Kind gift of THP-1 cells from Dr. Mark Chong and assistance in microfabrication from Dr. Yuejun Kang and Dr. Nishanth V. Menon (School of Chemical and Biomedical Engineering, Nanyang Technological University) were greatly acknowledged. This project was funded by NTU-Northwestern Institute of Nanomedicine (Nanyang Technological University). H.W.H. was supported by Lee Kong Chian School of Medicine (LKCMedicine) postdoctoral fellowship.

Mezenkimal Kök Hücre ve Monositler 1. Nanopartiküller (NPS) Etiketleme <ol><li> Dulbecco tadil edilmiş Eagle ortamı (DMEM) içinde kültür mezenkimal kök hücreler (MSC&#39;ler) Önceki etiketleme ≥80% konfluansa kadar% 10 cenin sığır serumu (FBS) ve antibiyotikler ile takviye edilmiştir. Benzer şekilde, Roswell Park Memorial Institute (RPMI) kültür, THP-1 hücreleri (ATCC), 1640 ortamında ~ 10 6 hücre / ml bir yoğunluğa,% 10 FBS ile takviye edilmiştir. </li><li> Yük silis NPS (~ 500 mikron) bir gecede karıştırma kullanarak kalsein boya çözeltisi (200 uM) ile. Daha önce açıklanan protokol kullanılarak PLGA-kalsein AM (CAM) Üretiyor. 22 <ol><li> 250 ug CAM ve 4 ° C&#39;de, kloroform içinde 100 mg PLGA (50:50) içinde çözülür. </li><li> Oda sıcaklığında yüksek hızlı bir homojenleştirici (13600 xg, 60 sn) kullanarak tek emülsiyon NPs oluşturun. (Çift DISTI, (3400 xg 5 dakika boyunca) santrifüj kullanılarak yıkama toplamadan önce bir kimyasal kaput (≥3 saat) kloroform buharlaşmasılled su), dondurarak kurutma ve saklama -20 ° C. </li></ol></li><li> 20 dakika - Cam-PLGA NP (1 mg) ya da 15, oda sıcaklığında,% 0.01 poli-L-lisin (PLL), çözelti içinde kalsein silika NPs (150 ug) inkübe edin. </li><li> 5 dakika boyunca 3400 x g&#39;de santrifüje ilgili kültür ortamının 1 ml NPS yeniden süspanse önce aşırı PLL süpernatan. </li><li> Yaklaşık 24 saat (0.1 mg · mi -1 markalama konsantrasyon) - hücreleri (toplam 2 x 10 6 hücre MSC veya THP-1, ~ 1), NPs inkübe edin. </li><li> Ayrıştırmaları ve% 0.25 tripsin (5 dakika, 37 ° C), 2 ml kullanılarak işaretli yapışma MKH&#39;lerin hasat ve DMEM 6 ml ile söndürün. Mikroakışkan oluşu 10 6 hücre / ml - 10 5 bir konsantrasyona hücreleri (1000 xg, 4 dakika) ve tekrar süspansiyon dönerler. Şirketinden Mikroakiskan saflaştırma için - (10 6 hücre / ml 10 5) etiketli, THP-1 hücreleri kullanır. </li></ol> 2. Mikroakışkan Cihaz Hazırlık <ol><li> cihaz İmalatı <ol><li> Mikroakışkan spiral cihazı Üretiyor (500 mikron 115 mikron (h) x (w)) standart yumuşak litografi adımları kullanarak bir ticari kit gelen polidimetilsiloksan (PDMS) ile. 23 </li><li> Baz prepolimerin 30 g ve tartı tekne iyice sertleştirici 3 g karıştırın. Gazlardan, 60 dakika boyunca bir kurutucuda karışımı herhangi bir hava kabarcıklarını çıkarmak için. </li><li> 10 mm - dikkatlice ~ 5 yüksekliğe sarmal kanal tasarımı ile desenli silikon gofret ana kalıp üzerine PDMS karışımı dökün. </li><li> De-gaz 60 dakika boyunca bir kurutucu vakum karışım tekrar herhangi bir hava kabarcıklarını çıkarmak için. tüm kabarcıklar giderilinceye kadar işlemi tekrarlayın. </li><li> PDMS ayarlanana kadar 2 saat için bir 80 ° C fırın içinde PDMS karışımı Cure. gofret sabit cihaz yüksekliği için kür sırasında eğik olmadığından emin olun. </li><li> Bir neşter kullanılarak PDMS spiral cihazı kesin ve dikkatli bir şekilde ana kalıp PDMS levha soyun. </li><li> Trbağlanma için düzgün bir yüzey sağlamak için bir neşter ile cihazın kenarları im. </li><li> ağızlığı için iki delik (1,5 mm) ve 1.5 mm biyopsi zımba kullanarak PDMS cihazda çıkış için iki delik (1,5 mm) yumruk. </li><li> 5 dakika boyunca 80 ° C fırın içinde cihazı kalıntıları çıkarmak ve kuruması için izopropanol (IPA) ile cihaz yıkanır. </li><li> maskeleme bandı kullanarak (kanal özellikleri ile) PDMS cihazın alt yüzeyini temizleyin. </li><li> maskeleme bandı kullanarak ( &quot;3 ile&quot; 2) cam slayt temiz bir tarafı. </li><li> Dikkatle yerleştirin ve bir plazma temizleyici odasında PDMS cihaz ve cam slayt temizlenmiş yüzeyler ortaya çıkarmak ve 60 saniye boyunca vakum onları tabi. Sonraki maksimum plazma gücünü açma ve oda rengi pembe açılana kadar kamara basıncını düşürmek. <ol><li> 60 saniye boyunca hava plazmaya yüzeyleri Açığa. fizik içine sokulduğunda, plazma sıkı bağlama sağlar PDMS ve cam maruz kalan yüzeylerde reaktif türler oluşturanal temas. Plazma gücünü kapatın ve cihazı ve cam slayt almak için plazma temizleyici basıncı serbest bırakın. </li></ol></li><li> sıkıca plazma maruz kalan yüzeyleri basarak ve hiçbir kabarcıklar iki yüzey arasında sıkışıp sağlayarak bir araya PDMS cihaz ve cam slayt Bond. </li><li> bağlama güçlendirmek için 2 saat boyunca 80 ° C&#39;de ayarlanmış bir ocak gözünü kullanılarak bağlanmış cihazı ısıtın. </li></ol></li><li> cihazı Çalıştırma <ol><li> giriş şırınga için 20 cm ve her hortumun bir ucunda bir şırınga ucunu (gösterge 23) takmak - ~ 15 borusunun iki adet (1.52 mm OD) kesin. </li><li> çıkışları için 10 cm ve PDMS cihazın çıkış delikleri eklemek - ~ 5 boru, iki adet (1.52 mm OD) kesin. </li><li> o çıkış boru akana kadar elle başbakan,% 70 etanol içeren bir şırınga ile cihazı çalışan örnek önce. Cihazı sterilize etmek için, 1 dakika 30 saniye için etanol oturmaya bırakın. </li><li> süzüldü ve 30 ml yük(0.2 um gözenek) kılıf tamponu, 60 ml şırıngaya (% 0.1 Sığır Serum Albumin (BSA) ile fosfat tamponlu tuz (PBS)) ve bir şırınga pompası şırınga sabitleyin. Doğru ayarlar için pompayı (60 ml, Cilt: 60.000 ul Şırınga boyutunu) ayarlayın. Not: PBS BSA ilave hücre-hücre bağlanması ve hücre ve PDMS cihaz arasında spesifik olmayan bağlanmayı en aza indirmektir. </li><li> Yük 3 ml şırınga içine işaretli hücrelerin 3 ml ve ayrı bir şırınga pompası şırınga güvenli. Doğru ayarlar için pompayı (: 3 ml, Cilt: 3.000 ul Şırınga boyutunu) ayarlayın. </li><li> hava kabarcıkları istikrarlı akışını sağlamak için şırınga içinde sıkışıp kalırlar emin olun. yavaşça memeden dışarı sıvı birkaç damla püskürtülmesiyle herhangi bir hava kabarcıklarını çıkarın. </li><li> şırınga giriş şırınga ipuçları ve tüp bağlayın ve cihazın (kılıf ve örnek giriş) ilgili girişler içine yerleştirin. boru boyunca hava kabarcığı olmadığından emin olun. </li><li> Bir invert üzerine cihazları montehücre sıralama işlemi sırasında gerçek zamanlı görüntüleme için ed faz-kontrast mikroskop. </li><li> yapıştırıcılar kullanılarak mikroskop sahnede cihaza yakın küçük bir atık beher ve iki 15 ml tüpler sabitleyin. </li><li> atık behere çıkış borulama yerleştirin. </li><li> 1:10 kılıf tamponuna numune akış oranını ayarlamak ve sıralama işlemini başlatmak için her iki şırınga pompaları başlar (Örnek: 120 ul / dk örnek şırınga ve 1200 ul / dak kılıf şırınga için, kanal akış hızı ~ 0.38 m / sn ). </li><li> Akış hızı stabilize etmek için 1.5 dakika süreyle cihazı çalıştırın. Bu yüksek hız kamerasını kullanarak faz kontrast ile parlak alan altında kanal iç duvarına yakın atalet odaklı hücrelerinin varlığı ile teyit edilebilir (~ 5000 - Saniyede 10.000 kare (fps), pozlama süresi: 10-50 mikro-sn). </li><li> Hücre çıkışına ve atık çıkışından yürütücüler toplamak için farklı tüpler içine çıkış borulama yerleştirin. </li></ol></li></ol>

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Yazarlar ifşa hiçbir şey yok.

Burada tarif edilen DFF hücre saflaştırma tekniği, yüksek verimli bir şekilde etiketlenmiş hücrelerin hızlı ve sürekli ayırma sağlar. Bu ayırma yaklaşımı büyük örneklem hacmi veya yüksek hücre konsantrasyonu numune işleme için idealdir ve uzun süreli kullanım sonrasında tıkanmaya meyilli olan klasik membran bazlı filtreleme daha iyidir. Benzer bir şekilde, afinite bazlı manyetik ayırma zahmetli ve pahalı ek hücre etiketleme adımları gerektirir. Saflaştırılmış hücreleri (kanal i?...

Ajan yüklü mikro / nano partiküller (NPS) hücreleri mühendislik bio yeteneği geliştirmek ve rejeneratif tıpta doğal tedavi edici özelliklere ek / artırmak için basit, genomik entegrasyon-free, ve çok yönlü bir yöntemdir. 1-3 Hücresel değişiklikler etiketleyerek elde edilir ajan yüklü NPS aşırı konsantrasyon ile plazma zarı ya da sitoplazma bağlayıcı siteleri doyurmak için. Bununla birlikte, bu yöntemin önemli bir sakıncası dönüştürücü ihtiva eden NP önemli potansiyel partikül işlenmiş hücrelerde tam kimlik bulandırabilir veya terapötik sonuçlar karmaşık hale getirmektedir. 4,5 Ayrıca hücre etiketleme işlemleri sonrası çözeltide kalan bağlanmamış parçacık miktarları, maruz olduğu aşırı derecede yüksek bir konsantrasyonda maddeler (büyüme faktörleri, kortikosteroidler, vs.) hedef olmayan hücreler üzerinde istenmeyen sonuçları indükleyebilir sitotoksisitesinin ve yanlış yönlendirilmiş riskini ortaya çıkarmaktadır. o içeren parçacıklı bir taşıyıcıf &quot;biyolojik olarak uyumlu&quot; malzemeler [örneğin, poli (laktik ko-glikolik asit), PLGA] güçlü bir bağışıklık hücresi, belirli koşullar altında yanıtlar da teşvik edebilir. 6 Bu bağışıklığı (örn, romatizmal artrit), potansiyel olarak gecikmeler olan kişilerde özellikle riskli sistemik nanopartikül izni. 7 Dolayısıyla, önceki parçacık işlenmiş hücrelerde giriş için serbest partiküllerin randımanlı bir şekilde çıkarılması toksisite profilini en aza indirmek ve in vivo madde yüklü parçacıklara yönlendirilmiş maruz kalmayı azaltmak için büyük bir önem taşımaktadır. Konvansiyonel gradient santrifüj genellikle ücretsiz parçacıklardan mühendislik hücreleri ayırmak için kullanılan, ancak zahmetli ve toplu modunda çalıştırılır. Ayrıca, yüksek hızda santrifüj sırasında hücreler ve hücre bütünlüğü ve / veya etkisi hücre davranışını bozabilir yoğunluk gradyan ortamının bileşenleri yaşadığı kayma gerilmeleri. 8 Mikroakiskan sev ile cazip bir alternatifdeterministik yanal deplasman (Anti Blokaj Sistemi) 9, 10,11 dieletrophoresis ve küçük partiküllerin ayrılması ve tampon değişimi uygulamaları için geliştirilmiş 12 acoustophoresis dahil taraflı ayırma teknolojileri. Ancak, bu yöntemler düşük verim muzdarip (1-10 ul · dk - 1) ve sorunları tıkanma yatkındır. Böyle Dielektroforez tabanlı yöntemler olarak aktif ayrımları da içsel dielektroforetik hücre fenotipleri veya ayrılmasını sağlamak için ek hücre etiketleme adımlarla farklılıkları gerektirir. Karşısında parçacıkların veya hücrelerin yan göç nedeniyle yüksek akış koşulları ve üstün boyut çözünürlüğü nedeniyle yüksek Reynolds sayısı (Re) 13 baskın asansör güçleri (F L) farklı pozisyonlarda odaklanmak düzene - Daha umut verici bir yaklaşım eylemsiz mikroakışkanları içerir. , genellikle boyut temelli hücre ayırma 14,15 ve de tampon değişimi uygulamaları için istifade edilmiştir. 16-18 Howeveayrılan hücreler genellikle özgün ve yeni tampon çözeltileri arasındaki sınıra yakın kaldığı sürece r, tampon değişimi performansı ~10-30% kirletici solüsyonu ile yetersiz kalmaktadır. 16-18 Daha da önemlisi, hedef hücrelerin boyut dağılımı elde etmek için benzer olmak zorundadır özellikle mezenkimal kök hücreleri gibi heterojen boyutlu hücre tipleri (MKH) işlenmesinde bir sorunu teşkil orijinal tampon çözeltisinden hassas odaklama atalet ve ayırma. 19 Biz daha önce, yeni bir eylemsizlik Mikroakiskan hücre sıralama tekniği 2-giriş, 2-çıkış spiral mikro cihazı kullanılarak dolaşımdaki tümör hücrelerinin (KTC) tam kandan 20 ve bakteriler 21 izole etmek için Dean akış ayırma (DFF) olarak adlandırılan geliştirdik. Bu video protokolü, biz etiketleme THP-1 (insan akut monositik lösemi hücre hattı) süspansiyonu monositik hücreleri (~ 15 mikron) ve kalsein-l MKH (10-30 mikron) sürecini anlatacağızoaded NPler, işaretlenmiş hücreleri ve bağlanmamış NP kaldırılması verimli geri kazanımı için DFF sarmal mikrocihazda imalat ve işlem takip eder. 22. Bu tek aşamalı saflaştırma stratejisi etiketli süspansiyon ve santrifüj olmadan, taze tampon çözelti içinde süspanse yapışan hücrelerin sürekli bir iyileşme sağlar. Ayrıca, 10 milyon hücreleri · ml -1, bir hücre yoğunluğu rejeneratif tıp uygulamalarında mükellef kadar işleyebilir.

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			<td height="21" style="height:21px;width:368px;">Scotch tape</td>
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microfluidic buffer exchange for interference-free micro/nanoparticle cell engineering

Aktif-madde yüklü mikro / nano partiküller ile mühendislik hücreleri (NPS), yerli terapötik özellikleri geliştirmek biyo görüntüleme sağlamak ve hücre fenotipi kontrol etmek için giderek daha popüler bir yöntem haline geliyor. Kritik henüz yetersiz giderilen sorun kolayca geleneksel santrifüj kaldırılamaz hücre etiketleme sonra ilişkisiz kalır parçacıkların önemli bir sayıdır. Biyo görüntüleme plan gürültüsünde bir artışa yol açar ve komşu olmayan hedef hücreler üzerinde dönüştürücü etkisi kazandırabilir. Bu protokol, biz verimli bir yüksek verimli bir şekilde serbest NP etiketli hücreleri ayırmak için Dean Akış Fraksiyonu (DFF) olarak adlandırılan bir atalet Mikroakiskan tabanlı tampon değişimi stratejisi sunuyoruz. Bağlanmamış bir floresan boya veya boya yüklü NP (SIL&gt;% 95 azalmasını elde ederken geliştirilmiş sarmal mikrovasıta, yeni bir tampon çözeltisi içinde süspansiyon haline saflaştınldı hücrelerinden (THP-1 ve MSC&#39;ler) sürekli toplama (&gt;% 90 hücre geri kazanım) kolaylaştırırica veya PLGA). Bu tek aşamalı, boyut tabanlı hücre arıtma stratejisi yüksek hücre işleme verimi (10 6 hücre / dak) sağlayan ve parazitsiz klinik uygulama sağlamak için mikro / nanoparçacık mühendislik hücrelerin büyük hacimli hücre arıtma için son derece yararlıdır.

Gece boyunca biyo görüntüleme maddesi yüklenmiş NP ile hücrelerin etiketlenmesi sonra etiketli hücreler (serbest partikülleri içeren) toplandı ve bir tek aşamalı işlem, (Şekil 1A) serbest NPs kaldırma DFF sarmal mikrocihazda ile saflaştırılmıştır. 2-giriş, 2-çıkış spiral mikrokanal SU-8 fotorezist kullanarak mühendislik yazılımları tarafından tasarlanan ve mikrofabrike edilir. Desenli silikon yonga plâkası sonra yumuşak litografi teknikl...

Watch this Scientific Journal Video about Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering at JoVE.com

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Bu protokol, bağlanmamış partiküllerin randımanlı bir şekilde azalması olan mikro / nanopartikül mühendislik hücrelerinin saflaştırılması için bir atalet Mikroakiskan tabanlı tampon değişimi stratejisi kullanımını tarif eder.

Parazit Mikro / Nanoparçacık Hücre Mühendisliği mikroakışkan Tampon Değişim

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

microfluidic-buffer-exchange-for-interference-free-micronanoparticle

Research

JoVE Journal

Bioengineering

9.0K Görüntüleme.  Nanyang Technological University.  Bu protokol, bağlanmamış partiküllerin randımanlı bir şekilde azalması olan mikro / nanopartikül mühendislik hücrelerinin saflaştırılması için bir atalet Mikroakiskan tabanlı tampon değişimi stratejisi kullanımını tarif eder. 

Video: Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

Siteye özgü Bakteriyel Kromozom Mühendislik: ΦC31 integraz Aracılı Kaset Borsası (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Biyomühendislik

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

Protein Katlama Öğrenmenin mikroakışkan Mikserler

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

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 ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Sağlam, mikroakışkan Hücre içi Delivery Platform olarak hücre Sıkma

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Mikroakışkan Tekniği Hücre deformabilitesi Probe

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Controlled Microfluidic Environment for Dynamic Investigation of Red Blood Cell Aggregation

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white blood cells and platelets that are suspended in blood plasma. Due to the abundance of the RBCs (40% to 45% of the blood volume), their behavior dictates the rheological behavior of blood especially in the microcirculation. At very low shear rates, RBCs are seen to assemble and form entities called aggregates, which causes the non-Newtonian behavior of blood. It is important to understand the conditions of the aggregates formation to comprehend the blood rheology in microcirculation. The protocol described here details the experimental procedure to determine quantitatively the RBC aggregates in microcirculation under constant shear rate, based on image processing. For this purpose, RBC-suspensions are tested and analyzed in 120 x 60 &#181;m poly-dimethyl-siloxane (PDMS) microchannels. The RBC-suspensions are entrained using a second fluid in order to obtain a linear velocity profile within the blood layer and thus achieve a wide range of constant shear rates. The shear rate is determined using a micro Particle Image Velocimetry (&#181;PIV) system, while RBC aggregates are visualized using a high speed camera. The videos captured of the RBC aggregates are analyzed using image processing techniques in order to determine the aggregate sizes based on the images intensities.

The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Kırmızı Kan Hücresi Toplama Dinamik Soruşturma Kontrollü mikroakışkan Çevre

Blood, as a non-Newtonian biofluid, represents the focus of numerous studies in the hemorheology field. Blood constituents include red blood cells, white ...

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 ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Yüksek verim Tek hücreli İzolasyonu ve Kültür mikroakışkan Platformu

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

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 ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.