Bu çalışma, ABD Ulusal Sağlık hibe NIH / NICHD # HD051998 (CAM) Enstitüleri tarafından da desteklenmiştir.

1. Kollajen Boncuk Preparation <ol><li> 3-boyutlu hücre kültürü için yükleme EVTs önce, bir Cytodex-3 microcarrier boncuk hazırlamak gerekir: <ol><li> Deney için gerekli Cytodex-3 boncuk uygun miktarda tartılır. Bu protokol, boncuk 0.05g gerekli olan 10ml RCC gemi için uyarlanmıştır. 50ml RCC bir gemi için, buna göre ölçekli. 50ml otoklavlanabilir konik tüp 12ml Dulbecco&#39;nun fosfat, 250 mg Cytodex-3 boncuklar bir arada çözüm (DPBS) tamponlu. Bu miktar 5 RCC gemiler için yeterli olacaktır. </li></ol></li><li> Otoklav süreci buharlaşma sonucu olarak yeterli hacmi, konik tüp içinde bulunur olun. Gevşek 110 tüp ve 10 dakika otoklav kapağı ° C </li><li> Otoklav döngüsü bitiminde konik tüp çıkarın ve Cytodex-3 boncuk çözüm soğumasını bekleyin. </li><li> Konik tüp oda sıcaklığına kadar soğuduktan sonra, 1X DP ile toplam hacmi 12.5mL getirmek için steril tekniği kullanmakBS. </li><li> Cap ve oda sıcaklığında hazırlanan Cytodex-3 boncuk saklamak. Hazırlanan boncuklar kullanmadan önce oda sıcaklığına kadar şişer ve serinlemek için izin verin. Yukarıdaki hazırlık Cytodex-3 boncuk, beş 10ml RCC gemiler için sağlayacaktır. Biz yaklaşık bir ay sonra kollajen istikrarsızlaştırmak Cytodex-3 boncuk oda sıcaklığında uzun depolama, performans azalması neden olacağını gözlemledik. 133-215 mikron boyutunda Cytodex-3 boncuk aralığı. </li></ol> 2. Medya Hazırlık <ol><li> Artı takviyeleri ve% 60 L-15 Leibovitz medya (22 uyarlanmıştır),% 40 MEM alfa-2 oluşur GTSF medya (22 uyarlanmıştır), optimize edilmiş RCC 1L hazırlayın. Birincisi, MEM alfa ile desteklenmiş toplam hacmi 400ml hazırlamak: 21.2mM sodyum bikarbonat % 0,06 Peptonlu 0.7mm Fruktoz 1.4mm Galaktoz 5.6mM Glikoz 1% Hepes % 1 L-glutamin % 0.5 &#39;DA % 10 FBS </li><li> ITS bir stok solüsyonu hazırlamak için, buzul asetik asit ilavesi (yaklaşık 0.05mL) tarafından hazırlanan 5 ml&#39;lik steril asitlendirilmiş H 2 O çözülür. Çözmek için Swirl 45mL steril su ile izleyin. </li><li> Doku kültürü notu H 2 O çözme orta 600ml için yeterince L-15 Leibovitz tozu tartılır </li><li> L-15 Leibovitz orta hücre kültür ortamı ile 1L toplam hacim kazandırın. Filtre sterilize ve 4 saklamak ° C karanlıkta. Kullanılan orta her kısım için ayrı ayrı% 1 Penisilin-Streptomisin ekleyin. </li><li> GTSF-2 medya daha başka birçok medya formülasyonları RCC başarıyla test edilmiştir. Bireysel laboratuvarlarda deney yapıldığı için en uygun olan orta kendileri için karar vermelisiniz. </li></ol> 3. Hücreler ve Boncuk Kuluçka <ol><li> ~ 80% confluency EVTs yayma, trypsinize ve kabul edilen hücre kültürü uygulamaları kullanarak saymak. </li><li> Ilık 4ml 1x10 6 EVTs Askıya Almaed medya. </li><li> Hazırlanan Cytodex-3 boncuk hafifçe karıştırın. Steril teknikleri kullanarak, geniş bir ucu, 10 ml serolojik pipet ve kullanılmayan bir 15 mL konik tüp transfer kullanılarak hazırlanan boncuk 2.5ml çıkarın. Pipet eklemek gibi küçük bir boncuk kaybı olabileceğini unutmayın. </li><li> Cytodex-3 boncuk tüp alt yerleşmek için izin verin. Sedimantasyon sonra Cytodex-3 boncuk bozmadan DPBS en üst tabakasını kaldırmak için bir pipet kullanın. </li><li> Hazırlanan Cytodex-3 boncuk ile medya 1x10 6 hazırlanan EVTs karıştırın. </li><li> Hücre-boncuk karışımı oda sıcaklığında 30 dakika inkübe edin. Periyodik yavaşça karıştırın. 37 ° C ve% 5 CO 2 &#39;de bir 30 dakika daha inkübe. Periyodik yavaşça karıştırın. </li></ol> 4. RWV yükleme <ol><li> Laminer akış kabini, ambalaj, 10 ml RCC kaldırmak ve istikrarı için steril, 6 gözlü bir kültür plakasına yerleştirin. RC etiketli diyagram Şekil 1 bakınCS. </li><li> Limandan büyük stoper çıkarın. </li><li> Kuluçka hücre-boncuk karışımı toplam hacmi 10 ml ile ısındı medya getirin. </li><li> Hücre / boncuk-karışımı büyük bir bağlantı noktası üzerinden RCC içine yükleyin. RCC az eğik olmalıdır 45 ° açılı (büyük port) olası hava kabarcıklarının alınması yardım yüklerken. Geminin yavaş yavaş ve sürekli doldurarak pozitif basınç oluşturmak önleyin. </li><li> Büyük liman stoper değiştirin. </li><li> 3 ml şırınga pistonları çıkarın ve küçük liman üzerine boş bir şırınga yerde. 1-3ml her şırınga medya ekleyin. Yavaş yavaş iki vanalarının açık. Yavaşça şırınga şırınga pistonları değiştirin. Tüm kabarcıklar iç odasının kaldırılana kadar bir şırınga medya ekleyin. </li><li> RCC Pick up ve kabarcıkları kontrol etmek için önünüzde dönerken yan hafifçe dokunun. Hava kabarcıkları oluşumu o engel olacak şekilde, herhangi bir kabarcıkları varsa, oda onları almak gerekirf hücre agrega ve kesme kuvvetleri tanıtmak. RCC kabarcıkları küçük liman altında olana kadar döndürerek kabarcıklarını çıkarın. Sonra yavaşça liman ve odanın içine ya da dışına baloncuklar zorlamak için, karşı tarafında şırınga aşağı doğru itin. </li><li> Bir tarafı bağlantı noktasını kapat. Şekillendirme kabarcıkları önler gemi, içine küçük bir miktar pozitif basınç tanıtmak için diğer şırınga pistonu yavaşça aşağı doğru itin. İkinci vanasını kapatın. </li><li> RCC rotor üzerine yerleştirin. 37 ° C CO 2 inkübatör 19rpm dönme başlayın. </li></ol> 5. Medya değiştirme <ol><li> Sonra her gün daha sonra, ilk üç gün boyunca her gün bir ortam. </li><li> Rotor kapatın ve RCC kaldırmak. Pistonlar, bazı emme oluşturmak, her bir küçük liman şırınga kaldırmak ve boncuklar, büyük bir liman tersi yerleşmek böylece bir açı RCC yere kadar çekin. </li><li> Boncuk yapıldıktan sonra, küçük vanaların birini açmak veRCC medya ve bir atık kabı içine akmasına izin verin. Bu şekilde 2 / 3 &#39;medya çıkarın. Boncuk rahatsız değil ve herhangi bir agrega atmayın emin olun. </li><li> Küçük bir kapak kapatın ve büyük bir liman açmak. Medyanın büyük bir bağlantı noktası üzerinden RCC içine ekleyin. Büyük liman stoper değiştirin. </li><li> 4.9 - 4.6 adımları tekrarlayın. </li><li> Agrega boyutu arttıkça, dönme hızını artırmak için en uygun hızda küçük bir dolaşım deseni, ideal olarak, her zaman süspansiyon onları tutmak için gerekir. Genel olarak, agrega, gözle görülür bir şekilde büyüyen başladıktan sonra her emzirmeden sonra 0,3-0,7 rpm arasında hız artışı. </li></ol> 6. Toplama Hücreler yayılma <ol><li> Rotor RCC çıkarın. Her bir küçük liman şırınga çıkarın ve RCC, boncuklar, büyük bir liman ters yerleşmek böylece bir açıyla yerleştirin. </li><li> Boncuk yapıldıktan sonra, küçük vanaların açık ve medya dışarı akmasına izinRCC ve bir atık kabı içine. Bu şekilde 1 / 3 medya çıkarın. </li><li> Küçük bir kapak kapatın ve büyük bir liman açmak. Yavaşça girdap gemi çözüm içine agrega dağıtmak için. Boş steril bir 50ml konik tüp içine sıvı karışımı. Toplanan büyüklükler agrega kurtarma en üst düzeye çıkarmak için kültür damarı iyice yıkamak için supernatant kullanmadan önce konik tüp yerleşmek için izin verin. Büyüklükler, akış sitometri, istila testleri, immunofloresan ve diğerleri gibi hemen aşağı deneyleri için kullanılan olabilir. </li></ol> 7. Temsilcisi Sonuçlar RCC Cytodex-3 boncuk üzerinde yetiştirilen EVT benzeri hücreler (trofoblast hücre hattı SGHPL-4) bir örneği Şekil 2&#39;de gösterilmiştir. EVT hücre hattı gibi ana küme uzak uzanan ve komşu kümeler takılarak projeksiyonları görüntüler. Birçok boncuk tamamen propaganda hücreleri ile kaplıdır. Bir kez RCC ve pla kaldırıldıted bir ekstrasellüler matriks, EVT gibi 3-D yetiştirilen hücreler, agresif ve / veya göç (Şekil 3) işgal . RT-PCR veriler geleneksel hücre kültürü mono tabakaları (Şekil 4) karşı 3-D agrega görülen MMP&#39;lerin artmış ekspresyonu onaylar. İlginçtir ki, işgali ile ilişkili olmayan genlerin de RCC upregüle (Tablo 1) ve işgalci trofoblast hücreleri ile bağışıklık etkileşimler gibi alanlarda tasvir yardımcı olabilir. <img alt="Şekil 1" src="/files/ftp_upload/3367/3367fig1.jpg" /> Şekil 1. Döner Hücre Kültürü Sistemi (RCC) Karikatür tasviri. <img alt="Şekil 2" src="/files/ftp_upload/3367/3367fig2.jpg" /> Şekil 2, 5 gün büyüme bir RCC sonra bir temsilci toplam faz kontrast mikrografı . Oklar işgalci hücreleri gösterir ve * Cytodex-3 microcarrier boncuk gösterir. <imgalt = &quot;Şekil 3&quot; src = &quot;/ files/ftp_upload/3367/3367fig3.jpg&quot; /&gt; Şekil 3 RCC Yetiştirilen agrega fibrin jel gömüldü. Fibrin jel ile Invasion, (A) 24 saat sonrası fibrin gömme gibi erken olarak görülen ve (B) 48 saat boyunca devam edildi. Oklar işgalci hücreleri gösterir * Cytodex-3 microcarrier boncuk (Ref izni ile uyarlanmıştır. 7) ifade eder. <img alt="Şekil 4" src="/files/ftp_upload/3367/3367fig4.jpg" /> Şekil 4, tek tabaka ve RCC Karşılaştırmalı jelatin zymogram SGHPL-4 EVT-benzeri hücreler yayılır . Pro-MMP-2 ve MMP-9 ve aktif formları RCC yetiştirilen trofoblast hücreleri (Ref 7 izni ile uyarlanmıştır) tarafından salgılanan. <img alt="Tablo 1" src="/files/ftp_upload/3367/3367table1.jpg" /> Tablo 1 bir RCC yetiştirilen SGHPL-4 hücreleri (Ref 7 izni ile uyarlanmıştır) mikroarray sonuçları özeti. <ol class="lalpha"><li3-D normalize gen değeri / normalize monlayer gen değeri:&gt; 3-D agrega Katlama indüksiyon aşağıdaki gibi hesaplanır. </li><li> Mono tabakaları algılama sınırının altında genler gösterir. </li></ol>

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Biz ifşa etmek başka bir şey var.

Burada sunulan kültür tekniği son derece invaziv EVT-benzeri hücreler ile araştırmacılar sağlar. Şimdi farklılaşma kaybı hücresel yanıtları, üç boyutlu (apikal, bazal ve lateral hücre yüzeylerinde) 10,13 kimyasal ve moleküler ipuçları inhibisyonu nedeniyle mono tabakaları oluşur kabul edilmiştir . Bu teknik EVT hücreleri istila in utero not özelliklerini yansıtır. Prosedür geleneksel tek tabakalı doku kültürü zaman kinetiği taklit eder, ancak diferansiyel ifadesi il...

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Cytodex microcarrier boncuklar </td><td> Sigma-Aldrich </td><td> C3275 </td><td></td></tr><tr><td> Döner Hücre Kültürü Sistemi (RCC) </td><td> Synthecon </td><td> RCC-D </td><td> Rotor tabanı, güç kaynağı, 4 tek RCC birimleri içerir </td></tr><tr><td> RCC Disposable Üniteleri </td><td> Synthecon </td><td> İletişim Synthecon </td><td></td></tr><tr><td> 3ml Luer-Lock ucu şırınga </td><td> BD </td><td> 309585 </td><td></td></tr><tr><td> 10ml geniş ucu serolojik pipet </td><td> BD </td><td> 357504 </td><td></td></tr><tr><td> MEM Alpha </td><td> Invitrogen </td><td> 12561-072 </td><td></td></tr><tr><td> Leibovitz L-15 orta, toz </td><td> Invitrogen </td><td> 41300-039 </td><td></td></tr><tr><td> H 2 O, Endotoksin ücretsiz </td><td> Balıkçı </td><td> MT-25-055-CM </td><td></td></tr><tr><td> Sodyum bikarbonat </td><td> Sigma-Aldrich </td><td> S-7795 </td><td></td></tr><tr><td> Pepton </td><td> Fisher Scientific </td><td> BP1420-100 </td><td></td></tr><tr><td> Fruktoz </td><td> Sigma-Aldrich </td><td> F3510-100 </td><td></td></tr><tr><td> Galaktoz </td><td> Sigma-Aldrich </td><td> G5388-100 </td><td></td></tr><tr><td> Glikoz </td><td> Sigma-Aldrich </td><td> G7528-250 </td><td></td></tr><tr><td> Hepes </td><td> Invitrogen </td><td> 15630-080 </td><td></td></tr><tr><td> L-Glutamine </td><td> Invitrogen </td><td> 25030 </td><td></td></tr><tr><td> İnsülin-Transferrin-sodyum selenit (ITS) </td><td> Sigma-Aldrich </td><td> I1884 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 10437 </td><td></td></tr><tr><td> Penisilin-Streptomisin </td><td> Invitrogen </td><td> 15140 </td><td></td></tr></tbody></table>

rotating cell culture systems for human cell culture: human trophoblast cells as a model

Insan trofoblast araştırma alanında plasenta sırasında kurulan karmaşık bir ortamda anlama yardımcı olur. Bu çalışmalar doğası gereği, in vivo deney insan mümkün değildir. Primer kültürlerinde, eksplant kültürler ve trofoblast hücre hatları 1 bir arada rahim duvarına 2 işgali ve gebelik başarılı bir şekilde kurulması için gerekli olan extravillous trofoblast hücreleri (EVTs), rahim spiral arterler 3,4 remodeling anlayışımızı desteklemektedir. Bu modellerin toplanan bilginin zenginlik rağmen, in vivo meslektaşları 5,6 ile kıyaslandığında EVT gibi hücre hatları kullanılarak in vitro hücre kültür modelleri hücresel özellikleri değişmiş ekran kabul edilir. Dönen hücre kültür sistemi (RCC) kültür hücreler u daha yakından ayırt taklit EVT hücre hatları gibi, morfolojik, fenotipik ve fonksiyonel özellikleri görüntülerişgali (örneğin matriks metalloproteinazların (MMP)) ve trofoblast farklılaşma 7,8,9 aracılık genlerin artmış ekspresyonu ile Tero EVTs. Saint Georges Hastanesi Plasental hücre Line-4 (SGHPL 4) (nazik Dr Guy Whitley ve Dr. Judith Cartwright tarafından bağışlanan) RCC test etmek için kullanılan bir EVT gibi hücre hattı. RCC kültür geminin tasarımı, organ ve dokuların üç boyutlu (3-D) bir ortamda işlev olduğu ilkesine dayanmaktadır. Fizyolojik ilgili kayma koşulları da dahil olmak üzere, üç boyutlu olarak yetişen gemi dinamik kültür koşulları, hücreler nedeniyle doğal hücresel yakınlık temelinde bir araya toplanırlar ve doku gibi meclisleri Organotipik 10,11,12 farklılaşırlar. In vivo koşullara benzer bir düşük kesme, düşük türbülans ortamında bir sıvı yörüngesinin bakım sağlar. Kültürlü hücrelerinin Sedimantasyon dönme ayarlayarak karşıhücrelerin sürekli bir serbest düşüş sağlamak için RCC hızı. Gaz değişimi biyoreaktör arkasında bulunan geçirgen bir hidrofobik zarından oluşur. Ebeveyn in vivo doku gibi, RCC yetiştirilen hücreler, gözenekli microcarrier boncuk yüzey kültüre, çünkü üç boyutlu (yani, apikal, bazal ve lateral yüzeylerde), kimyasal ve moleküler gradyanlar yanıt vermek mümkün. Plastik gibi geçirimsiz yüzeylerde iki boyutlu mono tabakaları gibi büyüdüğü zaman, hücreler bazal yüzeyindeki bu önemli iletişim yoksun. Sonuç olarak, çevreye dayattığı mekansal kısıtlamalar derinden, böylece 3-D ortam 13 için önemli bir rol ima hücreleri çevreleyen mikroçevresinin duygusu ve decode sinyalleri nasıl etkileyecek. Biz, çeşitli insan epitel dokuların 7,14,15,16 biyolojik olarak anlamlı bir 3-D modellerin mühendisi RCC kullandık . Gerçekten de, çok daha önceki raporlarda demRCC kültür hücreleri diğer modelleri 10,17-21 mümkün olmayan fizyolojik ilgili fenotipleri varsayabiliriz hücre dizilerindeki ardışık. Özet olarak, RCC kültür farklılaşmış hücrelerin çeşitli deneysel manipülasyon için uygun olan çok sayıda sağlayan kolay, tekrarlanabilir, yüksek verimli bir platformu temsil eder. Aşağıdaki protokol, bir örnek olarak EVTs kullanarak, açıkça RCC üç boyutlu kültür yapışık hücreleri için gerekli adımları tanımlamak.

Watch this Scientific Journal Video about Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model at JoVE.com

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Geleneksel iki boyutlu hücre kültür teknikleri genellikle farklılaşma belirteçleri, sitokinler ve büyüme faktörleri açısından değişmiş özellikleri. Extravillous trofoblast hücre hattı ile burada gösterildiği gibi dönen hücre kültür sistemi Üç boyutlu hücre kültürü (RCC), bu faktörlerin çoğunun ifade yeniden kurar.

İnsan Hücre Kültürü Döner Hücre Kültür Sistemleri: Bir Model Olarak İnsan trofoblast Hücreleri

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

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Research

JoVE Journal

Bioengineering

16.4K Görüntüleme.  Tulane University Medical School. Geleneksel iki boyutlu hücre kültür teknikleri genellikle farklılaşma belirteçleri, sitokinler ve büyüme faktörleri açısından değişmiş özellikleri. Extravillous trofoblast hücre hattı ile burada gösterildiği gibi dönen hücre kültür sistemi Üç boyutlu hücre kültürü (RCC), bu faktörlerin çoğunun ifade yeniden kurar.

Video: Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

Mekanik Dinamik Hücre Kültürü için Microfabricated platformlar

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Biyomühendislik

Shape Memory Polymers for Active Cell Culture

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent shape upon the application of a stimulus such as heat1-5. In a typical shape memory cycle, the SMP is first deformed at an elevated temperature that is higher than its transition temperature, Ttrans [either the melting temperature (Tm) or the glass transition temperature (Tg)]. The deformation is elastic in nature and mainly leads to a reduction in conformational entropy of the constituent network chains (following the rubber elasticity theory). The deformed SMP is then cooled to a temperature below its Ttrans while maintaining the external strain or stress constant. During cooling, the material transitions to a more rigid state (semi-crystalline or glassy), which kinetically traps or "freezes" the material in this low-entropy state leading to macroscopic shape fixing. Shape recovery is triggered by continuously heating the material through Ttrans under a stress-free (unconstrained) condition. By allowing the network chains (with regained mobility) to relax to their thermodynamically favored, maximal-entropy state, the material changes from the temporary shape to the permanent shape.
Cells are capable of surveying the mechanical properties of their surrounding environment6. The mechanisms through which mechanical interactions between cells and their physical environment control cell behavior are areas of active research. Substrates of defined topography have emerged as powerful tools in the investigation of these mechanisms. Mesoscale, microscale, and nanoscale patterns of substrate topography have been shown to direct cell alignment, cell adhesion, and cell traction forces7-14. These findings have underscored the potential for substrate topography to control and assay the mechanical interactions between cells and their physical environment during cell culture, but the substrates used to date have generally been passive and could not be programmed to change significantly during culture. This physical stasis has limited the potential of topographic substrates to control cells in culture.
Here, active cell culture (ACC) SMP substrates are introduced that employ surface shape memory to provide programmed control of substrate topography and deformation. These substrates demonstrate the ability to transition from a temporary grooved topography to a second, nearly flat memorized topography. This change in topography can be used to control cell behavior under standard cell culture conditions.

A method for developing cell culture substrates with the ability to change topography during culture is described. The method makes use of smart materials known as shape memory polymers that have the ability to memorize a permanent shape. This concept is adaptable to a wide range of materials and applications.

Aktif Hücre Kültürü Şekil Bellek Polimerler

Shape memory polymers (SMPs) are a class of "smart" materials that have the ability to change from a fixed, temporary shape to a pre-determined permanent ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Farklı soy İnsan Embriyonik Kök Hücreleri (hESC) Yayılması ve Farklılaşma için 3D Platformu olarak aljinat Mikrokapsül

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment 1. Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions 1-2. However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion3. Matrix density 4, stiffness 5-6, and structure 6-7, interstitial fluid pressure 8, and interstitial fluid flow 8 are all altered during cancer progression.
Interstitial fluid flow in particular is higher in tumors compared to normal tissues 8-10. The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 &mu;m s-1, depending on tumor size and differentiation 9, 11. This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability 12. Interstitial fluid flow has been shown to increase invasion of cancer cells 13-14, vascular fibroblasts and smooth muscle cells 15. This invasion may be due to autologous chemotactic gradients created around cells in 3-D 16 or increased matrix metalloproteinase (MMP) expression 15, chemokine secretion and cell adhesion molecule expression 17. However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts 18, (b) transporting of antigens and other soluble factors to lymph nodes 19, and (c) modulating lymphatic endothelial cell morphogenesis 20.
The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion 13-15, 17. By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Interstitial fluid flow is elevated in solid tumors and can modulate tumor cell invasion. Here we describe a technique to apply interstitial fluid flow to cells embedded in a matrix and then measure its effects on cell invasion. This technique can be easily adapted to study other systems.

Tümör Hücre Invasion üzerine İnterstisyel Akışkan Etkilerinin Ölçülmesi için üç boyutlu Hücre Kültürü Modeli

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Kütle Sitometri: Bir CyTOF Kütle Sitometre Daily Tuning ve Koşu Hücre Örnekleri için protokol

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Cell Co-culture Patterning Using Aqueous Two-phase Systems

Cell patterning technologies that are fast, easy to use and affordable will be required for the future development of high throughput cell assays, platforms for studying cell-cell interactions and tissue engineered systems. This detailed protocol describes a method for generating co-cultures of cells using biocompatible solutions of dextran (DEX) and polyethylene glycol (PEG) that phase-separate when combined above threshold concentrations. Cells can be patterned in a variety of configurations using this method. Cell exclusion patterning can be performed by printing droplets of DEX on a substrate and covering them with a solution of PEG containing cells. The interfacial tension formed between the two polymer solutions causes cells to fall around the outside of the DEX droplet and form a circular clearing that can be used for migration assays. Cell islands can be patterned by dispensing a cell-rich DEX phase into a PEG solution or by covering the DEX droplet with a solution of PEG. Co-cultures can be formed directly by combining cell exclusion with DEX island patterning. These methods are compatible with a variety of liquid handling approaches, including manual micropipetting, and can be used with virtually any adherent cell type.

Aqueous two-phase systems were used to simultaneously pattern multiple populations of cells. This fast and easy method for cell patterning takes advantage of the phase separation of aqueous solutions of dextran and polyethylene glycol and the interfacial tension that exists between the two polymer solutions.

Sulu İki fazlı sistemler kullanma Hücre Co-kültür desenlendirme

Cell patterning  technologies that are fast, easy to use and affordable will be required for the  future development of high throughput cell assays, ...

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Zehirli İnhale Kimyasal Test in vitro Hücre Kültürü Modeli

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

İnsan Meme Bezi Epitelinin Bir Hormon duyarlı 3D Kültür Modeli

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3D Hücre Kültürü için Pulmoner Ekstrasellüler Matrix Ayarlanabilir Hidrojellerinin

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

Kültür Ultrathin Porated elastik Hydrogels bir Biomimetic membran için çift olarak hücre

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...