Name: microfluidic flow chambers using reconstituted blood to model hemostasis and platelet transfusion in vitro
Uploaded: 2016-03-19T14:00:00
Description: Watch this Scientific Journal Video about Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro at JoVE.com

The authors have no acknowledgements.

Dieses Protokoll folgt die institutionellen ethischen Richtlinien für die Forschung an menschlichen Proben und informierte Zustimmung wurde von allen Spendern beteiligt erhalten. Zulassung für den hier beschriebenen Experimenten wurde von der Institutional Review Board der Universitätsklinik Antwerpen erhalten. Hinweis: Temperaturangaben sind immer Raumtemperatur, sofern nicht anders angegeben. 1. Vorbereitung Flusskammer-Setup <ol><li> Vorbereiten Lanes, Sc...

<ol>
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Mikrofluidik - Strömungskammer Experimente sind ein hervorragendes Instrument Thrombozytenfunktion im fließenden Blut zu untersuchen und verwendet werden , die Hämostase in vitro in verschiedenen experimentellen Kontexten zu bewerten. Trotz schlechter Ring Standardisierung 9 zeigen wir , dass in unserem Labor die experimentelle Variation akzeptabel ist. Dies ermöglicht es, zuverlässig zu vergleichen (gepaart) Proben innerhalb einer gegebenen Studie. Dies wurde mit der gut dokumentierten Phänome...

Hämostase erfordert die kombinierte Aktivität und regulierte von Zellen, Proteinen, Ionen und Gewebe in einem eingeschränkten Raum - Zeit - Rahmen 1. Unkontrollierte Aktivität kann in einem Spektrum von Erkrankungen zu Blutungen oder Thrombosen und Morbidität oder Mortalität führen zu Blutgerinnung im Zusammenhang. Ein mikrofluidischen Strömungskammer Experiment ist eine anspruchsvolle Technik , die Hämostase in vitro nachahmt. Dieser Ansatz ermöglicht Untersuchung des komplexen Zusammenspiel von Prozessen, die Teil der Hämostase mit einer führenden Rolle für Blutplättchen nehmen. Gefäßverletzung folgenden haften Blutplättchen an den exponierten subendothelialen Matrix (Glyko) Proteine ​​Blutverlust zu verhindern. Folgende Adhäsion, Blutplättchen zu aktivieren und zu aggregieren in Reaktion auf auto- und parakrine Signalisierung , die schließlich zur Bildung eines Plättchens Netzwerk führt, durch Fibrin stabilisiert , und was zu einer festen, gewickelten Thrombus 2 abdichtet. Im Gegensatz zu den meisten anderen Thrombozytenfunktionstests, Erfahgen mit Strömungskammern , um die physikalischen Parameter des Blutflusses und damit den Einfluss der Rheologie auf den beteiligten Zellen und Biomolekülen Berücksichtigung 3,4. Strömungskammer Experimente durch Variation Schlüsselparameter Grenzstein Einblicke in Hämostase und Thrombose erzeugt, die hämostatische (sub) beeinflussen Prozesse, einschließlich der Klebstoffmatrix, Rheologie und Strömungsprofile, zellulären Zusammensetzung, die Anwesenheit von Toxinen oder Medikamente, die Ionenstärke und viele mehr. In den vergangenen zwei Jahrzehnten geringen Durchsatz Flusskammer Experimente erfordern große Probenvolumina (10-100 ml) wurden zu mikrofluidischen Kammern oft entwickelt , bestehend aus kleinen Parallelplattenkammern und moderne Technologie auch für die Perfusion von Vollblut bei kontrollierter Wandscherbedingungen 5. Microscaling hat sich deutlich Assay Durchsatz meist erhöht, weil die Hardware-Setup vereinfacht und weniger (Blut) Volumen benötigt wird, wodurch das Experiment zugänglicher und Versatile. Zum Beispiel Blut von kleinen Labortieren kann nun ohne die Notwendigkeit verwendet werden, um Tiere zu opfern. Blutproben von gentechnisch veränderten Mäusen haben damit in der Identifizierung von Schlüsselmoleküle Förderung oder Hemmung Hämostase und in neue basische Erkenntnisse 6 unterstützt. Spezialisierte Forschungslaboratorien noch oft nach Maß Strömungskammern zum Beispiel aus Polydimethylsiloxan (PDMS) 7 verwenden , die auf lithogr Formen polymerisieren , die durch Software blueprinted werden kann. Die resultierende Kammer ist billig, Einweg und kann leicht für die Post-hoc-Analyse zerlegt werden. Darüber hinaus grundsätzlich jede Konstruktion von Schiffen, einschließlich Abzweigungen oder scharfe Kurven kann auf Befehl gebaut werden. Dieser Vorteil ist auch seine Nachteile, da die Standardisierung war bereits das primäre Problem mit Strömungskammer Experimente und PDMS Maß Kammern haben dies nicht unterstützt. Am Anfang dieser speziellen Frage, Beschichtung (Bedingungen), Fluoreszenzsonden, Antikoagulans, Temperatur und Zeit zwischen Probenahme und Analyse sind alle schlecht 8 standardisiert. Die Standardisierung dieser Variablen ist eine Herausforderung, aber dennoch den Vergleich der Ergebnisse zwischen den Laboratorien erforderlich zu ermöglichen. Dieses Thema ist das Hauptthema der Internationalen Gesellschaft für Thrombose und Hämostase in Wissenschaft und Standardisierung Unterausschuss für Biorheology 9,10. Thrombozytenkonzentrate (PC) sind bei Patienten mit verschiedenen Erkrankungen, die transfused Thrombozytopenie und / oder Blutungen verursachen. Aber Blutplättchen in PC sind bekannt zu desensibilisieren, insbesondere in Funktion der Lagerzeit 11 verbunden eine Verschlechterung Verfahren alterungs- und allgemein als Plättchenspeicher Läsion. Es wird manchmal behauptet , dass solche Thrombozyten bei Zirkulation wiederherzustellen einmal 12 transfused, aber Beweise dafür ist knapp. Ferner ist ein PC die Funktionalität von Plättchen bilden nicht routinemäßig getestet, weil die Beziehung zwischen solchen Assays undtherapeutische oder prophylaktische Wirksamkeit ist unklar , 13. Mikrofluidik-Strömungskammern bieten ein Mittel der Thrombozytenfunktion in PC zu untersuchen, um die Kette von Manipulationen zwischen Sammlung und die Ausstellung zu optimieren. Es ist ein leistungsfähiges Forschungswerkzeug für die direkte (gepaart) Vergleiche von PC , wie wir zuvor 14,15 veröffentlicht und wird hier beschrieben.

<table border="0" cellpadding="0" cellspacing="0" width="1113">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD vacutainer tube with EDTA&#160;</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368856</td>
			<td style="width:409px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD vacutainer tube with Heparin</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368480</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">BD vacutainer tube with Sodium Citrate</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">366575</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hirudin Blood tube</td>
			<td style="width:220px;">Roche</td>
			<td style="width:268px;">6675751 001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BD vacutainer Eclipse</td>
			<td style="width:220px;">Becton, Dickinson and Company</td>
			<td style="width:268px;">368650</td>
			<td>Blood collection needle with preattached holder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 100-1000</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">740290</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 2-200</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">739280</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pipette tips 1-10</td>
			<td style="width:220px;">Eppendorf</td>
			<td style="width:268px;">A08928</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tube 5mL</td>
			<td style="width:220px;">Simport</td>
			<td style="width:268px;">11691380</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tube 15mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">1888271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Conical tube 50mL</td>
			<td style="width:220px;">Greiner bio-one</td>
			<td style="width:268px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL Syringe</td>
			<td style="width:220px;">BD</td>
			<td style="width:268px;">309604</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Precision wipes</td>
			<td style="width:220px;">Kimtech</td>
			<td style="width:268px;">5511</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Fluoro+ Biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">188V8CF-400-100-02P10</td>
			<td>Named in figure S1 A as &#39;Biochip&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Tubing</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">TUBING-TYGON-B1IC-B1OC-ROLL 100F</td>
			<td>Named in figure S1 B as &#39;Disposable tubing&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vena8 Needles</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">SS-P-B1IC-B1OC-PACK200</td>
			<td>Named in figure S1 B as &#39;Pin&#39;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Connectors for single inlet cables of biochips</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">CONNECTORS-B1IC-PACK100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Multiflow8 connect</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">MF8-CONNECT-BIC3-N-THROMBOSIS</td>
			<td>Named in figure S1 B as &#39;Reusable tubing&#39; and &#39;Splitter&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Humidified box</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">HUMID-BOX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software microfluidic pump</td>
			<td style="width:220px;">Cellix</td>
			<td style="width:268px;">N/A</td>
			<td>Venaflux Assay</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Horm Collagen</td>
			<td style="width:220px;">Takeda/Nycomed</td>
			<td style="width:268px;">1130630</td>
			<td style="width:409px;">Native equine tendon collagen (type I) 
			Isotonic glucose solution to dilute collagen is supplemented</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HEPES buffered saline (HBS)</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td style="width:409px;">10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (0.9% (w/v) NaCl, pH 7.4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Blocking buffer</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td style="width:409px;">1.0% (w/v) bovine serum albumin and 0.1% (w/v) glucose in HBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcein AM</td>
			<td style="width:220px;">Molecular probes</td>
			<td style="width:268px;">C1430</td>
			<td style="width:409px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bleach 10%</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.1M NaOH</td>
			<td style="width:220px;">in house preparation</td>
			<td style="width:268px;">in house preparation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Denaturated alcohol</td>
			<td style="width:220px;">Fiers</td>
			<td style="width:268px;">T0011.5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mirus Evo Nanopump</td>
			<td>Cellix</td>
			<td>188-MIRUS-PUMP-EVO</td>
			<td>with Multiflow8. Named in figure S1 A as &#39;Pump&#39; and &#39;Manifold&#39;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope</td>
			<td>Zeiss</td>
			<td>Axio Observer Z1</td>
			<td>equipped with a colibri-LED and high resolution CCD camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Software microscope&#160;</td>
			<td style="width:220px;">Zeiss</td>
			<td style="width:268px;">N/A</td>
			<td>ZEN 2012</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematology analyzer</td>
			<td>Sysmex</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table Top Centrifuge</td>
			<td>Eppendorf</td>
			<td>521-0095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Platelet incubater</td>
			<td style="width:220px;">Helmer</td>
			<td style="width:268px;">PF-48i</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Incubation water bath</td>
			<td>GFL</td>
			<td>1013</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pipette</td>
			<td>Brand</td>
			<td>A03429</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tube Roller</td>
			<td>Ratek</td>
			<td>BTR5-12V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile docking device</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:268px;">TSCD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tubing Sealer</td>
			<td style="width:220px;">Terumo BCT</td>
			<td style="width:268px;">AC-155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vortex</td>
			<td style="width:220px;">VWR</td>
			<td style="width:268px;">58816-121</td>
			<td></td>
		</tr>
	</tbody>
</table>

microfluidic flow chambers using reconstituted blood to model hemostasis and platelet transfusion in vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Um zu zeigen , Variation intra-assay, drei identische rekonstituierten Vollblutproben wurden gleichzeitig über Kollagen - beschichteten Oberflächen (Abbildung 1) perfundiert. Dies führte zu einem Variationskoeffizienten von 8,7%. Diese Statistik deutet darauf hin, akzeptabel Intra-Assay und labor Variation zuverlässigen Vergleich zwischen verwandten Proben ermöglicht. Der Einlass der kommerziellen Strömu...

Watch this Scientific Journal Video about Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro at JoVE.com

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Platelet transfusion and hemostasis was modeled using blood reconstitution and microfluidic flow chambers to investigate the function of blood banking platelets. The data demonstrate the consequences of platelet storage lesion on hemostasis, in vitro.

Mikrofluidik-Strömungskammern Mit Rekonstituierte Blut Hämostaseologie und Transfusions Platelet zu Modell<em&gt; In Vitro</em

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of ...

Research

JoVE Journal

Bioengineering

Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

Microfluidic Device for Recreating a Tumor Microenvironment in Vitro

Mikrofluidik-Vorrichtung für die Neuerstellung eines Tumor-Mikroumgebung<em&gt; In Vitro</em

We have developed a microfluidic device that mimics the delivery and systemic clearance of drugs to heterogeneous three-dimensional tumor tissues in ...

Forschung

Biotechnik

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Die Trennung Beads und Zellen in Multi-Kanal-Mikrofluidiksysteme Mit Dielektrophorese und Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

Systematische Analyse von<em&gt; In-vitro-</em&gt; Zellroll Mit einer Multi-Well-Platte mikrofluidischen System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Mikrofluidik-Plattform zur Messung der Neutrophilen-Chemotaxis von Rohboden Vollblut

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

Test auf Augenreizung (EIT) zur Kennzeichnung der Gefahr von Augen Reizt Chemicals mit rekonstruierter menschlicher Hornhaut-ähnlichen Epithelial (RhCE) Gewebemodell

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

In-vitro-Modell von physiologischen und pathologischen Blutfluss mit Anwendung auf Untersuchungen der Gefäßzell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Ein Mikrofluidik-Flow-Kammer-Modell für Platelet Transfusions und Hämostase Maßnahmen Platelet Deposition und Fibrinbildung in Echtzeit

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Ein<em&gt; In Vitro</em&gt; Organ Culture Modell der Murine Bandscheiben

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

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

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 ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

Eine automatisierte Methode zur Durchführung des In-Vitro-Mikrokern-Assays mit multispektraler Bildflusszytometrie

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...