Die Autoren möchten sich Dirk Herrmann, Oliver Wendt, Siegfried Horn, Hartmut Gutzeit, und Jörg Bohn für ihre substantielle Hilfe über die Lösemitteldampf Schweißen danken. Darüber hinaus möchten wir Michael Hartmann, Alex Gerwald und Daniel Leisen für ihre technische Unterstützung zu bestätigen.

Prozess Sequence # 1: Hot Embossing, Bearbeitungs-und Solvent Vapour Welding Die CellChip in seine kubische Design ist durch Heißprägen oder Mikrospritzguss repliziert. Dafür verwenden wir eine mikromechanische Messing Form mit der inversen Geometrie des Chips. Die Container - in einer regelmäßigen Anordnung von bis bis 1156 Containern angeordnet - eine kubische Design mit einer Kantenlänge von 300 um. Für Heißprägen, ist die Replikation auf einem herkömmlichen WUM02 (Jenoptik Mikrotechnik, Deutschland) durchgeführt. Das Tool besteht aus zwei kreisförmigen Metallplatten. In einem ersten Schritt wird eine dünne PMMA-Platte (Lucryl, G77Q11, BASF) in der Mitte der unteren Platte des geöffneten Werkzeug eingelegt. Die mikrostrukturierten Formeinsatz ist mittig in der oberen Platte montiert. Dann wird das Werkzeug für die Evakuierung der Form geschlossen und auf eine Temperatur oberhalb der Glasübergangstemperatur des Polymers. Durch Drücken der Platten zusammen, ist das viskose Polymer in die evakuierte Hohlräume gedrückt, bis sie vollständig gefüllt sind genau die Replikation der Geometrie der Form. Nach dem Abkühlen des Werkzeugs, kann die mikrostrukturierte Polymer Teil entformt werden. Der Prozess erfordert mehr Polymermasse, als tatsächlich benötigt, um die Kavitäten zu füllen. Das Polymer Überschuss bildet eine Restschicht, die verwendet werden, um die Entformung des Teils erleichtern können. Um jedoch Poren mit einem Durchmesser kleiner als 3 um in den Boden der Container zu schaffen, hat die dicken Restschicht nach unten verdünnt werden, oder sogar komplett entfernt und durch eine poröse Membran abgelöst. Zur Vereinfachung des Prozesses der Pore Integration, ist die Rückseite der replizierten CellChip vollständig durch die Bearbeitung mit einem Diamanten Mühle entfernt. Hierzu werden die Teile auf einer gekühlten Montageplatte befestigt und zusätzlich die fragile Strukturen sind in VE-Wasser eingefroren, um sie vor Beschädigung zu schützen. In einem letzten Verfahrensschritt schließlich ein kommerzielles Ionen-track-geätzten Membran (Polycarbonat, Dicke 10 um, Porengröße 3 mu m, 2x10 6 Poren / cm ², Pieper Filter GmbH) ist auf der Rückseite des Arrays von Containern verbunden nun eröffnet sowohl auf oben und unten. Die Bonding-Prozess ist ein Lösungsmittel, Dampf-Schweißverfahren in einem gasdichten, beheizten Kammer [Abb. durchgeführt. 1], bestehend aus einem oberen Kolben und einer beweglichen unteren Platte mit integriertem Vakuum-Spannfutter. 4 Bis zu vier bearbeiteten CellChips und Track-geätzten Membranen sind parallel zu einer verdampfte Lösungsmittel ausgesetzt, nachdem die Kammer wurde evakuiert. Dann werden die Formteile und Membranen, die durch den oberen Kolben gepresst. Nach einer kurzen Exposition (&lt;15 s), wird die Kammer evakuiert wieder damit das Lösungsmittel entfernt. Aufgrund der kurzen Kontaktzeit, ist nur Oberfläche in der Nähe Material gelöst und eine Verformung des Bulk-Struktur durch die mechanische Belastung vermieden werden kann. Schließlich wird die Kammer geöffnet und das Lösungsmittel verschweißt CellChips entfernt werden können und bereit für die Zellkultur [Abb.. 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="Abbildung 1" width="100" /></a> Abbildung 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="Abbildung 2" width="100" /></a> Abbildung 2. Prozessablauf # 2: Schwerionen-Bestrahlung, microthermoforming und verfolgen Ätzen (SMART-Prozess) Der neue Prozess namens SMART ist eine neu entwickelte Technologie zur Herstellung von Mikro-funktionalisierte Membran Mikrostrukturen. 5 Die Technologie auf einer mikrotechnischen Thermoformen, genannt &quot;microthermoforming&quot; beruht. 6,7 In dieser zentralen Prozessschritt, die aus der makroskopischen angepasst wurde gefangen Blatt Thermoformen wird ein erhitzter dünnen Polymerfilm in seiner erweicht, gummielastischen Zustand durch Gasdruck in einen Formhohlraum [Abb. gebildet. 3]. Im Gegensatz zu Heißprägen oder Spritzgießen, ist dieses Verfahren nicht eine primäre Bildung und das Polymer wird nicht geschmolzen. Aufgrund der Tatsache, dass der Film noch in einen festen Zustand mit einer dauerhaften Material Zusammenhalt gebildet, können wesentliche Änderungen mit hoher lateraler Auflösung zunächst auf planaren Polymerfilmen erzeugt werden und sind während der Umformung erhalten. Nach dem microthermoforming Schritt können diese Modifikationen weiter selektiv bearbeitet werden, zB durch nasschemische Behandlung. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_9/Giselbrecht-Fig3.png" alt="Abbildung 3" width="400" height="217" /> Abbildung 3 Die SMART-Prozess besteht im Prinzip aus drei Prozessschritte: <ol><li> Erstellung von hoch aufgelösten Modifikation Muster auf planaren dünner Polymerschichten in einem Pre-Prozess </li><li> 3D ShapIng. Filme von microthermoforming ohne Verlust der (Muster) Änderungen </li><li> Post-Prozess (optional) für eine endgültige Funktionalisierung von dünnwandigen Teilen mikrostrukturierten </li></ol> Die SMART Prozess, den wir derzeit geltenden sind für die Herstellung von porösen CellChips umfasst die folgenden Verfahrensschritte [Abb.. 4]. Ein dünner Polymerfilm, z. B. aus Polycarbonat (Pokalon OG461Gl, 50 um, LOFO High Tech Film GmbH, Deutschland), wird mit einem beschleunigten schweren Ionen (z. B. Xe, Au oder U-Ionen) an den Beschleunigeranlagen der GSI bestrahlt (Darmstadt, Deutschland) mit Energien von rd. 1 GeV und Einflüsse in der Größenordnung von 106 8 Nach dem Abkühlen des Werkzeugs können die dünnwandigen Teil entformt werden. Ionen / cm ². Wenn durchdringt den Film, produziert jedes Ion einer nahezu geraden Strecke von verändertem Material genannte latente Spur. Das vorbehandelte Filme werden dann auf ein Array von 25x25 dünnwandigen Mikrocontainer, jede mit einem Durchmesser und Tiefe von 300 um tiefgezogen. Der Prozess ist derzeit auf einem modifizierten Heißprägen drücken Sie [Abb. durchgeführt. 5], wo der Polymerfilm in zwischen zwei Metallplatten eingespannt ist. Die obere Platte wird mit der mikromechanischen Werkzeug ausgestattet und die untere enthält die Druck-und Vakuum-Anschlüsse. Der Film ist in zuvor evakuierten Mikrokavitäten der Form von Stickstoff mit einem Druck von bis zu 5 MPa gestreckt. Die Filme sind in der Nähe ihrer Glasübergangstemperatur verhindert track Glühen gebildet. <img style="border: 1px solid black;" src="/files/ftp_upload/old/699_2008_5_12_10/Giselbrecht-Fig4.png" alt="Abbildung 4" width="400" height="119" /> Abbildung 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="Abbildung 5" width="100" /></a> Abbildung 5 In einer post-Verfahren werden die Ionen-Tracks selektiv an Poren, die durch das Eintauchen des gesamten Gefüges in ein geeignetes Ätzmedium (zB 5 mol / l NaOH, 10% w / v MeOH) geätzt. Durch die Steuerung der Ätzzeiten und Ätzen Bedingungen, wie Konzentration, Temperatur und speziellen Additiven (zB etch-Promotoren), kann die Größe und Form der entstehenden Poren [Abb. eingestellt werden. 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="Abbildung 6" width="100" /></a> Abbildung 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>

Obwohl etablierten Methoden der Polymer Mikroreplikation, wie Mikro-Spritzguss oder Heißprägen, geeignet zur Herstellung von Mikrostrukturen sind, sind sie nicht wirklich effektiv bei der Herstellung von Mikrostrukturen mit einem integrierten und sehr kontrollierter Porosität, wie es für die CellChip benötigt. Sperrige Strukturen, zB erfordern kostspielige Bearbeitung der Wanddicke für eine anschließende Laser-Perforation reduzieren oder Wände sind vollständig von Track-geätzten Membranen ersetzt werden. SMART ist eine neue und vi...

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

Mit Mikro-Herstellungsverfahren ist eine Voraussetzung für Gerüste reproduzierbarer Geometrie und konstante Qualität für dreidimensionale Zellkultivierung zu schaffen. Diese Technologien bieten ein breites Spektrum von Vorteilen nicht nur für die Fertigung, sondern auch für verschiedene Anwendungen. Die Größe und Form der gebildeten Zellhaufen kann durch die exakte und reproduzierbare Architektur des mikrofabrizierten Gerüst und damit die Verbreitung Weglänge von Nährstoffen und Gasen können controlled.1 werden Dies ist zweifellos ein nützliches Instrument zur Apoptose und Nekrose verhindern beeinflusst werden Zellen auf eine unzureichende Nährstoff-und Gasversorgung oder Entfernung von zellulären Metaboliten. Unser Polymer-Chip, genannt CellChip hat die äußeren Abmessungen von 2 x 2 cm mit einer zentralen mikrostrukturierten Bereich. Dieser Bereich ist in einem Array von bis auf das Jahr 1156 Mikrocontainer mit einer typischen Dimension von 300 m Kantenlänge der kubischen Design (cp-oder CF-Chip) oder von 300 m Durchmesser und Tiefe für die runde Form (r-chip) unterteilt. 2 Bisher war Heißprägen oder Mikro-Spritzguss (in Kombination mit weiteren mühsamen Bearbeitung der Teile) für die Herstellung der mikrostrukturierten Chips verwendet. Grundsätzlich Mikrospritzguss einer der einzigen Polymer basierte Replikation Techniken, die bis jetzt in der Lage ist für die Massenproduktion von Polymer microstructures.3 jedoch ist, haben beide Techniken bestimmte unerwünschte Einschränkungen aufgrund der Verarbeitung einer viskosen Polymerschmelze mit der Erzeugung von sehr dünnen Wänden oder durch Löcher integriert. Im Falle der CellChip werden dünne Bodenschichten notwendig, um das Polymer zu perforieren und bieten kleinen Poren definierter Größe, um Zellen mit Kulturmedium z. B. die Lieferung von mikrofluidischen Perfusion der Behälter. Um diese Einschränkungen zu überwinden und die Herstellkosten wir einen neuen mikrotechnischen Ansatz entwickelt auf der Grundlage eines nach unten skaliert Tiefziehverfahren zu reduzieren. Für die Herstellung von hochporösen und dünnwandigen Polymer-Chips verwenden wir eine Kombination von schweren Ionen Bestrahlung microthermoforming und verfolgen Ätzen. In diesem so genannten &quot;SMART&quot;-Verfahren (Substrat Änderung und Replikation durch Thermoformen) dünner Polymerschichten mit energiereichen, schweren Geschossen von einigen hundert MeV Einführung sogenannter bestrahlt werden &quot;latente Spuren&quot; Anschließend wird der Film in einer gummielastischen Zustand in drei gebildet dimensionalen Teilen ohne Änderung oder Glühen des Tracks. Nach der Umformung, selektive Ätzen schließlich wandelt die Tracks in zylindrische Poren von einstellbaren Durchmesser.

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

Wir präsentieren zwei Prozesse für die Mikrofabrikation von porösen Polymer-Chips für dreidimensionale Zellkultivierung. Die erste ist Heißprägen mit einem Lösemitteldampf Schweißverfahren kombiniert. Die zweite nutzt ein neu entwickeltes microthermoforming Prozess mit Ionen-Track-Technologie führt zu einer wesentlichen Vereinfachung der Herstellung kombiniert.

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 ...

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

Research

JoVE Journal

Biology

11.7K Aufrufe.  Karlsruhe Research Centre. Wir präsentieren zwei Prozesse für die Mikrofabrikation von porösen Polymer-Chips für dreidimensionale Zellkultivierung. Die erste ist Heißprägen mit einem Lösemitteldampf Schweißverfahren kombiniert. Die zweite nutzt ein neu entwickeltes microthermoforming Prozess mit Ionen-Track-Technologie führt zu einer wesentlichen Vereinfachung der Herstellung kombiniert.

Serie: 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-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 ...

Forschung

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: Bioreaktoren und Aufgetaucht-Modified 3D-Gerüste für Stammzellforschung

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.

Elektrospinnen Faserige Polymer Gerüste für Tissue Engineering und 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 ...

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 ...

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 ...

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: Ein Gewebe Clearing-Methode für die dreidimensionale Darstellung von Fettgewebe

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.

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 ...