Bu çalışma, JLI ve National Institutes Heath hibe R01AR056624 bir Amerikan Kalp Derneği predoctoral Bursu ile desteklenmiştir.

A. Tek Polimer elektrospinning <ol><li> Elektrospinning çözüm hazırlarken önce, birkaç gün boyunca 37 ° C eriterek photoinitiator 0.5 ağırlıkça% çözüm Irgacure 2959 (I2959), deiyonize su. Bu adım, bir photoreactive polimer kullanılıyor ise gerekli değildir. </li><li> 2 ağırlıkça% Meha, 3 ağırlıkça% nihai bir konsantrasyon ile bir çözüm hazırlamak için methacrylated hyaluronik asit (Meha, Burdick ve ark. Sentezi için), poli (etilen oksit) (PEO, 900 kDa) ve deiyonize su I2959 birleştirin PEO, ve 0.05 ağırlıkça% I2959. Netleşene kadar çözüm karıştırmak için bir girdap kullanın. Polimer tipi ve konsantrasyonu, kullanılan solvent gibi, bu aşamada istenilen iskele özellikleri bağlı olarak değiştirilebilir. </li><li> Çözümün bir şırınga içine aktarın ve sonuna kadar 18 gauge, 6 inç uzunluğunda, künt uçlu iğne takın. </li><li> 1.2 ml / saat hızında çıkarmak için bir şırınga pompası. Cihazın içine şırınga ve iğne takın. </li><li> Yüksek gerilim güç kaynağı topraklı kurşun toplama cihazı takın. Rastgele dağıtılan lifler düz bir metal plaka kullanan toplamak veya hizalanmış lifleri toplamak için ~ 10 m / s hızla dönen bir mandrel kullanmak için. Için pozitif yüklü kurşun iğne takın. Elektrospinning aparat şematik Şekil 1 Bkz. </li><li> Ikisi arasında 15 cm mesafe olduğu gibi iğne veya toplama cihazı, ayarlayın. </li><li> Şırınga pompası akışını başlatın. Sıvı iğne ucu görüntülendi olduğunda, güç kaynağı açın ve 22 kV gerilim. </li><li> Koleksiyonda (ince film için birkaç dakika, 24 saate kadar kalın mat) tamamlandıktan sonra, iskele toplama cihazı çıkarın ve solvent tümüyle kaldırılmasını sağlamak için gece boyunca vakum altında saklamak. </li><li> Taramalı elektron mikroskobu (SEM) kullanılarak lif morfolojisi gözünüzde canlandırın, bir temsilci iskele aynı hizaya ve non-hizalanmış yapıları Şekil 1&#39;de gösterilmiştir. </li></ol> Not: örnek akış hızı, toplama cihazı olan mesafe ve gerilim polimer ve solvent kombinasyonu bağımlı ve genellikle iskele morfolojisi SEM ile gözlemleyerek, her bir sistem için optimize edilmiş olmalıdır. B. Photocrosslinking ve Photopatterning <ol><li> Iskele mat 5mm x 5mm örnekleri kesin. </li><li> Her iskele, folyo kaplı bir cam slayt yerleştirme, iskele doğrudan photomask yerleştirerek, temiz bir cam slayt kaplama ve bağlayıcı klipler ile her iki ucunda kırpma crosslinking için hazırlayın. Not: dört örnekleri bir defada yapılabilir ve bir desen istenilen değilse bir desen olmayan bir şeffaflık crosslinking için kullanılabilir. </li><li> Temizle iskele azot odasında kurdu. Crosslinking inhibe edebilir oksijen, inşa serbest tutmak için önemlidir. </li><li> ~ 10 mW / cm 2, 5 dakika boyunca collimating adaptör ile 365 nm ışığı altında azot odasında Yeri iskele kurulum . Şekil 2&#39;de şematik bakınız. </li><li> 12 plaka her iskele ve yer çıkarın. </li><li> Her iyi parafilm plaka 37 ° C&#39;de 24 saat süreyle su yeri ve buharlaşmasını önlemek için 2 mL deiyonize su ekleyin. Üç kez su değiştirin. </li><li> Işık mikroskobu altında gözenek oluşumu (Şekil 2 örneğe bakın) gözünüzde canlandırın. Iskeleleri artık kullanıma hazır. </li></ol> Not: Photocrosslinking polimerlerin birçok türleri için, ancak photopatterning sadece photoreactive polimerler ile gerekli değildir. Floresan Fiber Görselleştirme C. Çift Polimer elektrospinning <ol><li> % 90 etanol içinde PEO (200 kDa) 5 ağırlıkça% çözelti hazırlayın. 50 ° C&#39;de en az 2 saat önce elektrospinning 700 rpm&#39;de karıştırın. </li><li> PEO çözüm bir şırıngaya aktarın ve elektrospinning çözüm son konsantrasyon için 10 mg / ml konsantrasyon DAPI ekleyin. Şırınga ışıktan korumak için alüminyum folyo ile sarın. </li><li> Elektrospinning çözüm 25 mcM son bir konsantrasyon için methacryloxyethyl thiocarbamoyl rodamin B (MeRho 683,24 g / mol) eklemek de dışında, adım A2 açıklanan aynı çözüm hazırlayın. Şırınga ışıktan korumak için alüminyum folyo ile sarın. </li><li> Mandrel yüzeyinde methacrylated cam lamelleri (Khademhosseini ve ark.) Güvence altına almak için dikkatlice seloteyip kullanın. Not: elyaf methacrylated cam lamelleri için gergin olacak. </li><li> Döner kilit ekleri ile bir şırınga silikon boru 5 mm uzunluğunda bir parça bir ucunu takın ve diğer ucunu bir iğne takın. Şırınga pompası içine şırınga takın ve vantilatör delikten iğne koymak. Mandrel ters sitesinde diğer şırınga ve vantilatör tekrarlayın. Fanners l çevirmekmandrel ength ve çıkan iskele, iki polimerlerin eşit dağılımını sağlamak için kullanılır. Şekil 3&#39;te şematik bakınız. </li><li> Bölüm A uygun Tablo 1&#39;e göre açıklanan parametrelerini ayarlayın. Iğne uçları mandrel merkezli emin olun. Tablo 1. Çift Polimer elektrospinning Parametreler <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Şırınga </td><td> Vantilatör İğne İpucu (cm) </td><td> İğne İpucu Mandrel (cm) için </td><td> Akış Hızı (ml / saat) </td><td> Uygulamalı Gerilimi (kV) </td></tr><tr><td> Meha </td><td> 6 </td><td> 15 </td><td> 1,2 </td><td> +22 </td></tr><tr><td> PEO </td><td> 6 </td><td> 10 </td><td> 1,2 </td><td> +15 </td></tr></tbody></table> </li><li> Her şeyin düzgün hizalanmış sonra, ışıkları kapatmak ve mili ve şırınga pompalar açmak. Şırıngalar alüminyum folyo çıkarın. Sıvı, hem de iğnelerin uçları görünür olduğunda, aynı anda fanners güç kaynakları ve fiş açmak. </li><li> Toplama işlemi tamamlandığında, güç kaynakları ve mandrel fanners kapatın ve fişten çekin. Bir jilet kullanarak lamelleri ve bant dikkatli bir şekilde kaldırın. </li><li> Rodamin ve DAPI filtreleri ile donatılmış bir floresan mikroskop kullanılarak lifleri gözünüzde canlandırın. Şekil 3 örneğe bakın. Not: süresi (&lt;3 dakika) kısa bir süre için electrospun, ancak, bu sürecin özellikle cam lamelleri kullanılmadan, kalın yapıları oluşturmak için bir zaman sınırsız miktarda uzatılabilir zaman görüntülemek için Floresan lifleri en net . </li></ol> Yapı iskeleleri D. Yayımlama Hücreler <ol><li> Electrospun polimer ile her bir birey haline uygun boyutta bir plaka takılı lamel yerleştirin. Not: lifleri ile hücresel etkileşimleri methacrylated cam lamelleri üzerine elektrospinning tarafından kolayca görülebilir. Ayrıca, MeRho lif görünüm için tekrar kullanılmamalıdır. </li><li> Solvent tamamen kaldırılması ve olası yan ürünleri salabilen sağlanması için PBS içinde iskeleler gece inkübe edin. </li><li> Kuyulardan PBS çıkarın. </li><li> Iskeleleri sterilize etmek için, 30 dakika boyunca bir laminer akış kaputu bir antiseptik lamba altında yerleştirin. Tam bir iskele numaralı seribaşı ise, bir antiseptik lamba altında bir 30 dakika içinde iskele ve yer çevirin. </li><li> Standart hücre kültürü gerçekleştirmek ve (örneğin, 6.000 hücreleri cm -2) istenilen hücre yoğunluğu konsantre bir hücre süspansiyonu hazırlamak. Örneğin, bir 22 mm X 22 mm lamel için 100 ml hücre süspansiyonu kullanın. 1 saat için kuvöz koyun. </li><li> Her kuyuya uygun miktarda hücre kültür ortamı ekleyin. Iskeleleri inkübatör içine yerleştirin. </li><li> Leke piyasada mevcut Live / Dead kitini kullanarak hücrelerin. Not: DAPI (hücre çekirdekleri) ve floresan etiketli Phalloidin (aktin stres lifleri) gibi hücre boyama diğer türdeki hücrelerin görselleştirmek için uygulanabilir. </li><li> TRITC ve FITC filtreleri ile donatılmış bir floresan mikroskop kullanılarak hücre ve lifleri gözünüzde canlandırın. Şekil 4&#39;te örneğe bakın. </li></ol> Temsilcisi Sonuçlar: <img src="/files/ftp_upload/1589/1589figure1tmb.jpg" border="0" alt="Şekil 1" /> Şekil 1. aygıt kurulum olmayan hizalanmış iskele oluşumu (üstte) ve hizalı iskele formasyonu (alt) gösteren şematik. Her tip iskele örneği taramalı elektron mikroskobu görüntüleri gösterilmiştir. Ölçek çubuğu = 5μm. Lütfen Şekil 1&#39;de bir büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1589/1589figure1.jpg">buraya tıklayınız</a> . <img src="/files/ftp_upload/1589/1589figure2tmb.jpg" border="0" alt="Şekil 2" /> Şekil 2 desenlendirme yönteminin şematik. Desenler photocrosslinking sırasında ışık kaynağı ve iskele arasında photomask yerleştirerek ve sonra uzakta girmemiş polimer yıkama oluşur. Gözenek oluşumu ve liyofilizasyon sonra iskeleleri Photomask ve SEM görüntüsü. Ölçeği bar = 100 mikron. Lütfen Şekil 2&#39;de büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1589/1589figure2.jpg">buraya tıklayın</a> . <img src="/files/ftp_upload/1589/1589figure3tmb.jpg" alt="Şekil 3" border="0" /> Şekil 3 multi-polimer elektrospinning kurulum ve gerekli işleme parametreleri şematik yanı sıra, iki adet elyaf nüfus (örneğin: kırmızı Meha ve mavi PEO) karışımı bir temsilcisi floresan görüntü, bir multi-polimer iskele oluşturacak şekilde aynı anda electrospun . Ölçeği bar = 100 mikron. Lütfen daha büyük bir versio görmek için <a href="https://www.jove.com/files/ftp_upload/1589/1589figure3.jpg">buraya tıklayın</a>Şekil 3 n. <img src="/files/ftp_upload/1589/1589figure4tmb.jpg" border="0" alt="Şekil 4" /> Şekil 4. Bir örnek insan mezenkimal kök hücreleri ve electrospun lifleri ile olan etkileşimleri Dead / Canlı boyama görüntü. Ölçeği bar = 100 mikron. Lütfen Şekil 4 büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1589/1589figure4.jpg">buraya tıklayın</a> .

<ol>
	<li>Burdick, J. A., Chung, C., Jia, X., Randolf, M. A., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+degradation+and+mechanical+behavior+of+photopolymerized+hyaluronic+acid+networks.">Controlled degradation and mechanical behavior of photopolymerized hyaluronic acid networks.</a> Biomacromolecules. 6, 386-391 (2005).</li><li>Baker, B. M., Gee, A. O., Metter, R. B., Nathan, A. S., Marklein, R. A., Burdick, J. A., Mauck, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+potential+to+improve+cell+infiltration+in+composite+fiber-aligned+electrospun+scaffolds+by+the+selective+removal+of+sacrificial+fibers.">The potential to improve cell infiltration in composite fiber-aligned electrospun scaffolds by the selective removal of sacrificial fibers.</a> Biomaterials. 29, 2348-2358 (2008).</li><li>Ifkovits, J. L., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review:+Photopolymerizable+and+degradable+biomaterials+for+tissue+engineering+applications.">Review: Photopolymerizable and degradable biomaterials for tissue engineering applications.</a> Tissue Engineering. 13, 2369-2385 (2007).</li><li>Khademhosseini, A., Eng, G., Yeh, J., Fukuda, J., Blumling, J., Langer, R., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micromolding+of+photocrosslinkable+hyaluronic+acid+for+cell+encapsulation+and+entrapment.">Micromolding of photocrosslinkable hyaluronic acid for cell encapsulation and entrapment.</a> J. Biomed Mater Res A. 79A, 522-532 (2006).</li><li>Mauck, R. L., Baker, B. M., Nerurkar, N. L., Burdick, J. A., Li, W. J., Tuan, R. S., Elliott, D. M., M, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+on+the+Straight+and+Narrow:+The+Mechanics+of+Nanofibrous+Assemblies+for+Fiber-Reinforced+Tissue+Regeneration.">Engineering on the Straight and Narrow: The Mechanics of Nanofibrous Assemblies for Fiber-Reinforced Tissue Regeneration.</a> Tissue Engineering B. 15, 171-193 (2009).</li><li>Sill, T. J., Von Recum, H. a. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospinning:+applications+in+drug+delivery+and+tissue+engineering.">Electrospinning: applications in drug delivery and tissue engineering.</a> Biomaterials. 29, 1989-2006 (2008).</li><li>Sundararaghavan, H. G., Metter, R. B., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospun+fibrous+scaffolds+with+multi-scale+and+photopatterned+porosity.">Electrospun fibrous scaffolds with multi-scale and photopatterned porosity.</a> , (2009).</li></ol>

Elektrospinning polimerler fibröz iskeleleri hazırlamak için kullanılmıştır. Photocrosslinkable ışığa maruz kalma çapraz bağlanma için gerekli olan bir örnek olarak kullanılan hyaluronik asit dayalı iskele. Meha olarak reaktif macromers, kullanımı ile, daha önce gelişmiş hücresel dağılım göstermiştir kanallar, makro ve mikro gözenekli iskeleleri formu photocrosslinking sırasında bir maske kullanımı ile iskeleler dahil edilmiştir. Ayrıca, aynı anda iki farklı polimerler bizim özel ap...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>DAPI</td>
<td> Reagent</td>
<td>Invitrogen</td>
<td>D1306</td>
<td>&nbsp;</td>
<tr>
<td>I2959</td>
<td>Reagent</td>
<td>Ciba Specialty Chemicals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PEO 200 kDa</td>
<td>&nbsp;</td>
<td>Polysciences</td>
<td>17503</td>
<td>&nbsp;</td>
<tr>
<td>PEO 900 kDa</td>
<td>Reagent</td>
<td>Sigma</td>
<td>189456</td>
<td>&nbsp;</td>
<tr>
<td>Methacryloxethyl thiocarbamoyl rhodamine B</td>
<td>Reagent</td>
<td>Polysciences</td>
<td>23591-100</td>
<td>Prepare stock solution in DMSO</td>
</tr>
<tr>
<td>Live/Dead Stain Kit</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>L3224</td>
<td>Contains Calcein (stains live cells green) and ethidium homodime (stains red dead cells)</td>
</tr>
<tr>
<td>Syringe Pump</td>
<td>Equipment</td>
<td>KD Scientific</td>
<td>KDS100</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Power Source</td>
<td>Equipment</td>
<td>Gamma High Voltage</td>
<td>ES30P-5W</td>
<td>Two are needed for dual polymer spinning</td>
</tr>
<tr>
<td>Motor</td>
<td>Equipment</td>
<td>Triem Electric Motors, Inc</td>
<td>0132022-15</td>
<td>Must attach to a custom built mandrel</td>
</tr>
<tr>
<td>Tachometer</td>
<td>Equipment</td>
<td>Network Tool Warehouse</td>
<td>ESI-330</td>
<td>Use to monitor mandrel speed</td>
</tr>
<tr>
<td>Omnicure UV Spot Cure System with collimating adapter</td>
<td>Equipment</td>
<td>Exfo Life Sciences Division</td>
<td>S1000</td>
<td>&nbsp;</td>
<tr>
<td>Silicone Tubing</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51135K151</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Female Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K293</td>
<td>&nbsp;</td>
<tr>
<td>Luer Lock Male Adapter</td>
<td>Equipment</td>
<td>McMaster-Carr</td>
<td>51525K143</td>
<td>&nbsp;</td>
<tr>
<td>Needles</td>
<td>Equipment</td>
<td>Fisher Scientific</td>
<td>14-825-16H</td>
<td>&nbsp;</td>
<tr>
<td>Coverslips</td>
<td>Equipment</td>
<td>Corning</td>
<td>2875-22</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

electrospinning fibrous polymer scaffolds for tissue engineering and cell culture

Doku mühendisliği alanında geliştikçe, daha karmaşık organ ve dokuların gereksinimlerini (örneğin, mekanik ve damarlanma) ele almak üzere daha uygun malzeme ve işleme teknikleri üretmek için çok büyük bir bir talep var. Elektrospinning yerli ekstraselüler matriks mimarisi ve büyüklük ölçeği taklit fibröz iskeleleri oluşturmak için popüler bir tekniktir. Lifler, kök hücre farklılaşma (bkz. Mauck tarafından geniş yorumlar da dahil olmak üzere, hücresel davranışı doğrudan kullanılabilir beri bu fibröz iskeleleri de hücre kültürü substrat olarak yararlı<em&gt; Ve ark.</em&gt; Ve eşik<em&gt; Ve ark.</em&gt; Daha fazla bilgi için). Bu makalede, polimerler elektrospinning genel süreci tanımlamak ve bir örnek olarak, bkz. Electrospin ışığa maruz kalma ile çapraz bağlanma yeteneğine sahip bir reaktif hyaluronik asit (Ifkovits<em&gt; Ve ark.</em&gt; Photocrosslinkable malzemeler üzerine bir inceleme). Biz de böyle photopatterning ve multi-polimer iskele oluşumu gibi daha fazla işleme yetenekleri tanıtmak. Photopatterning hücresel infiltrasyon ve doku dağıtımı arttırmak için kanallar ve çok ölçekli porozite iskeleleri oluşturmak için kullanılabilir. Multi-polimer iskeleler porozite hücresel infiltrasyon için özel bir iskele de dahil olmak üzere özellikleri (mekanik ve bozulma), daha iyi ayarlamak için yararlıdır. Ayrıca, bu teknikler, başarılı bir doku mühendislik yapılarının geliştirilmesi için gerekli ipuçlarını sağlayan karmaşık iskeleleri oluşturmak için polimerler ve reaktif macromers geniş bir yelpazeye dahil etmek için uzatılabilir.

Watch this Scientific Journal Video about Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture at JoVE.com

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

Polimerlerin doku mühendisliği ve hücre kültürü için elektrospinning süreci, bu makalede ele alınmaktadır. Özellikle multi-polimer elektrospinning photopatterning ve ek işleme yetenekleri ile photoreactive macromers elektrospinning açıklanmıştır.

Doku Mühendisliği ve Hücre Kültürü için Lifli Polimer Yapı iskeleleri elektrospinning

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

electrospinning-fibrous-polymer-scaffolds-for-tissue-engineering-cell

Research

JoVE Journal

Biology

21.4K Görüntüleme.  University of Pennsylvania. Polimerlerin doku mühendisliği ve hücre kültürü için elektrospinning süreci, bu makalede ele alınmaktadır. Özellikle multi-polimer elektrospinning photopatterning ve ek işleme yetenekleri ile photoreactive macromers elektrospinning açıklanmıştır.

Video: Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

Experimental Approaches to Tissue Engineering

Doku Mühendisliği Deneysel Yaklaşımlar

Biyoloji

Micro-scale Engineering for Cell Biology

Hücre Biyolojisi Mikro ölçekli Mühendisliği

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Aktin Co-Sedimantasyon Testi; F-Aktin Bağlayıcı Protein Analizi

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Üç boyutlu Hücre ekimi için Chip-ölçekli Yapı iskeleleri mikroimalat

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

Röportaj: Kök Hücre Araştırma ve Yüzey-Modifiye 3D-Yapı iskeleleri Biyoreaktörler

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Doku Mühendisliği 3D Hidrojeller Hücresel Kapsülleme

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Engineering Cell-permeable Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can make use of the translocating ability of so-called cell penetrating peptides (CPPs), also designated as protein transduction domains (PTDs). The TAT-CPP derived from the human immunodeficiency virus type 1 (HIV-1) Tat (trans-activator of transcription) protein has been widely used. The positively charged TAT promotes cell permeability thereby overcoming the barriers of the cellular membrane by endocytosis or/and direct membrane penetration2. In combination with a nuclear localization signal (NLS) fusion proteins are able to enter the nucleus exhibiting functionality. Our video presentation demonstrates, as an exemplification for the engineering of cell-permeable proteins, the construction, production and application of a cell-permeable version of the DNA-modifying enzyme Cre.
Cre is a site-specific recombinase that is able to recognize and recombine 34 base pair loxP sites in mammalian cells in vitro and in vivo. Therefore the Cre/loxP system is widely used to conditionally induce mutations in the genome of living cells3,4. The delivery of active Cre recombinase to cells, however, represents a limitation.
We describe the pSESAME vector system, which allows a direct insertion of the gene-of-interest and provides a platform to rapidly clone different domains and tags used within the vector in a convenient and standardized manner. Rearranging of the different tags has been shown to modify the biochemical properties of the fusion proteins providing a possibility to achieve higher yield and better solubility. We demonstrate how to express and purify recombinant cell-permeant proteins in and from E. coli. The functionality of the recombinant Cre protein is finally validated in cell culture by assessing its intracellular recombinase activity.

Protein transduction enables the direct delivery of biologically active proteins into cells. In contrast to conventional methods such as DNA transfection or viral transduction this non-invasive paradigm allows highly efficient cellular manipulation in a titratable manner circumventing cellular toxicity and the risk of oncogenic transformation by permanent genetic modification.

Mühendislik Hücre geçirgen Protein

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Newt Lens Rejenerasyon Eğitim Kültürleme İris Pigment Epitel Hücreler için Sistem

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Biyogüvenlik Geliştirilmiş Polimer complexed Ecotropic lentivirüs İnsan Hücreleri, İletimi

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hücre Kültürü ve Hipoksi Test İndüksiyon

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...