Les auteurs tiennent à remercier Dirk Herrmann, Oliver Wendt, Siegfried Horn, Hartmut Gutzeit, et Joerg Bohn pour leur aide substantielle concernant le soudage des vapeurs de solvants. Par ailleurs, nous tenons à remercier Michael Hartmann, Alex Gerwald, et Daniel Leisen pour leur assistance technique.

Séquence Process # 1: embossage à chaud, d&#39;usinage et de soudage de vapeur de solvant Le CellChip dans son design cubique est répliqué par estampage à chaud ou micro injection. Pour cela, nous utilisons un moule en laiton usinés avec la géométrie inverse de la puce. Les conteneurs - disposés en un réseau régulier de jusqu&#39;à 1156 conteneurs - ont un design cubique avec une longueur d&#39;arête de 300 um. Pour embossage à chaud, le processus de réplication est effectuée sur un WUM02 classiques (Jenoptik Mikrotechnik, Allemagne). L&#39;outil se compose de deux plaques métalliques circulaires. Dans un premier temps, une fine plaque de PMMA (Lucryl, G77Q11, BASF) est placé dans le centre de la plaque inférieure de l&#39;outil ouvert. L&#39;insert de moule microstructuré est centriquement monté dans la plaque supérieure. Ensuite, l&#39;outil est fermé pour l&#39;évacuation du moule et chauffé à une température supérieure à la température de transition vitreuse du polymère. En appuyant sur les plaques les unes, le polymère visqueux est poussé dans les cavités évacués jusqu&#39;à ce qu&#39;ils soient complètement remplis précisément reproduisant la géométrie du moule. Après refroidissement de l&#39;outil, la partie microstructurées polymère peut être démoulé. Le processus exige une plus grande masse de polymère que sont réellement nécessaires pour remplir les cavités du moule. L&#39;excédent polymère forme une couche résiduelle qui peut être utilisé pour faciliter le démoulage de la pièce. Toutefois, pour créer des pores de diamètre inférieur à 3 um dans le fond des récipients, l&#39;épaisse couche résiduelle doit être aminci, voire totalement supprimée et remplacée par une membrane poreuse. Pour simplifier le processus d&#39;intégration des pores, l&#39;arrière de la CellChip répliqué est complètement enlevée par usinage avec un moulin à diamant. Pour cela, les pièces sont fixées sur une plaque refroidie de montage et, en outre, les structures fragiles sont congelés dans de l&#39;eau déminéralisée pour les protéger contre les dommages. Dans une étape du processus dernière, enfin, un ion commerciales track-etched membrane (polycarbonate, épaisseur 10 microns, la taille des pores 3 um, 2x10 6 pores / cm ², Pieper Filter GmbH) est collée à l&#39;arrière du tableau de conteneurs maintenant ouvert à la fois sur haut et en bas. Le processus de collage est un procédé de soudage des vapeurs de solvants réalisés dans une étanche, chambre chauffée [Fig. 1], composé d&#39;un piston supérieure et une plaque mobile inférieure avec un mandrin à vide intégrée. 4 Jusqu&#39;à quatre CellChips usinées et track-etched membranes sont exposées en parallèle à un solvant vaporisé après la chambre a été évacuée. Ensuite, les pièces moulées et les membranes sont pressées par le piston supérieur. Après une courte période d&#39;exposition (&lt;15 s), la chambre est évacué à nouveau ce qui élimine le solvant. En raison de la courte durée de contact, seulement près de la surface du matériel est dissoute et une déformation de la structure en vrac à cause de la charge mécanique peut être évitée. Enfin, la chambre est ouverte et le solvant CellChips soudés peuvent être prélevés et préparés pour la culture cellulaire [Fig. 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="Figure 1" width="100" /></a> Figure 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="Figure 2" width="100" /></a> Figure 2. Séquence Process # 2: irradiation aux ions lourds, microthermoforming et gravure de piste (processus SMART) Le nouveau processus appelé SMART est une technologie récemment développée pour la fabrication de micro-fonctionnalisés de type membranaire microstructures. 5 La technologie est basée sur un processus de thermoformage microtechniques, appelé «microthermoforming &#39;6,7. Dans cette étape du processus central, qui a été adaptée de l&#39;échelle macroscopique piégées processus de feuille de thermoformage, une fine pellicule chauffée polymère est formé dans son adouci, caoutchouc état élastique par la pression du gaz dans une cavité du moule [Fig. 3]. Contrairement à gaufrage chaud ou le moulage par injection, ce processus n&#39;est pas une forme primaire et le polymère n&#39;est pas fondu. En raison du fait que le film est formé toujours dans un état solide avec une cohésion permanente du matériel, des modifications matérielles à haute résolution latérale peut d&#39;abord être générés sur des films polymères planaires et sont conservés tout au long du processus de formage. Après l&#39;étape de microthermoforming, ces modifications peuvent encore être traitées de manière sélective, par exemple, par un traitement chimique par voie humide. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_9/Giselbrecht-Fig3.png" alt="Figure 3" width="400" height="217" /> Figure 3 Le processus dans son principe SMART se compose de trois étapes: <ol><li> création de modèles de modification hautement résolues sur le plan des films de polymère mince dans un processus de pré- </li><li> 3D ShapING de films de microthermoforming sans perte de (motif) des modifications </li><li> post-traitement (en option) pour une fonctionnalisation finale de pièces à parois minces microstructurées </li></ol> Le processus de Smart, nous appliquons actuellement pour la fabrication de CellChips poreuse comprend les étapes suivantes [Fig. 4]. Un film mince de polymère, par exemple, à partir de polycarbonate (Pokalon OG461Gl, 50 um, Lofo High Tech Film GmbH, Allemagne), est irradié par des ions lourds (tels que les Xe, Au ou des ions U) à ses installations d&#39;accélérateur du GSI (Darmstadt, Allemagne) avec des énergies d&#39;env. 1 GeV et fluences de l&#39;ordre de 106 8 Après refroidissement de l&#39;outil, la partie à parois minces peuvent être démoulées. ions / cm ². En pénétrant à travers le film, chaque ion produit un sentier presque rectiligne de matière modifiée, appelée piste latente. Les films pré-traitées sont ensuite thermoformé à un tableau de 25x25 minces parois microcontainers, chacun avec un diamètre et une profondeur de 300 um. Le processus est actuellement réalisée sur une presse de gaufrage à chaud modifiés [Fig. 5], où le film de polymère est serré entre deux plaques de métal. La plaque supérieure est équipée de micro-usinés le moule et la partie inférieure contient la pression et connecteurs sous vide. Le film est étiré en microcavités préalablement évacués du moule par l&#39;azote avec une pression de 5 MPa. Les films sont formés près de leur température de transition vitreuse prévenir suivre recuit. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_10/Giselbrecht-Fig4.png" alt="Figure 4" width="400" height="119" /> Figure 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="Figure 5" width="100" /></a> Figure 5 Dans un post-traitement, les pistes d&#39;ions sont gravé sélectivement aux pores en immergeant la microstructure ensemble dans un milieu gravure approprié (par exemple, 5 mol / L NaOH, 10% p / v MeOH). En contrôlant le temps de gravure et la gravure des conditions, telles que la concentration, la température et des additifs spéciaux (par exemple, les promoteurs Etch), la taille et la forme des pores qui en résulte peut être ajustée [Fig. 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="Figure 6" width="100" /></a> Figure 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>

Bien que les méthodes établies de microreplication polymère, tels que les micro injection ou gaufrage à chaud, sont aptes à la production des microstructures, ils ne sont pas vraiment efficace dans la production de microstructures avec un système intégré et hautement porosité contrôlée, comme cela est nécessaire pour la CellChip. Structures encombrants par exemple nécessitent un usinage coûteux de réduire l&#39;épaisseur de mur pour une perforation au laser ultérieurs ou les murs doivent être complètement remplacés par d...

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

En utilisant des technologies de microfabrication est un prérequis pour créer des échafaudages de la géométrie reproductible et une qualité constante pour la culture cellulaire en trois dimensions. Ces technologies offrent un large éventail d&#39;avantages non seulement pour la fabrication mais aussi pour des applications différentes. La taille et la forme d&#39;amas cellulaires formés peuvent être influencés par l&#39;architecture exacte et reproductible de la microfabriqué échafaudage et, par conséquent, la longueur du chemin de diffusion des nutriments et des gaz peuvent être controlled.1 C&#39;est incontestablement un outil utile pour empêcher l&#39;apoptose et nécrose des cellules en raison d&#39;un nutriment insuffisant et la fourniture de gaz ou de l&#39;élimination des métabolites cellulaires. Notre puce polymère, appelé CellChip, a les dimensions extérieures de 2 x 2 cm avec une zone centrale microstructurée. Cette zone est subdivisée en un tableau d&#39;un maximum de 1156 microcontainers avec une dimension typique de 300 m longueur de bord pour la conception cubes (cp-ou CF-chip) ou de 300 m de diamètre et la profondeur de la conception ronde (r-chip). 2 Jusqu&#39;ici, l&#39;embossage à chaud ou à micro-injection (en combinaison avec d&#39;usinage laborieuses ultérieures des parties) a été utilisé pour la fabrication des puces microstructurée. Fondamentalement, la micro injection est une des techniques de polymère seul réplication basée que, jusqu&#39;à présent, est capable d&#39;une production de masse de polymère microstructures.3 Toutefois, les deux techniques ont certaines limitations indésirables dus au traitement d&#39;un polymère visqueux fondre avec la génération des parois très minces ou intégrés à travers les trous. Dans le cas de l&#39;CellChip, des couches minces en bas sont nécessaires pour perforer le polymère et de fournir de petits pores de taille définie à l&#39;approvisionnement des cellules avec par exemple un milieu de culture par perfusion microfluidique des conteneurs. Afin de surmonter ces limitations et de réduire les coûts de fabrication, nous avons développé une nouvelle approche microtechniques sur la base d&#39;un processus de bas-échelle de thermoformage. Pour la fabrication de très poreux et mince paroi puces polymères, nous utilisons une combinaison de l&#39;irradiation d&#39;ions lourds, microthermoforming et gravure piste. Dans ce soi-disant «SMART» du processus (modification du substrat et de la réplication par thermoformage) des films minces de polymère sont irradiés avec énergiques projectiles lourds de plusieurs centaines de MeV introduisant soi-disant «pistes latente» Par la suite, le film dans un état de caoutchouc élastique est formé en trois pièces dimensions sans modifier ou de recuit les pistes. Après le processus de formation, la gravure chimique sélective convertit enfin les pistes dans les pores cylindriques d&#39;un diamètre réglable.

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

Nous présentons deux procédés pour la microfabrication de puces polymère poreux pour la culture cellulaire en trois dimensions. Le premier est gaufrage à chaud combiné avec un processus de soudage à la vapeur de solvant. Le second utilise un procédé développé récemment microthermoforming combinée à la technologie d&#39;ions piste menant à une simplification significative de la fabrication.

La microfabrication de Chip entreprises Échafaudages pour la culture cellulaire en trois dimensions

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

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11.7K vues.  Karlsruhe Research Centre. Nous présentons deux procédés pour la microfabrication de puces polymère poreux pour la culture cellulaire en trois dimensions. Le premier est gaufrage à chaud combiné avec un processus de soudage à la vapeur de solvant. Le second utilise un procédé développé récemment microthermoforming combinée à la technologie d'ions piste menant à une simplification significative de la fabrication.

Vidéo: 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.

Chip à base de culture cellulaire tridimensionnelle dans perfusé micro-bioréacteurs

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

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Biologie

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.

Interview: bioréacteurs et de surfaçage-Modified-3D Echafaudages pour la recherche de cellules souches

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.

Électrofilage treillis polymères fibreux pour l&#39;ingénierie tissulaire et de la culture cellulaire

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.

Isolement des cellules épithéliales mammaires à partir de trois dimensions de cellules mixtes Sphéroïde 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 ...

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.

Deux et trois dimensions imagerie de cellules vivantes de protéines endommagent l&#39;ADN de réponse

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.

Souris foetal entier Intestin Système de culture pour<em&gt; Ex Vivo</em&gt; Manipulation des voies de signalisation et d&#39;imagerie en direct en trois dimensions du développement des villosités

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.

FRET Imaging en hydrogels en trois dimensions

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.

Reconstruction tridimensionnelle de l’Architecture vasculaire de l’ovaire de souris de clarté affranchis Passive

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 : Un tissu méthode de compensation pour l’imagerie en trois dimensions du tissu adipeux

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.

Imagerie tridimensionnelle à résolution unicellulaire des Organoïdes intacts

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