Die Autoren bedanken sich bei Dr. Peyman Hemmati (Department of Medicine, University of Wisconsin-Madison) für die großzügige Bereitstellung der H1 MSCs und Dr. Tim Hacker, Dr. Gouqing Song und Frau Jill Koch von der University of Wisconsin Kardiovaskuläre Physiologie danken Core Facility für die Durchführung von Operationen mit der Maus. Diese Arbeit wurde von der National Science Foundation durch eine Graduate Research Fellowship an Brian Freeman und NIH R21 HL089679 unterstützt.

1. Donor Zelltransfektion <ol><li> Ernte-mesenchymale Stammzellen (MSCs aus H1 embryonalen Stammzellen freundlicherweise von Dr. Peyman Hematti gespendet abgeleitet, alternativ jeden Zelltyp jeder Art angenommen, dass sie in vivo-Sicherung eingesetzt werden könnten), wenn 70 bis 80% konfluent mit 1X Trypsin (Mediatech, Manassas VA) für 5 min. inaktivieren Trypsin mit α-MEM Vollmedium (Antibiotikum frei, Invitrogen, Carlsbad CA) 18. Zentrifuge bei 300 xg für 5 min. </li><li> Sorgfältig Überstand absaugen und resuspendieren Pellet in 1 ml 1X PBS und Zählen von Zellen unter Verwendung eines Hämozytometers. </li><li> Transfer 1,5 x 10 6 Zellen in ein 1,5 ml Zentrifugenröhrchen. Zentrifuge bei 300 xg für 5 min. </li><li> Vorsichtig absaugen Überstand. Pellet in 300 ul R Buffer (Neon Transfektion System, Invitrogen) und 6 pg (2 ug / 5,0 x 10 5 Zellen) von P231 pCMVe-betaAc-STOP-luc (Addgene, Cambridge, MA). Platz 3 ml E-Puffer (Invitrogen) in die Elektroporation Docking-Anschluss per Protokoll des Herstellers (Neon Transfektion System, Invitrogen). </li><li> Transfer-Zell-Plasmid-Lösung für ein 100 ul Neon Pipettenspitze und elektroporieren mit einer Pulsdauer von 20 ms und einer Höhe von 1500 Volt. Legen Sie elektroporiert Zellen in ein 15 mL konische Röhrchen mit 9,7 mL α-MEM Vollmedium. </li><li> Wiederholen Sie Schritt 1.5 zwei weitere Male und Pool transfizierten Zellen auf ein Gesamtvolumen von 10 ml zu erhalten. Fügen Sie die 10 mL Zellsuspension (1,5 x 10 6 Zellen), eine T175 Flasche mit 10 ml α-MEM Vollmedium. Die Lebensfähigkeit der Zellen nach der Elektroporation ist etwa 30% auf ca. 4,5 x 10 5 vitale Zellen pro T175 liefern. </li><li> Ändern α-MEM Vollmedium 24 Stunden nach der Transfektion. </li><li> Ernte transfizierter Zellen bei 70 bis 80% konfluent (~ 2 bis 3 Tage nach der Elektroporation). Führen Zellzahl unter Verwendung eines Hämozytometers und Zellen in einer Konzentration von 1,0 x 10 6Zellen/50 ul von α-MEM Vollmedium. Minimieren der Zeit Zellen verbringen in Suspension zum Zelltod zu reduzieren, um vor Injektion. </li></ol> 2. Intramyokardialen Injection <ol><li> Induzieren Anästhesie durch Isofluran (Phoenix Pharmaceuticals, Inc., St. Joseph, MO) an transgenen Mäusen entwickelt, um konstitutiv Cre-Rekombinase in jeder Zelle (B6.C-Tg (CMV-cre) 1CGN / J, Jackson Laboratory in Bar Harbor, ME). </li><li> Entfernen Sie Haare aus Brustbereich mit Haarschneidemaschinen oder chemische Haarentferner. </li><li> Intubieren mit einem 18-Gauge-Katheter (Becton Dickinson &amp; Co, Franklin Lakes NJ) und am Maus-Ventilator bei 120 bis 130 Atemzüge pro Minute mit einem Hubvolumen von 150 ul. </li><li> Machen seitlichen Schnitt über dem vierten Interkostalraum wodurch eine Thorakotomie. </li><li> Visualisierung des Herzens, machen zwei 25 ul Injektionen von transfizierten Zellsuspension mit einer 1 ml-Spritze (Temuro Medical Corporation, Somerset, NJ) und 28-Gauge-Nadel (BECTauf Dickinson &amp; Co, Franklin Lakes NJ). Zur Erleichterung der intramyokardialen Injektion und verhindern, dass übermäßige Schäden an der Orgel, biegen Sie die Nadel Kopf ~ 90 Grad. </li><li> Nach der Injektion verwenden resorbierbares Nahtmaterial (zB Vicryl) an den Rippen und Muskelschichten zu schließen. Suture Haut geschlossen mit 4-0 Nylon oder Seide. </li><li> Lassen Sie Maus, um aus der Narkose und Extubation erholen. </li><li> Kontrollgruppen sollten Cre Mäusen, die mittel-Injektionen nur, Empfangen Cre Mäusen die gleiche Konzentration an transfizierten Zellen und Wildtyp-Mäusen, die transfizierten Zellen (alternativ erhalten Cre Mäusen transfizierten Zellen nicht anfällig für Sicherung). </li></ol> 3. Biophotonic Imaging <ol><li> Fünf bis 15 Minuten vor der Belichtung, intraperitoneal (IP) zu injizieren 10 l pro Gramm Maus Körpergewicht von 15 mg / mL D-Luciferin (Caliper Life Sciences, Hopkinton, MA). </li><li> Induzieren Anästhesie an Mäusen über Isofluran bei 4% für Induktion eind 1 bis 2% für die Wartung. </li><li> Legen Mäuse in Rückenlage in der Bildgebung Box mit Gesichtsmaske Verwaltung von 1 bis 2% Isofluran zur Aufrechterhaltung der Narkose (Xenogen Biophotonic Imaging System, Hopkinton, MA). Mehrere Mäuse können gleichzeitig abgebildet werden. Bild sham Kontroll-Maus mit experimentellen Mäuse für einen einfachen Vergleich der Lumineszenz-Signal. </li><li> Mit Lebens-Image Software (Xenogen), setzen entsprechende Belichtungszeit (in der Regel 60 Sekunden, siehe Ergebnisse). Set Bereich des Bildes, Mäuse fit und halten Bereich konsequent über Imaging, um Änderungen in der Empfindlichkeit zu verhindern. Set unterliegen Höhe von 4,5 cm. </li><li> Erwerben Sie Lumineszenzintensität entsprechendes Signal Maus oder Mäuse im Sichtfeld und speichern unveränderten Bilddateien. Bild bearbeiten zu entfernen Hintergrund entsprechendes Signal an eine Steuerung oder unmanipulierten Maus. Intensity-Werte über dem Hintergrund zu fusionierenden Zellen innerhalb des Tieres entsprechen. Intensity-Analyse kann unter Verwendung Wohnen Image Software (Xenogen) oder Open-Source-Bildanalyse-Software. Typically, einer Region of Interest (ROI) wird ausgewählt, um die Intensität von Daten zwischen Tieren und Experimenten zu vergleichen. </li></ol> 4. Repräsentative Ergebnisse Zur Bestimmung der Empfindlichkeit des Xenogen Biophotonic Imaging System, eine Zelllinie, die konstitutiv exprimiert Luziferase (231-LUC-D3H1, Xenogen) wurde in den Herzmuskel des C57/BL6-Mäusen (Jackson Laboratory) geliefert. Die Zellen wurden in einer Konzentration von 1 x gespritzt 10 6, 1 x 10 3, oder 1 x 10 1 Zellen. Sechs Stunden nach Zell Lieferung, Mäuse wurden intraperitoneal mit Luciferin injiziert und abgebildet mit den Xenogen System. Ein spezifisches Signal konnte mit 1.000 Zellen (2 von 6 Mäusen abgebildet, Abbildung 2) nachgewiesen werden, aber Detektion wurde mehr zuverlässig mit 10.000 Zellen (6 von 6 Mäusen abgebildet). Wichtig ist, dass diese Studie diente auch dazu, eine grobe Korrelation zwischen Anzahl der Luciferase-exprimierenden Zellen und Signalintensität zu etablieren. <p class="jove_content"&gt; Um den Nutzen des beschriebenen Protokolls bei der Aufdeckung und Verfolgung Zellfusion zu demonstrieren, wurden MSCs mit den loxP-Stop-loxP-Luciferase-Plasmid (Addgene) transfiziert und an das Myokard des Cre-exprimierenden Mäusen. Etwa eine Woche nach Zell Lieferung, wurden Mäuse ersten abgebildet mit den Xenogen System ohne D-Luciferin Injektion. Wie zu erwarten, ohne die enzymatische Substrat wurde keine Intensität detektiert (Abbildung 3). Als nächstes wurde D-Luciferin intraperitoneal und ein entsprechendes Signal Luciferase Intensität injiziert und damit Zellfusion wurde in zwei von vier Mäusen getestet erkannt. Ein ähnliches Signal gefunden wurde eine Woche später (Abbildung 3), was darauf hindeutet MSC-coupled Fusionsprodukte in vivo aufrechterhalten werden kann. In diesem Fall wird die Studie beendet wurde, für die Auswertung des Herzens und des umgebenden Gewebes ermöglichen, aber man könnte sich vorstellen, längerfristig Analysen und häufiger Bildgebung zur Erhaltung, Vermehrung Titel in derd vielleicht Migration von Fusion-Produkte in Mäusen. <img alt="Abbildung 1" src="/files/ftp_upload/3581/3581fig1.jpg" /> Abbildung 1. Schematische Darstellung der Technik to Cell Fusion in vivo nachzuweisen. Wenn Fusion zwischen Cre-exprimierenden Zellen der Maus und transplantierten Zellen, die ein floxed Luciferase Plasmid auftritt, Luciferase exprimiert werden. Luciferase kann durch die Injektion der enzymatischen Substrat, D-Luciferin, in die Maus und dann die Abbildung der Maus über einen Xenogen Biophotonic Imaging System (Übernommen von 19) erkannt werden <img alt="Abbildung 2" src="/files/ftp_upload/3581/3581fig2.jpg" /> Abbildung 2. Die Empfindlichkeit der Detektion von Luciferase-exprimierenden Zellen im Herzgewebe mit biophotonischen Bildgebung. Eine Zelllinie, die konstitutiv exprimiert Luziferase (231-LUC-D3H1, Xenogen) war es, die intramyokardialen Raum C57/BL6-Mäusen an verschiedenen insgesamt Zellzahlen geliefert. Repräsentative Bilder von Mäusen receiving 1 x 10 6, 1 x 10 3 und 1 x 10 1-Zellen (von links nach rechts) gezeigt wurde Bildgebung ca. 6 Stunden nach der Injektion durchgeführt. <img alt="Abbildung 3" src="/files/ftp_upload/3581/3581fig3.jpg" /> Abbildung 3. Die Quantifizierung der In-vivo-Lumineszenz Indikativ of Cell Fusion. MSCs wurden mit der loxP-Stop-loxP-Luciferase-Plasmid und an das Myokard des Cre-exprimierenden Mäusen transfiziert. Etwa eine Woche und zwei Wochen nach Lieferung Zelle wurden Cre Mäusen abgebildet mit den Xenogen Biophotonic Imaging System, um die Intensität der Lumineszenz bezeichnend für Zellfusion zu messen. (A) Überlagerung von Bild und Intensität der Lumineszenz von Schein-und Mäuse 1-4 (von links nach rechts) 17 Tage nach Lieferung Zelle. (B) Intensität der Lumineszenz von Schein-und Mäuse 1-4 (von links nach rechts) 17 Tage nach Lieferung Zelle. Eine Region von Interesse ausgewählt wurde (gelb) entspricht der Injektionsstelle und Intensität levels wurden mittels ImageJ (kostenlos source) Software 20. (C) Die Intensität der Lumineszenz wurde auf die gleiche Region von Interesse am sham Maus für alle experimentellen Bedingungen normalisiert. In einer Woche, zeigten die Mäuse 3 und 4 positive Lumineszenzsignal darauf hindeutet, spontane Fusion einer Maus Zelle und transplantierten MSC. Das Signal blieb in der Maus 3 auf 2 Wochen. Um festzustellen, Organ-spezifische Lokalisation des Signals entsprechend Maus 3 wurde die Brusthöhle ausgesetzt und primären Organe herausgeschnitten und abgebildet. (D) Überlagerung von Bild und Intensität der Lumineszenz der Maus 3. Hinweis Lokalisierung von Intensität Signal in den Dünndarm. (E) Intensität der Lumineszenz der Maus 3. (F) Überlagerung von Bild und Intensität der Lumineszenz von Schein-Maus. (G) Intensität der Lumineszenz von Schein-Maus.

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Die hier beschriebene Methode ermöglicht es, zum ersten Mal, diskrete Identifikation und zeitliche Analyse von Zell-Fusion in Organismen, einschließlich der kleinen Tiere. Der Ansatz kombiniert Cre-loxP Rekombination mit anschließender biophotonischen Bildanalyse. Der Ansatz ist offen für Tracking nicht nur Zell-Zell-Fusion, sondern auch Virus-Zell-Fusion und so könnte sich als nützlich erweisen für die Verfolgung von viralen Infektionen. Die Bildanalyse ist schnell und es ist möglich, Bild mehrere kleine Tiere ...

<table border="1"><tbody><tr><td> Name des Reagenz </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Neon Transfektionssystem </td><td> Invitrogen, Carlsbad, CA </td><td> MPK5000 </td><td></td></tr><tr><td> Neon 100 ul Kit </td><td> Invitrogen, Carlsbad, CA </td><td> MPK10025 </td><td> Enthält R und E Buffer </td></tr><tr><td> a-MEM-Pulver </td><td> Invitrogen, Carlsbad, CA </td><td> 12000-022 </td><td></td></tr><tr><td> Fetal Bovine Serum (FBS) </td><td> Hyclone, Logan UT </td><td> SH30070.03 </td><td></td></tr><tr><td> B6.C-Tg (CMV-cre) 1CGN / J </td><td> Jackson Laboratory in Bar Harbor, ME </td><td> 006054 </td><td></td></tr><tr><td> Trypsin 10X </td><td> Fisher Scientific, Forest Lawn, NJ </td&gt; <td> MT-25 bis 054-Cl </td><td></td></tr><tr><td> L-Glutamin </td><td> Fisher Scientific, Forest Lawn, NJ </td><td> 25030-081 </td><td></td></tr><tr><td> D-Luciferin </td><td> Caliper Life Sciences, Hopkinton, MA </td><td> 122796 </td><td></td></tr><tr><td> Xenogen Biophotonic Imaging System </td><td> Caliper Life Sciences, Hopkinton, MA </td><td> IVIS Spectrum </td><td></td></tr><tr><td> Sodium Biocarbonate </td><td> Sigma Aldrich, St. Louis, MO </td><td> S6014-500G </td><td></td></tr><tr><td> Nicht-essentielle Aminosäuren </td><td> Invitrogen, Carlsbad, CA </td><td> 11140-050 </td><td></td></tr></tbody></table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

Die Fähigkeit von zwei oder mehr Zellen des gleichen Typs zu verschmelzen hat in Metazoen im Laufe der Evolution verwendet worden, um viele komplexe Organe, einschließlich der Skelettmuskulatur, Knochen-und Plazenta bilden. Zeitgenössische Studien zeigen Fusion von Zellen des gleichen Typs verleiht erweiterte Funktion. Zum Beispiel, wenn die Trophoblastzellen der Plazenta Sicherung der Synzytiotrophoblasten Form ist die Synzytiotrophoblasten besser in der Lage, Nährstoffe und Hormone in der mütterlichen und fötalen Barriere als ungesichert Trophoblasten 1-4 transportieren. Neuere Studien zeigen, Verschmelzung von Zellen verschiedener Typen können Zellschicksal lenken. Die &quot;Rückkehr&quot; oder Modifikation von Zellen Schicksal durch die Fusion war einst als begrenzt an Zellkulturen. Aber das Aufkommen der Stammzelltransplantation führte zur Entdeckung von uns und anderen, dass Stammzellen mit somatischen Zellen in vivo Sicherung und dass die Fusion erleichtert Stammzell-Differenzierung 5-7. So ist Zellfusion einem geregelten Prozess capable fördern das Überleben der Zelle und die Differenzierung und somit könnte von zentraler Bedeutung für die Entwicklung, die Reparatur von Gewebe und auch die Pathogenese der Krankheit zu sein. Die Begrenzung der Untersuchung von Zell-Fusion, ist der Mangel an geeigneten Technologien zu 1) genau zu identifizieren Fusion Produkten und zu 2) verfolgen Fusion Produkte im Laufe der Zeit. Hier präsentieren wir einen neuen Ansatz für beide Einschränkungen über die Induktion von Biolumineszenz auf Fusion (Abbildung 1)-Adresse,. Biolumineszenz mit hoher Empfindlichkeit in vivo 8-15 erkannt werden wir nutzen ein Konstrukt, das die Firefly-Luciferase (Photinus pyralis)-Gen benachbart zu ein Stop-Codon durch loxP-Sequenzen flankiert. Wenn Zellen Expression dieses Gens Sicherung mit Zellen, die Cre-Rekombinase-Protein, das loxP-Stellen gespalten werden und das Stopp-Signal wird herausgeschnitten ermöglicht die Transkription von Luciferase. Da das Signal induciBLE, ist die Inzidenz von falsch-positive Signale sehr gering. Im Gegensatz zu bestehenden Verfahren, die die Cre / loxP-Systems 16, 17 zu nutzen, haben wir eine &quot;lebende&quot; Erfassungssignals integriert und damit zum ersten Mal die Gelegenheit, um die Kinetik der Zellfusion in vivo verfolgen leisten. Zur Demonstration der Vorgehensweise, Mäuse ubiquitär exprimiert die Cre-Rekombinase als Empfänger von Stammzellen mit einem Konstrukt zu Luciferase hinter einem floxed Stop-Codon ausdrückliche transfizierten serviert. Stammzellen wurden über intramyokardialen Injektion transplantiert und nach der Transplantation intravital Bildanalyse wurde durchgeführt, um die Anwesenheit von track Fusion-Produkte in das Herz und die umgebenden Gewebe im Laufe der Zeit. Dieser Ansatz könnte angepasst Zellfusion in jedem Gewebetyp analysieren in jedem Stadium der Entwicklung, Krankheit oder adultem Gewebe zu reparieren.

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Ein Verfahren zur Zellfusion in lebenden Organismen im Laufe der Zeit verfolgen wird beschrieben. Der Ansatz nutzt Cre-<em&gt; LoxP</em&gt; Rekombination Luciferaseexpression auf Zellfusion induzieren. Die Lumineszenz-Signal erzeugt wird, kann in lebenden Organismen mit biophotonischen Imaging-Systeme mit einer Nachweisempfindlichkeit von ~ 1.000 Zellen im peripheren Gewebe nachgewiesen werden.

Ein Cre-Lox P Rekombination Ansatz für den Nachweis von Cell Fusion In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

a-cre-lox-p-recombination-approach-for-detection-cell-fusion

Research

JoVE Journal

Bioengineering

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

18.7K Aufrufe.  University of Wisconsin-Madison. Ein Verfahren zur Zellfusion in lebenden Organismen im Laufe der Zeit verfolgen wird beschrieben. Der Ansatz nutzt Cre- LoxP Rekombination Luciferaseexpression auf Zellfusion induzieren. Die Lumineszenz-Signal erzeugt wird, kann in lebenden Organismen mit biophotonischen Imaging-Systeme mit einer Nachweisempfindlichkeit von ~ 1.000 Zellen im peripheren Gewebe nachgewiesen werden.

Serie: A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Biomolekulare Erkennung Einsatz der Interferometrische Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Forschung

Biotechnik

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

Herstellung von elektrochemischen DNA Biosensoren für die Reagenzienfreier Nachweis von Nukleinsäuren, Proteinen und kleinen Molekülen

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Erstellen Zweidimensionale Patterned Substrate für Protein-und Zell-Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Einfache mikrofluidischen Systemen für<em&gt; In vivo</em&gt; Imaging der<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; Und Zebrafisch

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

Mikroinjektion Wound Assay und<em&gt; In-vivo-</em&gt; Lokalisierung von Epidermal Wundreaktion Reporter in<em&gt; Drosophila</em&gt; Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Ein Vollhautdefekt Modell zur Vaskularisierung von Biomaterialien Bewerten<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Ein Markierungsfreie Technik für die Raum-zeitliche Imaging von Single Cell Sekrete

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Hohlfaser-Bioreaktoren für<em&gt; In Vivo</em&gt; -ähnlichen Mammalian Gewebekultur

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Herstellung von 3D-Kollagen-Gel und Mikrokanäle für das Studium der 3D-Interaktionen<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Ein Hochdurchsatz-Zellarray-Plattform für korrelative Analyse von Zelldifferenzierung und Zugkräften

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

Ein Cre-Lox P Rekombination Ansatz für den Nachweis von Cell Fusion In Vivo

Zusammenfassung

Weitere Videos entdecken

Kapitel in diesem Video

Biomolekulare Erkennung Einsatz der Interferometrische Reflectance Imaging Sensor (IRIS)

Herstellung von elektrochemischen DNA Biosensoren für die Reagenzienfreier Nachweis von Nukleinsäuren, Proteinen und kleinen Molekülen

Erstellen Zweidimensionale Patterned Substrate für Protein-und Zell-Confinement

Einfache mikrofluidischen Systemen für

Mikroinjektion Wound Assay und

Ein Vollhautdefekt Modell zur Vaskularisierung von Biomaterialien Bewerten

Ein Markierungsfreie Technik für die Raum-zeitliche Imaging von Single Cell Sekrete

Hohlfaser-Bioreaktoren für

Herstellung von 3D-Kollagen-Gel und Mikrokanäle für das Studium der 3D-Interaktionen

Ein Hochdurchsatz-Zellarray-Plattform für korrelative Analyse von Zelldifferenzierung und Zugkräften