Yazarlar, Ulusal Bilim Vakfı aracılığıyla Malzeme Araştırma Bilimi ve Mühendisliği Merkezi parçası olarak finanse, Johns Hopkins, yüzey analizi laboratuvarı kullanımı kabul etmiş sayılırsınız. LED IGERT stajyer ve Ulusal Bilim Vakfı Lisansüstü Üyesi. Bu araştırma, kısmen, Ulusal Sağlık Enstitüsü, hibe U54CA143868 tarafından desteklenen oldu.

1. Micropatterned Damga Fabrikasyon Standart Fotolitografi <ol><li> Etanol ile yeni bir silikon yonga hava akışı ile durulayın ve kurulayın. Gofret ele almak ve tüm süreç boyunca yüzey hasarları önlemek için forseps kullanın. </li><li> Lak spin ve SU-2025 negatif fotorezist gofret yüzeyini kaplayan gofret aktarın. Paslanmaz çeliğin en az% 80 gofret örtün. 3000rpm 30 saniye takip 600rpm az 10 saniye süreyle Spin kat. Fotorezist fotosensitivite nedeniyle, bu noktadan itibaren oda ışıkları kapatın. </li><li> 95 ° C&#39;de 3 dakika boyunca &quot;yumuşak fırında&quot; sıcak levha kaplamalı silikon gofret fotorezist aktarın. Sıcak levha çıkar ve 1 dakika soğumasını bekleyin. </li><li> Fotorezist kaplı silikon gofret üstüne istenilen desen prefabriklerin maske koyun ve 20 saniye için ultraviyole (UV) ışınları (350-450 nm) maruz bırakmayın. </li><li> Sıcak levha &quot;sabit fırında&quot; için 110 ° C 6 dakika Transfer silikon gofret. </li><li> Işıkları açın ve sıcak plaka gofret kaldırmak. Farklı desenleri bu noktada görünür olmalıdır. </li><li> SU-8 fotorezist geliştirici ile silikon gofret dikkatlice yıkayın. 3 geliştirici çözümü ile çalkalama sonra, etanol ile yıkayın. Herhangi bir beyaz kalıntı varsa, paslanmaz çeliğin geliştirici çözümü ile durulayın tekrar, ya da sadece batığın gofret geliştirici çözüm ve 2 dakika süreyle yıkayın etanol ile tekrar devam edebilir. Su ve kuru hava ile yıkayın. </li><li> Mix polidimetilsiloksan (PDMS) 10:1 ağırlık oranı elastomer çözüm ve sertleştirici. Hava kabarcıklarını çıkarmak için 5 dakika için 900 RPM santrifüjleyin. </li><li> Place Petri kabındaki gofret desenli PDMS ve silikon yonga üzerine dökün. Birkaç dakika için mevcut kabarcıkları, desicator yer ve vakum varsa kabarcıkları kayboluncaya kadar. </li><li> Tamamlayıcı bir elastomerik damgası oluşturmak için oda sıcaklığında (RT) bir gecede kurtulun. PDMS tamamen 60 ° C&#39;de 30 dakika fırında, yerde 12 saat, sonra tedavi değil ise. </li><li> Dikkatle silikon gofret PDMS çıkarın. Istediğiniz boyuta damga kesmek için bıçak kenar kullanın. 15 dakika boyunca etanol içinde sonikasyon. Gofret yeni elastomerik pullar oluşturmak için birden fazla kez kullanılabilir. </li></ol> 2. HA Micropatterning <ol><li> Plazma temiz bir cam, 5 dakika boyunca slaytlar. 5 dakika boyunca oda ve vakum tüm slaytlara yerleştirin. Sonra odasına girmek için düşük oksijen akışına izin verir. 5 dakika boyunca plazma temizleyici açın. Plazma temizleyici ve forseps kullanarak bir Petri kabındaki yerden çıkarın. </li><li> Plazma temizlik sırasında, 15 dakika boyunca etanol PDMS pulları sonikasyon. </li><li> 15 ml santrifüj tüpüne% 95 v / v etanol 3-aminopropyltrimethoxysilane (APTMS) taze% 3 v / v çözüm hazırlayın. Tepki silanolle formu RT az 5 dakika izin ver. </li><li> Yeri PDMS damga dönmeye coater Chuck desen ve kapak yüzeyi desenli APTMS çözüm. 30 saniye için 3500 Spincoat. PDMS damga, sadece iki tarafın damga yüzeyine dokunmayın. </li><li> Desenli yan bir ucunu aşağı bakacak şekilde set mühür ve 1 dakika için cam yüzeyi ile temas halinde tamamen yavaşça PDMS basarak kayma: Transfer plazma APTMS çözüm cam yüzeyler temizlenmelidir. </li><li> PDMS hafifçe damga çıkarın ve buzlu cam slayt RT 30 dakika boyunca inkübe izin. Desenli yüzeyler etanol ve kuru hava ile durulayın. Sonikasyon PDMS aşırı APTMS kaldırmak için 15 dakika boyunca damgası. </li><li> 115 1 saat fırında veya sıcak plaka üzerinde Isı yüzeyler ° C Etanol ile çalkalayın ve kuru hava ile. </li><li> [Metoksi (polyethyleneoxy) propil] toluende trimethoxysilane - taze polietilen glikol (PEG) silan% 20 v / v 2 çözüm hazırlayın. Bu desen çevreleyen yapışkan olmayan bir bölge olarak hizmet verecek. </li><li> 75 az 45 dakika boyunca bir bardak PEG-silan çözüm Petri kabı ve inkübe Transfer buzlu cam slayt ° C sıcak bir plaka. Toluen, su ve hava ile kuru ile durulayın. </li><li> Biyolojik güvenlik kabini UV antiseptik ışınlama kullanarak 1 saat süreyle sterilize edin. Biyolojik güvenlik kabini kısırlık korumak için bu noktadan devam edin. </li><li> Sulu steril HA çözeltisi hazırlayın. 1-Etil-3-(3-Gaz sistemlerinde) carbodiimide (EDC), 10mM N-hydroxysuccinimide, 5mM 50μg / ml flöresein etiketli HA </li><li> Steril koşullar HA çözüm buzlu cam yüzeye uygulayın ve ışıktan korunmalıdır. Desenli yüzeylerde 16-24 saat boyunca rahatsız olmalıdır. </li><li> HA kapalı aspire çözüm, bol suyla yıkayın fosfat tamponlu salin (PBS), ve 1 saat süreyle PBS (BSA)% 1 sığır serum albümini ile yüzeyini kaplayacak. Yüzeyi hücre kültürü için hazırdır. </li></ol> 3. HA Micropatterned Yüzeylerde Hücre Kültürü <ol><li> Expand MDA-MB-231 üreticinin talimatlarına göre insan meme kanseri hücreleri ve LS174T kolorektal karsinom hücreleri. </li><li> 0.4 ve 1x10 arasındaki yoğunlukları ile kanser hücrelerinin Tohum tek hücre süspansiyonları <sup&gt; 6 kanser hücrelerinin 18,75 cm 2 cam yüzey alanı başına ve HA üzerine yüzeyler immobilize. % 5 CO 2 ile korunur ortamlarda nemlendirilmiş inkübatör (37 ° C) inkübe edin. Hücre adezyon 24 saat içinde ortaya çıkar ve ters ışık mikroskobu kullanılarak görülebilir </li><li> Hücre kültürü yüzeyleri standart inkübatör koşullarında 5 gün kadar muhafaza edilebilir. Her gün medya değiştirin. Hücre morfolojisi ve büyüme ters ışık mikroskobu kullanılarak görülebilir. Ek hücre deneyleri HA yüzeye yanıtları analiz etmek için uygulanabilir. </li></ol> 4. Temsilcisi Sonuçlar: Kovalent APTMS buzlu cam slaytlara HA hareketsiz kullanılan carbodiimide kimya Şekil 1A gösterilmiştir. EDC HA karboksil grupları ile reaksiyona girer amin-reaktif ara oluşturmak için sıfır uzunlukta bir çapraz bağlanma ajandır. Bu ara hidroliz duyarlı olarak, NHS carbodiimide reaksiyon verimi artırmak için eklenir. HA çözüm APTMS micropatterns, HA ATPMS, orta ve primer amin arasında istikrarlı bir amid bağı formları ile etkileşim. HA desenli yüzeyinin bir örnek gösterilmiştir (Şekil 1B-C) bir floresan mikroskop ve bir ImageJ floresan analizi kullanarak HA desenli immobilizasyon göstermek görüntülendi. HA micropatterned yüzeyler expgenous HA ile kanser hücre etkileşimleri çalışma sağlar. Her ikisi de MDA-MB-231 insan meme ve LS174t insan kolon kanseri hücre dizilerinde tercihen HA micropatterned bölgeleri (Şekil 2) için uygun. Taramalı elektron mikroskobu kullanılarak yüksek çözünürlüklü görüntüleme HA hareketsiz yüzeyler (Şekil 3) ile kültür hücreleri etkileşimlerini analiz etmek için kullanılabilir. <img src="/files/ftp_upload/2413/2413fig1.jpg" alt="Şekil 1" /> Şekil 1 HA micropatterned yüzeyler anlatan HA fonksiyonel yüzeyler geliştirme (A) Şematik, (B) floresein floresan (yeşil) etiketli HA micropatterned yüzey. (C) 3D piksel dağılım haritası ayrık HA desen gösteren ImageJ yazılımı kullanarak. Ölçek çubuğu = 100μm. Elsevier izin 9 - Modifiye. <img src="/files/ftp_upload/2413/2413fig2.jpg" alt="Şekil 2" /> Şekil 2 HA micropatterned yüzeylerde Kanser hücre adezyon. Kolon (A) ve meme Her ikisi de (B) karsinom tercihen 24 saat kültür ile HA yamaları üzerine yapıştırılır. Elsevier izin 9 - Modifiye. <img src="/files/ftp_upload/2413/2413fig3.jpg" alt="Şekil 3" /> Şekil 3 HA ile Kanser hücrelerinin etkileşimi. (A), kolon kanseri hücrelerinin HA yüzeylerde düşük büyütme (solda) ve yüksek büyütmede (sağ, sol tarafta kareler) gösterilen (A) kültürlü elektron mikroskobu görüntüleri, meme kanseri hücrelerinin Tarama ve (B) gösterilen HA yüzeylerinin kültüre düşük büyütme (solda) ve yüksek büyütmede (sağ, sol tarafta kareler).

<ol>
	<li>Toole, B. P., Wight, T. N., Tammi, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan-cell+interactions+in+cancer+and+vascular+disease.">Hyaluronan-cell interactions in cancer and vascular disease.</a> Journal of Biological Chemistry. 277, 4593-4596 (2002).</li><li>Hamilton, S. 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+Hyaluronan+Receptors+CD44+and+Rhamm+(CD168)+Form+Complexes+with+ERK1,2+That+Sustain+High+Basal+Motility+in+Breast+Cancer+Cells.">The Hyaluronan Receptors CD44 and Rhamm (CD168) Form Complexes with ERK1,2 That Sustain High Basal Motility in Breast Cancer Cells.</a> Journal of Biological Chemistry. 282, 16667-16680 (2007).</li><li>G&#246;tte, M., Yip, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparanase,+Hyaluronan,+and+CD44+in+Cancers:+A+Breast+Carcinoma+Perspective.">Heparanase, Hyaluronan, and CD44 in Cancers: A Breast Carcinoma Perspective.</a> Cancer Research. 66, 10233-10237 (2006).</li><li>Lord, M. S., Pasqui, D., Barbucci, R., Milthorpe, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+adsorption+on+derivatives+of+hyaluronic+acid+and+subsequent+cellular+response.">Protein adsorption on derivatives of hyaluronic acid and subsequent cellular response.</a> Journal of Biomedical Materials Research. Part A 91, 635-646 (2009).</li><li>Barbucci, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+hyaluronic+acid+by+sulphate+groups+insertion+to+obtain+a+heparin-like+molecule.">Modification of hyaluronic acid by sulphate groups insertion to obtain a heparin-like molecule.</a> Gazz. Chim. Ital. 125, 169-180 (1995).</li><li>Morra, M., Cassinelli, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-fouling+properties+of+polysaccharide-coated+surfaces.">Non-fouling properties of polysaccharide-coated surfaces.</a> Journal of Biomaterials Science, Polymer Edition. 10, 1107-1124 (1999).</li><li>Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer-by-layer+deposition+of+hyaluronic+acid+and+poly-L-lysine+for+patterned+cell+co-cultures.">Layer-by-layer deposition of hyaluronic acid and poly-L-lysine for patterned cell co-cultures.</a> Biomaterials. 25, 3583-3592 (2004).</li><li>Ibrahim, S., Joddar, B., Craps, M., Ramamurthi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+surface-tethered+model+to+assess+size-specific+effects+of+hyaluronan+(HA)+on+endothelial+cells.">A surface-tethered model to assess size-specific effects of hyaluronan (HA) on endothelial cells.</a> Biomaterials. 28, 825-835 (2007).</li><li>Dickinson, L. E., Ho, C. C., Wang, G. M., Stebe, K. J., Gerecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+surfaces+for+high-resolution+analysis+of+cancer+cell+interactions+on+exogenous+hyaluronic+acid.">Functional surfaces for high-resolution analysis of cancer cell interactions on exogenous hyaluronic acid.</a> Biomaterials. 31, 5472-5478 (2010).</li><li>Gao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan+oligosaccharides+promote+excisional+wound+healing+through+enhanced+angiogenesis.">Hyaluronan oligosaccharides promote excisional wound healing through enhanced angiogenesis.</a> Matrix Biology. 29, 107-116 (2010).</li><li>Slevin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyaluronan-mediated+angiogenesis+in+vascular+disease:+Uncovering+RHAMM+and+CD44+receptor+signaling+pathways.">Hyaluronan-mediated angiogenesis in vascular disease: Uncovering RHAMM and CD44 receptor signaling pathways.</a> Matrix Biology. 26, 58-68 (2007).</li></ol>

Izni ile çoğaltılabilir video şematik figür; Copyright Wiley-VCH Verlag GmbH &amp; Co. KGaA. Dickinson LE, Kusuma S, Gerecht S. biyomateryalleri kullanılarak embriyonik kök hücrelerinin farklılaşması niş Yeniden. Macromol. Biosci. 2010; Oct 21. [Epub ahead of print].

Ekzojen HA HA micropatterning yöntemi ile hücre etkileşimleri çalışma sağlar. HA 1 kanserinin ilerlemesini önemli bir rol oynadığı bilinmektedir ancak iki boyutlu HA desenli yüzeyler üzerinde kanser hücrelerinin etkileşimi araştıran sınırlı sayıda çalışma yapılmış ve yapılmaktadır . Ekzojen HA micropatterns denetlenebilir bir çalışmada, kanser hücre adezyon, büyüme, göç ve yüksek çözünürlüklü görselleştirme sağlar ve kanser daha fazla temellerini aydınlatmak ola...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SU-2025 photoresist</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>Y111069</td>
<td>&nbsp;</td>
<tr>
<td>SU-8 developer</td>
<td>&nbsp;</td>
<td>MicroChem Corp.</td>
<td>Y020100</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3-aminopropyltrimethoxysilane (APTMS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>281778</td>
<td>&nbsp;</td>
<tr>
<td>2- [methoxy(polyethyleneoxy) propyl] trimethoxysilane (Peg-silane)</td>
<td>&nbsp;</td>
<td>Gelest Inc</td>
<td>SIM6492.7</td>
<td>&nbsp;</td>
<tr>
<td>1-Ethyl-3- (3-dimethylaminopropyl) carbodiimide</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>22980</td>
<td>&nbsp;</td>
<tr>
<td>N-hydroxysuccinimide (NHS)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>24500</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescein labeled hyaluronic acid (FL-HA)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrick</td>
<td>F1177</td>
<td>Reconstitute with 10ml of DI water </td>
</tr>
<tr>
<td>MDA-MB-231 breast carcinoma cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>HTB-26</td>
<td>&nbsp;</td>
<tr>
<td>LS174t colon carcinoma cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>Cl-188</td>
<td>&nbsp;</td>		</tbody>
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micropatterned surfaces to study hyaluronic acid interactions with cancer cells

Kanser işgali ve ilerlemesi tümör mikroçevresinin içinde büyüme faktörleri / sitokinler ve hücre dışı matriks (ECM) bileşenleri karmaşık düzenleme altında bir hareketli hücre fenotipi içerir. Hyaluronik asit (HA), artırılması işgali, büyüme ve anjiyogenez 1 tümör ilerlemesini kolaylaştırmak için bilinen bir stromal ECM bileşeni . Etkileşim HA hücre yüzey reseptörü CD44, tümör hücre büyümesini, hayatta kalma ve göç teşvik olaylar böylece metastatik yayılmasını 2-3 artan sinyalizasyon neden olur. HA, D-glukuronik asit ve DN-asetilglukozamin birimleri tekrar oluşan bir anyonik, nonsulfated glikozaminoglikan. Tekrarlayan disakkarit ünitelerinde karboksil ve hidroksil grupları, varlığı nedeniyle doğal HA büyük ölçüde hidrofilik ve sülfat photoreative immobilizasyon 4-5 grupları tanıtmak kimyasal değişiklikler için uygun. Önceki çalışmalar yüzeyler üzerine HA immobilizations içeren 6-7 yüzeylerinden hücre yapışmasını kontrol etmek HA bioresistant davranış ve sülfatlanmış türevi kullanmaktadır. Tercihen bu çalışmalar hücre adezyon-HA olmayan desenli bölgelerde oluşur. Ekzojen HA ile hücresel etkileşimleri analiz etmek, kontrol edilebilir bir çalışma ve HA ile kanser hücre etkileşimleri yüksek çözünürlüklü görselleştirme sağlayan desenli Fonksiyonlu yüzeyler geliştirdiler. Biz cam yüzeyler üzerinde HA ayrık desenli bölgeleri tanımlamak için kullanılan microcontact baskı (UCP). HA hareketsiz carbodiimide bağlayan kimya uygulanan &quot;bağlama&quot; yaklaşımı 8 kullanıldı. Cam yüzeyler microcontact aminosilane ile basıldı ve EDC ve NHS desenli dizileri HA immobilizasyon sağlamak için optimize edilmiş oranları HA çözüm tepki gösterdi. Carbodiimide kimya MCP ile ekleme in vitro uygulamaları için uygun yüzeyler oluşturmak, tanımlanmış bölgelere HA immobilizasyon etkin. Her iki kolon kanseri hücreleri ve meme kanseri hücrelerinin dolaylı HA micropatterned yüzeyler ile etkileşim. Kanser hücre adezyon proliferasyonu ile 24 saat içinde 48 saat oluştu. HA micropatterned yüzeyler kullanarak, kanser hücre adezyon HA reseptörü CD44 üzerinden gerçekleşir gösterdi. Ayrıca, HA desenli yüzeyler, tarama elektron mikroskobu (SEM) ve kanser hücresi yapışkan çıkıntılar izin verilen yüksek çözünürlüklü görüntüleme ve kanser hücre hareketi analiz etmek için ekzojen HA HA desen yayılan ile uyumlu idi.

Watch this Scientific Journal Video about Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells at JoVE.com

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Eksojen hyaluronik asit (HA) ile yüksek çözünürlüklü kanser hücre etkileşimleri analiz olanak sağlayan yeni bir yaklaşım açıklanmıştır. Desenli yüzeyleri carbodiimide kimya ve microcontact baskı birleştirerek imal edilir.

Micropatterned Yüzeyler Kanser Hücreleri ile Hyaluronik Asit Etkileşimleri Eğitim

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

micropatterned-surfaces-to-study-hyaluronic-acid-interactions-with

Research

JoVE Journal

Bioengineering

15.0K Görüntüleme.  Johns Hopkins University. Eksojen hyaluronik asit (HA) ile yüksek çözünürlüklü kanser hücre etkileşimleri analiz olanak sağlayan yeni bir yaklaşım açıklanmıştır. Desenli yüzeyleri carbodiimide kimya ve microcontact baskı birleştirerek imal edilir.

Video: Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

İlaç Etkisi Micropatterned Hücreler Geliştirilmiş Görselleştirme ve Kantitatif Analiz

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Biyomühendislik

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

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

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

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

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

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

Stretching Micropatterned Cells on a PDMS Membrane

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

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

PDMS Zarında Micropatterned Hücreler germe

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

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Kanser Hücre Invasion Uygulama ile Tümör Stromal mikroçevresinin Doku Mühendisliği

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Genetik olarak tadil edilmiş hücre transplantasyonu için Micropatterned kültür plakalarında Sferoid hücrelere karşı gene

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Doku Mühendisliği Thermoresponsive Nanostructured Yüzeylerin Hazırlanması

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D Etkileşimleri Çalışmaları 3D Kollajen jeller ve Mikrokanallarda Hazırlanması<em&gt; İn Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Site-Directed Immobilization of Bone Morphogenetic Protein 2 to Solid Surfaces by Click Chemistry

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present several limitations that have led to the use of biomaterials in combination with osteogenic growth factors, such as bone morphogenetic proteins (BMPs). Commonly used absorption or encapsulation methods require supra-physiological amounts of BMP2, typically resulting in a so-called initial burst release effect that provokes several severe adverse side effects. A possible strategy to overcome these problems would be to covalently couple the protein to the scaffold. Moreover, coupling should be performed in a site-specific manner in order to guarantee a reproducible product outcome. Therefore, we created a BMP2 variant, in which an artificial amino acid (propargyl-L-lysine) was introduced into the mature part of the BMP2 protein by codon usage expansion (BMP2-K3Plk). BMP2-K3Plk was coupled to functionalized beads through copper catalyzed azide-alkyne cycloaddition (CuAAC). The biological activity of the coupled BMP2-K3Plk was proven in vitro and the osteogenic activity of the BMP2-K3Plk-functionalized beads was proven in cell based assays. The functionalized beads in contact with C2C12 cells were able to induce alkaline phosphatase (ALP) expression in locally restricted proximity of the bead. Thus, by this technique, functionalized scaffolds can be produced that can trigger cell differentiation towards an osteogenic lineage. Additionally, lower BMP2 doses are sufficient due to the controlled orientation of site-directed coupled BMP2. With this method, BMPs are always exposed to their receptors on the cell surface in the appropriate orientation, which is not the case if the factors are coupled via non-site-directed coupling techniques. The product outcome is highly controllable and, thus, results in materials with homogeneous properties, improving their applicability for the repair of critical size bone defects.

Biomaterials doped with Bone Morphogenetic Protein 2 (BMP2) have been used as a new therapeutic strategy to heal non-union bone fractures. To overcome side effects resulting from an uncontrollable release of the factor, we propose a new strategy to site-directly immobilize the factor, thus creating materials with improved osteogenic capabilities.

Site yönettiği immobilizasyon kemik morfogenetik protein 2 katı yüzeyler için tıklayın kimya tarafından

Different therapeutic strategies for the treatment of non-healing long bone defects have been intensively investigated. Currently used treatments present ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Hyaluronik asit Hydrogels hasta kaynaklı Glioblastoma hücreleri 3 boyutlu kültür için dayalı.

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

İnsan kaynaklı Pluripotent Kök Hücre Türevli Kardiyomiyositlerde Tek Hücreli Optik Eylem Potansiyeli Ölçümü

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...