Wir danken Professor Wolf Frommer (Carnegie Institution for Science, Stanford University) für die freundliche Bereitstellung uns mit dem Glukose-FRET-Plasmid und Dr. Uwe Ludewig (Universität Hohenheim) für die freundliche Versorgung der Glutamin-FRET-Konstrukt in der pUTkan Pflanzenexpressionsvektor. AB wird durch die BBSRC Gezielte Priorität Studentprogramm finanziert. Sowohl CK und KP sind durch RCUK Fellowships in Biopharmaceuticals Verarbeitung unterstützt. CK möchte auch Lonza Biologics für die finanzielle Unterstützung bedanken. Das Zentrum für Synthetische Biologie und Innovation wird großzügig von der EPSRC unterstützt.

1. Zelllinie Revival und Wartung <ol><li> Revive CHO-Zellen in 9 ml CD-CHO-Medium mit 8 mM L-Glutamin und 10 ml / L 100x Hypoxanthin / Thymidin-Supplement (komplette Wachstumsmedium) ergänzt. </li><li> Zentrifuge bei 100 × g für 5 min. </li><li> Resuspendieren der Zellen in 10 ml frisches vollständiges Wachstumsmedium. </li><li> Entfernen einer 1 ml Probe und bestimmen die Lebendzellkonzentration mittels Lichtmikroskopie mit der Trypanblau-Ausschluss-Verfahren in einer Zählkammer. </li><li> Initiieren einer Kultur bei einer Aussaatdichte von 3 x 10 5 Zellen / ml in 125 ml-Schüttelkolben. </li><li> Erhaltung der Kultur im Brutschrank bei 37 ° C, einer befeuchteten Atmosphäre von 5% CO 2, auf einem Orbitalschüttler bei 120 Umdrehungen pro Minute dreht. </li><li> Subkultivierung alle 3-4 Tage in komplettem Wachstumsmedium bei einer Aussaatdichte von 2 x 10 5 Zellen / ml. </li></ol> 2. Transfektion der Zelllinie mit dem Plasmid Containing den Biosensor Gene <ol><li> Bereiten Plasmid-DNA mit einem großen Plasmid-Reinigungs-Kits. </li><li> Aufrechterhaltung Zellen an den entsprechenden Bedingungen (37 ° C, 5% CO 2) 12-24 Stunden vor der Transfektion, um sicherzustellen, dass die Zellen sich aktiv teil Zeitpunkt der Transfektion. </li><li> Zählen der Zellen unter Verwendung des Trypanblau-Ausschlussverfahrens, bereiten dann ein Arbeitsvolumen von 20 ml der Zellkultur in einem 125 ml Schüttelkolben in einer Konzentration von 1 x 10 6 Zellen / ml. </li><li> Verwenden Sie eine geeignete Transfektion Kit für die Zelllinie verwendet wird. Die Plasmid-DNA zu Transfektionsreagenzien Verhältnis wird abhängig von der Zelllinie und Vektor sein. Es wird empfohlen, dass diese durch Testen eines Bereichs von Verhältnissen innerhalb des durch den Hersteller vor der Durchführung der abschließenden Transfektionen angegeben optimiert. Auch halten mindestens eine negative Kontrollkultur, die Zellen aber keine DNA enthalten. </li><li> Inkubation für 4 Tage im statischen Modus und dann zu einem shaki übertragenng-Plattform bei 125 Umdrehungen pro Minute rotiert. </li><li> Fügen Sie den entsprechenden Antibiotikum für Plasmid-Selektion auf eine geeignete Konzentration, wie durch einen Kill-Kurve bestimmt. In dieser Studie wurde Zeocin bis zu einer Endkonzentration von 400 ug / ml für die Selektion von transfizierten Zellen zugegeben. </li><li> Ändern Sie Medien zu einem geeigneten Zeitintervall für die Zelllinie, das Hinzufügen Antibiotikum jedes Mal, bis die Zellen in Steuer gut sind gestorben. </li><li> Mit den meisten Brunnen konfluenten Kulturen Schütteln Fortschritt. </li><li> Schaffung eines Zellbank durch Einfrieren der Zellen in Kryoröhrchen mit 10 7 lebensfähigen Zellen in 1 ml Gefriermischung. In dieser Arbeit besteht diese aus 92,5% Wachstumsmedium und 7,5% Dimethylsulfoxid. </li></ol> 3. Batch-und Fed-Batch-Zellwachstumskurve <ol><li> Folgen Sie den Anweisungen für die Zellwartungs oben, um dreifach-Zellkulturen der transfizierten CHO-Zellen in 250 ml etablieren Schüttelkolben mit einem Arbeitsvolumen von 50 ml. </li><li> Pflegen Sie die kulAuflösung in einem befeuchteten Zell Inkubator bei 37 ° C mit 5% CO 2, auf einem Orbitalschüttler bei 125 Umdrehungen pro Minute dreht, und entfernen 4,1 ml Proben von jeder wachsenden Kultur bei 24 Stunden Intervallen. </li><li> Verwenden von 100 &amp;mgr; l der Probe, um die Konzentration lebensfähiger Zellen und die Lebensfähigkeit der Zellen mit Trypan-Blau-Ausschlussverfahren zu bestimmen. </li><li> Wiederholen, bis die Konzentration lebensfähiger Zellen auf Null reduziert wird. </li><li> Wiederholen für die Fed-Batch-Zellkulturen die Ergänzung mit der entsprechenden Menge an Glucose oder Glutamin am Tag 6 nach deren Konzentrationen auf ihre Anfangswerte von 36 mm und 4 mm wiederherzustellen sind. Es wird empfohlen, dass zusätzliche Kontrollkulturen werden auch erhalten, was mit dem gleichen Volumen (12 ml in diesem Fall) von reinem Wasser zugeführt werden. </li></ol> 4. FRET-Messungen Verhältnis <ol><li> Nehmen 2 ml der Proben täglich in Schritt 3.2 Zentrifuge entfernt und bei 100 × g für 5 min bei 4 ° C. </li><li> Resuspendete das Zellpellet in 2 ml eiskalter phosphatgepufferter Kochsalzlösung (PBS). Bringen Sie diese in eine 6-Well-Platte und fügen Sie eine Leerprobe, die keine Zellen, die man gut. </li><li> Messen Sie die Höhe der blauen und gelben Fluoreszenz sofort bei einer Anregungswellenlänge von 430/435 nm und Emissionswellenlängen von 465/435 nm (blau) und 520/510 nm (gelb). </li><li> Berechnen Sie die FRET-Ratios (Verhältnis von gelben Fluoreszenz über blaue Fluoreszenz erfasst wird). </li></ol> 5. Metabolite Assays <ol><li> Nehmen 2 ml der Proben täglich in Schritt 3.2 Zentrifuge entfernt und bei 100 × g für 5 min bei 4 ° C. Entfernen Sie den Überstand. </li><li> Zellpellet in 3 ml eiskaltem PBS gewaschen und erneut zentrifugieren bei 100 × g für 5 min. </li><li> Den Überstand und Zellpellet in 3 ml eiskaltem PBS wie oben. Beschallen der Probe 5x für jeden 3 min bei einem Puls von 15 Sek. auf 15 Sek. und ausgeschaltet. An diesem Punkt ist die Zelle zusätzlichects, falls gewünscht eingefroren werden. </li><li> Verwenden ein geeignetes Glucose-Assay-Kit nach den Anweisungen des Herstellers an Proben von Zellextrakten, die intrazellulären Glucosekonzentration zu ermitteln (siehe Tabelle der Materialien). Hinweis: Verwenden Sie geeignete Standards, um eine Standardkurve zu konstruieren. In dieser Arbeit, die Konzentration der Standards lag zwischen 0-100 mM. </li><li> Verwendung der Messwerte für die Standards, die Standardkurve zu konstruieren, und um die lineare Gleichung der besten Anpassung (in diesem Fall y = 1310.51x - 723,43 wobei y die Absorptionsmessung und x der Glucosekonzentration). </li><li> Berechnen Sie die Glucose-Konzentrationen anhand der Standardkurve und unter Berücksichtigung der Verdünnung. Die intrazellulären Glukosekonzentration kann dann berechnet werden unter Verwendung dieser Gleichung: <img alt="" fo:content-width="4.5in" src="/files/ftp_upload/51200/book_bg.png" width="500" /> Für CHO-Zellen, wurde die Einzelzellvolumen berechnet wird unter Verwendung eines Zelldurchmesser von 12 &amp;mgr; M 12 </sup&gt;. </li><li> Verwenden Sie eine geeignete Glutamin-Assay-Kit nach den Anweisungen des Herstellers an Proben von Zellextrakten, um die intrazellulären Glutamin-Konzentration zu bestimmen (siehe Tabelle der Materialien). Hinweis: Verwenden Sie geeignete Standards, um eine Standardkurve zu konstruieren. In dieser Arbeit, die Konzentration der Standards lag zwischen 0-2 mM. </li><li> Wie in 5.5 oben, Die Eichkurve (in diesem Fall y = + 17,1 203.1x wobei y die Absorptionswert ist und x die Glucosekonzentration) </li><li> Mit der Standardkurve, die Berechnung der Glutamin-Konzentration der Proben, wobei die Verdünnung berücksichtigt. Die intrazelluläre Konzentration Glutamin können unter Verwendung der obigen Gleichung 1 berechnet werden. </li></ol>

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Die Autoren haben nichts zu offenbaren.

Die FIBS ermöglichen in vivo und in situ Quantifizierung von Schlüsselmoleküle, in diesem Fall das Wachstum einschränkende Nährstoffe, die Unsicherheiten, die aus dem Abschrecken und Extraktionsverfahren. Die Ergebnisse legen nahe, dass es eine gute Korrelation zwischen der FIBS Signal und die intrazellulären Konzentrationen im Bereich von 1-5 mM für Glucose und 0,3-2 mM Glutamin. Im Batch-CHO-Zellkultur, sind diese Konzentrationen in den exponentiellen, stationär, und Anfang der Rückgang Phas...

Metabolite Überwachung hat verschiedene Anwendungen in der Bioverfahrenstechnik, einschließlich Prozessentwicklung, Medien-und Futtermittel-Design und Metabolic Engineering. Verschiedene Verfahren sind durch die Verwendung von enzymatischen 1, Chemie 2, 3 oder Bindungsassays für Konzentrationsmessungen zur Verfügung. Eine interessante Möglichkeit ist die Verwendung von Fluoreszenz-, Protein-basierte biologische in vivo-Sensoren, die Daten über die intrazelluläre Konzentration von Schlüsselmetaboliten. Im Ultra-Low-Volume-Assays, Fluoreszenz ist ein geeignetes Mittel, wie Miniaturisierung tatsächlich verbessert das Signal-zu-Rausch-Verhältnis von 4,5 und einer proteinbasierte Sensoren können genetisch codierten Sinne, dass keine exogenen Reagenzien zur Metabolitenanalyse notwendig sind. Förster Resonanz Energie Transfer (FRET) Biosensoren bestehen aus zwei fluoreszierenden Proteinen, die von einem Liganden-bindenden Domäne verbunden ist. FRET ist eine strahlungslose Energieübertragung von einem photoangeregten Donor zu einem Akzeptor-Fluoreszenzmolekül locat<html> in unmittelbarer Nähe (&lt;100) ed. Liganden oder Bindungsspaltung bewirkt eine Konformationsänderung in den Sensor, der wiederum induziert eine Änderung in der Nähe der Fluorophore, die zu einer Änderung im FRET-Effizienz durch die Änderung im Emissionsspektrum gemessen. FRET integrierten biologischen Sensoren (FIBS) konstitutiv in der Zelllinie hergestellt und ihre spektralen Eigenschaften ändern sich die Konzentration des Metaboliten in der Zelle. FIBS haben schnelle Reaktionszeiten machen sie ideal für Messungen für dynamische Modelle 6. Zurück Anwendungen umfassen die Überwachung und mehrfache Einzel 7-9 10 Metaboliten und Bereitstellung von Daten über raumzeitliche Verteilung 11. Apo-Max, wobei die Ligandenbindung stört die Nähe der Fluorophore Senkung der Energieübertragung und Apo-Min, wobei die Ligandenbindung bringt die beiden Fluorophore in engeren Kontakt (Fig. 1): FIBS kann in zwei Konfigurationen erzeugt werden. _content &quot;&gt; In dieser Arbeit wird ein Protokoll für die Konstruktion von Chinese Hamster Ovary (CHO)-Zelllinien, die konstitutiv ein FIBS ein Metabolit Glucose oder Glutamin dargestellt. eine Methodik für die Kalibrierung des Sensors in vivo eingerichtet, um künftige quantitative Freigabe Messungen der intrazellulären Metaboliten-Konzentration, wie in Fig. 2 dargestellt. Auf dieser Grundlage wird die intrazelluläre Konzentration von Glucose oder Glutamin in Fed-Batch-Kulturen von CHO-Zellen bestimmt werden, an denen die beiden Nährstoffe in hohen Konzentrationen in-Verfahren zugegeben. Die Ergebnisse zeigen, dass die Verwendung des Fluoreszenzsignals von den FIBS und dem zuvor konstruierten Eichkurve ist genau Vorhersage der intrazellulären Konzentration möglich, von einem unabhängigen enzymatischen Test bestätigt. Dieses Verfahren bietet wesentliche Vorteile gegenüber gegenwärtigen analytischen Techniken, da sie nicht invasiv ist, kostengünstige und schnell, was eine Echtzeit-Signal der FIBS, die mon seinwährend der Kultivierungs itored.

<table border="0" cellpadding="0" cellspacing="0" width="936">
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		<col />
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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">CHO-S Cells</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;">11619-012</td>
			<td style="width:459px;">Cell line will vary depending on the goals of the study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">pCDNA4/TO vector&#160;</td>
			<td style="width:120px;">Life Tecnologies</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">TransIT-PRO Transfection Reagent</td>
			<td style="width:120px;">Mirus Bio</td>
			<td style="width:120px;">MIR 5700</td>
			<td style="width:459px;">Transfections can be accomplished using any method suitable for the cell line under study</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Zeocin</td>
			<td style="width:120px;">Invivogen</td>
			<td style="width:120px;"></td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">CD-CHO medium</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">10743-011</td>
			<td style="width:459px;">Cell growth medium is dependent upon the cells under study</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">100X HT Supplement</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">11067-030</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">L-Glutamine 200 mM (100X), Liquid</td>
			<td style="width:120px;">Life Technologies</td>
			<td style="width:120px;">25030032</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">InfinitePRO 200 plate reader</td>
			<td style="width:120px;">Tecan</td>
			<td style="width:120px;">FLx800TBI</td>
			<td style="width:459px;">Any 96-well fluorescence plate reader that can access the required wavelengths can be substituted</td>
		</tr>
		<tr height="21">
			<td height="190" rowspan="6" style="height:190px;width:239px;">Filters for plate reader</td>
			<td rowspan="6" style="width:120px;">Tecan</td>
			<td align="right" style="width:120px;">30000463</td>
			<td rowspan="6" style="width:459px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(520NM BW 10NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022786</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:120px;">(430NM BW 35NM)</td>
		</tr>
		<tr height="21">
			<td align="right" height="21" style="height:21px;width:120px;">30022787</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:120px;">(465NM BW 35NM)</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Maxiprep Plasmid Purification Kit</td>
			<td style="width:120px;">Qiagen</td>
			<td style="width:120px;">12163</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Amplex Red glucose/glucose oxidase assay kit</td>
			<td style="width:120px;">Invitrogen</td>
			<td style="width:120px;">A22189</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">EnzyChrom&#160; Glutamine Assay Kit</td>
			<td style="width:120px;">BioAssay systems</td>
			<td style="width:120px;">EOAC-100</td>
			<td style="width:459px;">Any suitable kit can be substituted</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Improved Neubauer haemocytometer</td>
			<td style="width:120px;">Fisher Scientific</td>
			<td style="width:120px;">MNK-420-010N</td>
			<td style="width:459px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Incubator&#160;</td>
			<td style="width:120px;">Nuaire</td>
			<td style="width:120px;">NU-5510E</td>
			<td style="width:459px;"></td>
		</tr>
	</tbody>
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fibs-enabled noninvasive metabolic profiling

Im Zeitalter der Computerbiologie, neue Hochdurchsatz-Versuchssysteme, um zu füllen und zu verfeinern Modelle, so dass sie für Prognosezwecke validiert werden erforderlich. Idealerweise würden solche Systeme mit geringem Volumen, die Probenahme und destruktiven Analysen ausschließt, wenn die Zeit natürlich Daten erhalten werden sollen sein. Was benötigt wird, ist eine in-situ-Monitoring-Tool, das in Echtzeit und nichtinvasiv die notwendigen Informationen mitteilen können. Eine interessante Möglichkeit ist die Verwendung von Fluoreszenz-, Protein-basierte biologische in vivo-Sensoren als Reporter der intrazellulären Konzentrationen. Eine besondere Klasse von Biosensoren, die in vivo-Anwendungen in Metaboliten Quantifizierung gefunden ist, wird auf Förster Resonanz Energie Transfer (FRET) zwischen zwei fluoreszierenden Proteinen, die von einem Ligandenbindungsdomäne basierend verbunden. FRET integrierten biologischen Sensoren (FIBS) konstitutiv in der Zelllinie hergestellt, sie haben schnelle Reaktionszeiten und ihre spektralen chaschaften bezogen auf die Konzentration des Metaboliten in der Zelle ändern. In dieser Arbeit wird das Verfahren zur Konstruktion des chinesischen Hamsters (CHO)-Zelllinien, die konstitutiv ein FIBS für Glukose und Glutamin und die Kalibrierung der FIBS in vivo im Batch Zellkultur, um zukünftige Quantifizierung von intrazellulären Metaboliten-Konzentration zu ermöglichen beschrieben. Daten von Fed-Batch-Kulturen von CHO-Zellen zeigt, daß die FIBS konnte in jedem Fall, um die sich ergebende Änderung der intrazellulären Konzentration zu detektieren. Verwendung des Fluoreszenzsignals von den FIBS und der zuvor konstruierten Eichkurve wurde die intrazelluläre Konzentration genau bestimmt, wie durch einen unabhängigen enzymatischen Assay bestätigt.

Ein Überblick der Methoden ist in Fig. 2 dargestellt. In der hier vorgestellten Arbeit wurden CHO-Zellen mit dem Vektor FIBS und stabile Zelllinien transfiziert wurden bei einem Antibiotikum Druck von 400 ug / ml Zeocin selektiert. Daher wurden zwei verschiedene stabile Zelllinien, die konstitutiv die Glucose und Glutamin Sensoren geschaffen. Fig. 1 zeigt die Konfiguration der beiden Biosensoren in dieser Studie verwendet. Der Glukosesensor ist auf der Apo-Max-Prinzip und die entsprech...

Watch this Scientific Journal Video about FIBS-enabled Noninvasive Metabolic Profiling at JoVE.com

FIBS-enabled Noninvasive Metabolic Profiling

Eine Beschreibung, wie Förster Resonance Energy Transfer integriert biologischen Sensoren (FIBS) zur in-situ-Kalibrierung Metabolic Profiling vorgestellt. Die FIBS kann verwendet werden, um die intrazellulären Spiegel von Metaboliten nicht-invasiv Unterstützung bei der Entwicklung von Stoffwechselmodelle und Hochdurchsatz-Screening von Bioprozess-Bedingungen zu messen.

FIBS-fähigen Nichtinvasive Metabolic Profiling

In the era of computational biology, new high throughput experimental systems are necessary in order to populate and refine models so that they can be ...

fibs-enabled-noninvasive-metabolic-profiling

Research

JoVE Journal

Bioengineering

9.6K Aufrufe.  Imperial College London.  Eine Beschreibung, wie Förster Resonance Energy Transfer integriert biologischen Sensoren (FIBS) zur in-situ-Kalibrierung Metabolic Profiling vorgestellt. Die FIBS kann verwendet werden, um die intrazellulären Spiegel von Metaboliten nicht-invasiv Unterstützung bei der Entwicklung von Stoffwechselmodelle und Hochdurchsatz-Screening von Bioprozess-Bedingungen zu messen. 

Serie: FIBS-enabled Noninvasive Metabolic Profiling

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

High Throughput MicroRNA Profiling: Optimierte Multiplex qRT-PCR in Nanoliter-Maßstab auf dem Fluidigm Dynamische ArrayTM IFCs

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Forschung

Biotechnik

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a high resolution X-ray microscope, thus enabling visualization of internal structural elements under simulated physiological loads and wet conditions. Experimental specimens will include intact bone-periodontal ligament (PDL)-tooth fibrous joints. Results will illustrate three important features of the protocol as they can be applied to organ level biomechanics: 1) reactionary force vs. displacement: tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics: geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. from concentric to eccentric loads. Efficacy of the proposed protocol will be evaluated by coupling mechanical testing readouts to 3D morphometrics and overall biomechanics of the joint. In addition, this technique will emphasize on the need to equilibrate experimental conditions, specifically reactionary loads prior to acquiring tomograms of fibrous joints. It should be noted that the proposed protocol is limited to testing specimens under ex vivo conditions, and that use of contrast agents to visualize soft tissue mechanical response could lead to erroneous conclusions about tissue and organ-level biomechanics.

In situ Compressive Loading and Correlative Noninvasive Imaging of the Bone-periodontal Ligament-tooth Fibrous Joint

In this study, the use of an in situ loading device coupled with micro-X-ray computed tomography for fibrous joint biomechanics will be discussed. Experimental readouts identifiable with an overall change in joint biomechanics will include: 1) reactionary force vs. displacement, i.e. tooth displacement within the alveolar socket and its reactionary response to loading, 2) three-dimensional (3D) spatial configuration and morphometrics, i.e.&#160;geometric relationship of the tooth with the alveolar socket, and 3) changes in readouts 1 and 2 due to a change in loading axis, i.e. concentric or eccentric loads.

In-situ-Druckbelastung und Correlative Nichtinvasive Bildgebung des Knochen parodontalen Ligament-Zahn-Faser-Joint

This study demonstrates a novel biomechanics testing protocol. The advantage of this protocol includes the use of an in situ loading device coupled to a ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have witnessed a tremendous increase in interest in EVs. EVs have been shown to play important roles in various physiological and pathological processes, including coagulation, immune responses, and cancer. In addition, EVs have potential as therapeutic agents, for instance as drug delivery vehicles or as regenerative medicine. Because of their small size (50 to 1,000 nm) accurate quantification and size profiling of EVs is technically challenging.
This protocol describes how tunable resistive pulse sensing (tRPS) technology, using the qNano system, can be used to determine the concentration and size of EVs. The method, which relies on the detection of EVs upon their transfer through a nano sized pore, is relatively fast, suffices the use of small sample volumes and does not require the purification and concentration of EVs. Next to the regular operation protocol an alternative approach is described using samples spiked with polystyrene beads of known size and concentration. This real-time calibration technique can be used to overcome technical hurdles encountered when measuring EVs directly in biological fluids.

Extracellular vesicles play important roles in physiological and pathological processes, including coagulation, immune responses, and cancer or as potential therapeutic agents in drug delivery or regenerative medicine. This protocol presents methods for the quantification and size characterization of isolated and non-isolated extracellular vesicles in various fluids using tunable resistive pulse sensing.

Quantifizierung und Größe-Profilierung der extrazellulären Vesikeln Mit Tunable Resistive Pulse Sensing

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Easy and Accurate Mechano-profiling on Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate surroundings. Particularly, cell-substrate interactions are the main interest. Today micropost arrays are a well-characterized and established method with a broad range of applications that have been published over the last decade. However, there seems to be a reservation among biologists to adapt the technique due to the lengthy and challenging process of micropost manufacture along with the lack of easily approachable software for analyzing images of cells interacting with microposts. 
The force read-out from microposts is surprisingly easy. A micropost acts like a spring with the cell ideally attached at its tip. Depending on size a cell applies force from its cytoskeleton through one or multiple focal adhesion points to the micropost, thus deflecting the micropost. The amount of deflection correlates directly to the applied force in direction and in magnitude. The number of microposts covered by a cell and the post deflection patterns are characteristic and allow determination of values like force per post and many biologically relevant parameters that allow &#8220;mechano-profiling&#8221; of cell phenotypes.
A convenient method for mechano-profiling is described here combining the first generation of ready-to-use commercially available microposts with an in-house developed software package that is now accessible to all researchers. As a demonstration of typical application, single images of bone cancer cells were taken in bright-field microscopy for mechano-profiling of cell line models of metastasis. This combination of commercial traction force sensors and open source software for analysis allows for the first time a rapid implementation of the micropost array technique into routine lab work done by non-expert users. Furthermore, a robust and streamlined analysis process enables a user to analyze a large number of micropost images in a highly time-efficient manner.

Described are protocols for quantifying mechanical interactions between adherent cells and microstructured substrates. These interactions are closely linked to essential cell behaviors including migration, proliferation, differentiation, and apoptosis. The protocols present an open-source image analysis software called MechProfiler, which enables determination of involved forces for each micropost.

Einfache und genaue Mechanotherapie Profilierung Micropost Arrays

Cell culture substrates with integrated flexible microposts enable a user to study the mechanical interactions between cells and their immediate ...

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing termini of oligo- and polysaccharides, as well as its unique chemical characteristics, sialic acid is involved in a multitude of different receptor-ligand interactions. By modifying the expression of sialic acid on the cell surface, sialic acid-dependent interactions will consequently be influenced. This can be helpful to investigate sialic acid-dependent interactions and has the potential to influence certain diseases in a beneficial way. Via metabolic glycoengineering (MGE), the expression of sialic acid on the cell surface can be modulated. Herein, cells, tissues, or even entire animals are treated with C2-modified derivatives of N-acetylmannosamine (ManNAc). These amino sugars act as sialic acid precursor molecules and therefore are metabolized to the corresponding sialic acid species and expressed on glycoconjugates. Applying this method produces intriguing effects on various biological processes. For example, it can drastically reduce&#160;the expression of polysialic acid (polySia) in treated neuronal cells and thus affects neuronal growth and differentiation. Here, we show the chemical synthesis of two of the most common C2-modified N-acylmannosamine derivatives, N-propionylmannosamine (ManNProp) as well as N-butanoylmannosamine (ManNBut), and further show how these non-natural amino sugars can be applied in cell culture experiments. The expression of modified sialic acid species is quantified by high performance liquid chromatography (HPLC) and further analyzed via mass spectrometry. The effects on polysialic acid expression are elucidated via Western blot using a commercially available polysialic acid antibody.

Metabolic Glycoengineering of Sialic Acid Using N-acyl-modified Mannosamines

Sialic acid is a typical monosaccharide-unit found in glycoconjugates. It is involved in a plethora of molecular and cellular interactions. Here we present a method to modify cell surface sialic acid expression using metabolic glycoengineering with N-acetylmannosamine derivatives.

Metabolische Glykan der Sialinsäure Säure mit N-Acyl-Mannosamines geändert

Sialic acid (Sia) is a highly important constituent of glycoconjugates, such as N- and O-glycans or glycolipids. Due to its position at the non-reducing ...

Metabolic Support of Excised, Living Brain Tissues During Magnetic Resonance Microscopy Acquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic resonance (MR) microscopy data. While it is possible to perform MR collections on living, excised mammalian tissue, such experiments have traditionally been constrained by resolution limits and are thus incapable of visualizing tissue microstructure. Conversely, MR protocols that did achieve microscopic image resolution required the use of fixed samples to accommodate the need for static, unchanging conditions over lengthy scan times. The current protocol describes the first available MR technique that enables imaging of living, mammalian tissue samples at microscopic resolutions. Such data is of great importance to the understanding of how pathology-based contrast changes occurring at the microscopic level influence the content of macroscopic MR scans such as those used in the clinic. Once such an understanding is realized, diagnostic methods with greater sensitivity and accuracy can be developed, which will translate directly to earlier disease treatment, more accurate therapy monitoring and improved patient outcomes.
While the described methodology focuses on brain slice preparations, the protocol is adaptable to any excised tissue slice given that changes are made to the gas and perfusate preparations to accommodate the tissue&#39;s specific metabolic needs. Successful execution of the protocol should result in living, acute slice preparations that exhibit MR diffusion signal stability for periods up to 15.5 h. The primary advantages of the current system over other MR compatible perfusion apparatuses are its compatibility with the MR microscopy hardware required to attain higher resolution images and ability to provide constant, uninterrupted flow with carefully regulated perfusate conditions. Reduced sample throughput is a consideration with this design as only one tissue slice may be imaged at a time.

The current protocol describes a method by which users can maintain viability of acute hippocampal and cortical slice preparations during the collection of magnetic resonance microscopy data.

Metabolische Unterstützung der herausgeschnitten, lebendes Gehirn Gewebe während der Magnet-Resonanz-Mikroskopie-Akquisition

This protocol describes the procedures necessary to support normal metabolic functions of acute brain slice preparations during the collection of magnetic ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: Eine starke, nicht-invasive Ansatz zur zellulären Energiestoffwechsel untersuchen

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

Nanosensoren Protease-Aktivität In Vivo für die nicht-invasive Diagnostik zu erkennen

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Messung der globalen zellulären Matrix Metalloproteinase und metabolische Aktivität in 3D Hydrogele

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Ein menschliches 3D extrazelluläres Matrix-Adipozyten-Kulturmodell zum Studium der Matrix-Zell Metabolischen Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...