SynVivo Technologie wurde unter dem Förder # 2R44HL076034 vom NHLBI entwickelt.

1. Auffüllen der SynVivo-SMN Mikrofluidikvorrichtung <ol><li> Jeder Port (Einlass / Auslass) der Vorrichtung besteht aus zwei parallelen Ports umfaßt - eine für Oberflächenbeschichtungseinheiten fließt (Adhäsionsmoleküle, Wachstums Matrizen, etc.) und / oder Zellen zum Impfen und die andere für die Testdurchführung (1A ). </li><li> Komplett Tauchen Sie das SynVivo-SMN Mikrofluidik-Vorrichtung (Abbildung 1B) in einer Petrischale sterilem deionisiertem (DI), das Wasser und die Schale in einen Vakuumtrockenschrank. Erlauben der Exsikkator ausgeführt, bis die gesamte Luft aus den Kanälen der Vorrichtung entfernt. Dies sollte etwa 15 Minuten dauern. </li><li> Bevor Sie das Gerät aus dem Wasser, legen Tygon-Schlauch (OD von 0,06 &quot;und ID von 0,02&quot;) mit Wasser vorbereitet in jedes Port des Gerätes mit feinen Punkt Pinzette. Der Schlauch sollte etwa 1 cm lang sein. Das Gerät kann nun aus dem Wasser entfernt werden. 1C zeigt Bild des DevicE mit dem Schlauch. </li></ol> 2. Beschichtung der mikrofluidischen Vorrichtung mit gewünschten Protein (z. B. Avidin) <ol><li> Mit einer Pipette einen Tropfen Wasser (etwa 100 ul), um die Basis des einen Einlaßanschlußschlauch. Den Schlauch mit dem Gerät verwendet prime vorsichtig entfernen. Der Wassertropfen wird Luft in das Gerät zu verhindern. </li><li> Bereiten einer 1 ml Spritze mit Avidin beladen in einer Konzentration von 20 ug / ml. Schließen Sie die Spritze zu einer 24 G Nadel aus Edelstahl und Schläuche. Stecken Sie den Schlauch an einer der Einlassöffnungen des Gerätes. Klemmen Sie die Einlassöffnung nicht mit einem Backenschelle eingesetzt. </li><li> Injizieren Avidin bei einer Flussrate von 1 &amp;mgr; l / min für 10 Minuten, um eine vollständige Perfusion der Vorrichtung zu ermöglichen. Am Ende des Durchflusszeit, klemmen Sie den Schlauch mit dem Kieferklemme und stellen Sie das Gerät bei 4 ° C über Nacht. </li></ol> 3. Mitteln des biotinylierten Partikel Haftung für Experimente <ol><li> Lassen Sie das Gerät anauf Raumtemperatur kommen. Stellen Sie das Gerät auf einem inversen Fluoreszenzmikroskop mit einem motorisierten Bühne und einem Hochleistungs-Kamera ausgestattet. </li><li> Eine Lösung von 2 um biotinylierten Partikel in einer Konzentration von 5 x 10 6 Partikeln / ml in phosphatgepufferter Kochsalzlösung (PBS). Laden der Teilchen in einer 1 ml-Spritze. Bereiten Sie eine zweite 1-ml-Spritze von nur PBS. Legen Sie jede Spritze auf eine Spritzenpumpe und eine Verbindung zu Nadel und Schläuche. </li><li> Mit einer Pipette einen Tropfen Wasser (etwa 100 ul), um die Basis des Einlasses Schläuche. Den Schlauch zu beschichten das Gerät verwendet, vorsichtig entfernen. Der Wassertropfen wird Luft in das Gerät zu verhindern. </li><li> Vorsichtig den Schlauch für biotinylierten Teilchen und PBS aus Schritt 3.2 in jede der Einlassöffnungen. 2A zeigt Bild von der Set-up. </li><li> Einspritzen beginnen biotinylierten Partikel mit einer Durchflussrate von 2,5 ml / min. Überwachung der Einlaßöffnung auf dem Mikroskop. Beim ersten Anzeichenvon Partikeln, beginnen Sie den Timer und Fluss weiterhin für 3 min. </li><li> Am Ende der 3 min, Auslaufen des biotinylierten Partikel, während gleichzeitig die Strömung des starren PBS bei einer Flussrate von 2,5 ml / min. Lassen PBS in der Einrichtung für 3 min fließen zu waschen ungebundene Partikel. </li></ol> 4. Erwerb von Bildern und Erstellen Area of ​​Interest (AOI) Messungen mit Imaging-Software (NIKON Elements) <ol><li> Mit der Funktion &quot;Scan-große Bild&quot; in der Imaging-Software, um das Bild der gesamten Einrichtung zu erwerben. </li><li> Nacheinander die Anzahl Gabelungen im Gerät und eine kreisförmige AOI mit der doppelten Durchmesser der Kanäle. In diesem Fall den AOI Durchmesser bis 200 um, da die Kanaldurchmesser beträgt 100 um. </li><li> Verwenden Sie die automatisierte Zählung Funktion in der Bildbearbeitungssoftware, um die Anzahl der Teilchen in jedem AOI in eine MS Excel-Tabelle exportieren. </li><li> Ebenso nutzen die automatisierte Zählung-Funktion, um die Anzahl der Teilchen in der gesamten exportierenGerät. </li></ol> 5. Particle Flux Analyse mit Computational Fluid Dynamics (CFD) Modelle <ol><li> CFD-Simulationen werden mit kommerziell erhältlicher Software (CFD-ACE +, Inc. ESI) für den SynVivo-SMN Gerätetopologie laufen. Die Ergebnisse werden in einer Datenbank zur Analyse von experimentellen Beobachtungen gespeichert. Die Simulationsergebnisse speichern Informationen über Wandscherraten, Geschwindigkeit, Partikelfluss und Haftung im Gerät. </li><li> Die Simulationsergebnisse werden verwendet, um die Anzahl der Partikel, die in jeder AOI basierend auf einem gegebenen Eintrittspartikelkonzentration zu bestimmen. </li></ol> 6. Genescherhaft Karte <ol><li> Berechnen% der Adhäsion durch Dividieren der anhaftenden Teilchen in der Gabelung von den in der Verzweigung, wie in Gleichung 6.1 gezeigt fließenden Teilchen. <img alt="Gleichung 1" fo:content-width="3in" src="/files/ftp_upload/51025/51025eq1.jpg" /> wobei die Anzahl der Partikel haftet und den fließenden Teilchen aus p erhaltenrotocol Schritte 4.3 und 5.2 auf. </li><li> Zeichnen Sie die Scherfestigkeit Karte mit der Scherrate bei jeder Gabelung der von der Datenbank in Schritt (5.1) erhalten Netze und die aus Gleichung 6.1% erhalten Haftwerte. </li></ol>

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Veröffentlichungsgebühr für diesen Artikel von CFD Research Corporation gesponsert.

Parallelplattenflusskammer und bietet wesentliche Erkenntnisse über Zell-Zell-und Zell-Teilchen-Wechselwirkungen, leiden unter mehreren Einschränkungen wie hoher Verbrauch an Reagenzien und die Notwendigkeit für mehrere Versuchsdurchläufe, um eine Scherhaft Karte zu erzeugen. Die Verwendung von synthetischen Mikrovaskuläre SynVivo-Netzwerke (SynVivo-SMNS) ermöglicht die Erzeugung einer Scherhaft Karte aus einem einzigen Experiment unter Bedingungen in vivo-Bedingungen nachahmen. Zusätzlich wird deutliche...

Aktuelle Assays auf Zell-Zell-und Zell-Teilchen-Wechselwirkungen untersuchen typischerweise statische Well-Plattenformat, in welchem ​​Teilchen oder Zellen auf Protein-Matrices oder adhärente Zellen inkubiert. Am Ende der angegebenen Inkubationszeit werden die Zahlen der anhaftenden Teilchen oder Zellen unter Verwendung von 1-Mikroskopie quantifiziert. Auch wenn diese Untersuchungen liefern wichtige Einblicke in die biochemischen Prozesse hinter dieser Wechselwirkungen ist eine wesentliche Einschränkung, der Mangel an physiologischen Flüssigkeitsstrom (typisch für die Mikrozirkulation) und ihre Auswirkungen auf die Partikelhaftung. Um diese Einschränkung zu überwinden, wurden in-vitro-Strömungskammern in den letzten Jahren entwickelt worden. Ein gemeinsames Element dieser Strömungskammern ist ein transparentes Gerät bei niedrigen Reynolds-Zahlen an der Wand der Blutgefäße in vivo 2 beobachtet Scherraten entsprechen durchblutet. Die Behälterwand wird entweder durch Beschichtung von Biomolekülen oder das Wachstum von Zellen auf einer Oberfläche des Strömungs c modelliertHamber 3. Partikel oder Zellen 4-7 8-16 werden dann flossen bei gewünschten Durchsatzbereich, die Anzahl der anhaftenden Partikeln unter verschiedenen Scherraten zu quantifizieren. Jedoch ist die Verwendung von parallelen Plattenströmungskammern zu studieren und zu validieren die biochemische Phänomene ziemlich teuer und zeitaufwendig. Dies ist vor allem aufgrund der Tatsache, dass mehrere Versuche benötigen, um zum Erzeugen einer Karte von der Fluidscher gegen die Anzahl der Partikel / Zellen haften geführt werden. Außerdem Platte Strömungskammern erfordern große Mengen von Reagenzien aufgrund ihrer Größe (Höhe&gt; 250 um und einer Breite von&gt; 1 mm). Schließlich sind diese Geräte nicht genau modellieren geometrische Merkmale (zB Bifurkationen) und Durchflussbedingungen (z. B. gegen konvergierenden divergierenden Ströme), die in vivo vorhanden sind. Jüngste Fortschritte in der Lithographie Mikro 17-19 haben den Bereich der Lab-on-a-Chip beschleunigtGeräte 20-21. Diese Geräte waren maßgeblich an der Entwicklung eines miniaturisierten Version der Parallelplattenlaufkammer mit Abmessungen im Mikrometerbereich. Die Reduzierung der Dimension ergibt auch erhebliche Vorteile in Bezug auf die Mengen von Reagenzien, Zellen oder Partikel für die Experimente erforderlich. Eine wesentliche Einschränkung, die derzeit erhältlichen Vorrichtungen ist jedoch die Verwendung von linearen Kanälen Mikrogefäßen, die den in vivo beobachteten komplexen Mikrogefäß nicht zu imitieren ist modellieren. Wir haben kürzlich ein neues Verfahren zur Wieder mikrovaskulärer Netzwerke auf Einweg-Kunststoffsubstraten, was zu synthetischen Darstellung der in vivo-Bedingungen entwickelt. Diese Geräte bezeichnet SynVivo-Synthetic mikrovaskuläre Networks (SMN) werden mit PDMS-basierte Soft-Lithographie-Prozess entwickelt. SynVivo SMN-Geräte können verwendet werden, um die Scherhaft Karte von Zell / Partikelhaftung 22 zu erhalten, Studien gezielten Wirkstofftransport und 23 have gegen in-vivo-Daten 24-25 validiert. In diesem Beitrag stellen wir ein Protokoll, dass die Erzeugung der Schubhaft Karte aus einem einzigen Experiment in Volumen so klein wie 1-5 ul was zu erheblichen Einsparungen von Ressourcen und Zeit ermöglicht.

<table border="0" cellpadding="0" cellspacing="0" width="638">
	<tbody>
		<tr>
			<td>SynVivo-SMN</td>
			<td>CFD Research</td>
			<td>SMN-001</td>
			<td>Exclusive at CFDRC</td>
		</tr>
		<tr>
			<td>CFD-ACE+</td>
			<td>ESI Inc.</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Avidin</td>
			<td>Invitrogen</td>
			<td>43-4401</td>
			<td>Any avidin source will work for this assay</td>
		</tr>
		<tr>
			<td>Biotinylated Particles</td>
			<td>Polysciences</td>
			<td>24173-1</td>
			<td>Any source of biotinylated particles will work for the assay</td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>VWR</td>
			<td>63018-044</td>
			<td>Size is typical for use with SynVivo-SMN</td>
		</tr>
		<tr>
			<td>NIKON Elements</td>
			<td>NIKON Instruments</td>
			<td>N/A</td>
			<td>Any other imaging software can be used</td>
		</tr>
	</tbody>
</table>

generation of shear adhesion map using synvivo synthetic microvascular networks

Handy / Partikelhaftung Tests sind entscheidend für das Verständnis der biochemischen Interaktionen in Krankheit Pathophysiologie beteiligt und haben wichtige Anwendungen in der Suche nach der Entwicklung neuartiger Therapeutika. Tests mit statischen Bedingungen nicht um die Abhängigkeit der Haftung auf Scher erfassen, was ihre Korrelation mit in vivo-Umgebung. Parallelplattenflusskammern, die die Haftung unter physiologischen Fluidstrom zu quantifizieren müssen mehrere Experimente zur Erzeugung einer Scherhaft Karte. Darüber hinaus müssen sie nicht unbedingt die in vivo-Skala und Morphologie und erfordern große Volumina (~ ml) Reagenzien für die Experimente. In dieser Studie zeigen wir die Erzeugung von Scherhaftung Karte aus einem einzigen Experiment unter Verwendung einer mikrovaskulären Netzwerk basierend Mikrofluidikvorrichtung SynVivo-SMN. Das Gerät erstellt die komplexen in vivo Gefäßsystem einschließlich geometrischen Maßstab, morphologische Elemente, Strömungsmerkmale und zellulären Interaktionen in einIn-vitro-Format, wodurch eine biologisch realistische Umgebung für grundlegende und angewandte Forschung in Zellverhalten, Drug Delivery und Drug Discovery. Der Test wurde durch die Untersuchung der Wechselwirkung der 2 um Biotin-beschichteten Teilchen mit Avidin-beschichteten Oberflächen der Mikrochip gezeigt. Der gesamte Bereich der Scherung in der Mikrovaskulatur beobachtet in einem einzelnen Assay ermöglicht Adhäsion gegenüber der Scher Karte für die Partikel unter physiologischen Bedingungen erhalten wird.

1A zeigt eine schematische und eine helle Bildfeld SynVivo SMN-Gerät. 1B zeigt die SynVivo SMN-Gerät auf einem Glasträger montiert. 1C zeigt das Gerät mit folgenden Rohr im Vakuumtrockenschrank Grundierung mit Wasser. 2A zeigt ein Bild der Versuchs-Einrichtung. 2B zeigt eine typische Avidinbeschichtete SynVivo SMN-Gerät nach der Bindung von 2 um biotinylierten Partikel. Beachten Sie, dass Partikel bevor...

Watch this Scientific Journal Video about Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks at JoVE.com

Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables.

Erzeugung von Scherhaft Karte Mit SynVivo Synthetische mikrovaskuläre Networks

Cell/particle adhesion assays are critical to understanding the biochemical interactions involved in disease pathophysiology and have important ...

generation-shear-adhesion-map-using-synvivo-synthetic-microvascular

Research

JoVE Journal

Bioengineering

8.8K Aufrufe.  CFD Research Corporation. Flow chambers used in adhesion experiments typically consist of linear flow paths and require multiple experiments at different flow rates to generate a shear adhesion map. SynVivo-SMN enables the generation of shear adhesion map using a single experiment utilizing microliter volumes resulting in significant savings in time and consumables. 

Serie: Generation of Shear Adhesion Map Using SynVivo Synthetic Microvascular Networks

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Plasma Lithography Oberflächenstrukturierung für die Erzeugung von Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

Forschung

Biotechnik

Endothelialized Microfluidics for Studying Microvascular Interactions in Hematologic Diseases

Advances in microfabrication techniques have enabled the production of inexpensive and reproducible microfluidic systems for conducting biological and biochemical experiments at the micro- and nanoscales 1,2. In addition, microfluidics have also been specifically used to quantitatively analyze hematologic and microvascular processes, because of their ability to easily control the dynamic fluidic environment and biological conditions3-6. As such, researchers have more recently used microfluidic systems to study blood cell deformability, blood cell aggregation, microvascular blood flow, and blood cell-endothelial cell interactions6-13.However, these microfluidic systems either did not include cultured endothelial cells or were larger than the sizescale relevant to microvascular pathologic processes. A microfluidic platform with cultured endothelial cells that accurately recapitulates the cellular, physical, and hemodynamic environment of the microcirculation is needed to further our understanding of the underlying biophysical pathophysiology of hematologic diseases that involve the microvasculature.
Here, we report a method to create an "endothelialized" in vitro model of the microvasculature, using a simple, single mask microfabrication process in conjunction with standard endothelial cell culture techniques, to study pathologic biophysical microvascular interactions that occur in hematologic disease. This "microvasculature-on-a-chip" provides the researcher with a robust assay that tightly controls biological as well as biophysical conditions and is operated using a standard syringe pump and brightfield/fluorescence microscopy. Parameters such as microcirculatory hemodynamic conditions, endothelial cell type, blood cell type(s) and concentration(s), drug/inhibitory concentration etc., can all be easily controlled. As such, our microsystem provides a method to quantitatively investigate disease processes in which microvascular flow is impaired due to alterations in cell adhesion, aggregation, and deformability, a capability unavailable with existing assays.

A method to culture an endothelial cell monolayer throughout the entire inner 3D surface of a microfluidic device with microvascular-sized channels (&lt;30 &mu;m) is described. This in vitro microvasculature model enables the study of biophysical interactions between blood cells, endothelial cells, and soluble factors in hematologic diseases.

Endothelialisierten Mikrofluidik für ein Studium Mikrovaskuläre Wechselwirkungen in Hämatologische Krankheiten

Advances in  microfabrication techniques have enabled the production of inexpensive and  reproducible microfluidic systems for conducting biological and ...

Induction of Adhesion-dependent Signals Using Low-intensity Ultrasound

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from regulation of cell migration and proliferation, to secretion and even differentiation. The signals underlying each of these complex processes arise from the molecular interactions of extracellular matrix receptors on the surface of the cell. Integrins are the prototypic receptors and provide a mechanical link between extracellular matrix and the cytoskeleton, as well as initiating some of the adhesion-dependent signaling cascades. However, it is becoming increasingly apparent that additional transmembrane receptors function alongside the integrins to regulate both the integrin itself and signals downstream. The most elegant of these examples is the transmembrane proteoglycan, syndecan-4, which cooperates with &alpha;5&beta;1-integrin during adhesion to fibronectin. In vivo models demonstrate the importance of syndecan-4 signaling, as syndecan-4-knockout mice exhibit healing retardation due to inefficient fibroblast migration1,2. In wild-type animals, migration of fibroblasts toward a wound is triggered by the appearance of fibronectin that leaks from damaged capillaries and is deposited by macrophages in injured tissue. Therefore there is great interest in discovering strategies that enhance fibronectin-dependent signaling and could accelerate repair processes.
The integrin-mediated and syndecan-4-mediated components of fibronectin-dependent signaling can be separated by stimulating cells with recombinant fibronectin fragments. Although integrin engagement is essential for cell adhesion, certain fibronectin-dependent signals are regulated by syndecan-4. Syndecan-4 activates the Rac1 protrusive signal3, causes integrin redistribution1, triggers recruitment of cytoskeletal molecules, such as vinculin, to focal adhesions4, and thereby induces directional migration3. We have looked for alternative strategies for activating such signals and found that low-intensity pulsed ultrasound (LIPUS) can mimic the effects of syndecan-4 engagement5. In this protocol we describe the method by which 30 mW/cm2, 1.5 MHz ultrasound, pulsed at 1 kHz (Fig. 1) can be applied to fibroblasts in culture (Fig. 2) to induce Rac1 activation and focal adhesion formation. Ultrasound stimulation is applied for a maximum of 20 minutes, as this combination of parameters has been found to be most efficacious for acceleration of clinical fracture repair6. The method uses recombinant fibronectin fragments to engage &alpha;5&beta;1-integrin, without engagement of syndecan-4, and requires inhibition of protein synthesis by cycloheximide to block deposition of additional matrix by the fibroblasts., The positive effect of ultrasound on repair mechanisms is well documented7,8, and by understanding the molecular effect of ultrasound in culture we should be able to refine the therapeutic technique to improve clinical outcomes.

This protocol describes the stimulation of cultured fibroblasts with low-intensity pulsed ultrasound, which drives focal adhesion formation and Rac1 activation by mimicking engagement of the transmembrane matrix receptor, syndecan-4. This approach allows investigation of a successful clinical technique at the cellular level, thereby providing opportunities for refinement of the therapy.

Die Induktion von Adhäsions-abhängigen Signale mit geringer Intensität Ultraschall

In multicellular organisms, cell behavior is dictated by interactions with the extracellular matrix. Consequences of matrix-engagement range from ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Benutzen Sie die Cell-Substrat-Impedanz und Live Cell Imaging, um Echtzeit-Änderungen in der Zelladhäsion und De-Haftung durch Matrix Änderung Induzierte Messen

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

This paper presents a series of protocols for developing engineered cells and functionalized surfaces that enable synthetically engineered E. coli to control and manipulate programmable material surfaces.

Unter Verwendung der synthetischen Biologie lebenden Zellen zu entwickeln, die Schnittstelle mit programmierbarer Materialien

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is ...

Gene Expression Analysis of Endothelial Cells Exposed to Shear Stress Using Multiple Parallel-plate Flow Chambers

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate flow chambers. Endothelial cells form the inner cellular lining of blood vessels and are chronically exposed to the frictional force of blood flow called shear stress. Under physiological conditions, endothelial cells function in the presence of various shear stress conditions. Thus, the application of shear stress conditions in in vitro models can provide greater insight into endothelial responses in vivo. The parallel-plate flow chamber previously published by Lane et al.9 is adapted to study endothelial gene regulation in the presence and absence of steady (non-pulsatile) laminar flow. Key adaptations in the set-up for laminar flow as presented here include a large, dedicated environment to house concurrent flow circuits, the monitoring of flow rates in real-time, and the inclusion of an exogenous reference RNA for the normalization of quantitative real-time PCR data. To assess multiple treatments/conditions with the application of shear stress, multiple flow circuits and pumps are used simultaneously within the same heated and humidified incubator. The flow rate of each flow circuit is measured continuously in real-time to standardize shear stress conditions throughout the experiments. Because these experiments have multiple conditions, we also use an exogenous reference RNA that is spiked-in at the time of RNA extraction for the normalization of RNA extraction and first-strand cDNA synthesis efficiencies. These steps minimize the variability between samples. This strategy is employed in our pipeline for the gene expression analysis with shear stress experiments using the parallel-plate flow chamber, but parts of this strategy, such as the exogenous reference RNA spike-in, can easily and cost-effectively be used for other applications.

Here, a workflow for the culture and gene expression analysis of endothelial cells under fluid shear stress is presented. Included is a physical arrangement for simultaneously housing and monitoring multiple flow chambers in a controlled environment and the use of an exogenous reference RNA for quantitative PCR.

Gen Expressionsanalyse von Endothelzellen ausgesetzt um Stress mit mehreren Parallel-Platte Fluss Kammern Scheren

We describe a workflow for the analysis of gene expression from endothelial cells subject to a steady laminar flow using multiple monitored parallel-plate ...

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.

Live Cell Analysis of Shear Stress on Pseudomonas aeruginosa Using an Automated Higher-Throughput Microfluidic System

Here, we describe the use of a higher-throughput microfluidic bioreactor coupled with a fluorescent microscope for the analysis of shear stress effects on Pseudomonas aeruginosa biofilms expressing green fluorescent proteins, including instrument set up, the determination of biofilm coverage, growth rate, and morphological properties.

Pseudomonas Aeruginosa mit einem automatisierten höheren Durchsatz mikrofluidischen System Zelle Analyse der Schubspannung weiterleben

A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, ...

Generation of Transgenic Rats using a Lentiviral Vector Approach

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

This article aims to provide the methodology for lentiviral transgenesis in rat embryos using multiple injections of a virus suspension into the zygote perivitelline space. Female rats that are mated with a fertile male strain with a different dominant fur color is used to generate pseudopregnant foster mothers.

Generierung transgener Ratten mit einem Lentiviral Vector Ansatz

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

Herstellung von Proteinproduktion synthetischer Zellen mit zellfreien bakteriellen Extrakten, Liposomen und Emulsionstransfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

Herstellung von Mehrkomponenten-Lipid-Nanoröhrchen-Netzwerken unter Verwendung des Gliding Kinesin Motility Assays

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...