Wir möchten Mechthild Herschbach und Anke Dech für hervorragende technische Unterstützung danken.

Dieses Papier beschreibt die Verwendung eines Chip-basierte Plattform (Abb. 1) für die dreidimensionale Kultivierung von Zelllinien sowie primäre Zellen. Da viele Zellen exprimieren organotypischen Funktionen nur in einer 3D-Umgebung zu tun, haben wir ein Polymer-Chip, ein Gerüst, auf die die Zellen können in alle Raumrichtungen haften bietet entwickelt, und das kann in einem Bioreaktor Gehäuse für die Steuerung der Strömung montiert werden , Sauerstoffspannung etc. Je nach Versuchsanordnung kann die Oberfläche des Polymers durch verschiedene Techniken, zB UV-Bestrahlung, PECVD, γ-Pfropfen oder konventionelle Nasschemie geändert werden. <img src="/files/ftp_upload/old/564_2008_9_19_6/564_Figure-01.png" alt="564_Figure-01.png" width="546" height="386" /> Abbildung 01 1. De-Be-und Hydrophilierung des Chips Vor Gebrauch muss der Chip entlüftet und hydrophilisiert werden. Dafür ist ein Alkohol-Serie durchgeführt. Isopropanol-Lösungen, bestehend aus 100%, 70%, 50%, 30% Isopropanol in DMPC-behandeltem Wasser hergestellt werden und der Chip ist in jeder Konzentration getaucht, beginnend mit dem 100%-Lösung für bis zu 30s. Der letzte Schritt der Serie besteht aus reinem Dimethyl pyrocarbonat (DMPC)-behandeltem Wasser. Von diesem Punkt an ist es wichtig, dass der Chip nass. 2. Collagen I Beschichtung Nach dem Alkohol-Serie ist der Chip in der Regel mit einer Kollagen-I-Lösung von Rattenschwanz beschichtet. Von der Kollagen-Stammlösung von 2 mg / ml in 0,2% Essigsäure ein Aliquot entsprechend 30 ug Kollagen-Protein ist mit DMPC-behandeltem Wasser auf ein Endvolumen von 150 ul verdünnt. Dies führt zu einer Kollagen-Beschichtung von der Chip-Oberfläche mit einer Dichte von 10 ug Kollagen I pro cm 2 Fläche. 3. Inokulation von hepatozellulären Karzinom-Zellen Hepatozellulären Karzinom-Zellen der Linie HepG2 sind trypsiniert und gezählt. Für kurzfristige Versuche (1 bis 6 Tage) 5 * 10 6 Zellen sind in jedem Chip und die entsprechende Steuerung 6 cm Gewebekultur-Petrischalen ausgesät. Um inoculte sind die Chip-5 * 10 6 Zellen in 150 ul Kulturmedium resuspendiert und auf der mikrostrukturierten Fläche des Chips (Abb. 2). Danach wird es in einem Inkubator für 2-3 Stunden platziert. Während dieser Inkubationszeit der Zellen Sediment in der Mikro-Behälter und haften an der Kollagen I-beschichteten Gerüst. <img src="/files/ftp_upload/old/564_2008_9_19_7/564_Figure-02.png" alt="564_Figure-02.png" width="300" height="225" /> Abbildung 2 4. Einsetzen des Chips in den Bioreaktor Gehäuse Nach der Inkubationszeit wird der Chip aus dem Brutschrank entnommen und montiert in den Bioreaktor Gehäuse. Für diese, unter der Sterilbank wird die vormontierte Bioreaktor aus der sterilen Verpackung entnommen und zerlegt zu einem gewissen Grad, dass für das Einsetzen des Chips ermöglicht. Der Chip wird vorsichtig mit einer sterilen Pinzette gehandhabt und in die Nut, dass die Dichtung, die Dichtungen der Chip und die Ergebnisse in der Erzeugung von einer oberen und unteren Kompartiment in dem Bioreaktor enthält. Dann wird der Bioreaktor wieder zusammengebaut übertragen in den Inkubator, wo sie mit der Pumpe verbunden ist, die Gasversorgung und die Sauerstoff-Analysator. 5. Befüllen der Anlage Sobald der Bioreaktor auf das Medium Reservoir verbunden ist, Förderpumpe und Gas den geschlossenen Kreislauf mit Medium gefüllt. Dies wird durch die Positionierung der 3-Wege-Anschlüsse in einer Weise, dass Superfusion, die als die Strömung des Mediums über die Oberseite des Chips definiert ist, erreicht wird getan. Dies führt zu einer Entlastung der eingeschlossenen Luft aus dem Bioreaktor Kreislauf, ohne die Zellen aus dem Schafott. Nachdem das System ist komplett mit Medium gefüllt ist, sind die 3-Wege-Anschlüsse in einer Weise, dass die Perfusion, die als die Strömung von unten den Chip durch das Gewebe definiert ist, erreicht wird eingeschaltet. In der Perfusion Konfiguration ist die Strömung in die Zelle die Bedürfnisse, die für Hepatozyten liegt typischerweise im Bereich von 60 bis 500 ml / min eingestellt. 6. Stichprobenverfahren Während des Experiments Medium Proben gezogen werden können. Dazu werden Spritzen in die sterile Anschlüsse auf der Oberseite des Mediums Reservoir verbunden. Nach der Probenahme werden die Ports mit 70% Isopropanol sterilisiert. 7. Isolierung von intakten Zellen aus dem Chip für nachgelagerte Anwendungen Am Ende des Experiments sind die Bioreaktoren aus der Gasversorgung und die Rollerpumpe getrennt, übertragen auf die saubere Werkbank und zerlegt, wie oben beschrieben. Mit einer sterilen Pinzette wird der Chip aus dem Bioreaktor Gehäuse entfernt, platziertin einen 3,5 cm Petrischale mit PBS gespült. Anschließend wird der Chip mit Trypsin / EDTA (0,25% / 0,53 mm) für 5-15 min im Brutschrank inkubiert, um die Zellen aus der mikrostrukturierten Bereich zu lösen. Die gesammelten Zellsuspension für 5 min bei 600 g zentrifugiert Die Zellen können dann für die konventionelle Downstream-Anwendungen, zB Gesamt-RNA oder Protein Isolierung verwendet werden. Routinemäßig isolieren wir Gesamt-RNA (PARIS-Kit, Ambion Inc., Austin, Texas, USA) für die Microarray-Analyse und real-time RT-PCR. Die Proteinexpression wird nach immunhistochemischer Färbung der Zellen im Inneren des Chips mit einem Laser-Scanning-Mikroskop analysiert, kann aber auch anderweitig ausgewertet werden, zB mittels Durchflusszytometrie.

<ol>
	<li>Berry, M. N., Friend, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-yield+preparation+of+isolated+rat+liver+parenchymal+cells:+A+biochemical+and+fine+structural+study.">High-yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structural study.</a> J. Cell Biol. 53, 506-520 (1969).</li><li>Seglen, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+rat+liver+cells.+3.+Enzymatic+requirements+for+tissue+dispersion.">Preparation of rat liver cells. 3. Enzymatic requirements for tissue dispersion.</a> Exp. Cell Res. 82, 391-398 (1973).</li><li>Giselbrecht, S., Gietzelt, T., Gottwald, E., Trautmann, C., Truckenmueller, 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. Microdev. 8, 191-199 (2006).</li><li>Welle, A., Gottwald, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UV-based+patterning+of+polymeric+substrates+for+cell+culture+applications.">UV-based patterning of polymeric substrates for cell culture applications.</a> Biomed. Microdev. 4, 33-41 (2002).</li><li>Gottwald, E., Giselbrecht, S., Augspurger, C., Lahni, B., Dambrowsky, N., Truckenmueller, 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>Eschbach, E., Chatterjee, S. S., Noldner, M., Gottwald, E., Dertinger, H., Weibezahn, K. -. F., Knedlitschek, G., G, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructured+scaffolds+for+liver+tissue+with+high+density:+Morphological+and+biochemical+characterization+of+tissue+aggregates.">Microstructured scaffolds for liver tissue with high density: Morphological and biochemical characterization of tissue aggregates.</a> J. Cell. Biochem. 95, 243-255 (2005).</li></ol>

The authors have nothing to disclose.

Wir haben eine Chip-basierte Plattform für die dreidimensionale Kultivierung von Zellen in aktiv durchströmten micro Bioreaktoren entwickelt. Die Chips können aus biologisch nicht abbaubaren sowie biologisch abbaubare Polymere, die durch Mikro-Spritzguss, Heißprägen sowie Mikro Warmformtechniken 3 hergestellt werden. Je nach Versuchsanordnung kann die Oberfläche des Polymers durch UV-Bestrahlung 4 modifiziert werden. Hepatocyte Zelllinien sowie primäre Rattenhepatozyten erfolgreich in diese Geräte angebaut werd...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Cells</td>
<td>Other</td>
<td>ATCC</td>
<td>HB-8065</td>
<td>&nbsp;</td>
<tr>
<td>Collagen I from rat tail</td>
<td>Reagent</td>
<td>Roche Diagnostics</td>
<td>11 179 179 001</td>
<td>&nbsp;</td>
<tr>
<td>PARIS kit</td>
<td>Reagent</td>
<td>Ambion Inc.</td>
<td>AM1921</td>
<td>&nbsp;</td>
<tr>
<td>Syto16</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>S7578</td>
<td>&nbsp;</td>
<tr>
<td>anti cytokeratin 18</td>
<td>Antibody</td>
<td>Abcam plc</td>
<td>ab668</td>
<td>Primary Ab, Mouse monoclonal, used 1/100 in PBS</td>
</tr>
<tr>
<td>Anti E-cadherin</td>
<td>Antibody</td>
<td>Abcam plc</td>
<td>ab1416</td>
<td>Primary Ab, Mouse monoclonal, used 1/50 in PBS.</td>
</tr>
<tr>
<td>Goat anti-albumin</td>
<td>Reagent</td>
<td>Bethyl Laboratories</td>
<td>E80-129</td>
<td>Primary Ab, goat anti-human Albumin, used 1/200 in PBS</td>
</tr>
<tr>
<td>Rabbit anti-mouse IgG1</td>
<td>Antibody</td>
<td>Invitrogen</td>
<td>A11059</td>
<td>Secondary Ab, Alexa Flour 488 conjugated, used 1/100 in PBS + 0.5 % BSA</td>
</tr>
<tr>
<td>Cy3 anti-goat IgG</td>
<td>Reagent</td>
<td>Jackson ImmunoResearch Lab</td>
<td>705-165-003</td>
<td>Cy3 AffiniPure donkey a-goat IgG Ab, used 1/700 in PBS + 0.5% BSA</td>
</tr>		</tbody>
		</table>
		</div>

chip-based three-dimensional cell culture in perfused micro-bioreactors

Wir haben eine Chip-basierte Zellkultur-System für die dreidimensionale Kultivierung von Zellen entwickelt. Der Chip ist in der Regel von nicht-biologisch abbaubaren Polymeren, zB Polycarbonat oder Polymethylmethacrylat durch Mikro-Spritzgießen, Mikro Heißprägen oder Mikro-Tiefziehen hergestellt. Aber kann es auch aus biologisch abbaubaren Polymeren hergestellt werden. Seine Abmessungen sind 0,7 1 x 20 x 20 x 0,7 1 mm (HxBxT). Die wichtigsten Merkmale der verwendeten Chips werden entweder ein Raster von bis bis 1156 Kubik Mikro-Containern (cf-Chip) jedes von der Größe 120-300 x 300 x 300 μ (HxBxT) oder runde Vertiefungen mit einem Durchmesser von 300 μ und einer Tiefe von 300 μ (r-Chip). Das Gerüst kann Haus 10 Mio. Zellen in einer dreidimensionalen Konfiguration. Für eine optimale Nährstoff-und Gasversorgung, ist der Chip in einem Bioreaktor Gehäuse eingesetzt. Der Bioreaktor ist Teil eines geschlossenen Kreislauf steril, dass in der einfachsten Konfiguration, zusätzlich von einer Walze Pumpe und ein Medium-Reservoir mit einer Gasversorgung besteht. Der Bioreaktor kann in Perfusion, Superfusion, oder sogar ein Mischbetrieb gefahren werden. Wir haben erfolgreich Zelllinien sowie primäre Zellen über einen Zeitraum von mehreren Wochen kultiviert. Für Ratten primären Leberzellen konnten wir einen Erhalt der organotypischen Funktionen für mehr als 2 Wochen zeigen. Für hepatozellulären Karzinom-Zelllinien konnten wir zeigen die Induktion von Leber-spezifische Gene nicht oder nur geringfügig in Standard-Monolayer-Kultur zum Ausdruck gebracht. Das System könnte auch nützlich sein, wie eine Stammzelle Anbausystem seit der ersten Differenzierung Experimente mit Stammzell-Linien waren viel versprechend.

Watch this Scientific Journal Video about Chip-based Three-dimensional Cell Culture in Perfused Micro-bioreactors at JoVE.com

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

Wir beschreiben eine Chip-basierte Plattform für die dreidimensionale Kultivierung von Zellen in Mikro-Bioreaktoren. Ein Chip kann bis zu 10 Mio. Zellen, die unter genau definierten Bedingungen im Hinblick auf die Strömung, Sauerstoffspannung etc. in einem sterilen, geschlossenen Kreislauf kultiviert werden können.

Chip-basierte dreidimensionale Zellkultur in perfundierten Micro-Bioreaktoren

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

chip-based-three-dimensional-cell-culture-perfused-micro

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.3K Aufrufe.  Forschungszentrum Karlsruhe. Wir beschreiben eine Chip-basierte Plattform für die dreidimensionale Kultivierung von Zellen in Mikro-Bioreaktoren. Ein Chip kann bis zu 10 Mio. Zellen, die unter genau definierten Bedingungen im Hinblick auf die Strömung, Sauerstoffspannung etc. in einem sterilen, geschlossenen Kreislauf kultiviert werden können.

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

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

Using microfabrication technologies is a prerequisite to create scaffolds of reproducible geometry and constant quality for three-dimensional cell cultivation. These technologies offer a wide spectrum of advantages not only for manufacturing but also for different applications. The size and shape of formed cell clusters can be influenced by the exact and reproducible architecture of the microfabricated scaffold and, therefore, the diffusion path length of nutrients and gases can be controlled.1 This is unquestionably a useful tool to prevent apoptosis and necrosis of cells due to an insufficient nutrient and gas supply or removal of cellular metabolites.
Our polymer chip, called CellChip, has the outer dimensions of 2 x 2 cm with a central microstructured area. This area is subdivided into an array of up to 1156 microcontainers with a typical dimension of 300 m edge length for the cubic design (cp- or cf-chip) or of 300 m diameter and depth for the round design (r-chip).2
So far, hot embossing or micro injection moulding (in combination with subsequent laborious machining of the parts) was used for the fabrication of the microstructured chips. Basically, micro injection moulding is one of the only polymer based replication techniques that, up to now, is capable for mass production of polymer microstructures.3 However, both techniques have certain unwanted limitations due to the processing of a viscous polymer melt with the generation of very thin walls or integrated through holes. In case of the CellChip, thin bottom layers are necessary to perforate the polymer and provide small pores of defined size to supply cells with culture medium e.g. by microfluidic perfusion of the containers.
In order to overcome these limitations and to reduce the manufacturing costs we have developed a new microtechnical approach on the basis of a down-scaled thermoforming process. For the manufacturing of highly porous and thin walled polymer chips, we use a combination of heavy ion irradiation, microthermoforming and track etching. In this so called "SMART" process (Substrate Modification And Replication by Thermoforming) thin polymer films are irradiated with energetic heavy projectiles of several hundred MeV introducing so-called "latent tracks" Subsequently, the film in a rubber elastic state is formed into three dimensional parts without modifying or annealing the tracks. After the forming process, selective chemical etching finally converts the tracks into cylindrical pores of adjustable diameter.

We present two processes for the microfabrication of porous polymer chips for three-dimensional cell cultivation. The first one is hot embossing combined with a solvent vapour welding process. The second one uses a recently developed microthermoforming process combined with ion track technology leading to a significant simplification of manufacture.

Microfabrication der Chip-Größe Gerüste für die dreidimensionale Kultivierung der Zellen

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

Forschung

Biologie

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hallo-C: eine Methode, um die dreidimensionale Architektur des Genoms Study.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

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.

Isolierung von Brustepithelzellen aus dreidimensionalen Mixed-Zelle Spheroid Co-Kultur

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.

Zwei-und dreidimensionale Live Cell Imaging der DNA-Reparatur-Proteine

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.

Maus Fetal ganze Darm-System für Kultur<em&gt; Ex-vivo-</em&gt; Manipulation von Signalwegen und dreidimensionale Live-Imaging von Zotte Entwicklung

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 im dreidimensionalen Hydrogelen

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 ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

Die Maus isoliert perfundierten Nieren-Technik

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

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.

Dreidimensionale Rekonstruktion der vaskulären Architektur des Eierstocks Passive Klarheit geclearte Maus

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 ...

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.

Single-Cell Resolution Dreidimensionale Bildgebung intakter Organoide

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

Staining and High-Resolution Imaging of Three-Dimensional Organoid and Spheroid Models

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and disease modeling, drug discovery, and regenerative medicine. To fully exploit these models, it is crucial to study them at cellular and subcellular levels. However, characterizing such in vitro 3D cell culture models can be technically challenging and requires specific expertise to perform effective analyses. Here, this paper provides detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed in vitro 3D cell culture models ranging from 100 &#181;m to several millimeters. These protocols are applicable to a wide variety of organoids and spheroids that differ in their cell-of-origin, morphology, and culture conditions. From 3D structure harvesting to image analysis, these protocols can be completed within 4-5 days. Briefly, 3D structures are collected, fixed, and can then be processed either through paraffin-embedding and histological/immunohistochemical staining, or directly immunolabeled and prepared for optical clearing and 3D reconstruction (200 &#181;m depth) by confocal microscopy.

Here, we provide detailed, robust, and complementary protocols to perform staining and subcellular resolution imaging of fixed three-dimensional cell culture models ranging from 100 &#181;m to several millimeters, thus enabling the visualization of their morphology, cell-type composition, and interactions.

Färbung und hochauflösende Bildgebung von dreidimensionalen Organoid- und Sphäroidmodellen

In vitro three-dimensional (3D) cell culture models, such as organoids and spheroids, are valuable tools for many applications including development and ...