The authors have no acknowledgements.

Bu protokol, insan numuneler üzerinde araştırmalar için kurumsal etik kuralları takip ve bilgilendirilmiş onam ilgili tüm vericilerden elde edildi. Burada tarif edilen deneyler için onay Antwerp Üniversitesi Hastanesi kurumsal inceleme kurulu elde edilmiştir. Not: belirtilmediği sürece Sıcaklık göstergeleri, her zaman oda sıcaklığı vardır. 1. Hazırlık Akış Odası Kurulumu <ol><li> Hazırlama Lanes, Boru ve iğneler <ol><li> Vorteks kuvvetli bir şekilde kollajen süspansiyonu ve 50 ug / ml&#39;lik bir nihai konsantrasyona kadar sağlayıcı tarafından sağlanan izotonik glikoz çözeltisinde 1/20 seyreltin. Not: Biz esas olarak tip I fibriller oluşur, at tendon kollajen kullanın. At kollajen tip I sık sık &quot;Horm&quot; kolajen olarak adlandırılır ve tarihsel yanı sıra biyolojik nedenlerle hem tahlil 9 bu tür altın standardı mesafesindedir. İnsan tip III kollajen de kullanılabilir, ancak inci edilebilire daha az kat fibrillerinde ve trombosit cevabı kadar güçlü değildir. Diğer kaplama yüzeyleri de, örneğin von Willebrand Faktörü (vWF), fibrinojen, fibronektin, laminin, vitronektin, trombospondin-1 ya da bu 16 kombinasyonları için, kullanılabilir. </li><li> sağlayıcının kaptan yeni bir atılabilir Bioçip alın. Burada kullanılan bioçipler boyutları mm 3 0.4 W x 0.1 H x 20L vardır. </li><li> Pipetleyin çıkış olarak çip ve işaretinin bir ucunda mikroakışkan Biochip kulvar (lar) 0.8 ul. şerit 1.1.1 hazırlanan kaplama çözeltisi içeren kollajen ile 5 / 6th dolu olduğundan emin olun. hiç hava kabarcığı olmadığından emin olun. Not: Kanallar kısmen (tartışma) kanalı girişinde kollajen liflerinin birikmesini önlemek için kaplanır. </li><li> nemlendirilmiş ve kapalı bir kap içinde 4 saat veya gece boyunca 4 ° C&#39;de inkübe edin. </li><li> / H) inek serumu albumin (w bloke edici tampon (% 1.0 pipetleme kaplanmış kanallarını blokediğer ucu ile gösterirken, d,% 0.1 (a / h) glukoz, 10 mM 4- (2-hidroksietil) -1-piperazinetansülfonik asit (HEPES) tamponlu tuzlu su (hacim / hacim) NaCl, pH 7.4% 0.9 (HBA) &#39;de giriş olarak. şerit tamamen hava kabarcıkları kaçınarak engelleme tamponu ile dolu olduğundan emin olun. </li><li> eşit uzunlukta (12 cm) hortumu kesilir. kulvar başına birini kullanın ve bir iğne ile her tüp bağlayın. Örneğin, iki nüsha halinde çalışan iki koşulları karşılaştıran bir deney, dört boru uzanıyor ve dört pim hazırlanacak gerektirecektir. </li><li> bir şırınga ve bir 26 G iğne ya da eşlik eden bağlayıcıları kullanılarak saf su ile boru durulayın. </li><li> bloklama tamponu ile boru doyurabilecek. 1 saat arasında, en az bir kapalı ve nemli bir kap içinde depolayın. </li></ol></li><li> Pompa ve Manifold hazırlama <ol><li> hava kabarcıklarını çıkarmak, pompayı durulayın ve damıtılmış su ile manifoldu. </li><li> çıkışında sabit 10 ul ucunu kullanarak biyoçip şeritli (ler) engelleme tampon aspire. yüzeyini temizleyinhassas silip toz ve denatüre alkol ile Biochip baskılar ve tozunu alın. </li><li> otomatik mikroskop sahnede Bioçip sabitleyin. Birden fazla şeritli bir seferde aynı anda kullanılırsa, biyoçip çıkışına sekiz şerit manifoldu ayırıcı bağlanır. Not: Sekiz şeritli manifoldu ayırıcı donanım parçasıdır (Şekil S1) pompa ve Biochip bağlı. Bu Bioçip veya operatör tanımlı eden yazılım olabilir şerit kombinasyonuna tüm (sekiz) şerit çalışmasını sağlar. </li><li> biochipin girişinde bunları düzeltmek için tüp işaretçilerine kullanın. HBS ile doldurulmuş 1.5 ml konik biçiminde bir test tüpü içinde (PIN olmadan) hortumun diğer ucu yerleştirin. kalan tampon engelleme ve kötü kolajen yapışmış kaldırmak amacıyla, tüm boru ve 1 ml HBS pompa kullanılarak onların bağlı şerit durulayın. </li></ol></li></ol> Kan Örneklerinin 2. Hazırlık <ol><li> Toplama ve ayırmaSağlıklı bir gönüllüden taze tam kan. 17 <ol><li> Bir pıhtılaşma önleyici olarak etilendiamintetraasetik asit (EDTA) ihtiva eden bir tahliye borusu kan ilk mililitre toplamak ve özel olarak otomatik bir hematoloji analiz cihazı ile tam kan sayımı (CBC), bu örnek kullanın. </li><li> tahliye tüpler kullanılarak uygun bir antikoagülan kan hacmi toplayın. oluşumunu fibrin çalışma protokolüne ve sodyum sitrat parçası değilken akış odası deneyleri için standart antikoagülan heparin ya da hirudindir vardır. Not: Heparin açıklanan tüm deneyler için bir antikoagulan olarak kullanılmıştır. Not: kan miktarı yapılacak deneyler sayısına bağlıdır. Yaklaşık 3 şerit 1 borusu (7 mi). </li><li> Bir rotator bekleyen kan sulandırma üzerinde tüpler yerleştirin. Not: Tahlil Flebotomi 3 saat içinde tamamlanmalıdır. </li><li> Trombosit bakımından zengin plazma 250 g&#39;da 15 dakika boyunca santrifüj (PRP). gevşekçe paketlenmiş pelet olumsuz yönde etkilenmemesi için santrifüj mola kullanmayın. <ol><li> Birden fazla tüp toplandı zaman, tek bir konik santrifüj tüpü içinde kan havuzu. Not: Santrifüj PRP verim ve ayırıcı hücre &quot;kontaminasyon&quot; bağlı, daha yavaş ya da daha az uzun yapılabilir tercih. </li></ol></li><li> Çıkarın ve birkaç trombositler ile dolu kırmızı kan hücrelerinin veren PRP ve buffy coat atın. Bizim elimizde ortalama ul başına 13 ± 5 x 10 3 paketlenmiş kırmızı hücre fraksiyonunda trombosit sayısı ise (ortalama ± SD, n = 12): Not. </li></ol></li><li> kan Sulandırma <ol><li> 5 dakika ve 4 ml başına 20 saniye süreyle, 37 ° C&#39;de çözülme kan grubu AB (Rhesus D negatif), plazma. </li><li> otomatik bir hematoloji analiz cihazı kullanılarak 2.1.5 hazırlanan paketlenmiş kırmızı kan hücrelerinin hematokriti belirler. </li><li> Kan bankasında trombosit konsantrasyonunu belirlemek trombosit konsantresi tha hazırlanmışT, yukarıda kırmızı hücre fraksiyonu tekrar oluşturmak için kullanılır. </li><li> 1 ml&#39;lik bir örnek% 40 hematokrit ve 250 x 10³ trombosit / ul verecektir eritrosit, trombosit konsantresi hacmini hesaplar. Not: Hücrelerin Diğer hedef titreleri çalışma protokolüne bağlı olarak, keyfi olarak ayarlanabilir. </li><li> Transfer kırpılmış bir pipet ucu kullanılarak ve 1 ml lik bir numune hacmi elde edilene kadar trombosit konsantresi ilave taze tüpe kırmızı kan hücreleri ve plazma paketlendi. Not: Plazma trombüs oluşumu oranı üzerinde önemli bir etkiye sahip olduğu değişkenler okudu bağlı olarak, plazma fraksiyonu tüm sulandırılmış örneklerde eşit olmalıdır. Örneğin, sonuç etkileyebilir farklı donörden veya farklı antikoagülan alınan donma-çözülme ya da plazma tekrarladı. </li><li> yavaşça ters çevirerek yeniden kan karıştırın ve bir CBC gerçekleştirin. </li><li> trombosit fraksiyonun hacmi ikame edildiği bir &quot;boş&quot; kontrol numunesi hazırlanması% 0.9 su (kütle / hacim) sodyum klorür, aynı hacimde bir CBC kullanılarak yeniden kan endojen trombosit konsantrasyonu (yani, kan bankası trombositler) belirlemek gerekmektedir. </li></ol></li><li> etiketleme <ol><li> Pipet 1 mi 1 ul 5 mM Calcein AM (5 uM nihai konsantrasyon) ihtiva eden bir test tüpüne kan yeniden. Not: Diğer hücre boyalar 14 kullanılabilir. </li><li> ters çevirerek yavaşça karıştırın. </li><li> kullanımdan önce, 37 ° C&#39;de 5 dakika süreyle inkübe edilir. </li></ol></li></ol> 3. Perfüzyon Testi <ol><li> şerit dibinde yapıştırılır kollajen lifleri üzerinde nesnel odaklanın. İdeal olarak, bu odaklanma stratejisi için faz-kontrast veya diferansiyel girişim kontrast (DIC) ayarlarını kullanın. dijital seçilen Z-pozisyonları düzeltmek için deneme yazılımı &#39;seçilen çini bölgeler için ayarlayın akım Z&#39; seçeneğini seçin. </li><li> m deney yazılımı şeritli (xy) ilgi (ROI) bir bölge seçinDeney sırasında kaydedilir icroscope. Not: ROI keyfi bir perfüzyon şeritte içinde seçilen herhangi bir yüzey alanı olabilir. Nedenle bu göreceli olarak küçük olsa da, bu bölgede değişken akış profilinin yan etkilerden kaçınmak için yakın bir şeritli giriş ve çıkışına trombüs oluşumunu analiz için tavsiye edilir. YG yüzey alanı trombositlerin önemli sayıda içeren ya da sinyal tesviye izin trombüs gerekir. Bu protokolde YG 2 cm uzunluğundaki Lane ortasında 0.62 mm 2&#39;de elde edilen üç eşit yan tarafında bir görüntü dijital olarak dikilmiş toplamıdır. </li><li> yavaşça tersini örnekleri karıştırın ve otomatik sahnede Biochip bu sonraki yerleştirin. </li><li> sulandırılmış kan örnekleri içeren test tüplerinde Biochip girişi ile bağlantılı olan boru yerleştirin. </li><li> Test tüplerine bağlı olan kanallar için (istediğiniz gibi ya da diğer kesme stresleri) 50 din / cm2 pompayı başlatınpompa yazılımı kullanılarak yeniden kan örnekleri ihtiva etmektedir. Not: Diğer kayma gerilmeleri kullanılabilir. </li><li> Tutanak görüntüleri mikroskop edinimi ve deney yazılımı kullanarak gerçek zamanlı olarak 5 dakika boyunca her 15 sn. Not: Diğer zaman serisi deneysel set-up bağlı kullanılabilir. Not: Genellikle 100X büyütme (10X objektif ve 10X lens) kullanır, ancak daha yüksek (veya daha düşük) büyütme kolayca bir alternatif olarak kullanılabilir. </li></ol> 4. Yıkama Out <ol><li> sodyum hipoklorit (çamaşır suyu)% 0.5, ardından her tüp çıkışına bağlı ve damıtılmış su kullanılarak çok kanallı manifoldu veya pompaya bağlı yıkayın (h / h) ve su son olarak 0.1 M NaOH. tehlikeli atık olarak biyoçip girişine tutturulmuş boru atın. </li></ol> 5. Veri Analizi <ol><li> görüntü analiz yazılımı ile trombüs büyüme kinetikleri belirleyin. aşağıdaki komutlar ZEN2012 özgüdür. <ol&gt; <li> Trombositlerin yüzey kapsama belirlemek için eklenti Görüntü Analizi açın. </li><li> Olumlu bir sinyal ile ilişkilidir piksel yoğunluğunu belirlemek için İnteraktif sekmesini Analiz, yani bir yapışık trombosit ya da yapışan trombositler floresan eşiğini ayarlayın. </li><li> Otomatik olarak seçilen eşik arasında bir sinyal ile pikselleri ihtiva eden &quot;nesnelerin&quot; nin (mikron 2) ayrı yüzey alanlarını içerecek bir elektronik tablo oluşturmak için Tablolar oluşturma kullanın. Bu, her bir zaman noktası için gerçekleştirilir. Not: floresan eşikleri seçti edildikten sonra, analiz yazılımı otomatik olarak kriterleri yerine görünüm alanına &#39;nesneleri&#39; olarak algılar. Bu nesneler trombüsler, küçük trombosit agrega veya tek trombositler ve piksel sayısını kapsamaktadır. Her nesne ayrı ayrı e-tabloda listelenir. </li><li> xml formatında bu elektronik tabloları kaydedin ve bir elektronik tablo programı bunları açıksonraki hesaplamalar için. </li><li> Toplamı ile seçilen nesnelerin toplam yüzey alanları ve ölçüm alanına (mikron 2) toplam alanın tarafından sonucu bölün. Bu göreceli yüzey kaplama (%) verecektir. Her zaman noktası için bunu. </li><li> Perfüzyon, zamanın bir fonksiyonu olarak, bu yüzey Kapsamlı Konu ve söz konusu durumun trombüs büyümesi kinetiğini sonuçta, doğrusal regresyon ile eğimi hesaplanır. </li></ol></li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+for+quality+control+of+platelets+for+transfusion.">Assays for quality control of platelets for transfusion.</a> ISBT Sci Series. 8, 221-224 (2013).</li><li>Andre, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticoagulants+(thrombin+inhibitors)+and+aspirin+synergize+with+P2Y12+receptor+antagonism+in+thrombosis.">Anticoagulants (thrombin inhibitors) and aspirin synergize with P2Y12 receptor antagonism in thrombosis.</a> Circulation. 108, 2697-2703 (2003).</li><li>Casari, C., 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=von+Willebrand+factor+mutation+promotes+thrombocytopathy+by+inhibiting+integrin+alphaIIbbeta3.">von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin alphaIIbbeta3.</a> J Clin Invest. 123, 5071-5081 (2013).</li><li>Gitz, E., 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=Improved+platelet+survival+after+cold+storage+by+prevention+of+Glycoprotein+Ib&#945;+clustering+in+lipid+rafts.">Improved platelet survival after cold storage by prevention of Glycoprotein Ib&#945; clustering in lipid rafts.</a> Haematologica. , (2012).</li><li>Maurer-Spurej, E., Labrie, A., Brown, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+Quality+Testing+of+Blood+Platelet+Transfusions+with+Dynamic+Light+Scattering.">Routine Quality Testing of Blood Platelet Transfusions with Dynamic Light Scattering.</a> Part Part Syst Char. 25, 99-104 (2008).</li><li>Van Kruchten, R., Cosemans, J. M., Heemskerk, J. W. <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+whole+blood+thrombus+formation+using+parallel-plate+flow+chambers+-+a+practical+guide.">Measurement of whole blood thrombus formation using parallel-plate flow chambers - a practical guide.</a> Platelets. 23, 229-242 (2012).</li><li>Zwaginga, 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=Flow-based+assays+for+global+assessment+of+hemostasis.+Part+2:+current+methods+and+considerations+for+the+future.">Flow-based assays for global assessment of hemostasis. Part 2: current methods and considerations for the future.</a> J Thromb Haemost. 4, 2716-2717 (2006).</li><li>Zwaginga, 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=Flow-based+assays+for+global+assessment+of+hemostasis.+Part+1:+Biorheologic+considerations.">Flow-based assays for global assessment of hemostasis. Part 1: Biorheologic considerations.</a> J Thromb Haemost. 4, 2486-2487 (2006).</li></ol>

The authors have nothing to disclose.

Mikroakışkan akış odası deneyleri kan akan trombosit fonksiyonlarını araştırmak için mükemmel bir araçtır ve deneysel bağlamlarda farklı in vitro hemostaz değerlendirmek için kullanılır. Fakir laboratuvarlar arası standardizasyonun 9 rağmen bizim laboratuvar içinde deneysel varyasyon kabul edilebilir olduğunu göstermektedir. Bu güvenilir, belirli bir çalışma içinde (eşli) örnekleri karşılaştırmak için izin verir. Bu kan bankası koşullarında 11 trombos...

Hemostaz kısıtlı zamanmekansal bağlamda 1 hücreleri, proteinler, iyonlar ve dokuların kombine ve düzenlenmiş aktivite gerektirir. Kontrolsüz etkinliği kan pıhtılaşması ile ilgili bozuklukların bir spektrum içinde kanama ve tromboz ve morbidite ve mortaliteye neden olabilir. Bir mikroakışkan akış odası deneyi in vitro hemostazisi taklit zorlu bir tekniktir. Bu yaklaşım, kan trombositlerin için öncü bir role sahip hemostaz katılan süreçlerin karmaşık etkileşimin soruşturma sağlar. vasküler hasar ardından, trombositler, kan kaybını önlemek için maruz subendotelyal matris (gliko) proteinlere yapışır. Yapışma ardından, trombositler aktive ve yanıt olarak agrega matik ve nihayet bir trombosit ağının oluşumu, fibrin tarafından stabilize ve bir firmaya neden neden parakrin sinyal için, trombüs 2 sızdırmazlık yara. Diğer çoğu trombosit fonksiyon testleri, deney aksineAkış odaları ile ments dikkate kan akımının fiziksel parametre ve bu nedenle katılımcı hücreler ve biyomoleküllerin 3,4 tarihinde reolojinin etkisini alır. Akış odası deneyleri yapışkan matrisi, reoloji ve akış profilleri, hücresel kompozisyon, toksinler veya ilaç, iyonik kuvvet ve daha birçok varlığı da dahil olmak üzere süreçler hemostatik (alt) etkileyen önemli parametreler değiştirilerek hemostaz ve tromboz dönüm noktası anlayışlar yarattı. Son yirmi yılda, büyük örneklem hacimleri (10-100 ml) gerektiren düşük verim akım odası deneyleri mikroakışkan odaları genellikle küçük paralel plaka odadan oluşan ve kontrollü duvar kesme koşullarında 5 de tam kan perfüze için modern teknolojiyi dahil etmek gelişmiştir. donanım kurulum basitleştirilmiş ve daha az (kan) hacmi gereklidir çoğunlukla nedeniyle Microscaling önemli ölçüde deneme daha erişilebilir ve Versati render tahlil verimi arttıle. Örneğin, küçük laboratuar hayvanlarından kan Böylece hayvanlar kurban gerek kalmadan kullanılabilir. Genetiği değiştirilmiş farelerin kan örnekleri böylece hemostaz teşvik veya inhibe anahtar moleküller belirlenmesinde ve yeni temel anlayışlar 6 yardımcı olmaktadır. Özel araştırma laboratuarları çoğu hala yazılım tarafından blueprinted edilebilir Baskılı kalıplara polimerize polidimetilsiloksan (PDMS) 7 den, örneğin özel yapılmış akış odaları kullanın. Ortaya çıkan odası, ucuz tek kullanımlık ve kolayca post hoc analizi için demonte edilebilir. Ayrıca, temelde gemiler de dahil olmak üzere bifurkasyonlarında veya keskin dönüşler herhangi bir tasarım komutu üzerine inşa edilebilir. Bu avantaj da standardizasyon zaten akış odası deneyleri ile birincil sorun bu yana olumsuz ve PDMS özel odalar bu destekli değil yaptı. Bu belirli bir sorunun üstüne kaplama (şartlar), floresan probları, antikoagülan, geçici üzerindenumune alma ve analiz arasındaki kısa süreli olarak ve zaman tüm kötü 8 standardize edilmiştir. Bu değişkenlerin Standardizasyon zorlu ama yine de laboratuarlar arasında sonuçların karşılaştırılmasına olanak gerekmektedir. Bu konu Biorheology 9,10 Bilimsel ve Standardizasyon alt komite Tromboz ve hemostaz üzerine International Society önemli bir konudur. Trombosit konsantreleri (PC) trombositopeni ve / veya kanamaya neden çeşitli hastalıklardan mustarip hastalarda transfüzyon. Ama PC trombositler özellikle depolama süresinin 11 işlevinde, duyarsız bilinen bir bozulma süreci yaşlanma ve yaygın olarak trombosit saklama lezyon olarak anılacaktır bağlantılı. Bazen bu tür plateletler kez 12 transfüzyon dolaşımda geri iddia edilmektedir, ancak bunun için kanıt azdır. Ayrıca, bir PC oluşturan trombosit işlevselliği rutin test değil çünkü bu tür tahliller arasındaki ilişkiterapötik ya da profilaktik etkisi açık 13&#39;tür. Mikroakışkan akış odaları toplama ve ihracı arasındaki manipülasyonlar zincirini optimize etmek için PC trombosit fonksiyonlarını araştırmak için bir araç sunuyoruz. Biz daha önce 14,15 yayınlanmış ve burada tarif edildiği gibi PC doğrudan (eşli) karşılaştırmalar için güçlü bir araştırma aracıdır.

<table border="0" cellpadding="0" cellspacing="0" width="1113">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD vacutainer tube with EDTA&#160;</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368856</td>
			<td style="width:409px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD vacutainer tube with Heparin</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368480</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD vacutainer tube with Sodium Citrate</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">366575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hirudin Blood tube</td>
			<td style="width:220px;">Roche</td>
			<td style="width:268px;">6675751 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD vacutainer Eclipse</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368650</td>
			<td>Blood collection needle with preattached holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 100-1000</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">740290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 2-200</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">739280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 1-10</td>
			<td style="width:220px;">Eppendorf</td>
			<td style="width:268px;">A08928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tube 5mL</td>
			<td style="width:220px;">Simport</td>
			<td style="width:268px;">11691380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tube 15mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">1888271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tube 50mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL Syringe</td>
			<td style="width:220px;">BD</td>
			<td style="width:268px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Precision wipes</td>
			<td style="width:220px;">Kimtech</td>
			<td style="width:268px;">5511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Fluoro+ Biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">188V8CF-400-100-02P10</td>
			<td>Named in figure S1 A as &#39;Biochip&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Tubing</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">TUBING-TYGON-B1IC-B1OC-ROLL 100F</td>
			<td>Named in figure S1 B as &#39;Disposable tubing&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Needles</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">SS-P-B1IC-B1OC-PACK200</td>
			<td>Named in figure S1 B as &#39;Pin&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Connectors for single inlet cables of biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">CONNECTORS-B1IC-PACK100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multiflow8 connect</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">MF8-CONNECT-BIC3-N-THROMBOSIS</td>
			<td>Named in figure S1 B as &#39;Reusable tubing&#39; and &#39;Splitter&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Humidified box</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">HUMID-BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software microfluidic pump</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">N/A</td>
			<td>Venaflux Assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Horm Collagen</td>
			<td style="width:220px;">Takeda/Nycomed</td>
			<td style="width:268px;">1130630</td>
			<td style="width:409px;">Native equine tendon collagen (type I) 
			Isotonic glucose solution to dilute collagen is supplemented</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HEPES buffered saline (HBS)</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td style="width:409px;">10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (0.9% (w/v) NaCl, pH 7.4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Blocking buffer</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td style="width:409px;">1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in HBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcein AM</td>
			<td style="width:220px;">Molecular probes</td>
			<td style="width:268px;">C1430</td>
			<td style="width:409px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach 10%</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.1M NaOH</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Denaturated alcohol</td>
			<td style="width:220px;">Fiers</td>
			<td style="width:268px;">T0011.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mirus Evo Nanopump</td>
			<td>Cellix</td>
			<td>188-MIRUS-PUMP-EVO</td>
			<td>with Multiflow8. Named in figure S1 A as &#39;Pump&#39; and &#39;Manifold&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer Z1</td>
			<td>equipped with a colibri-LED and high resolution CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:268px;">N/A</td>
			<td>ZEN 2012</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematology analyzer</td>
			<td>Sysmex</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge</td>
			<td>Eppendorf</td>
			<td>521-0095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Platelet incubater</td>
			<td style="width:220px;">Helmer</td>
			<td style="width:268px;">PF-48i</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubation water bath</td>
			<td>GFL</td>
			<td>1013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette</td>
			<td>Brand</td>
			<td>A03429</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube Roller</td>
			<td>Ratek</td>
			<td>BTR5-12V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile docking device</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:268px;">TSCD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing Sealer</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:268px;">AC-155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex</td>
			<td style="width:220px;">VWR</td>
			<td style="width:268px;">58816-121</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic flow chambers using reconstituted blood to model hemostasis and platelet transfusion in vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Analiz içi varyasyon göstermek için, üç aynı yeniden tam kan numuneleri kollajen kaplı yüzeylerin (Şekil 1) üzerinde eşzamanlı olarak perfüze edildi. Bu% 8.7 bir farklılaşma katsayısı ile sonuçlanmıştır. Bu istatistik ile ilgili örnekler arasında kıyas izin kabul edilebilir içi tahlil ve laboratuvarda varyasyon göstermektedir. Burada tarif ticari akış odasının giriş ölçüm göz...

Watch this Scientific Journal Video about Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro at JoVE.com

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Mikroakışkan Akış Chambers Hemostaz ve Trombosit Transfüzyon Model Sulandırılarak Kan kullanma<em&gt; İn Vitro</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

microfluidic-flow-chambers-using-reconstituted-blood-to-model

Research

JoVE Journal

Bioengineering

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

9.3K Görüntüleme.  Belgium Red Cross-Flanders. Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro. 

Video: Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in vitro. Nutrients delivered by vasculature fail to reach all parts of tumors, giving rise to heterogeneous microenvironments consisting of viable, quiescent and necrotic cell types. Many cancer drugs fail to effectively penetrate and treat all types of cells because of this heterogeneity. Monolayers of cancer cells do not mimic this heterogeneity, making it difficult to test cancer drugs with a suitable in vitro model. Our microfluidic devices were fabricated out of PDMS using soft lithography. Multicellular tumor spheroids, formed by the hanging drop method, were inserted and constrained into rectangular chambers on the device and maintained with continuous medium perfusion on one side. The rectangular shape of chambers on the device created linear gradients within tissue. Fluorescent stains were used to quantify the variability in apoptosis within tissue. Tumors on the device were treated with the fluorescent chemotherapeutic drug doxorubicin, time-lapse microscopy was used to monitor its diffusion into tissue, and the effective diffusion coefficient was estimated. The hanging drop method allowed quick formation of uniform spheroids from several cancer cell lines. The device enabled growth of spheroids for up to 3 days. Cells in proximity of flowing medium were minimally apoptotic and those far from the channel were more apoptotic, thereby accurately mimicking regions in tumors adjacent to blood vessels. The estimated value of the doxorubicin diffusion coefficient agreed with a previously reported value in human breast cancer. Because the penetration and retention of drugs in solid tumors affects their efficacy, we believe that this device is an important tool in understanding the behavior of drugs, and developing new cancer therapeutics.

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

We present the procedure for fabrication and operation of a microfluidic device that recreates heterogeneous tumor microenvironments in vitro. The variability in apoptosis within tumor tissue was quantified using fluorescent stains and the effective diffusion coefficient of the chemotherapeutic drug doxorubicin into tumor tissue was evaluated.

Bir Tümör Mikroçevre yeniden oluşturmak için mikroakışkan Cihazı<em&gt; In vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Biyomühendislik

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Çok kanallı mikroakışkan Cihazlar ayırmak Boncuk ve Hücreler Dielectrophoresis ve Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-&#945; (TNF-&#945;), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

This study used a multi-well plate microfluidic system, significantly increasing throughput of cell rolling studies under physiologically relevant shear flow. Given the importance of cell rolling in the multi-step cell homing cascade and the importance of cell homing following systemic delivery of exogenous populations of cells in patients, this system offers potential as a screening platform to improve cell-based therapy.

Sistematik Analizi<em&gt; In Vitro</emÇok-çukurlu plaka mikroakışkan Sistemi ile&gt; Hücre Rolling

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

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 ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

Vasküler Hücre Remodeling Soruşturma Fizyolojik in vitro Modeli ve Uygulaması ile Patolojik Kan Akış

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Trombosit Transfüzyon ve Hemostaz için bir mikroakışkan Akış Odası Modeli Gerçek zamanlı Trombosit Birikimi ve Fibrin oluşumu Tedbirleri

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

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 ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

Stres birden çok paralel-plaka akışı Chambers kullanarak yamultmak için maruz endotel hücrelerinin gen ifade Analizi

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

Multispectral görüntüleme akışı sitometrisi kullanarak In vitro micronucleus assay gerçekleştirmek için otomatik bir yöntem

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Production of Autologous Platelet-Rich Plasma for Boosting In Vitro Human Fibroblast Expansion

There is currently great clinical interest in the use of autologous fibroblasts for skin repair. In most cases, culture of skin cells in vitro is required. However, cell culture using xenogenic or allogenic culture media has some disadvantages (i.e., risk of infectious agent transmission or slow cell expansion). Here, an autologous culture system is developed for the expansion of human skin fibroblast cells in vitro using a patient&#8217;s own platelet-rich plasma (PRP). Human dermal fibroblasts are isolated from the patient while undergoing abdominoplasty. Cultures are followed for up to 7 days using a medium supplemented with either fetal bovine serum (FBS) or PRP. Blood cell content in PRP preparations, proliferation, and fibroblast differentiation are assessed. This protocol describes the method for obtaining a standardized, non-activated preparation of PRP using a dedicated medical device. The preparation requires only a medical device (CuteCell-PRP) and centrifuge. This device is suitable under sufficient medical practice conditions and is a one-step, apyrogenic, and sterile closed system that requires a single, soft spin centrifugation of 1,500 x g for 5 min. After centrifugation, the blood components are separated, and the platelet-rich plasma is easily collected. This device allows a quick, consistent, and standardized preparation of PRP that can be used as a cell culture supplement for in vitro expansion of human cells. The PRP obtained here contains a 1.5-fold platelet concentration compared to whole blood together, with a preferential removal of red and white blood cells. It is shown that PRP presents a boosting effect in cell proliferation compared to FBS (7.7x) and that fibroblasts are activated upon PRP treatment.

This protocol presents a device that produces PRP to boost the in vitro expansion of cells in a 100% autologous fibroblast culture system.