Yazarlar Dirk Herrmann, Oliver Wendt, Siegfried Horn, Hartmut Gutzeit ve Joerg Bohn çözücü buhar kaynağı ile ilgili önemli yardım için teşekkür etmek istiyorum. Ayrıca, kendi teknik yardım için Michael Hartmann, Alex Gerwald, Daniel Leisen kabul etmek istiyorum.

İşlem Sırası # 1: Sıcak Kabartma, Talaşlı İmalat ve Solvent Buhar Kaynak Kübik tasarımı CellChip sıcak kabartma veya mikro enjeksiyon ile çoğaltılır. Bunun için çip ters geometri Mikroişlenmiş pirinç kalıp kullanın. Konteynerleri - 1156 kaplar kadar düzenli bir dizi düzenlenen bir kenar uzunluğu 300 mm olan bir küp tasarımı var. Sıcak kabartma için geleneksel bir WUM02 (Jenoptik Mikrotechnik, Almanya), çoğaltma işlemi yapılır. Aracı iki yuvarlak metal plaka oluşur. İlk adım olarak, ince bir PMMA plaka (Lucryl, G77Q11, BASF) açılan aracın alt plaka merkezinde yer almaktadır. Microstructured kalıp eklemek merkez üst plaka monte edilir. Sonra aracı kalıp tahliye için kapalı ve polimerin camsı geçiş sıcaklığı üzerinde bir sıcaklığa kadar ısıtılır. Plakalar birlikte basarak, tam kalıp geometrisi kopyalayan tamamen dolana kadar viskoz polimer tahliye boşlukları içine itilir. Aracı Soğuduktan sonra, microstructured polimer parçası demoulded olabilir. Bu süreç aslında kalıp boşlukları doldurmak için gerekli olan daha fazla polimer kitle gerektirir. Polimer fazlalık parça kalıptan çıkarma kolaylığı için kullanılabilir kalan bir tabaka oluşturur. Ancak, konteynerlerin altında 3 mm daha küçük çaplı gözenekler oluşturmak için, kalın kalan tabaka inceltilerek, hatta tamamen gözenekli bir zar ile kaldırılır ve yerine. Gözenek entegrasyon sürecini kolaylaştırmak için, çoğaltılmış CellChip tamamen geri bir elmas değirmeni ile işleme tarafından kaldırılır. Bunun için, parça soğutmalı bir montaj plakası sabit ve buna ek olarak, kırılgan yapıları hasara karşı onları korumak için deiyonize su dondurulur. Bir son süreç adım, nihayet bir ticari iyon parça-kazınmış membran (polikarbonat, kalınlığı 10 mikron gözenek boyutu 3 mm, 2x10 6 gözenekleri / cm ², Pieper Filtre GmbH) konteyner dizinin arkasına yapıştırılır. Şimdi hem de açıldı üst ve alt. Yapıştırma işlemi, gaz geçirmez, ısıtmalı hazneli [Şekil yapılan solvent buhar kaynak işlemidir. 1], entegre bir vakum mandreni ile bir üst piston ve hareketli bir alt tablası oluşan dört işlenmiş CellChips ve track-kazınmış membranlar 4 odanın tahliye edildikten sonra buharlaşarak çözücü paralel olarak maruz kalmaktadır. Daha sonra üst piston, kalıp parçaları ve membranlar birlikte bastırılır. Pozlama (&lt;15 s) kısa bir süre sonra tekrar böylece solvent kaldırma, oda boşaltıldı. Kısa temas süresi nedeniyle, sadece yüzey malzemesi yakın çözülür ve toplu yapı nedeniyle mekanik yük deformasyon önlenebilir. Son olarak, oda açıldı ve solvent kaynaklı CellChips silinebilir ve hücre kültürü [Şekil için hazırlanmıştır. 2]. <a href="/files/ftp_upload/old/699_2008_5_12_7/Giselbrecht-Fig1.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_7/Giselbrecht-Fig1.png" alt="Şekil 1" width="100" /></a> Şekil 1. <a href="/files/ftp_upload/old/699_2008_5_12_8/Giselbrecht-Fig2.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_8/Giselbrecht-Fig2.png" alt="Şekil 2" width="100" /></a> Şekil 2. İşlem Sırası # 2: ağır iyon ışınlama, microthermoforming ve parça gravür (SMART süreci) SMART olarak adlandırılan yeni bir süreç Fonksiyonlu membran benzeri mikro üretim için son zamanlarda geliştirilen mikro teknoloji 5 &#39;microthermoforming&#39; adında bir mikroteknik termoform süreç, teknoloji tabanlı. Makroskopik uyarlanan bu merkezi işlem adımında 6,7 , sac termoform işlemi sıkışıp, ısıtmalı ince polimer film, kalıp boşluğuna [Şekil içine gaz basıncı ile yumuşatılmış, kauçuk elastik devlet oluşur. 3]. Sıcak kabartma veya enjeksiyon aksine, bu sürecin birincil bir oluşum değildir ve polimerin eritilerek değildir. Film kalıcı bir malzeme uyum hala sağlam bir devlet oluşmuş olduğu gerçeği nedeniyle, yüksek yanal kararları malzeme modifikasyonlar düzlemsel polimer filmlerin ilk oluşturulur ve şekillendirme süreci boyunca korunur. Microthermoforming adımdan sonra, bu değişiklikler daha seçici ıslak kimyasal arıtma, örneğin, işlenmiş olabilir. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_9/Giselbrecht-Fig3.png" alt="Şekil 3" width="400" height="217" /> Şekil 3 SMART ilkesine süreci üç süreç adımları oluşur: <ol><li> bir ön işlem düzlemsel ince polimer film çok çözülmesi değişiklik desen oluşturma </li><li> 3D Shapkaybı (desenli) değişiklikler olmadan microthermoforming filmlerin ing </li><li> son bir fonksiyonelleştirilmesi, ince duvarlı microstructured parçalar için işlem sonrası (isteğe bağlı) </li></ol> SMART süreci şu anda gözenekli CellChips fabrikasyon için başvuran, aşağıdaki işlem adımları [Şekil içerir. 4]. Ince polimer film, örneğin, polikarbonat (Pokalon OG461Gl, 50 mikron, LoFo High Tech Film GmbH, Almanya), (Darmstadt, GSI hızlandırıcı tesislerinde hızlandırılmış ağır iyonları (örneğin Xe, Au veya U iyonları gibi) ile ışınlanmış yaklaşık enerjiler ile Almanya). 1 GeV ve aracı soğutma sonra 106 8 amacıyla fluence, ince duvarlı bir parçası demoulded olabilir . iyonları / cm ² &#39;dir. Film aracılığıyla nüfuz zaman, her iyon gizli track denilen değiştirilmiş materyalinin neredeyse düz bir iz üretir. Önişlemden filmler sonra 25x25, ince duvarlı microcontainers, çapı 300 mm ve derinliği her bir dizi için ısıyla. Bu süreç şu anda değiştirilmiş bir sıcak kabartma basın [Şekil yapılır. 5], polimer filmin, iki metal plaka arasına sıkıştırılır. Mikroişlenmiş kalıp üst plakası ile donatılmıştır ve düşük basınç ve vakum konnektörleri içerir. Film, 5 MPa kadar bir basınç ile azot kalıp, daha önce tahliye microcavities gerilir. Filmler tavlama parça önlenmesi cam geçiş sıcaklığına yakın oluşur. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_10/Giselbrecht-Fig4.png" alt="Şekil 4" width="400" height="119" /> Şekil 4 <a href="/files/ftp_upload/old/699_2008_5_12_11/Giselbrecht-Fig5.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_11/Giselbrecht-Fig5.png" alt="Şekil 5" width="100" /></a> Şekil 5 Bir süreç sonrası, tüm mikroyapı uygun bir gravür orta (örneğin, 5 Mol / L NaOH,% 10 w / v MeOH) içine çeker seçici iyon parçaları gözenekleri kazınmış. Gravür kez kontrol ve konsantrasyon, sıcaklık ve özel katkı maddeleri (örneğin, aşındırma yararlanıcı grup) olarak koşulları, gravür, gözeneklerin boyutunu ve şeklini [Şekil ayarlanabilir. 6]. <a href="/files/ftp_upload/old/699_2008_5_12_12/Giselbrecht-Fig6.png"><img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_12/Giselbrecht-Fig6.png" alt="Şekil 6" width="100" /></a> Şekil 6

<ol>
	<li>Knedlitschek, G., Schneider, F., Gottwald, E., Schaller, T., Eschbach, E., Weibezahn, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tissue-like+culture+system+using+microstructures:+influence+of+extracellular+matrix+material+on+cell+adhesion+and+aggregation.">A tissue-like culture system using microstructures: influence of extracellular matrix material on cell adhesion and aggregation.</a> J Biomech Eng. 121, 35-39 (1999).</li><li>Gottwald, E., Giselbrecht, S., Augspurger, C., Lahni, B., Dambrowsky, N., Truckenm&#252;ller, R., Piotter, V., Gietzelt, T., Wendt, O., Pfleging, W., Welle, A., Rolletschek, A., Wobus, A. M., Weibezahn, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chip-based+platform+for+the+in+vitro+generation+of+tissues+in+three-dimensional+organization.">A chip-based platform for the in vitro generation of tissues in three-dimensional organization.</a> Lab Chip. 7, 777-785 (2007).</li><li>Heckele, M., Schomburg, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+on+micro+molding+of+thermoplastic+polymers.">Review on micro molding of thermoplastic polymers.</a> Journal of Micromechanics And Microengineering. 14, (2004).</li><li>Giselbrecht, S., Gietzelt, T., Guber, A. E., Gottwald, E., Trautmann, C., Truckenm&#252;ller, R., Weibezahn, K. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microthermoforming+as+a+novel+technique+for+manufacturing+scaffolds+in+tissue+engineering+(CellChips.">Microthermoforming as a novel technique for manufacturing scaffolds in tissue engineering (CellChips.</a> IEE Proc.-Nanobiotechnol. 151, 151-157 (2004).</li><li>Giselbrecht, S., Gietzelt, T., Gottwald, E., Trautmann, C., Truckenm&#252;ller, R., Weibezahn, K. -. F., Welle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tissue+culture+substrates+produced+by+microthermoforming+of+pre-processed+polymer+films.">3D tissue culture substrates produced by microthermoforming of pre-processed polymer films.</a> Biomed Microdevices. 8, 191-199 .</li><li>Truckenm&#252;ller, R., Rummler, Z., Schaller, T., Schomburg, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-cost+thermoforming+of+micro+fluidic+analysis+chips.">Low-cost thermoforming of micro fluidic analysis chips.</a> Journal of Micromechanics and Microengineering. 12, 375-379 (2002).</li><li>Truckenm&#252;ller, R., Giselbrecht, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microthermoforming+of+flexible,+not+buried+hollow+microstructures+for+chip-based+life+sciences+applications.">Microthermoforming of flexible, not buried hollow microstructures for chip-based life sciences applications.</a> IEE Proc.-Nanobiotechnol. 151, 163-166 (2004).</li><li>Fleischer, R. L., Price, P. B., Walker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+tracks+in+solids.">Nuclear tracks in solids.</a> , .</li></ol>

, Mikro enjeksiyon kalıplama veya sıcak kabartma olarak polimer microreplication, kurulan yöntemleri, mikro üretimi için uygun olmasına rağmen, onlar CellChip için gerekli olduğu gibi, gerçekten entegre ve çok kontrollü bir gözenekli mikro üreten etkili değildir. Büyük hacimli yapılar, örneğin bir sonraki lazer perforasyon için duvar kalınlığı azaltmak için pahalı işleme gerektiren veya duvarları tamamen yerine parça kazınmış membranlar olmak zorunda. SMART, bu sorunların üstesinden gelmek ve seri üretim ...

microfabrication of chip-sized scaffolds for three-dimensional cell cultivation

Mikroimalat teknolojileri kullanarak, üç-boyutlu hücre kültürü için tekrarlanabilir geometri ve sürekli kalite iskeleleri oluşturmak için bir ön koşuldur. Bu teknolojiler sadece üretim için değil, aynı zamanda farklı uygulamalar için avantajları çok geniş bir yelpaze sunuyoruz. Oluşan hücre kümeleri boyutu ve şekli, kesin ve tekrarlanabilir microfabricated iskele mimarisi ve bu nedenle, besin ve gaz difüzyon yolu uzunluğu Bu tartışmasız apoptoz ve nekroz önlemek için yararlı bir araçtır controlled.1 olabilir etkilemiş olabilir Yetersiz besin ve gaz kaynağı ya da hücresel metabolitleri kaldırılması nedeniyle hücreler. CellChip olarak adlandırılan polimer çip, microstructured merkezi bir alana sahip 2 x 2 cm dış boyutları vardır. Bu alan, kübik tasarımı (cp-cf-chip) ya da yuvarlak tasarımı (r-chip) için 300 m çapında ve derinliği 300 m kenar uzunluğu tipik bir boyut ile 1156 microcontainers kadar bir dizi bölünmüştür. 2 Şimdiye kadar, sıcak kabartma veya mikro enjeksiyon kalıplama (sonraki zahmetli işleme parçaları ile birlikte) microstructured yongalarının üretiminde kullanılmıştır. Temel olarak, mikro enjeksiyon Ancak, bugüne kadar polimer microstructures.3 seri üretim kapasitesine sahip, tek bir polimer esaslı çoğaltma teknikleri, her iki tekniğin nesil eritebilir viskoz bir polimerin işlenmesi nedeniyle bazı istenmeyen sınırlamalar var , çok ince duvarlar veya deliklerden entegre. CellChip durumda, ince alt katmanları polimer perfore ve tanımlanan boyutu küçük gözenekleri konteyner mikroakışkan perfüzyon kültür ortamı, örneğin hücre kaynağı sağlamak için gereklidir. , Bu sınırlamaları aşmak için ve aşağı ölçekli termoform sürecinin temelinde yeni mikroteknik bir yaklaşım geliştirdik üretim maliyetleri azaltmak için. Çok gözenekli ve ince duvarlı polimer çiplerin üretim için, ağır iyon ışınlama, microthermoforming ve parça gravür bir kombinasyonunu kullanabilirsiniz. Bu sözde &quot;SMART&quot; sürecinde sözde tanıtan birkaç yüz MeV enerjik ağır mermiler ile ışınlanmış (Yüzey Ve Değişiklik Termoform tarafından çoğaltma) ince polimer film &quot;gizli parça&quot; Daha sonra, kauçuk elastik devlet film üçe oluşur boyutlu parça parça değiştirerek veya tavlama olmadan. Şekillendirme işleminden sonra, seçici kimyasal aşındırma nihayet, ayarlanabilir çaplı silindirik gözeneklerin içine parça dönüştürür.

Watch this Scientific Journal Video about Microfabrication of Chip-sized Scaffolds for Three-dimensional Cell cultivation at JoVE.com

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

Biz, üç-boyutlu hücre kültürü için gözenekli polimer cips mikroimalat için iki süreç sunuyoruz. Bunlardan birincisi, solvent buhar kaynak işlemi ile birlikte sıcak kabartma. Ikincisi ise, kısa bir süre önce geliştirilen iyon izleme teknolojisi ile üretim önemli bir basitleştirme giden kombine microthermoforming süreci kullanır.

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 ...

microfabrication-chip-sized-scaffolds-for-three-dimensional-cell

Research

JoVE Journal

Biology

11.7K Görüntüleme.  Karlsruhe Research Centre. Biz, üç-boyutlu hücre kültürü için gözenekli polimer cips mikroimalat için iki süreç sunuyoruz. Bunlardan birincisi, solvent buhar kaynak işlemi ile birlikte sıcak kabartma. Ikincisi ise, kısa bir süre önce geliştirilen iyon izleme teknolojisi ile üretim önemli bir basitleştirme giden kombine microthermoforming süreci kullanır.

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

Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from non-biodegradable polymers, e.g., polycarbonate or polymethyl methacrylate by micro injection molding, micro hot embossing or micro thermoforming. But, it can also be manufactured from bio-degradable polymers. Its overall dimensions are 0.7 1 x 20 x 20 x 0.7 1 mm (h x w x l). The main features of the chips used are either a grid of up to 1156 cubic micro-containers (cf-chip) each the size of 120-300 x 300 x 300 &mu; (h x w x l) or round recesses with diameters of 300 &mu; and a depth of 300 &mu; (r-chip). The scaffold can house 10 Mio. cells in a three-dimensional configuration. For an optimal nutrient and gas supply, the chip is inserted in a bioreactor housing. The bioreactor is part of a closed steril circulation loop that, in the simplest configuration, is additionaly comprised of a roller pump and a medium reservoir with a gas supply. The bioreactor can be run in perfusion, superfusion, or even a mixed operation mode. We have successfully cultivated cell lines as well as primary cells over periods of several weeks. For rat primary liver cells we could show a preservation of organotypic functions for more than 2 weeks. For hepatocellular carcinoma cell lines we could show the induction of liver specific genes not or only slightly expressed in standard monolayer culture. The system might also be useful as a stem cell cultivation system since first differentiation experiments with stem cell lines were promising.

We describe a chip-based platform for the three-dimensional cultivation of cells in micro-bioreactors. One chip can house up to 10 Mio. cells that can be cultivated under precisely defined conditions with regard to fluid flow, oxygen tension etc. in a sterile, closed circulation loop.

Perfüze Mikro-biyoreaktörler Chip tabanlı üç boyutlu Hücre Kültürü

We have developed a chip-based cell culture system for the three-dimensional cultivation of cells. The chip is typically manufactured from ...

Biyoloji

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,  ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

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 ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

Üç boyutlu Karışık-hücre Sfero Co-kültürden Meme Epitel Hücrelerinin İzolasyonu

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Two- and Three-Dimensional Live Cell Imaging of DNA Damage Response Proteins

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate mutations and chromosomal aberrations1. To prevent this trauma from catalyzing genomic instability, it is crucial for cells to detect DSBs, activate the DNA damage response (DDR), and repair the DNA. When stimulated, the DDR works to preserve genomic integrity by triggering cell cycle arrest to allow for repair to take place or force the cell to undergo apoptosis. The predominant mechanisms of DSB repair occur through nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) (reviewed in2). There are many proteins whose activities must be precisely orchestrated for the DDR to function properly. Herein, we describe a method for 2- and 3-dimensional (D) visualization of one of these proteins, 53BP1.
The p53-binding protein 1 (53BP1) localizes to areas of DSBs by binding to modified histones3,4, forming foci within 5-15 minutes5. The histone modifications and recruitment of 53BP1 and other DDR proteins to DSB sites are believed to facilitate the structural rearrangement of chromatin around areas of damage and contribute to DNA repair6. Beyond direct participation in repair, additional roles have been described for 53BP1 in the DDR, such as regulating an intra-S checkpoint, a G2/M checkpoint, and activating downstream DDR proteins7-9. Recently, it was discovered that 53BP1 does not form foci in response to DNA damage induced during mitosis, instead waiting for cells to enter G1 before localizing to the vicinity of DSBs6. DDR proteins such as 53BP1 have been found to associate with mitotic structures (such as kinetochores) during the progression through mitosis10.
In this protocol we describe the use of 2- and 3-D live cell imaging to visualize the formation of 53BP1 foci in response to the DNA damaging agent camptothecin (CPT), as well as 53BP1's behavior during mitosis. Camptothecin is a topoisomerase I inhibitor that primarily causes DSBs during DNA replication. To accomplish this, we used a previously described 53BP1-mCherry fluorescent fusion protein construct consisting of a 53BP1 protein domain able to bind DSBs11. In addition, we used a histone H2B-GFP fluorescent fusion protein construct able to monitor chromatin dynamics throughout the cell cycle but in particular during mitosis12. Live cell imaging in multiple dimensions is an excellent tool to deepen our understanding of the function of DDR proteins in eukaryotic cells.

This protocol describes a method for visualizing a DNA double-strand break signaling protein activated in response to DNA damage as well as its localization during mitosis.

DNA Hasarı Tepki Proteinler İki ve Üç Boyutlu Canlı Hücre Görüntüleme

Double-strand breaks (DSBs) are the most deleterious DNA lesions a cell can encounter. If left unrepaired, DSBs harbor great potential to generate ...

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

Için Fare Fetal Tüm Bağırsak Kültür Sistemi<em&gt; Ex vivo</em&gt; Sinyal facebook ve villus Kalkınma Üç boyutlu Canlı Görüntüleme Manipülasyon

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

Üç boyutlu Hidrojeller içinde Görüntüleme FRET

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Three-dimensional Reconstruction of the Vascular Architecture of the Passive CLARITY-cleared Mouse Ovary

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine system, but the complex structural relationships and three-dimensional (3D) vasculature architectures of the ovary are not well described. In order to visualize the 3D connections and architecture of blood vessels in the intact ovary, the first important step is to make the ovary optically clear. In order to avoid tissue shrinkage, we used the hydrogel fixation-based passive CLARITY (Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/ Immunostaining/In situ-hybridization-compatible Tissue Hydrogel) protocol method to clear an intact ovary. Immunostaining, advanced multiphoton confocal microscopy, and 3D image-reconstructions were then used for the visualization of ovarian vessels and follicular capillaries. Using this approach, we showed a significant positive correlation (P &#60;0.01) between the length of the follicular capillaries and volume of the follicular wall.

Here we present an adaptation of the passive CLARITY and 3D reconstruction method for visualization of the ovarian vasculature and follicular capillaries in intact mouse ovaries.

Pasif AÇIKLIĞI temizlenmiş Mouse ovarium vasküler mimarisinin üç boyutlu yeniden yapılandırma

The ovary is the main organ of the female reproductive system and is essential for the production of female gametes and for controlling the endocrine ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-Clear: Bir doku Yöntem yağ dokusu üç boyutlu görüntüleme için temizlenmesi

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Bozulmamış Organoidlerin Tek Hücreli Çözünürlükte Üç Boyutlu Görüntülemesi

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...