriched on selective media using appropriate stress gradients to recover most promising isolates through dilution plating. Isolates are screened on various hydrolyzate types and ranked using a novel procedure involving dimensionless relative performance index (RPI) transformations of the xylose uptake rate and ethanol yield data. Using the RPI statistical parameter, an overall relative performance average is calculated to rank isolates based on multiple factors, including culture conditions (varying in nutrients and inhibitors) and kinetic characteristics. Through application of these techniques, derivatives of the parent strain had the following improved features in enzyme saccharified hydrolyzates at pH 5-6: reduced initial lag phase preceding growth, reduced diauxic lag during glucose-xylose transition, significantly enhanced fermentation rates, improved ethanol tolerance and accumulation to 40 g/L.

Adaptive evolution and isolation techniques are described and demonstrated to yield derivatives of Scheffersomyces stipitis strain NRRL Y-7124 that are able to rapidly consume hexose and pentose mixed sugars in enzyme saccharified undetoxified hydrolyzates and to accumulate over 40 g/L ethanol.

Etanol ile lignoselüloz biyo dönüşümünü için Sağlam Pentoz-fermente Maya Evrimi Teknikleri

Lignocellulosic biomass is an abundant, renewable feedstock useful for production of fuel-grade ethanol and other bio-products. Pretreatment and enzyme ...

Measuring and Modeling Contractile Drying in Human Stratum Corneum

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both cleansing agents and daily variations in ambient moisture; both of which can alter the water content of the tissue. Reductions in water content from severe barrier dysfunction or low humidity environments can alter SC stiffness and cause a build-up of drying stresses. In extreme conditions, these factors can cause mechanical rupture of the tissue. We have established a high throughput method of quantifying dynamic changes in the mechanical properties of SC upon drying. This technique can be employed to quantify changes in the drying behavior and mechanical properties of SC with cosmetic cleanser and moisturizer treatments. This is achieved by measuring dynamic variations in spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. In-plane radial displacements acquired during drying are azimuthally averaged and fitted with a profile based on a linear elastic contractility model. Dynamic changes in drying stress and SC elastic modulus can then be extracted from the fitted model profiles.

This article describes a method of quantifying the dynamic drying behavior and mechanical properties of stratum corneum by measuring spatially resolved in-plane drying displacements of circular tissue samples adhered to an elastomer substrate. This technique can be used to measure how different chemical treatments alter drying and tissue mechanical properties.

Ölçme ve Modelleme Kontraktil Kurutma İnsan stratum korneumdaki

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

bir<em&gt; İn Vitro</emKemirgen İntervertebral Disk&gt; Organ Kültürü Modeli

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

Uzun Süreli Nanomalzeme Maruziyetini Takiben İn vitro Genotoksisite Testi için Gelişmiş 3D Karaciğer Modelleri

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An In vitro System to Gauge the Thrombolytic Efficacy of Histotripsy and a Lytic Drug

Deep vein thrombosis (DVT) is a global health concern. The primary approach to achieve vessel recanalization for critical obstructions is catheter-directed thrombolytics (CDT). To mitigate caustic side effects and the long treatment time associated with CDT, adjuvant and alternative approaches are under development. One such approach is histotripsy, a focused ultrasound therapy to ablate tissue via bubble cloud nucleation. Pre-clinical studies have demonstrated strong synergy between histotripsy and thrombolytics for clot degradation. This report outlines a benchtop method to assess the efficacy of histotripsy-aided thrombolytic therapy, or lysotripsy.
Clots manufactured from fresh human venous blood were introduced into a flow channel whose dimensions and acousto-mechanical properties mimic an iliofemoral vein. The channel was perfused with plasma and the lytic&#160;recombinant tissue-type plasminogen activator. Bubble clouds were generated in the clot with a focused ultrasound source designed for the treatment of femoral venous clots. Motorized positioners were used to translate the source focus along the clot length. At each insonation location, acoustic emissions from the bubble cloud were passively recorded, and beamformed to generate passive cavitation images. Metrics to gauge treatment efficacy included clot mass loss (overall treatment efficacy), and the concentrations of D-dimer (fibrinolysis) and hemoglobin (hemolysis) in the perfusate. There are limitations to this in vitro design, including lack of means to assess in vivo side effects or dynamic changes in flow rate as the clot lyses. Overall, the setup provides an effective method to assess the efficacy of histotripsy-based strategies to treat DVT.

Histotripsy-aided lytic delivery or lysotripsy is under development for the treatment of deep vein thrombosis. An in vitro procedure is presented here to assess the efficacy of this combination therapy. Key protocols for the clot model, image guidance, and assessment of treatment efficacy are discussed.