Nöron Hücre Kültürü için mikroakışkan Cihazlar hazırlanması 1) Temiz Corning No.1 24 mm X 40 mm cam slaytlar <ul><li> 30 dakika boyunca bir su banyosu sonikatör cam slaytlar sonikasyon </li><li> Cam slaytlar,% 70 EtOH ile durulayın </li><li> Yıkama dH 2 O ile üç kez slaytlar </li><li> Tercihen bir gecede, birkaç saat için bir doku kültürü davlumbaz, Kuru slaytlar </li></ul> Not: Biz özel metal tepsiler slaytlar yük var. Slaytları tek tek ayrılmış ve bu metal raf yuvalarına yerleştirilir. Daha sonra bir cam tabak, su, su banyosu sonikatör yer ile doldurun bu metal yükleme tepsisi yerleştirin ve 30 dakika sonikasyon. Sonraki yıkar bu yemeğin yürütülmektedir. 2) PDMS cihazları hazırlanması <ul><li> PDSM döküm ve çeyrek bu silikon gofret PDMS kalıp kesip, delgeç </li><li> PDMS cihazlar onları genelinde ilk ve inert gaz (argon veya Azot) üfleyerek temizleyin. Daha sonra kalan tüm enkaz kaldırmak için 3M Scotch Marka 471 bandı kullanın </li></ul> Not: cihazların yüzü yukarı tutmak için önemlidir. Başlangıçta, silikon gofret uzak PDMS kesildiğinde, gofret ile temas tarafında temiz tarafında: mikroakışkan kanallarını içerir. Biz biz de cihaza zarar verebilir ve plazma yapıştırılması için hazır olana kadar temiz yüzleri yetişmek için olabildiğince dikkatli olmaya çalışıyorum. <ul><li> Plastik kaplar Otoklav cihazlar </li><li> Otoklav, temiz, No: 1 Corning cam slaytlar ve harrick marka plazma temizleyici, plazma tedavi cihazları <a href="http://www.harrickplasma.com/" target="_blank">sonra</a> www.harrickplasma.com </li><li> Cihazları cam slayt tarafı aşağı temiz bir yerde. </li></ul> Cihaz şimdi cam slayt gümrüklü plazma. 3) Kaplama Poli L-Lizin (PLL) ile Cihazlar <ul><li> PLL cihazın her iki tarafında iyi eklenir ve bir sonraki kuyuya akmasına izin verilir </li></ul> =&gt; 150 ul büyük küçük kuyuları için kuyu ve 30 μ l PLL PLL. Bu sürece kuyuları tam olarak tam olarak bulunmamış. Not: Bir aygıt bakarsanız 4 kuyu göreceksiniz: her iki bağlanır. Siz de bu yüzden sonraki kuyuya cihaz üzerinden akacak bir PLL koymak istiyorum. <ul><li> PLL yaklaşık 10 dakika boyunca cihaz üzerinden akış ve daha sonra bunları doldurmak için kuyu daha PLL eklemek için izin ver </li><li> (Gece ​​tercih edilir) 37 ° C&#39;de en az 4 saat için bir kuluçka PLL içeren cihazlar yerleştirin </li><li> Tedavi sonrası aşırı PLL Vakum, ancak cihazın tüm sıvı emmek için dikkatli olun </li></ul> Not: kanalları ya da odasına hava kabarcıkları tanıtmak istemiyorum. Sadece kuyularda aşırı PLL vakum. <ul><li> Cihazın her iki tarafında her bir kuyu için otoklava dH 2 O ve kılcal eylem diğer iyi akmasına izin </li></ul> Şekil 1 mikroakışkan bir cihazın bir diyagram. Mavi (Soma tarafı) nasıl bağlandığına dikkat edin ve Kızıl (Aksonal tarafı) bağlanır. Cihazın her iki tarafında iyi bir PLL, ya su ya da medya, demekle, ne demek istediğimizi, örneğin: Yeri otoklava dH biri Blue Peki o yerde 150 ul otoklava dH 2 O 150 ul Peki bir Kırmızı içine 2 0. Su (ya da PLL, Medya, veya Hücreler) cihaz üzerinden diğer ilgili iyi akacaktır. <img src="/files/ftp_upload/old/410_2008_8_27_3/410_Figure-01.png" alt="Şekil 1" width="362" height="216" /> Şekil 1 Not: Lütfen artık, diyorum &quot;TOP kuyularda medya, hücreleri, su veya PLL&quot;, Soma, soldaki nerede olurdu Yukarıdaki diyagram ve Aksonlar kastediyorum, not büyüyecek hakkını saklı tutar. <ul><li> Su (daha dikkatli olmak tam olarak tüm sıvı cihazdan çıkarmak için değil) aspire, sonra otoklava dH 2 O başka bir 150 ul de bağlı her birine ekleyin ve iyi karşılık akmasına izin verin . </li><li> 37 ° dH 2 O içeren bir kuluçka cihaz yerleştirin ° C 1 saat sonra tekrar yıkama adımı tekrarlayın. DH 2 O cihazları, üç kez yıkayın toplam bir saat, her biri . </li></ul> Not: PLL borat Jeon Lab i bir çift sonra otoklava dH 2 O gecede cihazlar kuluçka tavsiye PDMS emebilir olasılığı nedeniylenitial hızlı durular. Bu, tüm serbest borat önce hücre yükleme PDMS liç olacağını garanti eder. Bu yapılmadığı takdirde, medya içine borat bir açıklaması, sitotoksisiteye neden olabilir zaman içinde olabilir. <ul><li> Üst kuyuları için gerekli faktörleri (glutamax, B27, PenStrep) Sinir Bazal Ortam (NBM) </li></ul> Not: cihazlar inkübe, gece boyunca ve ertesi gün hücreleri ile kaplı veya hücreleri kaplama önce NMB + faktörler ile en az 3 saat inkübe edilebilir. Nöronlar Into Aygıtlar yüklenmesi için hazırlanması Biz 18 gün ya Beyin Bit kampüs ya da bizim için burada hazırlanan adında bir şirket satın eski fetal sıçan korteks kullanın. Biz taze doku almak için tercih ederler. <ol><li> Hazırda Bekleme E saklanan 1 fetal beynin korteks (2 adet doku) edinmek </li><li> Alkol lamba kullanarak 3 uzun cam pipetler alın ve gittikçe daha küçük boyutlarda her yangın </li><li> NMB + Faktörler 2 ml 15 ml steril bir tüp içinde bir cam pipet ve yer Hazırda Bekleme E dokuların çıkarın. </li><li> Öğütme: korteks yaklaşık 5 ile 10 kat aşağı yukarı geçti ve bir ampul ve cam pipet kullanarak . Ardından, sonraki küçük pipet doku aşağı yukarı pipet ve kullanılır. Son olarak, küçük pipet doku aşağı yukarı pipet ve kullanılır. Biz hiçbir görünür parçaları olmadığından emin olmak istiyorum. Not: Son zamanlarda, Jeon Lab, başlangıçta% 50,% 0.125 tripsin ile tedavi Doku hazırlanan hücreler Hazırda Bekleme E saklanan doku hücreleri karşılaştırarak başladı Ca + + ve Mg + + ücretsiz diseksiyonu tampon. Konsensus tripsin verim doku hemen kullanmak için DEĞİLDİR Hazırda Bekleme E. başlangıçta yerleştirilen doku daha canlı hücreler kullanarak hazırlanan doku, daha sonra Hazırda Bekleme E. doku saklamak gerekir Eğer hemen doku kullanmak için gidiyoruz vardır ve artan canlılığı istiyorsanız Eğer aşağıdaki gibi, daha sonra mekanik öğütme önce tripsin ile doku tedavisi: 1) 1 ml Ca + + ücretsiz Mg + + ücretsiz 1 ml% 0.25 tripsin (Gibco, Invitrogen) sulandırmak diseksiyonu tampon içine 15 ml tüp (buz tutmaya) (son tripsin =% 0.125), 2) yer tampon doku ve 8 dakika 37 ° C az hemen inkübe 3) durdurmak için 10 ml% DMEM/10 FBS eklemek tripsin reaksiyon ve aşağıda protokol devam. </li><li> 1 dakika için 1100 rpm&#39;de Santrifüj hücreleri </li><li> Kapalı hücre pelletini dikkatle aspire medya </li><li> NMB + faktörler kadar pipetleme pelet ve yeniden askıya 1 ml ve aşağı </li><li> (BD Falcon hücre süzgecinden 40 um naylon) herhangi bir kümeleri kaldırmak için bir filtre üzerinden yerçekimi akışı ile yeniden askıya hücreleri Pass </li><li> Sayımı ve plaka hücreleri: <ul><li> Bir mikrofuge&#39;de tüpe, 60 ul NMB + faktörler, hücre süspansiyonu ul, 20 ve 20 ul tripan mavi karışımı </li><li> Bu çözüm yaklaşık 10 ul bir hemasitometre ekleyin ve canlı hücrelerin sayısı (mavi) </li><li> Konsantrasyonu 2.5 ayarlama - 4,5 x10 6 hücre / ml Not: Biz genellikle yaklaşık 2,5 &#39;lik bir konsantrasyon elde - 4.5 x 106 hücre / ml. hacmine bağlı olarak hücrelerin (normalde 1 ml NBM + B27/Glutamax/PenStrep percortex) yeniden süspanse. Doku tripsin ile hazırlanmış ise, verim muhtemelen artacaktır. </li></ul></li></ol> Cihazlar Kaplama <ol><li> Birine cihazın Yük hücreleri (primer nöronlar) </li><li> Plaka küçük bir cihaz başına 5 ul ve büyük cihaz başına 20 ul Not: cihazın üst ve alt cihazın (yarım toplam hacmi) 10 ul 10 ul hücre yükü, ya da üst warfarin iyi hücrelerin doğrudan 20 ul (5 ul de üst warfarin Küçük cihazlar için). </li><li> 10 dakika 37 ° C inkübatör inkübe hücreleri eklemek başlayabilirsiniz </li><li> NMB + faktörlerin iyi cihaz boyutuna bağlı olarak her biri için yaklaşık 150 / 30 ul Not: Hacimler PDMS kalınlığına bağlı olarak değişebilir. Tüm bu konularda kuyu tam olmasıdır. </li></ol> Plazma Yapıştırma olmadan Cihazlar Açık Ek Plazma tedavisi olmadan, plazma olmaksızın her hafta başarıyla Christina Tu, bir teknisyen, sadece hücreler hemen yüklemek için söylüyor. Soma tarafında iki kuyu (medya bırakırsanız akson tarafı önemli değil) medya kaldırmak unutmayın. Bu hücrelerin ana kanal girmek için izin sıvı akış. Kuyuların medya varsa, sıvı akışını almazsınız.

<ol>
	<li>Park, J. W., Vahidi, B., Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nat Protoc. 1 (4), 2128-2136 (2006).</li><li>Taylor, A. M., Blurton-Jones, M., Rhee, S. W., Cribbs, D. H., Cotman, C. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nat Methods. 2 (8), 599-605 (2005).</li></ol>

The authors have nothing to disclose.

Burada plazma temizleyici gerek kalmadan nöron mikroakışkan cihaz nasıl kullanılacağını gösterdi. Biz bu gibi non-plazma bağı bakın.

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>PDMS</td>
<td>Reagent</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>Sylgard 184 with curing agent Please consult Dow Corning to find a vendor near you</td>
</tr>
<tr>
<td>Cornig No. 1 Cover Glass</td>
<td>Cover Glass</td>
<td>Corning</td>
<td>Corning No. X2935 244</td>
<td>Available through Fisher Scientific, Fish Catalog number 12-531D</td>
</tr>
<tr>
<td>B27</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>17504-044</td>
<td>B27 is a proprietary supplement available through Invitrogen under their Gibco line of cell culture reagents.</td>
</tr>
<tr>
<td>Glutamax</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>35050-061</td>
<td>Glutamax is available through Invitrogen under their Gibco line of celll culture reagents.</td>
</tr>
<tr>
<td>60 mm Petri Dish</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>08-757-13A</td>
<td>60 mm polystyrene sterile petri dishes are used to house the device bound to glass.</td>
</tr>
<tr>
<td>Poly-L-Lysine</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-1274</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon 50 ml Tube</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>Falcon No. 352098</td>
<td>Available through Fisher Scientific catalog number 14-959-49A</td>
</tr>
<tr>
<td>BD Falcon 15 ml tube</td>
<td>Tool</td>
<td>BD Falcon</td>
<td>Falcon No. 352097</td>
<td>Available through Fisher Scientific catalog number 14-959-70C</td>
</tr>		</tbody>
		</table>
		</div>
			<div>
					</div>

non-plasma bonding of pdms for inexpensive fabrication of microfluidic devices

Bu video, plazma bağı olmayan nöron mikroakışkan cihazın nasıl kullanılacağını göstermektedir. Bazı durumlarda Corning No 1 kapak cam geri dönüşümlü bond cihazlar istenebilir. Bu plazma temizleyici kullanılabilir değil, belki de bağlı olabilir. Diğer durumlarda, bu nöronlar cihaz lekeli olabilir çünkü immün aygıtı kaldırmak için gerekli olmasa da, mikroskopi veya immün için belirli türleri için kültür nöronların sonra cam aygıtı kaldırmak için arzu olabilir. Bazı araştırmacılar, ancak hala aygıtı kaldırmak için tercih ediyor. Bu durumda, cihazın kapağı cam reversibl bağlanma mümkün kılar. Olmayan, bir mühür gibi sıkı değil oluşur cihazların plazma bağı bazı dezavantajları vardır. Bazı durumlarda aksonlar ziyade onların aracılığıyla oluk altında büyür. Ayrıca, cam ve PDMS hidrofobik olduğu için, sıvıların kolaylıkla medya ve diğer reaktiflerin cihazın içine zorlamak için zaman zaman gerekli cihaz girmek yok. Sıvılar cihaz kapiller eylem yoluyla girecek ama önemli ölçüde daha uzun plazma gümrüklü cihazlara kıyasla sürüyor. Plazma temizleyici, cam ve cihaz sonra cihaz üzerinden saniyeler içinde yapıştırma sıvıların akışını kolaylaştırmak geçici hidrofilik ücretleri oluşturur. Cihazları ile sıvı akış non-plazma bağlı cihazlar için, birkaç dakika sürer. Cihazları plazma temizleyici sterilize edecektir, çünkü plazma tedavi cihazlar kullanımdan önce sterilize edilecek gerekmez iken, önce sterilize edilecek bağ olmayan plazma ile kullanılmak üzere olduğunu da not etmek önemlidir.

Watch this Scientific Journal Video about Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices at JoVE.com

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

Bu video plazma bağı olmayan nöron mikroakışkan cihazın nasıl kullanılacağını göstermektedir.

Ucuz mikroakışkan Cihazlar İmalat PDMS Non-plazma Yapışma

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

non-plasma-bonding-pdms-for-inexpensive-fabrication-microfluidic

Research

JoVE Journal

Biology

34.2K Görüntüleme.  University of California, Irvine (UCI). Bu video plazma bağı olmayan nöron mikroakışkan cihazın nasıl kullanılacağını göstermektedir.

Video: Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

Nöron Soma ve Aksonlar compartmentalization mikroakışkan Aygıt Fabrikasyon

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Biyoloji

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

Hücre Biyolojisi Degrade üreten mikroakışkan Aygıt

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

PDMS Device Fabrication and Surface Modification

PDMS Cihaz İmalat ve Yüzey Modifikasyonu

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

Termoplastik mikroakışkan Kanallar imalatı

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

Microfluidic Applications for Disposable Diagnostics

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new diagnostics.

Tek kullanımlık Diagnostics mikroakışkan Uygulamaları

In this interview, Dr. Klapperich discusses the fabrication of thermoplastic microfluidic devices and their application for development of new ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Amperometrik Elektrotlar imalatı

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

MİYOJEN Engineered Doku oluşturur imalatı

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

Mikroakışkan Cihazlar Pasif pompalanması için Yüksek Hızlı Damlacık tabanlı Dağıtım Sistemi

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

Automated Microfluidic Blood Lysis Protocol for Enrichment of Circulating Nucleated Cells

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and endothelial cells, provide an ideal platform for an updated status on the immune condition of an individual. The microfluidic protocol allows for enrichment of CNCs without selective cell loss and sample preparation variability due to user-mediated steps. Briefly, the protocol includes device fabrication, sample collection, device setup, and running blood through the microfluidic chamber. Within the device whole blood is rapidly mixed with deionized water for approximately 10 seconds in a 50 micron x 150 micron microfluidic channel. In this time span erythrocytes are lysed due to hypotonic conditions. Herringbone structures on the bottom of the channel ensure thorough mixing and exposure of cells to a constant environment. Remaining cells are returned to isotonic conditions at the exit of the device, fixed using 2% paraformaldehyde, centrifuged to separate erythrocyte debris from CNCs, and suspended in flow buffer for staining and analysis by flow cytometry. Results show clean flow cytometry scatter plots with CNC populations saved. Significance of this device and protocol comes in the study and understanding of disease pathogenesis by analysis of CNC populations. Hence, automation, effectiveness, and simplicity of the microfluidic protocol are demonstrated.

An automated microfluidic device was developed for circulating nucleated cell enrichment from peripheral blood via erythrocyte lysis that ensures isolation of high quality sample without cell loss.

Çekirdekli Hücreler Sirkülasyon zenginleştirilmesi için Otomatik mikroakışkan Kan Lizis Protokolü

In this report the protocol for an automated microfluidic blood lysis device is detailed. Circulating nucleated cells (CNCs), including leukocytes and ...

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures 

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. The hypoxic chamber offers a simple system to control oxygenation in standard culture vessels, but lacks precise temporal and spatial control over the oxygen concentration at the cell surface, preventing its application in studying a variety of physiological phenomena. Other systems have improved upon the hypoxic chamber, but require specialized knowledge and equipment for their operation, making them intimidating for the average researcher. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. The device is simple to use and is connected to gas cylinders that provide the pressure to introduce the desired oxygen concentration into the platform. Fabrication involves a combination of standard SU-8 photolithography, replica molding, and defined PDMS spinning on a silicon wafer. The components of the device are bonded after surface treatment using a hand-held plasma system. Validation is accomplished with a planar fluorescent oxygen sensor. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The device can be sterilized for cell culture using common methods without loss of function. The device's applicability to studying the in vitro wound healing response will be demonstrated.

Fabrication and Operation of an Oxygen Insert for Adherent Cellular Cultures

Fabrication and validation of an add-on platform that offers enhanced control over the spatial and temporal oxygenation in a 6-well plate. The device is adaptable to a number of culture systems and can be used to investigate the effects of oxygen on wound healing.

İmalat ve Operasyon yapışkan Hücresel Kültürler Oksijen Ekle

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical ...