Die Studie wurde von der Artères Stiftung, der Astellas Foundation Europe und der Boninchi Foundation unterstützt. CT wurde von der Swiss National Science Foundation (SCORE Zuschuss 3.232.230-126.233) unterstützt.

1. Griff: Konzeption und Gestaltung <ol><li> Sichern einer Nr. 10 C-Skalpell auf einem Holzbrett mit einem Band. </li><li> Schild am Zentrum des Rings unter dem Mikroskop. Es sollte 1,2 cm direkt aus dem Hain des Skalpell entfernt werden. </li><li> Bohren Sie ein Loch 2 mm mit einer sphärischen Diamant-Bit, montiert auf einem High-Speed-Bohrer. </li><li> Widen das Loch mit fusiform oder konischen Diamant-Bits. Der Enddurchmesser variiert je nach Rattengewicht wie in Tabelle 1 gezeigt. </li><li> Entwerfen Sie die Form des Griffes mit einem Permanent-Marker. Abbildungen 1 und 2 können als Schablonen verwendet werden. </li><li> Rechnen Sie den Bohrer in einen Mini Schleifbock und entfernen Sie den Teil der Klinge außerhalb der Form. </li><li> Runden Sie das Stück mit einer niedrigen Geschwindigkeit rotierende Bürste. </li></ol> 2. Approximator: Konzeption und Gestaltung <ol><li> Die Approximator wird durch Modifizieren gerade chirurgischen Klemmer oder Kocher forcep erhaltens. </li><li> Schneiden Sie die Hälfte von einem Zweig unter dem Mikroskop mit dem High-Speed ​​Mini Schleifbock. </li><li> Als symmetrische Nuten auf beiden Seiten für den Griff zu rutschen in vollständig zu ermöglichen. </li><li> Schneiden Sie einen Längsschlitz in dem langen Arm und überprüfen, dass die Mitte des langen Arms und ein gut aufgestelltes Griff auf der gleichen Achse sind. </li><li> Entwerfen Sie eine Kerbe auf dem langen Arm vor der Mitte des Griffs. </li><li> Machen Sie den Zweig so dünn wie möglich und runden das Stück mit der niedrigen Drehzahl rotierende Bürste. </li></ol> 3. Manschetten: Konzeption und Gestaltung <ol><li> Stulpe Bremssättel je nach Gewicht der Ratte und Dehnung. Im Gegensatz dazu sollte deren Länge etwa 4 mm gehalten werden. </li><li> Nehmen feinen Bohrung Polyethylenschlauch und runden eine Kante mit einem Skalpell Nr. 10. </li><li> Entwerfen Sie einen Hain 0,5 mm von der Kante, und sorgfältig ausgeschnitten eine Steigung hinunter zum Hain. </li><li> Schneiden Sie ein oder zwei Full-Dicke nicks in den Schlauch. Diese werdenhelfen, die Manipulation der Manschette während Transplantat Vorbereitung und Transplantation. </li><li> Schneiden Sie die Manschette bei 4 mm von der Kante. </li></ol>

<ol>
	<li>Oldani, G., Lacotte, S., Morel, P., Mentha, G., Toso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+Liver+Transplantation+in+Rats.">Orthotopic Liver Transplantation in Rats.</a> J. Vis. Exp. (65), e4143 (2012).</li><li>Hori, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+and+innovative+techniques+for+liver+transplantation+in+rats:+A+surgical+guide.">Comprehensive and innovative techniques for liver transplantation in rats: A surgical guide.</a> World Journal of Gastroenterology., WJG. 16, 3120 (2010).</li><li>Kashfi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+various+techniques+of+orthotopic+liver+transplantation+in+the+rat.">A review of various techniques of orthotopic liver transplantation in the rat.</a> Transplantation Proceedings. 37 (1), 185-188 (2005).</li><li>Kamada, N., Calne, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+liver+transplantation+in+the+rat.+Technique+using+cuff+for+portal+vein+anastomosis+and+biliary+drainage.">Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage.</a> Transplantation. 28, 47 (1979).</li><li>Oldani, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+technique+for+rat+liver+transplantation+using+Quick+Linker+system:+a+preliminary+result.">A novel technique for rat liver transplantation using Quick Linker system: a preliminary result.</a> Journal of Surgical Research. 149, 303-309 (2008).</li></ol>

Die Autoren haben nichts zu offenbaren.

Der vorliegende Bericht beschreibt den Entwurf und Bau der Schnell-Linker-System zum leichteren Ratte Lebertransplantation. Rat Lebertransplantation ist ein beliebtes Modell mit 125-180 verwandten Papieren jährlich seit den frühen 1990er Jahren (veröffentlicht <a href="http://www.pubmed.com" target="_blank">www.pubmed.com</a> ). Es ermöglicht eine breite Palette von Experimenten einschließlich der Erforschung von immunologischen und Transplantat Erhaltung Fragen. Die Verwendung des &quot;Quick-Linker&quot;-Kit ermöglicht eine einfachere und schnellere Anastomosen mit garantierten Intima-to-Intima Kontakt. Eine detaillierte Beschreibung und Diskussion der technischen Schritte einer Ratte Lebertransplantation wurden bereits 1 beschrieben. Der Quick-Linker Kit kann bei niedrigen Preis von gemeinsamen Labor Material gebaut werden. Die größte Herausforderung begegnet wurde den Abbau von einem Griff in der letzten Bürsten zusammenhängen und dass Problem ist, indem Sie den schmalsten par gelöstt des Griffs Ring bei 0,5 mm (Abbildungen 1 und 2 zeigen entsprechende Größen). In Bezug auf die Qualität Klinge sollte nur Edelstahl in Betracht gezogen werden. Obwohl etwas weniger flexibel, um die durchschnittliche Klinge aus Kohlenstoffstahl Vergleich haben Klingen aus rostfreiem Stahl Rost nicht entwickeln, so dass eine lebenslange Nutzung der Griffe. Zu beachten ist, ist das Design der aktuellen Griffen und Manschetten das Ergebnis eines Prozesses, der auf die vorgeschlagenen Größen geführt hat, so dass für den effizientesten Anastomosen. Manschette Durchmesser variieren entsprechend der Ratte Gewichte (Tabelle 1). Minimale Variationen in der Länge toleriert werden, mit einer üblichen Länge von etwa 4 mm (Abbildungen 3 und 4). In Bezug auf die Approximator sollte der ausgewählten Kocher forcep haben Griffe mindestens 4 cm vom Drehpunkt. Dies ermöglicht einen sicheren Verschluss der Zahnstange außerhalb des Tieres Bauch. InsgesamtDie vorgeschlagene Vorrichtung kann einfacher Vene Anastomosen für die Transplantation von Leber und andere Arten von Transplantaten bei Ratten zu ermöglichen.

Eine Technik der orthotopen Lebertransplantation Ratte hat in einer aktuellen Journal of Visualisierte Experimente Papier 1 gezeigt. Pfortader und infra-Leber Hohlvene Anastomosen wurden mit dem Quick-Linker assisted Manschette Technik. Diese Technik wurde unter anderem Änderungen des ursprünglichen Kamadas Technik 2-4 gewählt, da es einfacher und schneller für Anastomosen, führt zu einer besseren hämodynamischen erlaubt und kann mit einer kürzeren Lernkurve 1,5 umgesetzt werden. Der Quick-Linker Technik erfordert die Verwendung eines Griffs, ein Approximator und Manschetten, können alle davon aus gemeinsamen Labor gemäß der Technik im vorliegenden Bericht beschrieben gestaltet werden.

<table border="1">
<tbody>
 <tr>
 <td>Tool / Reagent</td>
 <td>Company</td>
 <td>Catalogue n.</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>Porta cuff</td>
 <td>altecweb or 
 smiths-medical</td>
 <td>Coil 01-96-1729 
 800/100/420</td>
 <td>Bore : variable 
 Wall : 0.30 mm 
 Length: 3.8-4.2 mm 
 <a href="http://altecweb.com" target="_blank">altecweb.com</a> or 
 <a href="http://smiths-medical.com" target="_blank">smiths-medical.com</a></td>
 </tr>
 <tr>
 <td>IVC cuff</td>
 <td>altecweb or 
 smiths-medical </td>
 <td>Coil 01-96-1733 
 800/100/540</td>
 <td>Bore : variable 
 Wall : 0.30 mm 
 Length: 3.8-4.2 mm 
 <a href="http://altecweb.com" target="_blank">altecweb.com</a> or 
 <a href="http://smiths-medical.com" target="_blank">smiths-medical.com</a></td>
 </tr>
 <tr>
 <td>Mini-drill </td>
 <td>Dremel</td>
 <td>100 series</td>
 <td>35,000 rpm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Diamond wheel point</td>
 <td>Dremel</td>
 <td>7103</td>
 <td>2 mm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Diamond wheel point</td>
 <td>Dremel</td>
 <td>7105</td>
 <td>4.4 mm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Diamond wheel point</td>
 <td>Dremel</td>
 <td>7144</td>
 <td>2.4 mm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Cut-off wheels</td>
 <td>Dremel</td>
 <td>420</td>
 <td>dia: 23.8 mm 
 thickness: 0.5 mm 
 <a href="http://dremel.com" target="_blank">dremel.com</a></td>
 </tr>
 <tr>
 <td>Quick-linker handles</td>
 <td>dltchiropody (DLT)</td>
 <td>PAR 10</td>
 <td>modified scalpel blades 
 <a href="http://dltchiropody.co.uk" target="_blank">dltchriopdy</a></td>
 </tr>
 <tr>
 <td>Quick-linker approximator</td>
 <td>pharmap</td>
 <td>UD-04-275</td>
 <td>modified Kocher's forceps 
 <a href="http://pharmap.ch" target="_blank">pharmap.ch</a></td>
 </tr>
 </tbody>
</table>

manufacturing devices and instruments for easier rat liver transplantation

Orthotopen Lebertransplantation Ratte ist ein beliebtes Modell, das vor kurzem in einem JoVE Papier wurde mit dem Einsatz der &quot;Quick-Linker&quot; Gerät gezeigt. Diese Technik ermöglicht eine leichtere venösen Manschette-anatomoses nach einer angemessenen Einarbeitungszeit. Das Gerät besteht aus zwei Griffe, die aus Skalpell Klingen sind geschnitzt, eine Approximator, die durch Modifizieren Kocher Pinzette erhalten wird, und Manschetten aus feinen Bohrung Polyethylenschlauch entworfen zusammen. Der gesamte Prozess kann in einem Low-Cost-mit gemeinsamen Labor Material durchgeführt werden. Der vorliegende Bericht enthält eine Schritt-für-Schritt-Protokoll für die Gestaltung der benötigten Teile und umfasst Schablonen.

Fünf Kits, darunter fünf Approximatoren und 11 Griffe hergestellt wurden. Ein Griff brach während der letzten Bürsten. Dieses Problem resultiert aus der Tatsache, dass der Ring von dem Griff zu klein war, und es wurde durch Einstellen der engsten Stelle der Griffring bei 0,5 mm gelöst. Keine Probleme traten während der Gestaltung der Approximatoren. <table border="1"><tbody><tr><td> Ratte Gewicht (g) </td><td> Porta Griff (Innen-Ø) (mm) </td><td> Porta Manschette (Außen-Ø) (mm) </td><td> IVC Griff (Innen-Ø) (mm) </td><td> IVC Manschette (Außen-Ø) (mm) </td></tr><tr><td> 120-180 </td><td> 2.2 </td><td> 1.7 </td><td> 2.6 </td><td> 2,08 </td></tr><tr><td> 180-270 </td><td> 2.6 </td><td> 2,08 </td><td> 3,4-3,7 </td><td> 3,0-3,25 </td></tr><tr><td> &gt; 270 </td><td> 2.9<html></td><td> 2,42 </td><td> 4.2 </td><td> 3.7 </td></tr></tbody></table> Tabelle 1. <img src="/files/ftp_upload/50380/50380fig1.jpg" alt="Abbildung 1" fo:content-width="6in" fo:src="/files/ftp_upload/50380/50380fig1highres.jpg"/> Abbildung 1. Porta Griff Schablone. Wird mit einem 1:1 verwendet, um den Griff der Gestaltung führen kann ausgedruckt werden. <a href="https://www.jove.com/files/ftp_upload/50380/50380fig1large.jpg" target="_blank">Klicke hier, um eine größere Abbildung anzuzeigen</a> . <img src="/files/ftp_upload/50380/50380fig2.jpg" alt="Abbildung 2" fo:content-width="6in" fo:src="/files/ftp_upload/50380/50380fig2highres.jpg"/> Abbildung 2. IVC Griff Schablone. Wird mit einem 1:1 verwendet, um den Griff der Gestaltung führen kann ausgedruckt werden. <a href="https://www.jove.com/files/ftp_upload/50380/50380fig2large.jpg" target="_blank">Klicke hier, um eine größere Abbildung anzuzeigen</a> . Inhalt &quot;&gt; <img src="/files/ftp_upload/50380/50380fig3.jpg" alt="Abbildung 3" fo:content-width="4.6in" fo:src="/files/ftp_upload/50380/50380fig3highres.jpg" /> Abbildung 3. Porta und IVC Manschetten Seitenansicht. Graft V.Porta sollte der zentrale Nut gesichert werden, während Empfängers Schiff in den seitlichen eins. Diese Maßnahme ermöglicht eine optimale Intima-to-Intima Kontakt. <img src="/files/ftp_upload/50380/50380fig4.jpg" alt="Fig. 4" fo:content-width="4.1in" fo:src="/files/ftp_upload/50380/50380fig4highres.jpg"/> Abbildung 4. Porta und IVC Manschetten Vorderansicht. Bremssättel geeignet für Ratten mit einem Gewicht von 180 bis 270 g vertreten sind.

Watch this Scientific Journal Video about Manufacturing Devices and Instruments for Easier Rat Liver Transplantation at JoVE.com

Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

Wir beschreiben das Design des &quot;Quick-Linker&quot; Gerät zur leichteren orthotopen Lebertransplantation Ratte.

Fertigung von Geräten und Instrumenten für eine einfachere Rat Liver Transplantation

Orthotopic rat liver transplantation is a popular model, which has been shown in a recent JoVE paper with the use of the "quick-linker" device. This ...

manufacturing-devices-instruments-for-easier-rat-liver

Research

JoVE Journal

Bioengineering

8.6K Aufrufe.  University of Geneva Hospitals. Wir beschreiben das Design des "Quick-Linker" Gerät zur leichteren orthotopen Lebertransplantation Ratte.

Serie: Manufacturing Devices and Instruments for Easier Rat Liver Transplantation

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

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

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

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

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

Forschung

Biotechnik

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 &#956;l. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

Auf Papier basierende Vorrichtungen zur Isolierung und Charakterisierung von extrazellulärer Vesikel

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.

Verfahren für Dezellularisierung von Rattenlebern in einer oszillierenden Druck Perfusionsvorrichtung

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

Die Herstellung von Baumwolle Analytische Geräte

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps compared to standard metallographic sample preparation procedures. The paper proposes a novel bespoke rig for dynamic domain imaging with in situ BH (magnetic hysteresis) measurements and elaborates the protocols for the sensor preparation and the use of the rig to ensure accurate BH measurement. The protocols for static and ordinary dynamic domain imaging (without in situ BH measurements) are also presented. The reported method takes advantage of the convenience and high sensitivity of the traditional Bitter method and enables in situ BH measurement without interrupting or interfering with the domain wall movement processes. This facilitates establishing a direct and quantitative link between the domain wall movement processes&#8211;microstructural feature interactions in ferritic steels with their BH loops. This method is anticipated to become a useful tool for the fundamental study of microstructure&#8211;magnetic property relationships in steels and to help interpret the electromagnetic sensor signals for non-destructive evaluation of steel microstructures.

Optimized Setup and Protocol for Magnetic Domain Imaging with In Situ Hysteresis Measurement

This paper elaborates the sample and sensor preparation procedures and the protocols for using the test rig particularly for dynamic domain imaging with in situ BH measurements in order to achieve optimal domain pattern quality and accurate BH measurements.

Optimierte Setup und ein Protokoll für magnetische Domäne Bildgebung mit In Situ Hysterese-Messung

This paper elaborates the sample preparation protocols required to obtain optimal domain patterns using the Bitter method, focusing on the extra steps ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

Eine neuartige Operationstechnik als Grundlage für In Vivo teilweise Leber Engineering in der Ratte

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Rapid Fabrication of Custom Microfluidic Devices for Research and Educational Applications

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to submillimeter scales. A rapid increase in the use of this technology in the biological sciences has prompted a need for methods that are accessible to a wide range of research groups. Current fabrication standards, such as PDMS bonding, require expensive and time consuming lithographic and bonding techniques. A viable alternative is the use of equipment and materials that are easily affordable, require minimal expertise and allow for the rapid iteration of designs. In this work we describe a protocol for designing and producing PET-laminates (PETLs), microfluidic devices that are inexpensive, easy to fabricate, and consume significantly less time to generate than other approaches to microfluidics technology. They consist of thermally bonded film sheets, in which channels and other features are defined using a craft cutter. PETLs solve field-specific technical challenges while dramatically reducing obstacles to adoption. This approach facilitates the accessibility of microfluidics devices in both research and educational settings, providing a reliable platform for new methods of inquiry.

Here we present a protocol to design and fabricate custom microfluidic devices with minimal financial and time investment. The aim is to facilitate the adoption of microfluidic technologies in biomedical research laboratories and educational settings.

Schnelle Herstellung von kundenspezifischen mikrofluidischen Geräten für Forschungs- und Bildungsanwendungen

Microfluidic devices allow for the manipulation of fluids, particles, cells, micro-sized organs or organisms in channels ranging from the nano to ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Elektrische und magnetische Feldgeräte zur Stimulation biologischer Gewebe

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Formulierung und Charakterisierung von Lipid-Nanopartikeln für die Genabgabe mit einer mikrofluidischen Mischplattform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

Evaluierung der kardialen Kontraktilitätsmodulationstherapie in 2D-Kardiomyozyten aus humanen Stammzellen

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...