Bu çalışma Sayısal Biosciences için California Institute (QB3), Rogers Aile Vakfı, Cephe Biyomedikal Araştırma, BASF&#39;den bir hibe için UCSF / Sandler Vakfı Programı Bridging Gap Ödülü ve NSF aracılığıyla bir Araştırma Ödülü ile desteklenmiştir Fakülte Erken Kariyer Gelişimi (KARİYER) Programı (DBI-1253293).

1. SU8 Usta Fabrikasyon <ol><li> AutoCAD yazılımı kullanılarak iki katmanlı imalat için mikroakışkan yapıları tasarım ve 10 mikron çözünürlükte devre kartı film üzerine bir satıcı tarafından basılan tasarımları var. Cihaz tasarımı detayları ekli referans 11 de verilmiştir ve kanal geometrileri, Şekil 1 &#39;de gösterilmiştir. Tabakaların her biri, imalat tabaka 12 özelliklerini düzenlemek yardımcı hizalama işaretleri içermelidir. </li><li> Spin kaplayıcı bir ön temizlenmiş 3 inç çaplı silisyum gofret yerleştirin ve aynasının bunu tutturmak için vakum açın. 50 um arası bir tabaka kalınlığına sağlayan 2000 rpm&#39;de 30 saniye daha sonra, 500 rpm&#39;de 20 saniye boyunca gofret ve dönüş merkezinde SU8-3035 1 ml uygulanır. </li><li> Gofret çıkarın ve 30 dakika süre ile 135 ° C sıcak plaka üzerinde pişirilir. Gofret sonraki adıma geçmeden önce oda sıcaklığına kadar soğumasını bekleyin. </li><li> 1. katman maskesine kaplı gofret (Şekil 2A Açığa </strong&gt;) 90 sn için LED nm bir paralel 190 mW, 365 altında. Maruz kaldıktan sonra, bir sonraki adıma geçmeden önce, 1 dakika boyunca RT sonra serin bir 135 ° C ocak gofret yerleştirin. </li><li> Spin kaplayıcı üzerine gofret yerleştirin ve aynasının bunu tutturmak için vakum açın. 135 um&#39;lik bir kalınlık sağlayan bir ek tabaka ile sonuçlanan, 1375 rpm&#39;de, sonra 30 saniye, 500 rpm&#39;de 20 saniye boyunca gofret ve dönüş merkezinde SU8-2050 1 ml uygulanır. </li><li> Bir sonraki aşamaya geçmeden önce, 30 dakika süre ile 135 ° C sıcak plaka üzerinde oda sıcaklığına kadar daha sonra soğuk gofret ve fırında çıkarın. </li><li> 1.3 desenli geometri üzerine 2. katman maskesi (Şekil 2B) hizalayın ve 3 dakika boyunca LED nm bir paralel 190 mW, 365 kaplanmış gofret maruz. Maruz kaldıktan sonra, bir sonraki adıma geçmeden önce, 1 dakika süreyle 135 ° C ocak RT sonra serin yerleştirin. </li><li> 30 dakika boyunca, propilen glikol monometil eter asetat içinde karıştırılan bir banyosu içinde daldırılması yoluyla maske geliştirir. Gofret yıkayın1 dakika süreyle 135 ° C ocak izopropanol ve fırında. PDMS kalıplama için 100 mm Petri kabındaki geliştirilen usta yerleştirin. </li></ol> 2. PDMS Cihaz İmalatı <ol><li> Plastik bir kap içinde sertleştirici 5 g silikon baz 50 g birleştirerek 1 PDMS: 10 hazırlayın. Bir karıştırma çubuğu ile donatılmış bir döner aracı ile içeriği karıştırın. Tüm hava kabarcıkları bertaraf edilmiştir, 30 dakika boyunca, ya kadar bir desikatör içinde karışım gaz çıkışına. </li><li> Master üzerinde 3 mm kalınlığında vermek ve daha fazla gaz alma için desikatöre geri yerleştirmek için PDMS dökün. Bir kez tüm kabarcıklar kaldırılır, 2 saat süre ile 60 ° C &#39;de fırında cihazı. </li><li> Desenli yüzü yukarı bakacak şekilde temiz bir yüzey üzerinde bir neşter ve yer kullanarak kalıp cihazı kesin. Usta 2 (Şekil 3a) dan master 1 ayırmak için bir jilet ile yarım PDMS kalıp kesin. Usta 1 tarafından basılmış 50 mikron sıvı taşıma geometrisini içeren parçasına, bir 0.7 ile akışkan giriş ve çıkış yumruk5 mm biyopsi yumruk. </li><li> Plazma 300 W plazma temizleyici 60 saniye boyunca 1 mbar O 2 plazması cihazları tedavi. Geçici PDMS PDMS bağlama geciktirir ve bir kayganlaştırma maddesi olarak hizmet etmek DI su damla PDMS deliksiz parçasının yüzeyi ıslatılır. Master 2 yüzey üzerinde bir stereo mikroskop, yer Master 1 ile görüntüleme ve mekanik kilit elde edilene kadar, nispeten yüzeyleri kaydırın ederken zaman girintili çerçeveleri ve Şekil 3A arkadaşı çıkıntılı çerçeveler. </li><li> 60 ° C&#39;lik bir fırında Cihazı ve su ve yapışmasını tam buharlaşmaya 60 ° C &#39;de iki gün süre ile birleştirilmiş cihaz (Şekil 3B) fırında. </li></ol> Reaktiflerin 3. Hazırlık <ol><li> İç faz damıtılmış su ile 1 ml şırınga doldurun. </li><li> 1 wt ile HFE 7500 florlu yağ ile 1 ml şırınga doldurun. Orta faz için% biyo-uyumlu bir yüzey aktifi 13. </li><li> 10 wt ile 10 ml şırınga doldurun. % Polietilen1 kadarını ihtiva eden su çözeltisi içinde e glikol (PEG) içerebilir. % Tween 20 ve 1 wt. % Sürekli bir faz için sodyum dodesil sülfat. </li></ol> 4. Sistem Hazırlama <ol><li> &lt;100 mikro-saniye deklanşör hızları yeteneğine sahip dijital kamera ile birleştiğinde bir ters mikroskop sahnede mikroakışkan çip yerleştirin. </li><li> Şırınga pompaları tüm şırınga monte ve 27 G iğne takın. Iğne ~ PE-2 boru 30 cm uzunlukta takın ve uygun cihazda delik delinmiş içine gevşek uçları takın. </li><li> Aracının çıkış portuna PE-2 bir 10 cm uzunluğunda yerleştirin ve bir atık toplama kabı diğer ucunu yerleştirin. </li><li> Prime boru kesimleri sıvı kadar hızlarda yüksek oranda şırınga pompaları (2000 ul / dakika) çalıştırarak cihaz, giriş kapısı ulaşır. </li></ol> 5. Emülsiyon Üretimi <ol><li> 50 mm x 50 mm delik ve içeren bir bölgeye mikroskop odaklanınaşağı çıkış kanalı. </li><li> Sürekli bir faz için, 250 ul / İç faz için saat, 100 ul / orta aşama için saat ve 700 ul / saat akış hızında çift emülsiyon üretecine akışkanı sevk ve dengeyi 10 dakika beklemek şırınga pompaları ayarlayın. </li><li> Sırasıyla 250 ul / saat ve 100 ul / st&#39;de iç ve orta fazın akış oranları muhafaza edin. 1050 ul / saat, dış fazının akış hızını ayarlayın. Çift emülsiyonlar nesil akış koşulları bu seti altında stabilize etmek için 3-5 dakika bekleyin. </li><li> Manuel görüntü analizi ile çevrimdışı işleme için 30 Hz video görüntülerinin 5 sn edinin. </li><li> Tablo 1 &#39;de verilen bir akış oranı ile 5.3 ve 5.4 tekrarlayın. Iç ve orta fazı akış oranlarının sabit tutulması ve taşıyıcı fazının akış hızı, bir şırınga pompası ayar ayarlayarak değiştirilir. </li></ol>

<ol>
	<li>Van Der Graaf, S., Schro&#235;n, C. G. P. H., Boom, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+double+emulsions+by+membrane+emulsification+-+A+review.">Preparation of double emulsions by membrane emulsification - A review.</a> J. Membrane Sci. 251 (1-2), 7-15 (2005).</li><li>Laugel, C., Baillet, A. P., Youenang Piemi, M., Marty, J., Ferrier, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oil-water-oil+multiple+emulsions+for+prolonged+delivery+of+hydrocortisone+after+topical+application:+comparison+with+simple+emulsions.">Oil-water-oil multiple emulsions for prolonged delivery of hydrocortisone after topical application: comparison with simple emulsions.</a> Int. J. Pharm. 160 (1), 109-117 (1998).</li><li>Kim, S. H., Kim, J. W., Cho, J. C., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-emulsion+drops+with+ultra-thin+shells+for+capsule+templates.">Double-emulsion drops with ultra-thin shells for capsule templates.</a> Lab Chip. 11 (18), 3162-3166 (2011).</li><li>Lim, S. W., Abate, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrahigh-throughput+sorting+of+microfluidic+drops+with+flow+cytometry.">Ultrahigh-throughput sorting of microfluidic drops with flow cytometry.</a> Lab Chip. 13 (23), 4563-4572 (2013).</li><li>Bernath, K., Hai, M., Mastrobattista, E., Griffiths, A. D., Magdassi, S., Tawfik, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+compartmentalization+by+double+emulsions:+sorting+and+gene+enrichment+by+fluorescence+activated+cell+sorting.">In vitro compartmentalization by double emulsions: sorting and gene enrichment by fluorescence activated cell sorting.</a> Anal. Biochem. 325 (1), 151-157 (2004).</li><li>Seemann, R., Brinkmann, M., Pfohl, T., Herminghaus, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+based+microfluidics.">Droplet based microfluidics.</a> Rep. Prog. Phys. 75 (1), 016601 (2012).</li><li>Bauer, W. A. C., Fischlechner, M., Abell, C., Huck, W. T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+PDMS+microchannels+for+high-throughput+formation+of+oil-in-water+microdroplets+and+water-in-oil-in-water+double+emulsions.">Hydrophilic PDMS microchannels for high-throughput formation of oil-in-water microdroplets and water-in-oil-in-water double emulsions.</a> Lab Chip. 10 (14), 1814-1819 (2010).</li><li>Utada, A. S., Lorenceau, E., Link, D. R., Kaplan, P. D., Stone, H. A., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monodisperse+double+emulsions+generated+from+a+microcapillary+device.">Monodisperse double emulsions generated from a microcapillary device.</a> Science. 308 (5721), 537-541 (2005).</li><li>Chang, F. C., Su, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+double+emulsification+utilizing+3D+PDMS+microchannels.">Controlled double emulsification utilizing 3D PDMS microchannels.</a> J. Micromech. Microeng. 18 (6), 065018 (2008).</li><li>Romanowsky, M. B., Abate, A. R., Rotem, A., Holtze, C., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+production+of+single+core+double+emulsions+in+a+parallelized+microfluidic+device.">High throughput production of single core double emulsions in a parallelized microfluidic device.</a> Lab Chip. 12 (4), 802-807 (2012).</li><li>Tran, T. M., Cater, S., Abate, A. 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The authors have nothing to disclose.

Burada anlatılan çift emülsiyon üreten geometri cam kılcal cihazlarının 8 fiziğini taklit için tasarlanmıştır. Bunlarda, hizalanmış silindirik cam kılcal kısımların düzgün çift emülsiyon damlacıkları haline kesilmiş olan bir üç fazlı koaksiyal püskürmesi oluşturmak için kullanılır. Bizim 3D PDMS cihazın işlevi toplam yüksekliği 320 mm olan taşıyıcı faz kanallarını 50 mikron boyunda imalat ile oluşturulan küçük özellikler merkez hizalama bağlıdır. Maskeleri d...

<html> Çift emülsiyonlar nedeniyle, endüstriyel, ilaç potansiyel kullanımı ve biyolojik uygulamalarda 1 bir ara, karışmayan sıvı tabaka ile, bir taşıyıcı faz ayrılır damlacıklarının oluşur ve özellikle ilgi çekicidir. Bazı durumlarda, çift emülsiyonun iç değeri yüksek bileşikleri kapsüllemek yeteneği malzemesi korunması ve kontrollü bir şekilde serbest kalmasını sağlar. Örnek vermek gerekirse, dış taşıyıcı akışkan 2 için uygun değildir çözünürlük koşullar altında kapsüllenebilir. Buna ek olarak, ara-ürün yağ tabakası ilaç, kozmetik, ve besin 3 kapsüllenmesi ve dağıtımı için bir kapsül şablon olarak kullanılabilir. Bunlar alt nanoliter deneylerin büyük bir sayı daha sonra, yürütülen algılanabilir ve (FACS) aleti 4,5 sıralama bir floresan-aktive edilmiş hücre kullanarak kriteri izin için biyolojide çift emülsiyonlar, ayrıca yüksek verimli tarama teknikleri de yararlıdır. ent &quot;&gt; istenen performans özellikleri ile çift emülsiyonlar tasarımı çift emülsiyon boyutu, kompozisyonu ve tekdüzelik hassas kontrolünü gerektirir. Böyle zar emülsifikasyonu olarak toplu emülsifikasyon süreçleri, sanayide kullanılmasına rağmen, ortaya çıkan emülsiyonlar, son derece polidispers olduğu bir sergileme işlevsel özellikleri 1. çeşitli. damlacık Mikroakiskan alan doğal dikkatle kontrol kompozisyon 6 monodispers emülsiyonlar nesil uygundur. Mikroakışkan çift emülsiyon üretimi odaklanan iki ana stratejiler, sıralı damla yapma ve cam kılcal akışı ile elde edilmiştir. Çift emülsiyonlar can verme süreci, iki aşamalı bir düşüş ile düzlemsel PDMS cihazlar üretilebilir. İlk olarak, sulu bir yağ-içinde-su emülsiyonları, bir yağ-içinde-açılan verme hidrofobik kanal duvarları ile bir cihazın bölgesi. İleri kullanılarak oluşturulur, emülsiyon olabilir akıyordu ya da su içinde yağ için uygun hidrofil duvarlı bir damla yapma bölgeye tekrar enjektedamla yapma 4. Bununla birlikte, pmds hidrofilik yüzey işlemi ek bir üretim aşamasını gerektirmekte ve 7 geçici genellikle. Çift emülsiyonlar oluşturmak için en kontrollü ve tekrarlanabilir bir yöntem eş eksenel akış odaklanarak olduğunu bir tekniktir üç faz içeren bir konsantrik jet monodispers damlacıklar 8 üretmek için küçük bir delikten makaslanmış sayede cam kılcal mikroakışkanları kullanarak öncülük etmiştir. Bu teknik, her fazının akış oranlarının bir fonksiyonu olduğu, çift emülsiyon kesin boyutu ve bileşimi ile, kanal boyutları çok daha küçük damlacıkların üretimine olanak sağlamaktadır. Damlacık ve kanal boyutu ve koruyucu dış kılıf akışı arasındaki büyük fark, kanal duvarlarını temas gereksiz yüzey işleme render gelen damlacıkları engeller. Ancak, bu tür cam cihazları dikkatli montaj ve sızdırmazlık birlikte, konik kılcal ipuçları özel imalat gerektirir. Önceki araştırmacılar 3D yumuşak litho kullanmışgrafisi fizik odaklama akış kullanılarak çift emülsiyonlar oluşturmak için, ancak bu cihazlar çapları&gt; 150 mikron 9,10, genellikle FACS ile sıralanır nesnelerden daha büyük boyutlu kabaca bir sipariş ile emülsiyonlar üretti. Cazip bir alternatif PDMS yumuşak litografi üretim kolaylığı ile odaklama cam kapiller koaksiyel akışının güçlü işlevler ve küçük damlacık nesil içerir. Bu yazıda 50 mikron emülsiyon ≤ üretmek için odaklama koaksiyel akışını kullanır ve tamamen 3D yumuşak litografi 11 kullanılarak inşa edilmiştir çift emülsiyon jeneratör açıklar. Bizim cihaz çekilmiş bir cam kılcal memesinde emülsiyon oluşumu süreçlerine yaklaştığı küçük kesme kanalı (Şekil 1) ihtiva cihazları imal etmek için bir kapaklı bir yaklaşım kullanır. Daha da önemlisi, bu cihazlar özel yüzey işlemi gerekebilir ve tüm polimer yapı kolay ve tekrarlanabilir üretim sc sağlarÇift cihaz çok sayıda alable. Burada, çift emülsiyon jeneratör tasarımı, imalatı ve test özetlemektedir. Çift emülsiyon üretimi 14 um damlacık çapları kadar sağlam ve tekrarlanabilir olduğu gösterilmektedir. Imalat kolaylığı ile fonksiyonellik bağlama bu cihaz yeni çift emülsiyon uygulamalarının geliştirilmesi için cazip bir seçenek haline getiriyor.

<table border="0" cellpadding="0" cellspacing="0" width="691">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Photomasks</td>
			<td style="width:135px;">CadArt Servcies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">3&#34; silicon wafers, P type, virgin test grade</td>
			<td style="width:135px;">University Wafers</td>
			<td style="width:119px;">447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 3035</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y311074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">SU-8 2050</td>
			<td style="width:135px;">Microchem</td>
			<td style="width:119px;">Y111072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:135px;">Krayden</td>
			<td style="width:119px;">4019862</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">1 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">10 ml syringes</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">27 gaugue needles</td>
			<td style="width:135px;">BD</td>
			<td style="width:119px;">305109</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">PE 2 polyethylene tubing</td>
			<td style="width:135px;">Scientific Commodities, Inc.</td>
			<td style="width:119px;">B31695-PE/2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Novec 7500</td>
			<td style="width:135px;">Fisher Scientific</td>
			<td style="width:119px;">98-0212-2928-5</td>
			<td>Commonly knowns as HFE 7500</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Biocompatable surfactant</td>
			<td style="width:135px;">Ran Biotechnologies</td>
			<td style="width:119px;">008-FluoroSurfactant</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">35,000 MW PEG</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">1546660</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Tween 20</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">P1369</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sodium dodecyl sulfate&#160;</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td style="width:119px;">L3771</td>
			<td></td>
		</tr>
	</tbody>
</table>

double emulsion generation using a polydimethylsiloxane (pdms) co-axial flow focus device

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper presents a polydimethylsiloxane (PDMS) microfluidic device capable of generating water/oil/water double emulsions using a coaxial flow focusing geometry that can be fabricated entirely using soft lithography. Similar to emulsion devices using glass capillaries, double emulsions can be formed in channels with uniform wettability and with dimensions much smaller than the channel sizes. Three dimensional flow focusing geometry is achieved by casting a pair of PDMS slabs using two layer soft lithography, then mating the slabs together in a clamshell configuration. Complementary locking features molded into the PDMS slabs enable the accurate registration of features on each of the slab surfaces. Device testing demonstrates formation of double emulsions from 14 &#181;m to 50 &#181;m in diameter while using large channels that are robust against fouling and clogging.

Çift emülsiyon jeneratör 3D PDMS Fabrikasyon (Şekil 1A) kullanılarak oluşturulan bir eş eksenel akış odaklama aygıtın oluşur. Geometrisi üç fazlı koaksiyal jet oluşumunun su / yağ / su emülsiyonları, çift (Şekil 1B, Şekil 1C) oluşumuna izin veren, bir kare, x, 50 um menfez 50 um olarak kesilmiş olanak sağlar. İç sulu faz ve orta yağ fazı 10 um kanal boyutları olan bir birleşme kısmında bir araya getirilir...

Watch this Scientific Journal Video about Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device at JoVE.com

Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 &#181;m.

Bir Polidimetilsiloksan (PDMS) Koaksiyel Akış Odak Aygıtı Kullanma Çift Emülsiyon Üretimi

Double emulsions are useful in a number of biological and industrial applications in which it is important to have an aqueous carrier fluid. This paper ...

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Research

JoVE Journal

Bioengineering

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

15.9K Görüntüleme.  University of California, San Francisco. Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

Video: Double Emulsion Generation Using a Polydimethylsiloxane PDMS Co-axial Flow Focus Device

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Fizyolojik Akış Koşullarında Sirkülasyon Hücre Kemokin-yönettiği Ekstravazasyon Eğitim Bir Roman Üç boyutlu Akış Odası Cihazı

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

Biyomühendislik

PLGA Nanoparticles Formed by Single- or Double-emulsion with Vitamin E-TPGS

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular choice for drug delivery applications, particularly since it is already FDA-approved for use in humans in the form of resorbable sutures. Hydrophobic and hydrophilic drugs are encapsulated in PLGA particles via single- or double-emulsion. Briefly, the drug is dissolved with polymer or emulsified with polymer in an organic phase that is then emulsified with the aqueous phase. After the solvent has evaporated, particles are washed and collected via centrifugation for lyophilization and long term storage. PLGA degrades slowly via hydrolysis in aqueous environments, and encapsulated agents are released over a period of weeks to months. Although PLGA is a material that possesses many advantages for drug delivery, reproducible formation of nanoparticles can be challenging; considerable variability is introduced by the use of different equipment, reagents batch, and precise method of emulsification. Here, we describe in great detail the formation and characterization of microparticles and nanoparticles formed by single- or double-emulsion using the emulsifying agent vitamin E-TPGS. Particle morphology and size are determined with scanning electron microscopy (SEM). We provide representative SEM images for nanoparticles produced with varying emulsifier concentration, as well as examples of imaging artifacts and failed emulsifications. This protocol can be readily adapted to use alternative emulsifiers (e.g. poly(vinyl alcohol), PVA) or solvents (e.g.&#160;dichloromethane, DCM).

We describe the production and characterization of nanoparticles and microparticles composed of poly(lactic-co-glycolic acid) using vitamin E-TPGS as an emulsifier. By varying formulation parameters such as the concentration of emulsifier, it is possible to produce nanoparticles with mean diameters ranging from 220 nm to&#160;1.98 &#181;m.

Poly(lactic-co-glycolic acid) (PLGA) is a biocompatible member of the aliphatic polyester family of biodegradable polymers. PLGA has long been a popular ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

PDMS Zarında Micropatterned Hücreler germe

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Mikroakışkan jeti kullanarak çift katlı lipid Vesikül Üretimi

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

Bir polidimetilsiloksan İnce Film Kullanarak Otomatik çift katlı lipid Membran Oluşumu

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Sıvı özleri geleneksel sigara duman ve elektronik sigara Aerosol gelen nesil için aygıt mikroişlemci işletilen

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 &#181;m thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Bir Ultra-thin Polydimethylsiloxane mikrosıvısal çip su tutma ile kullanarak canlı Caenorhabditis elegans bireylerin immobilizasyon

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means ...

Generation of Transgenic Rats using a Lentiviral Vector Approach

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

Lentiviral Vektör Yaklaşımı Kullanarak Transgenik Sıçanların Üretimi

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is ...

Generation of Dynamical Environmental Conditions using a High-Throughput Microfluidic Device

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and organs are either highly expensive, like robotics, or lack nanoliter volume and millisecond time accuracy in liquid manipulation. We herein present the design and fabrication of a microfluidic system, which consists of 1,500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. To demonstrate the capacities of the microfluidic device, neural stem cell (NSC) spheres are maintained in the proposed system. We observed that when the NSC sphere is exposed to CXCL in day 1 and EGF in day 2, the round-shaped conformation is well maintained. Variation in the input order of 6 drugs causes morphological changes to the NSC sphere and the expression level representative marker for NSC stemness (i.e., Hes5 and Dcx). These results indicate that dynamic and complex environmental conditions have great effects on NSC differentiation and self-renewal, and the proposed microfluidic device is a suitable platform for high throughput studies on the complex life machinery.

We present a microfluidic system for high throughput studies on complex life machinery, which consists of 1500 culture units, an array of enhanced peristaltic pumps and an on-site mixing modulus. The microfluidic chip allows for the analysis of the highly complex and dynamic micro-environmental conditions in vivo.

Yüksek Verimli Mikroakışkan Cihaz Kullanarak Dinamik ÇevreSel Koşulların Üretilmesi

Mimicking in vivo environmental conditions is crucial for in vitro studies on complex life machinery. However, current techniques targeting live cells and ...

Contrast-Enhanced Subharmonic Aided Pressure Estimation (SHAPE) Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound imaging are gas-filled, encapsulated microbubbles (diameter &#60; 10 &#956;m) that traverse the entire vasculature and enhance signals by up to 30 dB. These microbubbles also produce nonlinear oscillations at frequencies ranging from the subharmonic (half of the transmit frequency) to higher harmonics. The subharmonic amplitude has an inverse linear relationship with the ambient hydrostatic pressure. Here an ultrasound system capable of performing real-time, subharmonic aided pressure estimation (SHAPE) is presented. During ultrasound contrast agent infusion, an algorithm for optimizing acoustic outputs is activated. Following this calibration, subharmonic microbubble signals (i.e., SHAPE) have the highest sensitivity to pressure changes and can be used to noninvasively quantify pressure. The utility of the SHAPE procedure for identifying portal hypertension in the liver is the emphasis here, but the technique has applicability across many clinical scenarios.

Contrast-Enhanced Subharmonic Aided Pressure Estimation SHAPE Using Ultrasound Imaging with a Focus on Identifying Portal Hypertension

A protocol for noninvasively estimating ambient pressures utilizing subharmonic ultrasound imaging of infused contrast microbubbles (following appropriate calibration) is described with examples from human patients with chronic liver disease.

Portal Hipertansiyonu Tanımlamaya Odaklanan Ultrason Görüntüleme Kullanarak Kontrastlı Subharmonik Yardımlı Basınç Tahmini (SHAPE)

Noninvasive, accurate measurement of pressures within the human body has long been an important but elusive clinical goal. Contrast agents for ultrasound ...