Biz H. Tsutsui ve ö. Kurihara T. fournieriRPS5Ap::H2B-tdTomato hattı ve A. thaliana UBQ10pro::H2B-kes hattı, sırasıyla gibi transgenik bitkiler ile bize verdiğiniz için teşekkür ederiz. Bu eser Enstitüsü, dönüştürücü biyo-moleküller tarafından Nagoya Üniversitesi ve Japonya gelişmiş bitki bilim ağ destek verdi. Bu eser için mali destek sağlanan hibe tarafından Japonya bilim ve teknoloji Ajansı (ERATO proje vermek yok. T.H. için JPMJER1004), yenilikçi alanlar ('ları üzerinde bilimsel araştırma için bir Grant-in-Aid JP16H06465 ve JP16H06464 T.H. için) ve Japonya toplum için zorlu araştırmacı araştırma (hibe No 26600061 için N.Y. ve grant NOS 25650075 ve 15 K 14542 YS için) promosyon bilim (JSP'ler) Grants-in-Aid.

1. polen tüpler büyüyen incelemek için PDMS Microdevice imalatı ve yosun Protonemata
Not: Silikon gofret üzerinde PDMS kalıpları hazırlamak için bir maskless fotolitografi aleti kullanılmış. Sistemin çalışması ile ilgili detayları bu el yazması göz ardı edilir. Bir photomask kullanarak bir standart fotolitografi teknik9 bu el yazması açıklanan PDMS kalıpları oluşturmak için de kullanılabilir.
<ol><li>11 g ön polimer PDMS karışımı dökün (elastomer temel: Ajan 10:1 oranında kür) her 4 inçlik kalıp içine.</li>
	<li>Adım 1.1 vakum odasında 20 dakika için hazırlanan kalıp degas.</li>
	<li>Bir sigara konveksiyon fırın içinde 90 dk 65 ° C'de kür sonra PDMS katman kalıp kapalı soyma ve biyopsi yumruklar kullanarak sıvı kanallarına erişim delikler. Not: polen tüpü aygıt için deliğin boyutunu pistil çapını yansıtacak şekilde ayarlanması gerekir. Bu değer bu nedenle türler tarafından değişir. Bu deneyde, pistil alıcılar için 1 mm delik ve sıvı orta rezervuarlar için 1.5 mm delik yumruk attı. Yosun protonemata aygıt için 4 mm delik örnek rezervuar için kullanıldı.</li>
	<li>Plazma 50 için hava için PDMS katmanı ve bir cam alt çanak (3.5-5 cm çapında) maruz s.</li>
	<li>PDMS katman cam alt çanak ve 65 ° C ısıda 30 dk içinde tamamen mikrosıvısal ağ oturumunu kapatma imzalamaya bir sigara konveksiyon fırın için basın.</li>
</ol>2. kök tüyleri için PDMS Microdevice imalatı
<ol><li>1.1-1.3 kök ve kök saç microdevices için iki PDMS katman hazırlamak için kalıpları kullanarak adımları yineleyin. Not: Biz kök microdevice sıvı orta rezervuar 2 mm delik kullandık.</li>
	<li>Plazma 50 için hava ile her iki PDMS katmanı açığa s.</li>
	<li>Özel yapım bir masaüstü aligner kullanarak bir stereomicroscope altında iki PDMS katmanları birleştirin. Not: Bu PDMS katmanlardaki mikro birbirine yüz gerekir. Montaj önce her iki katmanı hizalama izleri aynı olmasına dikkat edin.</li>
	<li>30 dk içinde tamamen mikrosıvısal ağ oturumunu kapatma imzalamaya bir sigara konveksiyon fırın için 65 ° C'de ısı.</li>
	<li>Kapak notu inşa microdevice çıkarın ve cihazın 5 cm çapında bir cam alt yemek üzerine yerleştirin.</li>
</ol>3. Hazırlık In Vitro hücre kültür orta polen tüpler (Torenia fournieri)
<ol><li>15 ve basınçlı kap daha önce açıklandığı gibi değiştirilmiş Nitsch'ın orta orta 121 ° C'de 20 min için hazır olun. Not: NH4modifed Nitsch'ın orta bileşimi olduğunu KNO3 (125 mg/L)3 (80 mg/L), Ca (NO3)2‧4H2O (500 mg/L), MgSO4‧7H2O (125 mg/L), KH2PO4 (125 mg/L ), MnSO4‧4H2O (3 mg/L), ZnSO4‧7H2O (0,5 mg/L), H3BO3 (10 mg/L), CuSO4‧5H2O (0.025 mg/L) Na2MoO4‧2H2O (0.025 mg/L) sukroz (50.000 mg/L) ve kazein (500 mg/L). Hazır orta 4 ° C'de en az 4 ay depolanabilir.</li>
	<li>% 26 (w/v) Polietilen glikol autoclaved deiyonize su kullanarak hazırlamak ve çözüm 0.3 µm gözenek filtre ile filtre. Not: Hazırlanan orta 4 ° C'de 1 ay boyunca saklanır.</li>
	<li>Adım 3.1 ve 3.2 1:1 (v/v) oranında hazırlanan reaktifler karıştırın. Not: Bu orta her kullanım için taze hazırlanmalıdır.</li>
</ol>4. kök tüyleri (Arabidopsis thaliana) için hazırlık In Vitro hücre kültür orta
<ol><li>%0.215 (w/v) Murashige & Skoog orta, % 0.05 (w/v) MES, %1 (w/v) sükroz ve %1 (w/v) agar deiyonize su çözeltisi hazırlamak. Otoklav 20 dk için 121 ° C'de çözüm.</li>
</ol>5. Hazırlık In Vitro hücre kültür orta olarak Moss Protonemata (Physcomitrella patens)
<ol><li>Yosun protonemata BCDAT orta16 bir petri içinde önceden kuluçkaya. Not: BCDAT orta kompozisyon ise 1 mM MgSO4, 10 mM KNO3, 45 µM FeSO4, 1.8 mM KH2PO4 (pH) 6.5 KOH ile düzeltilmiş, trace öğe çözüm (0,22 µM CuSO4, 0,19 µM ZnSO4, 10 µM H3BO 3, 0,1 µM Na2MoO4, 2 µM MnCl2, 0,23 µM CoCl2ve 0.17 µM KI), 1 mM CaCl2ve 5 mM diammonium (+)-tartarat.</li>
	<li>Kültür microdevice BCDATG orta olarak moss. Not: %0,5 (w/v) glikoz ile BCDAT orta BCDATG ortamdır. Hazır orta 4 ° C'de 1 ay boyunca saklanır.</li>
</ol>6. T. fournieri polen tüplerinin Microdevice in Vitro Culturing
<ol><li>20 dakikadır degas ve polen tüpü microdevice bir vakum odasında yerleştirin.</li>
	<li>Microdevice vakum odasından kaldırın ve bir micropipette ile çok iyi bir ipucu kullanarak pistil giriş büyüme orta tanıtmak. Üst kısımları için kalan wells ile aynı orta doldurun. Tüm mikro mikro içinde oluşturulan vakum tarafından orta ile doldurulur kadar birkaç dakika bekleyin.</li>
	<li>Bazı ıslak kağıt havlu çanak nem korumak için cam alt çanak yerleştirin.</li>
	<li>Polen taneleri vahşi türünden aktarım T. fournieri 'mavi ve beyaz' çiçek diseksiyon iğne kullanarak onun stigma için. Not: Biz de bu transgenik T. fournieri ile deney ( RPS5Ap::H2B-tdTomato hattı17) 'mor taç' çiçek, polen fluorescently etiketli sperm hücresi ve bitkisel çekirdek içeren tüpler.</li>
	<li>Kesim tozlanan stil (1 cm uzunluğunda) bir bıçak kullanarak.</li>
	<li>Kesim tarzı cihazın giriş Şekil 2' de gösterildiği gibi yerleştirin.</li>
	<li>Çanak üzerinde bir kapak koyun ve bant ile kapatın.</li>
	<li>Aygıt için 5-6 h 28 ° C'de bir kuluçka karanlıkta yerleştirin.</li>
</ol>7. A. thaliana kök kıllar Microdevice in Vitro Culturing
Not: Adımları 7.1-7,9 (7,3 ve 7,5 dışında) bir laminar akış mahallede gerçekleştirilmesi gerekiyor.
<ol><li>A. thaliana Columbia (Col-0) tohumlarından sterilize ve transgenik (floresan nükleer işaretçiyi içeren) UBQ10pro::H2B-kes satır 5 min için steril sıvı (%5 (v/v) ev çamaşır suyu ve %0.02 (v/v) Triton X-100) içinde ıslatarak.</li>
	<li>İyice sterilize tohum autoclaved su ile durulayın.</li>
	<li>Durulanır tohumları suda 4 ° C'de 2 gün boyunca karanlıkta saklayın.</li>
	<li>UV ışığı altında microdevice gecede sterilize.</li>
	<li>20 dakikadır degas ve microdevice bir vakum odasında yerleştirin.</li>
	<li>Büyüme orta wells için yapılan bir micropipette kullanarak aygıtı tanıtmak. Orta ile tüm mikro vakum doldurur kadar birkaç dakika bekleyin.</li>
	<li>Autoclaved ıslak kağıt havlu çanak nem korumak için cam alt çanak yerleştirin.</li>
	<li>Steril bir tohum cihazın giriş aktarın.</li>
	<li>Çanak üzerinde bir kapak koyun ve bant ile kapatın.</li>
	<li>Cihazın bir kuluçka 22 ° c sürekli beyaz ışık altında dikey olarak yerleştirin.</li>
	<li>Kök saç büyüme kuluçka 4-10 gün sonra kontrol edin. Parlak alan ve floresan görüntüleri ters bir mikroskop kullanarak elde edilir.</li>
</ol>8. P. patens (moss) mikrosıvısal cihazın Protonemata in Vitro Culturing
Not: Adımları 8.2-8,6 (8.3 dışında) bir laminar akış mahallede gerçekleştirilmesi gerekiyor.
<ol><li>P. patens gerilme Gransden 200418 25 ° C'de sürekli beyaz ışık altında bir hafta bir petri selofan kaplı BCDAT orta preculture</li>
	<li>Microdevice gecede bir laminar akış başlıklı UV ışık altında sterilize.</li>
	<li>20 dakikadır degas ve microdevice bir vakum odasında yerleştirin.</li>
	<li>Büyüme orta bir micropipette kullanarak wells tanıtmak. Orta ile tüm mikro vakum doldurur kadar birkaç dakika bekleyin.</li>
	<li>Autoclaved su çanak nem korumak için microdevice çevreleyen çanak içine ekleyin.</li>
	<li>Yosun protonemata doku küçük bir parça microdevice koya aktarmak ve sürekli beyaz ışık altında 25 ° C'de bir kuluçka doku kültür.</li>
	<li>Yosun protonemata büyüme kuluçka 2-3 hafta sonra kontrol edin. Parlak alan görüntü bir mikroskop kullanarak elde.</li>
</ol>9. T. fournieri polen tüpü büyüme hızlandırılmış görüntüleme
<ol><li>Microdevice görüntü edinme donanım ile donatılmış bir ters floresans mikroskobu yerleştirin (Örn., CCD kamera) ve yazılım. Microgap yerleri bulabilirsiniz. Not: görüntü alma yazılımı, biz ticari bir ürün (Tablo reçetesi) kullanılır. Açık kaynak mikroskopi µManager gibi aynı zamanda kullanılabilir çevrimiçi (https://micro-manager.org/wiki/Micro-Manager) yazılımdır. Bunun bir mikroskop kondansatör yüksek çözünürlüklü görüntüler elde etmek için mikroskobunun yüklemeniz önerilir.</li>
	<li>Parlak alan her 10 çekim s mikroskop görüntü alma yazılımı kullanarak.</li>
	<li>Fluorescently etiketli sperm hücresi ve polen tüpü RPS5Ap::H2B-tdTomato satırının bitkisel çekirdeğinde gözlemlemek için numune 561 nm lazer ile ışınlatayım ve bant optik filtre kullanın (578/105 nm). Not: her ne kadar polen tüpü büyüme hızlandırılmış görüntülerini sık sık ele geçirildi, görüntüleme büyüme etkilemeye görünmedi. Ancak, her zaman lazer yoğunluğu, çekim hızı ve hızlandırılmış aralığı fototoksisite ve fluorophore photobleaching en aza indirmek için en aza indirmek için tavsiye edilir.</li>
	<li>Parlaklık ve kontrast görüntülerin ve ImageJ yazılımı (https://imagej.nih.gov/ij/) kullanarak bir video dosyasını hazırlamak.</li>
</ol>

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Yazarlar onlar rakip hiçbir mali çıkarları var bildirin.

Protokol birkaç kritik adımda tam olarak yukarıda sunulan sonuçlar elde etmek için takip edilmesi gerekiyor. İlk olarak, katman ve cam alt çanak PDMS yüzeyler her ikisi de plazma ile yapıştırma önce yeterli süreyi için tedavi edilmelidir. Aksi takdirde, ipucu büyüyen hücreler microgaps geçiş yaparken PDMS katman yerel olarak cam yüzeyinden ayırmak. Başka bir önemli kök saç ve yosun protonemata protokol microdevice sterilizasyon adımdır. Normalde, kök tüyleri ve yosun protonemata hücreleri bi...

Polen taneleri üzerinde bir leke çimlenme sonra her tahıl sperm hücresi yumurta ve çift döllenme için ovule merkezi hücreye taşır tek bir polen tüp üretir. Polen tüpler tarzı uzamış ve sonunda birden çok kılavuz ipuçları onların yolu1boyunca algılama tarafından ovule ulaşmak. Uzama sırasında polen tüpler fiziksel engeller bir dizi karşılaşırsanız; verici parça hücreleri ile doldurulur ve polen tüpler onların hedef (Şekil 1A)2ulaşmak için ovule dakika micropylar açılışı girmeniz gerekir. Bu nedenle, polen tüpler çevreleri basınç stresten tolere ederken fiziksel engeller, nüfuz yeteneği olması gerekir. Kök tüyleri ortamında paketlenmiş toprak parçacıklar (Şekil 1B) şeklinde fiziksel engelleri dayanmak zorundadır ipucu büyüyen bitki hücre başka bir türüdür.
Turgor basıncı ve yeni başlayan plasmolysis Yöntem3,4 ve hücresel kuvvet mikroskobu kullanılarak ölçülebilir hücre apikal bölgede sertliği de dahil olmak üzere çeşitli mekanik özelliklerini polen tüpü incelenmiştir (CFM) 5 , 6, anılan sıraya göre. Ancak, bu yöntemlerin yalnız polen tüpler aracılığıyla onların büyüme yol üzerindeki fiziksel engeller elongating yeteneğine sahip olup olmadığını ortaya çıkarmak değil. İzlenen vivo içinde olmak polen tüpü uzaması sağlayan bir alternatif iki foton mikroskobu7tekniğidir. Ancak, bu yöntemle, bireysel polen tüpler morfolojik değişimler gözlemlemek zor öyle içten içe ovule doku. Ayrıca, toprakta kök saç büyüme x-ışını bilgisayarlı tomografi (BT) ve manyetik rezonans görüntüleme (MRG)8, kullanarak düşük çözünürlüklü de olsa görüntülenmeyecektir. Burada, bir hücrenin deformasyon işlemi geleneksel mikroskop yüksek çözünürlüklü görüntüleri elde etmek için kullanılan bir yöntem mevcut.
Burada açıklanan yöntemi genel amacı ipucu büyüyen bitki hücreleri polen tüpler, kök tüyleri, dahil olmak üzere, uzama kapasitesini görselleştirmek ve protonemata, son derece küçük alanlarda moss etmektir. Bu el yazması sunulan poli-dimethylsiloxane (PDMS) microdevices optik şeffaf ve hava geçirgen, biz canlı hücreler cihazın içinde kültür ve büyüme davranışları bir mikroskop altında gözlemlemek. Ayrıca mikro oluşturmak mümkündür ~ nanometre ölçek alanlarda kalıpları kullanımı ile yumuşak litografi tekniği9 . Bu özellikler bize ipucu büyüyen bitki hücreleri fiziksel olarak kapalı bir ortamda uzama yeteneğini çalışmaya olanak sağlar.
Bu çalışmada, 1 µm geniş boşluklar (yüksekliği 4 µm) mikrosıvısal cihazlarda inşa ve silindirik polen tüpü (yaklaşık 8 µm) çapından küçüktür bu yapay engelleri nüfuz yeteneği polen tüplerinin muayene etti. Bu deneysel platform polen tüpü yanıt-e doğru microgaps görselleştirmek ve hücrenin deformasyon işlemi izlemek belgili tanımlık yanıt, hızlandırılmış görüntülerini yakalamak bize sağlar. Ayrıca cihazın penetrasyon özelliği kök tüyleri ve yosun protonemata araştırmak için kullanılan microdevices geliştirdi. Birkaç microdevices bitki kök10,11,12,13 ve yosun protonemata14 büyüme yüksek çözünürlükte görselleştirme etkinleştirme tarihi bildirilmiştir. Bizim cihaz kök saç büyüme kanalları bir dizi dik bir kök büyüme odasına bağlı ve bireysel kök tüyleri (çapı yaklaşık 7 µm) geniş bir boşluk 1 µm ile sıvı kanallara yönlendirilir. Biz de yosun kültürlü protonemata (çapı yaklaşık 20 µm) Bu fiziksel engellerin yanıtlarını incelemek için microgaps içeren bir microdevice içinde. Önerilen mikrosıvısal dayalı yaklaşım çeşitli ipucu büyüyen bitki hücreleri aracılığıyla herhangi bir mevcut diğer yöntemle incelendiğinde çok küçük alanlarda uzamış yeteneği keşfetmek için bize izin verir.

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			<td height="21" style="height:21px;width:216px;">Murashige &#38; Skoog Medium</td>
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			<td style="width:119px;">392-00591</td>
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			<td height="21" style="height:21px;width:216px;">MES</td>
			<td style="width:165px;">Dojindo</td>
			<td style="width:119px;">345-01625</td>
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			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:165px;">Wako Pure Chemical</td>
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			<td height="21" style="height:21px;width:216px;">50 mm glass-bottom dish</td>
			<td style="width:165px;">Matsunami Glass</td>
			<td style="width:119px;">D210402</td>
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			<td height="21" style="height:21px;width:216px;">35 mm glass-bottom dish</td>
			<td style="width:165px;">Iwaki&#160;</td>
			<td style="width:119px;">3971-035</td>
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			<td height="21" style="height:21px;width:216px;">Surgical blade</td>
			<td style="width:165px;">Feather</td>
			<td style="width:119px;">No.11</td>
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			<td height="21" style="height:21px;width:216px;">biopsy punches</td>
			<td style="width:165px;">Harris</td>
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			<td>Uni-Core</td>
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			<td height="21" style="height:21px;width:216px;">Gel loading tips</td>
			<td style="width:165px;">Bio-Bik</td>
			<td style="width:119px;">124-R-204</td>
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			<td height="21" style="height:21px;width:216px;">Inverted Microscope</td>
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			<td height="21" style="height:22px;width:216px;">CSU-W1</td>
			<td style="width:165px;">Yokogawa Electric</td>
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			<td>No Catalog number is avairable for this customized microscope</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MetaMorph imaging software</td>
			<td style="width:165px;">Molecular Devices</td>
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development of microfluidic devices to study the elongation capability of tip-growing plant cells in extremely small spaces

Vivo, ipucu büyüyen bitki hücreleri fiziksel engeller bir dizi aşmak gerekir; Ancak, araştırmacılar bu kısıtlayıcı koşullarda hücresel davranış görselleştirmek için metodoloji eksikliği. Bu sorunu gidermek için biz bir poli-dimethylsiloxane (PDMS) substrat büyüme salonları dar, mikro fabrikasyon boşluklar (~ 1 µm) bir dizi içeren ipucu büyüyen bitki hücreleri için geliştirdik. Bu şeffaf malzeme ipucu uzama işlemlerde tek tek hücreler tarafından hızlandırılmış görüntüleme microgap penetrasyon sırasında izlemek için kullanıcı sağlar. Onlar microgap nüfuz olarak deneysel bu platformu kullanarak, biz polen tüpler morfolojik değişimler görülmektedir. Bu işlem sırasında fluorescently etiketli bir bitkisel çekirdek şeklinde dinamik değişiklikler ve sperm hücresi bir polen tüp ele geçirdi. Ayrıca, kök tüyleri ve yosun protonemata 1 µm Aralık nüfuz yeteneğini gösterdi. Bu vitro platform nasıl tek tek hücreleri yanıt fiziksel olarak kısıtlı alanlarda çalışmak için kullanılan ve anlayışlar ipucu-büyüme mekanizmaları içerebilir.

Şekil 1' de gösterildiği gibi onların büyüme yolları içinde vivoboyunca fiziksel engeller bir dizi ipucu büyüyen bitki hücreleri karşılaşma. Bu çalışmada sunulan mikrosıvısal vitro hücre kültür platformlar muayene etkin ipucu büyüyen bitki hücreleri (polen tüpler, kök tüyleri ve yosun protonemata) arasındaki 1 µm yapay geçitlerden (Şekil 3, üç tür süreç ?...

Watch this Scientific Journal Video about Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces at JoVE.com

Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

İpucu büyüyen bitki hücreleri polen tüpler, kök tüyleri, dahil olmak üzere, kapasitesini araştırmak ve protonemata son derece dar geçitlerden (~ 1 µm) uzamış, bir mikrosıvısal cihazda moss bir yöntem açıklanmaktadır.

İpucu büyüyen bitki hücreleri son derece küçük alanlarda uzama yeteneğini çalışmaya mikrosıvısal cihazların geliştirme

In vivo, tip-growing plant cells need to overcome a series of physical barriers; however, researchers lack the methodology to visualize cellular behavior ...

development-microfluidic-devices-to-study-elongation-capability-tip

Research

JoVE Journal

Bioengineering

7.3K Görüntüleme.  Nagoya University. İpucu büyüyen bitki hücreleri polen tüpler, kök tüyleri, dahil olmak üzere, kapasitesini araştırmak ve protonemata son derece dar geçitlerden (~ 1 µm) uzamış, bir mikrosıvısal cihazda moss bir yöntem açıklanmaktadır.

Video: Development of Microfluidic Devices to Study the Elongation Capability of Tip-growing Plant Cells in Extremely Small Spaces

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 ...

Biyomühendislik

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Devirli Basınç Biyoreaktör Tasarım Ex vivo</emAort Kalp Vanalar> Eğitim

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Için basit Mikroakışkan Cihazlar<em&gt; In vivo</emVe&gt; Görüntüleme<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; Ve Zebra balığı

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Microtissue Sabitleme için mikroakışkan Cihazlar Katman-katman Kolajen Biriktirme

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Tanımlı İnsan Engineered Kalp Dokuların inşaatı Kardiyak Hücre Tedavisinin Mekanizmaları Eğitim için

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

3D Kağıt mikroakışkan Cihazlar Construct Yapışkan Desenlendirme kullanma

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

Bir Küçük Hayvan Modelinde Arteriovenöz (AV) Döngü Anjiogenez ve Vaskülarize Doku Mühendisliği Eğitim için

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; represented by &#34;devices&#34; created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction.
Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols.
The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation.

Here, an automated workflow to perform modular DNA &#34;device&#34; assembly using a modular cloning DNA assembly method on liquid-handling robots is presented. The protocol uses an intuitive software tool for generating liquid handler picklists for combinatorial DNA device library generation, which we demonstrate using two liquid handling platforms.

Robot sıvı işleme derleme modüler DNA cihazların otomatik

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available &#34;design space&#34; ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Ventilli Katmanlı mikroakışkan Cihazlar için çok adımlı Değişken Yükseklik Fotolitografi

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

İmmüno için üç boyutlu Paper-based mikroakışkan Cihazlar İmalat

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...