Gli autori desiderano ringraziare Dirk Herrmann, Oliver Wendt, Siegfried Horn, Hartmut Gutzeit, e Joerg Bohn per il loro aiuto fondamentali riguardanti la saldatura vapori di solvente. Inoltre, vorremmo riconoscere Michael Hartmann, Alex Gerwald, e Daniel Leisen per la loro assistenza tecnica.

Processo Sequenza # 1: goffratura a caldo, lavorazione e saldatura di vapore solvente Il CellChip nel suo design cubico è replicato da caldo o goffratura micro stampaggio ad iniezione. Per questo, usiamo uno stampo micromachined ottone con la geometria inversa del chip. I contenitori - disposte in una serie regolare di fino a 1156 contenitori - hanno un design cubico, con una lunghezza dello spigolo di 300 micron. Per goffratura a caldo, il processo di replica viene eseguito su un tradizionale WUM02 (Jenoptik Mikrotechnik, Germania). Lo strumento è costituito da due piastre metalliche circolari. In una prima fase, una sottile lastra in PMMA (Lucryl, G77Q11, BASF) si trova nel centro della piastra inferiore dello strumento aperto. L&#39;inserto stampo microstrutturata centrico è montato la piastra superiore. Allora il programma è chiuso per l&#39;evacuazione dello stampo e riscaldato ad una temperatura superiore alla temperatura di transizione vetrosa del polimero. Premendo i piatti insieme, il polimero viscoso viene spinto nelle cavità evacuate fino a quando sono completamente riempito con precisione replicando la geometria dello stampo. Dopo il raffreddamento dello strumento, la parte microstrutturata polimero può essere sformati. Il processo richiede più di massa di polimeri di quanto sia effettivamente necessario per riempire la cavità dello stampo. L&#39;avanzo polimero forma uno strato residuo che può essere utilizzato per facilitare la sformatura del pezzo. Tuttavia, per creare pori di diametro inferiore a 3 micron sul fondo dei contenitori, lo spesso strato residuo deve essere assottigliato verso il basso, o addirittura completamente rimosso e sostituito da una membrana porosa. Per semplificare il processo di integrazione dei pori, la parte posteriore del CellChip replicato viene completamente rimosso dalla lavorazione con un mulino diamante. Per questo, le parti sono fissate su una piastra di raffreddamento di montaggio e, inoltre, le strutture fragili sono congelati in acqua deionizzata per proteggerli da eventuali danni. In una fase di processo scorso, infine, un commerciale ioni traccia incisa a membrana (policarbonato, spessore 10 micron, diametro dei pori 3 micron, 2x10 6 pori / cm ², Pieper Filter GmbH) è legato alla parte posteriore della serie di contenitori ora aperto sia a superiore e inferiore. Il processo di incollaggio è un solvente processo di saldatura vapore eseguito in una tenuta di gas, riscaldato da camera [fig. 1], composto da un pistone superiore e un piatto inferiore mobile con un mandrino vuoto integrato. 4 Fino a quattro CellChips lavorare e tenere traccia incisa a membrane sono esposti in parallelo ad un solvente vaporizzato dopo la camera è stato evacuato. Poi, i pezzi stampati e le membrane sono premuti insieme per lo stantuffo in alto. Dopo un breve periodo di esposizione (&lt;15 s), la camera viene evacuata nuovamente eliminando il solvente. A causa del tempo di contatto breve, solo in prossimità della superficie materiale è sciolto e una deformazione della struttura del blocco a causa del carico meccanico può essere evitato. Infine, la camera è aperta e il CellChips solvente saldato può essere rimosso e preparato per colture cellulari [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="Figura 1" width="100" /></a> Figura 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="Figura 2" width="100" /></a> Figura 2. Processo Sequenza # 2: irradiazione di ioni pesanti, microthermoforming e incisione traccia (processo SMART) Il nuovo processo chiamato SMART è una tecnologia sviluppata di recente per la produzione di micro-funzionalizzati membrana come microstrutture. 5 La tecnologia si basa su un processo di termoformatura microtecnica, chiamato &#39;microthermoforming&#39;. 6,7 In questa fase del processo centrale, che è stato adattato dal macroscopico intrappolati processo di termoformatura foglio, una riscaldata sottile pellicola polimerica è formata nella sua ammorbidito, lo stato di gomma elastica di pressione del gas in una cavità dello stampo [fig. 3]. A differenza di goffratura a caldo o stampaggio ad iniezione, questo processo non è una forma primaria e il polimero non è sciolto. A causa del fatto che il film è formato ancora in uno stato solido con una coesione materiale permanente, modifiche materiale con alta risoluzione laterale può anche essere generata a planare film polimerici e sono conservate in tutto il processo di formatura. Dopo il passo microthermoforming, queste modifiche possono essere ulteriormente elaborati selettivamente, per esempio, mediante trattamento chimico. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_9/Giselbrecht-Fig3.png" alt="Figura 3" width="400" height="217" /> Figura 3 Il processo di SMART in linea di principio si compone di tre fasi del processo: <ol><li> creazione di modelli di modifica altamente deliberato planare film sottili di polimeri in un pre-processo </li><li> 3D Shapzione dei film di microthermoforming senza perdita di (fantasia) modifiche </li><li> post-processo (optional) per una funzionalizzazione finale delle parti microstrutturate pareti sottili </li></ol> Il processo di SMART stiamo applicando per la realizzazione di CellChips poroso comprende le seguenti fasi di processo [fig. 4]. Una sottile pellicola polimerica, per esempio, dal policarbonato (Pokalon OG461Gl, 50 micron, LoFo High Tech Film GmbH, Germania), viene irradiato con ioni pesanti accelerati (come Xe, Au o ioni U) presso le strutture acceleratore del GSI (Darmstadt, Germania) con energie di ca. 1 GeV e fluenze nell&#39;ordine di 106 8 Dopo il raffreddamento dello strumento, la parte con pareti sottili possono essere sformati. ioni / cm ². Quando penetrare attraverso la pellicola, ogni ione produce una scia quasi retta di materiale modificato, chiamato traccia latente. La pre-trattati film sono poi termoformato ad un array di 25x25 microcontainers con pareti sottili, ciascuno con un diametro e una profondità di 300 micron. Il processo è attualmente eseguito su una pressa modificata goffratura a caldo [fig. 5], in cui è bloccato il film polimerico tra due lastre di metallo. La piastra superiore è dotata di stampo microlavorati e quello inferiore contiene la pressione e connettori vuoto. Il film è teso in microcavità preventivamente evacuati dello stampo con azoto con una pressione fino a 5 MPa. I film sono formate vicino alla loro temperatura di transizione vetrosa prevenire traccia ricottura. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_10/Giselbrecht-Fig4.png" alt="Figura 4" width="400" height="119" /> Figura 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="Figura 5" width="100" /></a> Figura 5 In un post-processo, le tracce di ioni sono selettivamente incise a pori immergendo il tutto in una microstruttura medio incisione appropriato (per esempio, 5 Mol / L di NaOH, il 10% w / v MeOH). Controllando i tempi di incisione e acquaforte condizioni, quali la concentrazione, la temperatura e speciali additivi (ad esempio, promotori etch), la dimensione e la forma dei pori risultante può essere regolato [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="Figura 6" width="100" /></a> Figura 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>

Anche se i metodi stabiliti di microreplicazione polimero, come micro-stampaggio ad iniezione o goffratura a caldo, sono adatte per la produzione di microstrutture, non sono realmente efficaci nella produzione di microstrutture con un sistema integrato ed altamente porosità controllata, in quanto è necessario per la CellChip. Strutture ingombranti per esempio richiedono lavorazioni costose per ridurre lo spessore della parete per una perforazione laser successivo o pareti devono essere completamente sostituita da track-incise membrane. SM...

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

Utilizzando le tecnologie di microfabbricazione è un prerequisito per creare impalcature della geometria riproducibile e qualità costante per tridimensionali coltivazione delle cellule. Queste tecnologie offrono un ampio spettro di vantaggi non solo per la produzione ma anche per applicazioni diverse. Le dimensioni e la forma dei cluster di cellule formate può essere influenzato da quello relativo all&#39;architettura e riproducibile della microfabbricazione patibolo e, di conseguenza, la lunghezza del percorso di diffusione di nutrienti e dei gas può essere controlled.1 Questo è senza dubbio uno strumento utile per prevenire l&#39;apoptosi e necrosi del cellule a causa di una sostanza nutritiva insufficiente e la fornitura di gas o la rimozione di metaboliti cellulari. Il nostro chip polimero, chiamato CellChip, ha le dimensioni esterne di 2 x 2 cm con una zona centrale microstrutturata. Questa zona è suddivisa in una serie di microcontainers fino al 1156 con una dimensione tipica di 300 m di lunghezza bordo per la progettazione cubi (cp-o cf-chip) o di 300 m di diametro e profondità per la progettazione round (r-chip). 2 Fino ad ora, a caldo o goffratura micro stampaggio ad iniezione (in combinazione con la conseguente lavorazione laboriosa delle parti) è stato utilizzato per la fabbricazione dei chip microstrutturate. In pratica, i micro stampaggio ad iniezione è una delle solo tecniche replica a base polimerica che, fino ad ora, è in grado per la produzione di massa di polimeri microstructures.3 Tuttavia, entrambe le tecniche sono alcune limitazioni indesiderati dovuti al trattamento di un polimero viscoso si fondono con la generazione di pareti molto sottili o integrati attraverso i fori. In caso di CellChip, sottili strati di fondo sono necessari per perforare il polimero e di fornire piccoli pori di dimensioni definite per la fornitura di cellule, ad esempio con terreno di coltura per perfusione microfluidica dei contenitori. Per superare queste limitazioni e per ridurre i costi di produzione, abbiamo sviluppato un nuovo approccio microtecnica sulla base di una down-scalati processo di termoformatura. Per la produzione di chip a parete altamente porosa e sottili di polimeri, si usa una combinazione di irradiazione di ioni pesanti, microthermoforming e incisione pista. In questo cosiddetto processo &quot;SMART&quot; (Modifica substrato e la replicazione di termoformatura) sottili pellicole polimeriche sono irradiati con energica proiettili pesanti di diverse centinaia di MeV introducendo le cosiddette &quot;tracce latenti&quot; Successivamente, il film in uno stato di gomma elastica è formata in tre parti dimensionale senza modificare o ricottura le tracce. Dopo il processo di formatura, attacchi chimici selettivi converte definitivamente le tracce in pori cilindrici di diametro regolabile.

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

Vi presentiamo due processi per la microfabbricazione di chip polimerici porosi tridimensionali coltivazione delle cellule. Il primo è goffratura a caldo combinato con un solvente processo di saldatura vapore. La seconda utilizza un processo recentemente sviluppato microthermoforming combinata con la tecnologia agli ioni di pista che porta ad una significativa semplificazione di fabbricazione.

Microfabbricazione di chip di dimensioni Ponteggi per tridimensionale coltura cellulare

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 Views.  Karlsruhe Research Centre. Vi presentiamo due processi per la microfabbricazione di chip polimerici porosi tridimensionali coltivazione delle cellule. Il primo è goffratura a caldo combinato con un solvente processo di saldatura vapore. La seconda utilizza un processo recentemente sviluppato microthermoforming combinata con la tecnologia agli ioni di pista che porta ad una significativa semplificazione di fabbricazione.

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.

Basato su chip tridimensionale coltura cellulare in perfuso Micro-bioreattori

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

Ricerca

Biologia

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.

Intervista: Bioreattori e nobilitati-Modified 3D-Ponteggi per la ricerca sulle cellule staminali

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.

Electrospinning Ponteggi polimeri fibrosi per l&#39;ingegneria tissutale e colture cellulari

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.

Isolamento di cellule epiteliali mammarie da tridimensionale Mixed-cell Spheroid Co-cultura

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.

Due-e tridimensionale Live Cell Imaging di proteine ​​danno al DNA di risposta

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.

Mouse fetale Whole Intestino Sistema Cultura per<em&gt; Ex Vivo</em&gt; La manipolazione delle vie di segnalazione e tridimensionale Imaging in diretta di Sviluppo del villo

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 in idrogel tridimensionali

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.

Ricostruzione tridimensionale dell'architettura vascolare dell'ovaia passiva sdoganate per la chiarezza del Mouse

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 tessuto metodo di compensazione per l'Imaging tridimensionale del tessuto adiposo

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.

Risoluzione a una cellula di immagini tridimensionali di organoidi intatti

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