Diese Studie wurde teilweise durch die NASA Weltraum Nebraska Grant und National Science Foundation unter dem Förderkennzeichen 0926880 Nr. unterstützt.

1. Herstellung des Latex Schiff <ol><li> Um beide Enden des Latex Schiff Schlauchanschluss Verbindungen, die auf einem stabilen Werkbank befestigt sind. </li><li> Man misst die Fläche von Zinsen auf Latex Schiff das Blickfeld zu bestimmen. Der interessierende Bereich für einen Stent Test sollte zwischen den Widerhaken Schlauchanschlüsse zentriert werden und umfassen etwa einem Zoll auf jeder Seite des Stents, um die Stämme außerhalb der Stent-Bereich zu beobachten. </li><li> Datensatz den Abstand von der äußeren Kante von einem Schlauchanschluss Stecker an der zentralen Stelle zwischen den Verbindern, die auch die ungefähre Mitte des Latex Behälter. Übersetzen der Abstand auf den Katheter durch die Messung von der Mitte des Stents bis der Katheter. Dann markieren Sie den Katheter mit einem Marker. </li><li> Entfernen Sie das Gefäß Latex aus den Widerhaken Schlauchanschlüsse. </li><li> Bereiten Sie die Latex-Schiff durch Besprühen der Bereich von Interesse mit einem stochastischen Muster aus weißen und schwarzen Sprühlack oder Kennzeichnungder interessierende Bereich mit zufälligen Punkten mit Filzstift. Für kleinere und feinere Muster stochastischen Muster ist nicht erforderlich. </li></ol> 2. In vitro Testsystem und Kalibrierung von ARAMIS-System <ol><li> Auswählen der Kalibriertemperatur Platte, die geringfügig größer als der Bereich von Interesse in Schritt 1 gemessen wird. </li><li> Legen Sie die Kalibrierung Panel zwischen den Widerhaken Schlauchanschlüsse auf dem Gebiet von Interesse und sicherzustellen, dass der interessierende Bereich gut beleuchtet ist. </li><li> Den Abstand zwischen zwei Kameras, die Entfernung von der Probe und der Kamera auf der Höhe Kalibrierfeld ausgewählt wurden. Jede Kalibrierung Panel ist anders, daher ist das ARAMIS Bedienungsanleitung müssen konsultiert werden, um diese Entfernungen zu bestimmen. </li><li> Öffnen Sie ein neues Projekt in ARAMIS, indem Sie &quot;Datei&quot;, dann &quot;Neues Projekt&quot;. Anschließend klicken Sie auf der &quot;Sensor&quot; und wählen Sie &quot;Kalibrierung&quot;, dann &quot;Full Calibration&quot;. </li><li> Das ARAMIS-Software wird nun zu Fuß den Benutzer durch die Schritte, um t kalibrieren<html> er Kameras. </li><li> Mit der Blende vollständig geöffnet ist, konzentrieren sich die Kamera auf dem Panel-Kalibrierung durch Lösen der Stellschraube an der Kamera und drehen Sie das Objektiv. Einmal scharfgestellt, ziehen Sie die Stellschraube und schließen Sie die Blende. </li><li> Nehmen Sie das erste Bild von der Kalibrierung. Verschieben oder Drehen der Kalibrierung Platte nach der Demonstration auf dem Computer, bis das Bild auf dem Bildschirm fokussiert wird. Nehmen Sie das zweite Bild. Wiederholen Sie diesen Vorgang für den Rest der Kalibrierung Bilder. </li><li> Wenn alle der Kalibrierung aufgenommenen Bilder sind, wird die Bildanalyse-Software ARAMIS berechnen die Kalibrierungseinstellungen. Die Kalibrierung wiederholt, wenn die Kalibrierung Abweichung größer als 0,04 ist. Etwaige Anpassungen vorgenommen, um den Fokus der Kamera oder der Abstand zwischen Kameras machen die Kalibrierung ungültig. </li><li> Entfernen Sie die Kalibrierung Panel und legen Sie die bemalten Latex Schiff wieder auf den gezackten Schlauchanschlüsse. </li></ol>Titel &quot;&gt; 3. Pretest zu Übermäßige Hintergrund Lärm zu vermeiden <ol><li> Bestimmen Sie die Anzahl der Frames pro Sekunde, die für den Test gewünscht wird. Erhöhte Bildern pro Sekunde produziert gleichmäßigere Belastung ergibt. </li><li> Stellen Sie die Verschlusszeit auf weniger als 1 Bild pro Sekunde und damit kein Rot auf dem Bild angezeigt wird. </li><li> Nehmen Sie sich 5 Bilder. </li><li> Fügen Sie starten Punkten auf der Bildserien und berechnen Sie den Test. </li><li> Halten Sie &quot;Strg&quot;, auf der Mitte der Probe klicken, um die Hintergrundgeräusche zu beobachten. Wenn der Vortest Lärm über 75 Mikrodeformation die Kalibrierung Prozess muss wiederholt werden. </li></ol> 4. Stententfaltung <ol><li> Wählen Sie die gewünschte Anzahl der Bilder während der Prüfung zu nehmen. 200 Bilder werden für die Expansion des Stents genügen. </li><li> Nach den Katheter in den Latex Gefäß und mit dem Marker-Anzeige auf dem Katheter, um die Insertion des Stents zu leiten, bis sie die zentrale Stelle erreicht. </li><li> Fangen Sie an, Bilder mit ARAMIS. </li><li> Für den Ballon-expandierbaren Stent, schrittweise Erhöhung der Ballondruck zu erweitern Stent, bis der Ballon vollständig ausgedehnt ist, dann allmählich verringert den Druck des Ballons auf Null und der Ballon wird entleert und zusammen mit dem Katheter zurückgezogen. </li><li> Für des selbst-expandierenden Stent, nach und nach beseitigt die Hülle, bis der Stent vollständig expandiert wird; dann allmählich zurückgezogen des Katheters. </li></ol> 5. Bilder-Analyse <ol><li> Der Stamm der Geschichte eines bestimmten Punktes auf dem Schiff <ol><li> Erstellen Sie eine Bühne, durch Drücken der &quot;Strg&quot;-Taste und Mausklick auf den Bereich von Interesse. </li><li> Wählen Sie den Stamm, die erwünscht ist, dh Stamm in der X, Y, XY, Hauptformänderung, Nebenformänderung oder Mises-Stamm. </li><li> Das Grundstück in der rechten unteren Ecke zeigt die Belastung an der Stelle, über die Dauer des Tests ausgewählt. </li></ol></li><li> Räumliche Stamm entlang einer bestimmten Bahn des Schiffes <ol><li> Create ein mehrstufiges Punkte-Linie durch Klicken auf die &quot;Abschnitte&quot;, dann &quot;schaffen Abschnitt&quot;. Internationale Linie auf dem Bild parallel zu der X-Achse mit Y gleich Null ist. Dies wird eine Reihe von Punkten der Bühne in einer Linie. </li><li> Nach dem mehrstufigen Linie erstellt wird die Handlung in der unteren Ecke wird eine Reihe von Linien auf einem Grundstück anzuzeigen. Jede Zeile repräsentiert die Spannung an einem Fall in der Zeit entlang der Länge des Abschnitts. </li></ol></li><li> Erstellen einer Best-Fit-Zylinder zu Expansionsrate und Radius des Schiffes zu analysieren <ol><li> In der oberen Menüleiste wählen Sie &quot;Primitiven&quot;, dann &quot;Best-Fit-Zylinder&quot;. </li><li> Wählen Sie einen kleinen Ausschnitt des Bildes mit der &quot;Auswahl durch Fläche&quot;-Tool auf der rechten Werkzeugleiste. </li><li> Das ARAMIS-Software generiert eine dreidimensionale Best-Fit-Zylinder. </li><li> Die Bilder können dann wieder eingeschaltet werden durch zu beobachten, wie der Durchmesser des Latex Behälter variiert. </li></ol></li><li> Auswertung der Abstand zwischenzwei Punkte <ol><li> Unter der &quot;Analyse&quot;-Registerkarte, klicken Sie auf &quot;Punkt zu Punkt Abstand&quot;. </li><li> Internationale Länge auf das Bild, die für die Analyse durch die zwei Punkte gewünscht wird. </li><li> Die Bilder können dann wieder eingeschaltet werden durch die Änderung des Abstands zwischen den beiden Punkten im Laufe der Zeit zu beobachten. </li></ol></li></ol> 6. Repräsentative Ergebnisse Die Stentstreben erweitern die Gefäßwand nach außen, wird im Allgemeinen höher sein Stämmen rund um Stents Standort. Abbildung 1 ist ein Beispiel für Stamm-Mapping während der Rückstoß Prozess der Ballon-Stent, sowie große Belastung Geschichte an einem bestimmten Punkt. Die schwarzen Punkte in Abbildung 1 sind Bezugspunkte, die von den High-Speed-Kameras verwendet wurden, zu erfassen und zu verfolgen, die Verschiebungen dieser Referenzpunkte auf der Leitung. Basierend auf den erfassten Bewegung der Bezugspunkte, die Software wird dann verwendet, um die Stämme von der Leitung oder ein zu berechnenny andere zielgerichtete Objekt. Wichtige Stamm, der auch als der maximale Hauptspannung bezeichnet wird, wird wie folgt berechnet: <img alt="Gleichung 1" src="/files/ftp_upload/3945/3945eq1.jpg" /> Es ist klar, dass die implantierten Stents zu einer ungleichmäßigen Spannungsverteilung auf dem Schiff Oberfläche geführt. Dies könnte durch den Rückstoß beim Laden von Enden-Latex eingeschränkt Leitung und der Netzstruktur des Stents erläutert. Dieser Stamm Feld entspricht der Anfangsphase der Stent Rückstoß, wie von der Rot-Kreuz-Marker in der unteren Bild von Abbildung 1 identifiziert. Der große Stamm-Geschichte Kurve von einem bestimmten Punkt 10 demonstriert unterscheidbare Stadien der Stent-Implantation. Die Expansion des Ballons tritt von etwa 10 bis 12 Sekunden und Stent Rückstoß nach dem Entleeren des Ballons tritt zwischen 12 und 14 Sekunden. <img alt="1" src="/files/ftp_upload/3945/3945fig1.jpg" /> Abbildung 1. </strong&gt; Versuchsaufbau (oben); ungleichmäßige Spannungsverteilung auf der Oberfläche gestenteten Conduit (Mitte), die große Belastung der Geschichte an der Nummer 10 (unten).

<ol>
	<li>Fischman, D. L., Leon, M. B., Baim, 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=A+randomized+comparison+of+coronary-stent+placement+and+balloon+angioplasty+in+the+treatment+of+coronary+artery+disease.+Stent+Restenosis+Study+Investigators.">A randomized comparison of coronary-stent placement and balloon angioplasty in the treatment of coronary artery disease. Stent Restenosis Study Investigators.</a> N. Engl. J. Med. 331, 496-501 (1994).</li><li>Abul Hasan Muhammad Bashar, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+Properties+of+Various+Z-Stent+Designs:+An+Endovascular+Stent-Grafting+Perspective.">Mechanical Properties of Various Z-Stent Designs: An Endovascular Stent-Grafting Perspective.</a> Artificial Organs. 27, 714-721 (2003).</li><li>Nuutinen, J. u. h. a. -. P. e. k. k. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+and+in+vitro+degradation+of+bioabsorbable+self-expanding+braided+stents.">Mechanical properties and in vitro degradation of bioabsorbable self-expanding braided stents.</a> Journal of Biomaterials Science -- Polymer Edition. , 255-266 (2003).</li><li>C. Schulz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+stent+symmetry+and+vascular+injury+determine+experimental+restenosis.">Coronary stent symmetry and vascular injury determine experimental restenosis.</a> Heart. 83, 462-467 (2000).</li><li>Jiménez, J. M., Davies, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamically+Driven+Stent+Strut+Design.">Hemodynamically Driven Stent Strut Design.</a> Annals of Biomedical Engineering. 1483, (2009).</li><li>Johnston, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Mechanical+Properties+of+Endovascular+Stents:+An+In+Vitro+Assessment.">The Mechanical Properties of Endovascular Stents: An In Vitro Assessment.</a> Cardiovascular Engineering: An International Journal. 10, 128-135 (2010).</li><li>Mejia, J. u. a. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effect+of+stent+strut+profile+on+shear+stress+distribution+using+statistical+moments.">Evaluation of the effect of stent strut profile on shear stress distribution using statistical moments.</a> Biomedical Engineering Online. , 1-10 (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ARAMIS User Manual. , (2009).</li><li>Chesler, N. C., Thompson-Figueroa, J., Millburne, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+Mouse+Pulmonary+Biomechanics.">Measurements of Mouse Pulmonary Biomechanics.</a> Journal of Biomechanical Engineering. 126, 309-314 (2004).</li><li>de Korte, C. L., Sierevogel, M. J., Mastik, F., Strijder, C., Schaar, J. A., Velema, E., Pasterkamp, G., Serruys, P. W., van der Steen, A. F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+Atherosclerotic+Plaque+Components+With+Intravascular+Ultrasound+Elastography+In+Vivo+A+Yucatan+Pig+Study.">Identification of Atherosclerotic Plaque Components With Intravascular Ultrasound Elastography In Vivo A Yucatan Pig Study.</a> Circulation. 105, 1627-1630 (2002).</li></ol>

Keine Interessenskonflikte erklärt.

Der Stereo-optischen Oberfläche Dehnungsmessung System wird verwendet, um die lokalen Stämme über die Oberfläche verformt sowohl für die in-und out-of-Plane-Bewegungen zu messen, ohne Kontakt mit der Probe. Dieses System verwendet zwei optischen Hochgeschwindigkeits-Kameras Bilder von einem zufälligen Muster kontrastierender indem auf der Oberfläche, genaue Messungen der Bewegung jedes Punkts zu konstruieren, mit einer hohen Genauigkeit zu lösen Oberflächenspannungen zu nehmen. Es s...

<table border="1"><tbody><tr><td> Anlage gebraucht </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> ARAMIS-Kamera-System </td><td> GOM: Optische Messtechnik </td><td></td><td></td></tr><tr><td> PALMAZ Genesis transhepatische Gallenstentsystem </td><td> Cordis Corporation </td><td> PG5910B </td><td> Ballon-Stent </td></tr><tr><td> Z-MED Ballondilatationskatheters </td><td> B. Braun Medical Inc. </td><td> PDZ336 </td><td> Ballondilatationskatheter </td></tr></tbody></table>

monitoring the wall mechanics during stent deployment in a vessel

Klinische Studien haben unterschiedliche Restenose Raten für verschiedene Stent-Designs 1 angegeben. Es wird spekuliert, dass Stent-induzierte Spannung Konzentrationen auf die Arterienwand zu Gewebeschäden, die Restenose 2-7 initiiert führen. Diese Hypothese bedarf weiterer Untersuchungen einschließlich einer besseren Quantifizierung von ungleichmäßiger Spannungsverteilung auf der Arterie nach einer Stentimplantation. Ein Nicht-Kontaktfläche Dehnungsmessung Methode für die Stent-Arterie wird in dieser Arbeit vorgestellt. ARAMIS Stereo optischen Oberfläche Dehnungsmessung System verwendet zwei optische High-Speed-Kameras, um die Bewegung eines jeden Bezugspunkt zu erfassen und zu lösen dreidimensionale Stämme über die Oberfläche verformt 8,9. Als ein Netz Stents in ein Gefäß mit einem Latex-zufällige kontrastierenden Musters aufgesprüht oder auf seiner äußeren Oberfläche gezogen bereitgestellt wird, wird die Oberfläche Stamm zu jedem Zeitpunkt der Verformung aufgezeichnet. Die berechneten Dehnungsverteilungen kann dann verwendet werden, um die lo verstehensche Läsion Reaktion, Validierung der Rechenmodelle und Hypothesen für weitere in-vivo-Studie.

Watch this Scientific Journal Video about Monitoring the Wall Mechanics During Stent Deployment in a Vessel at JoVE.com

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Stent-induzierte arterielle Dehnungsverteilungen sind gekennzeichnet mit einer optischen Oberfläche Dehnungsmessung System. Diese Visualisierung Technik wird verwendet, um Einblicke in die Auswirkungen der Stentimplantation auf dem Host-Schiff zu gewinnen.

Die Überwachung der Wand Mechanik Während der Entfaltung des Stents in einem Gefäß

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

monitoring-the-wall-mechanics-during-stent-deployment-in-a-vessel

Research

JoVE Journal

Bioengineering

9.2K Aufrufe.  University of Nebraska-Lincoln. Stent-induzierte arterielle Dehnungsverteilungen sind gekennzeichnet mit einer optischen Oberfläche Dehnungsmessung System. Diese Visualisierung Technik wird verwendet, um Einblicke in die Auswirkungen der Stentimplantation auf dem Host-Schiff zu gewinnen.

Serie: Monitoring the Wall Mechanics During Stent Deployment in a Vessel

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Magnet-Resonanz-Elastographie Methodik für die Beurteilung von Tissue Engineering gewonnenen Konstrukts Wachstum

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Forschung

Biotechnik

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Messe Cytometry: Protokoll für die tägliche Tuning und Ausführen von Zellproben auf einem CyTOF Messe Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

Eine vielseitige automatisierte Plattform für Mikro-Skala Handy Stimulation Experimente

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

Experimental Investigation of Secondary Flow Structures Downstream of a Model Type IV Stent Failure in a 180&#176; Curved Artery Test Section

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and tortuosity). Secondary flow structures are vortical flow patterns that occur in curved arteries due to the combined action of centrifugal forces, adverse pressure gradients and inflow characteristics. Such flow morphologies are greatly affected by pulsatility and multiple harmonics of physiological inflow conditions and vary greatly in size-strength-shape characteristics compared to non-physiological (steady and oscillatory) flows 1 - 7.
Secondary flow structures may ultimately influence the wall shear stress and exposure time of blood-borne particles toward progression of atherosclerosis, restenosis, sensitization of platelets and thrombosis 4 - 6, 8 - 13. Therefore, the ability to detect and characterize these structures under laboratory-controlled conditions is precursor to further clinical investigations.
A common surgical treatment to atherosclerosis is stent implantation, to open up stenosed arteries for unobstructed blood flow. But the concomitant flow perturbations due to stent installations result in multi-scale secondary flow morphologies 4 - 6. Progressively higher order complexities such as asymmetry and loss in coherence can be induced by ensuing stent failures vis-&#224;-vis those under unperturbed flows 5. These stent failures have been classified as &#34;Types I-to-IV&#34; based on failure considerations and clinical severity 14.
This study presents a protocol for the experimental investigation of the complex secondary flow structures due to complete transverse stent fracture and linear displacement of fractured parts (&#34;Type IV&#34;) in a curved artery model. The experimental method involves the implementation of particle image velocimetry (2C-2D PIV) techniques with an archetypal carotid artery inflow waveform, a refractive index matched blood-analog working fluid for phase-averaged measurements 15 - 18. Quantitative identification of secondary flow structures was achieved using concepts of flow physics, critical point theory and a novel wavelet transform algorithm applied to experimental PIV data 5, 6, 19 - 26.

Stent implants in stenosed arterial curvatures are prone to "Type IV" failures involving the complete transverse fracture of stents and linear displacement of the fractured parts. We present a protocol for detection of secondary flow (vortical) structures in a curved artery model, downstream of clinically relevant "Type IV" stent failures.

Experimentelle Untersuchung von Sekundärströmungsstrukturen hinter einem Modell Typ IV Stent Failure in einem 180 ° Curved Artery Test Sektion

The arterial network in the human vasculature comprises of ubiquitously present blood vessels with complex geometries (branches, curvatures and ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Anpassung der Elektrospinnverfahren für einen Dreischicht stellen drei einzigartige Umgebungen<em&gt; In-vitro-</em&gt; Modell der Atemwegswand

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

This video and manuscript describe an emulsion-based method to encapsulate mammalian cells in 0.5% to 10% alginate beads which can be produced in large batches using a simple stirred vessel. The encapsulated cells can be cultured in vitro or transplanted for cellular therapy applications.

Säugetierzellverkapselung in Alginatperlen unter Verwendung eines einfachen gerafften Gefäßes

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet ...

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved through the contraction of the ciliary body and relaxation of zonule fibers, which results in the thickening of the lens necessary for near vision. Here, we present a novel, simple method in which accommodation is replicated by tensing the zonules connected to the lens capsule via a manual lens stretcher (MLS). This method monitors the radial stretching achieved by a lens when subjected to a consistent force and allows for a comparison of accommodating lenses, which can be stretched, to non-accommodating lenses, which cannot be stretched. Importantly, the stretcher couples to the zonules directly, and not to the sclera of the eye, thus only requiring the lens, zonules, and ciliary body rather than the entire globe sample. This difference can significantly decrease the cost of acquiring donor cadaver lenses by about 62% compared to acquiring an entire globe.

We present an efficient method of studying lens accommodation by using a manual lens stretcher. The protocol mimics physiological accommodation by pulling the zonules connected around the lens capsule, thereby, stretching the lens.

Die Mechanik der Linse Unterkunft über einen manuellen Objektiv Stretcher simulieren

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved ...

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

Zerstörungsfreie Überwachung der abbaubaren Gerüst-basierten Tissue-Engineering Blutgefäß Entwicklung mit optischen Kohärenztomographie

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

Ein Tripeptid-stabilisierten Nanoemulsion Ölsäure

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Musterung der Geometrie menschlicher embryonaler Stammzellkolonien auf konformen Substraten zur Kontrolle der Gewebe-Level-Mechanik

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...