Die Autoren bedanken sich bei Leica Microsystems für ihre wertvollen Ratschläge Bildgebung Titan Abschnitte und Gemini Bio-Produkte danken für die Bereitstellung der Schweine-Serum in dieser Studie verwendet. Wir kommen auch gerne zu den NIH für ihre Unterstützung danken durch Grant &quot;Autologe EPC-Futter auf die Biokompatibilität von Herz-Kreislauf verbessern assist devices&quot;, RC1HL099863-01. Darüber hinaus sind wir dankbar für die National Science Foundation Graduate Research Fellowship der Unterstützung von Alexandra Jantzen. Wir kommen auch gerne zu George Quick, Mike Lowe und Ianthia Parker für die Unterstützung mit vielen Aspekten des chirurgischen Eingriffs und Handhabung unserer Versuchstiere danken. Steven Owen wurde in der Bearbeitung viele Teile unserer Seeding-Gerät von unschätzbarem Wert und Schneiden Titanrohre.

<ol>
	<li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+bleeding+and+clotting+in+the+cardiac+surgery+patient:+from+vascular+endothelium+to+circulatory+assist+device+surface.">Pathophysiology of bleeding and clotting in the cardiac surgery patient: from vascular endothelium to circulatory assist device surface.</a> Circulation. 122, 2068-2077 (2010).</li><li>Achneck, H. E., Sileshi, B., Lawson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+biology+of+bleeding+and+clotting+in+the+surgical+patient.">Review of the biology of bleeding and clotting in the surgical patient.</a> Vascular. 16, 6-13 (2008).</li><li>Arvidsson, S., Askendal, A., Tengvall, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+plasma+contact+activation+on+silicon,+titanium+and+aluminium.">Blood plasma contact activation on silicon, titanium and aluminium.</a> Biomaterials. 28, 1346-1354 (2007).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biocompatibility+of+titanium+cardiovascular+devices+seeded+with+autologous+blood-derived+endothelial+progenitor+cells:+EPC-seeded+antithrombotic+Ti+Implants.">The biocompatibility of titanium cardiovascular devices seeded with autologous blood-derived endothelial progenitor cells: EPC-seeded antithrombotic Ti Implants.</a> Biomaterials. 32, 10-18 (2011).</li><li>Kang, C., Bonneau, M., Brouland, J. P., Bal dit Sollier, C., Drouet, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+pig+models+of+venous+thrombosis+mimicking+human+disease.">In vivo pig models of venous thrombosis mimicking human disease.</a> Thromb Haemost. 89, 256-263 (2003).</li><li>Ueberrueck, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+ovine+and+porcine+animal+models+for+biocompatibility+testing+of+vascular+prostheses.">Comparison of the ovine and porcine animal models for biocompatibility testing of vascular prostheses.</a> J Surg Res. 124, 305-311 (2005).</li><li>Velik-Salchner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+values+for+thrombelastography+(ROTEM)+and+selected+coagulation+parameters+in+porcine+blood.">Normal values for thrombelastography (ROTEM) and selected coagulation parameters in porcine blood.</a> Thromb Res. 117, 597-602 (2006).</li><li>Yoder, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redefining+endothelial+progenitor+cells+via+clonal+analysis+and+hematopoietic+stem/progenitor+cell+principals.">Redefining endothelial progenitor cells via clonal analysis and hematopoietic stem/progenitor cell principals.</a> Blood. 109, 1801-1809 (2007).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> American Heart Association Scientific Sessions, Abstract Oral Sessions, Medical Aspects End Stage Heart Failure: Transplantation and Device Therapies. , (2010).</li><li>Achneck, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerating+titanium+ventricular+assist+device+surfaces+after+gold/palladium+coating+for+scanning+electron+microscopy.">Regenerating titanium ventricular assist device surfaces after gold/palladium coating for scanning electron microscopy.</a> Microsc Res Tech. 73, 71-76 (2010).</li><li>Ford, J. W., Welling, T. H., Stanley, J. C., Messina, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PKH26+and+125I-PKH95:+characterization+and+efficacy+as+labels+for+in+vitro+and+in+vivo+endothelial+cell+localization+and+tracking.">PKH26 and 125I-PKH95: characterization and efficacy as labels for in vitro and in vivo endothelial cell localization and tracking.</a> J Surg Res. 62, 23-28 (1996).</li></ol>

Keine Interessenskonflikte erklärt.

Die Methode der Zell-Aussaat Ti Rohre hier vorgestellten ermöglicht Ärzten und Wissenschaftlern, um schnell und gleichmäßig endothelialize Blut-Kontaktflächen von Implantaten. Da wir und zu isolieren, zu erweitern EPCs aus dem peripheren Blut-Proben, kein großer Eingriff erforderlich, um diese Zellen zu ernten. Darüber hinaus sind die EPCs autologen, also das Risiko einer Immunreaktion gegen die Zellen ausgesät Implantat wird eliminiert. Die Grundsätze in seinem Protokoll nachgewiesen werden kann, nicht nur für Ti Rohre, sondern auch für viele andere Biomaterialien, die in der kardiovaskulären Medizin genutzt werden genutzt werden. Kritische Schritte in diesem Protokoll sind die sorgfältige Reinigung der Ti Rohre, wie wir festgestellt, dass eine festhaftende Verunreinigungen Kompromisse Zelladhärenz. Weiterhin ist eine langsame Drehzahl (umgekehrt proportional zum Rohrdurchmesser) wesentlich bei der Aussaat Prozess ist, kann, so dass EPCs langsam absetzen und an der Oberfläche wie die Ti Rohr bewegt. Unsere Methode der Aussaat unmittelbar vor der Implantation vermeidet unpraktisch ex vivo Kultur Zeiten; Zellen einzeln haften und bilden später eine konfluente Blatt in vivo, die Vermeidung der Möglichkeit der Embolisation wie ein Blatt unmittelbar nach der Wiederherstellung der Strömung. Unsere früheren Studien zeigen, dass, sobald die EPCs auf eine konfluente Schicht aufgewachsen sind, sie eine extra-zelluläre Matrix, an die sie fest haften zu machen, zusätzlich Minimierung möglicher Ablösung einer endothelialen Blatt. Obwohl die Möglichkeit der embolischen Ablösung kann nicht vollständig ausgeschlossen werden, so scheint es multifold geringeres Risiko als Thrombose der gesamten Oberfläche der Vorrichtung, das Problem, dass diese Therapie entwickelt, um zu verhindern sein. Unser Ansatz der Implantation in den geringer Scherung, nutzt prothrombotischen Umfeld der unteren Hohlvene eines der am meisten vertraut großen Tiermodelle für die Erforschung Blutverträglichkeit und Thrombose von Geräten 5,6. Beachten Sie, dass alle Tierpflege und Experimente in Übereinstimmung mit dem National Institute of Health Richtlinien für die Pflege und Verwendung von Labortieren und nur nach Genehmigung des Duke University Institutional Animal Care und Verwenden Ausschusses durchgeführt wurde. Um erfolgreich zu nutzen diese Implantation Methode, um Biomaterialien und Geräten zu testen, ist es wichtig, geduldig Skelettierung der IVC-Segment und ligieren alle venösen Zweig Schiffe in Vorbereitung für ein Bestücken der Schaltung, so dass keine Blutung um das Gerät in einen &quot;falschen Lumen&quot; stammt. Ein weiterer wichtiger Schritt ist die Zugabe von DPBS in das Gerät Lumen, so dass die Zellen auf der Innenseite Rohroberfläche feucht bleiben während des Schließens des veinotomy und vor der Reperfusion eingeleitet. Wenn das Gerät nicht in der Lage, wo es implantiert wurde gefunden werden, kann es migriert &quot;Upstream&quot; in der IVC haben. 3 mm Abschnitt der PVC-Schlauch so, dass das Rohr fest in ihren jetzigen Standort verankert - Dies kann durch eine Naht (4-O Prolene) durch die Venenwand und durch ein 2 verhindert werden. Hält sich der Forscher Schwierigkeiten, die Fluoreszenz-pre-markierten Zellen in Abbildung 8 nach der Explantation in einer ansonsten Patent Rohr gezeigt haben, ist es wahrscheinlich, dass die Zellen aus als ein zusammenhängendes Blatt geschält. Dies kann durch sehr sanfte Präparation der umliegenden Venen-und Demontagearbeiten der 3 Ti Rohrabschnitte verhindert werden, nach der Fixierung der Vene zusammen mit dem Rohr. Unsere Technologie bietet Proof-of-concept zur Verhinderung von Herz-Kreislauf-Gerät Thrombose durch EPC-Aussaat. Diese Technologie kann in der Entwicklung von &quot;biogenen&quot; Implantate mit patienteneigenen endothelialen Vorläuferzellen gefüttert werden. Die Machbarkeitsstudie in unserer Schweine Tiermodell bietet für die ersten Schritte in Richtung Umsetzung dieses &quot;personalisierten Medizin&quot; in die klinische Praxis.

<table border="1"><tbody><tr><td> Reagens </td><td> Name des Unternehmens </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Acepromazine </td><td> Boehringer-Ingelheim </td><td> BIC670025 </td><td> NAC # 10280002 </td></tr><tr><td> Alconox Powered Precision Reiniger </td><td> Alconox </td><td> 1104 </td><td></td></tr><tr><td> Balfour Wund-Spreizer </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Baytril </td><td> Bayer </td><td> APVMA 46028/0705 </td><td></td></tr><tr><td> Butterfly-Nadel (19G) </td><td> Terumo </td><td> SV19CLK </td><td></td></tr><tr><td> Chlorhexidin </td><td> 3M </td><td> 9200 </td><td></td></tr><tr><td> Schellen (45-Grad) </td><td> Aesculap </td><td> FC339T </td><td></td></tr><tr><td> DPBS (-/-) </td><td> Gibco </td><td> 14190-144 </td><td></td></tr><tr><td> DPBS (+/+) </td><td> Gibco </td><td> 14040-133 </td><td></td></tr><tr><td> DuraPrep </td><td> 3M </td><td> 8635 </td><td></td></tr><tr><td> EBM-2 Medium </td><td> Lonza </td><td> CC-3156 </td><td> Basis für beide Serum freie und volle Wachstumsmedium </td></tr><tr><td> EGM-2 SingleQuots </td><td> Lonza </td><td> CC-3162 </td><td> Gebrauchte mit EBM-2 für beide Serum freie und volle Wachstumsmedium </td></tr><tr><td> Elektrokauter-Tool </td><td> Valleylab </td><td> SurgII-20 </td><td></td></tr><tr><td> Schmirgelleinen </td><td> 3M </td><td> 60-0700-0425-8 </td><td> 240 grit </td></tr><tr><td> Euthasol Euthanasie-Lösung </td><td> Virback Tiergesundheit </td><td></td><td> ANADA #: 200-071 </td></tr><tr><td> Fentanyl-Pflaster </td><td> Actavis </td><td></td><td> NDC # 67767-120-18 </td></tr><tr><td> Flunixin </td><td> Schering-Plough </td><td></td><td> NAC #: 10470183 </td></tr><tr><td> Foley-Katheter (16F) </td><td> Barde </td><td> 730116 </td><td></td></tr><tr><td> HBSS </td><td> Sigma </td><td> H8264-500ML </td><td></td></tr><tr><td> Heißluftgebläse </td><td> Milwaukee </td><td> 8988-20 </td><td></td></tr><tr><td> Heparin </td><td></td><td></td><td> NDC #: 25021 </td></tr><tr><td> Histopaque-1077 </td><td> Sigma </td><td> H8889-500ML </td><td></td></tr><tr><td> Intubationstubus </td><td> Mallinckrodt </td><td> 86113 </td><td></td></tr><tr><td> Isofluran </td><td> MWI, Meridian </td><td></td><td> NDC # 13985-030 </td></tr><tr><td> IV-Katheter (18G) </td><td> Becton-Dickinson </td><td> 381547 </td><td></td></tr><tr><td> Ketamin </td><td> Fort Dodge </td><td></td><td> NDC # 0856-2013 </td></tr><tr><td> Ebene </td><td> Swanson </td><td> LLA001 </td><td></td></tr><tr><td> Luer-Lock Spitzenkappe </td><td> CML Versorgung </td><td> 909-001 </td><td></td></tr><tr><td> Marcain </td><td> Hospira </td><td></td><td> NDC #: 0409-1560-10 </td></tr><tr><td> Metzenbaum Scissors </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Micro-Einführhilfe (5F) </td><td> Galt </td><td> KIT 002-01 </td><td></td></tr><tr><td> Mosquito Zange </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Motor </td><td> Herbach und Rademan </td><td> H1-08 </td><td></td></tr><tr><td> Oxymorphon </td><td> Endo Labs </td><td></td><td> NDC: 63481-624-10 </td></tr><tr><td> PKH26 Dye Kit </td><td> Sigma </td><td> PKH26GL-1KT </td><td></td></tr><tr><td> Porcine Serum </td><td> Gemini-Bio-Produkte </td><td> 100-115 </td><td> Konzentration von 2% in voller Wachstumsmedium </td></tr><tr><td> Potts Scissors </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Präzise Vista Haut Hefter </td><td> 3M </td><td> 3998 </td><td></td></tr><tr><td> PVC-Schlauch </td><td> McMaster-Carr/Insultab </td><td> 7132K117 </td><td> erweitert ID 15,88 mm, erholt ID 7,95 mm </td></tr><tr><td> Rechtwinklig Medium Size </td><td> Adler </td><td> N / A </td><td></td></tr><tr><td> Skalpell (# 10-15) </td><td> Barde </td><td> 373910 </td><td></td></tr><tr><td> Silikonschlauch </td><td> McMaster-Carr </td><td> 51735K26 </td><td> 16,64 mm OD, 9,52 mm ID </td></tr><tr><td> Spritze (5 cc) </td><td> Becton-Dickinson (BD) </td><td> 309603 </td><td></td></tr><tr><td> Tegaderm </td><td> 3M </td><td> 90001 </td><td></td></tr><tr><td> Drei-Wege-Hahn </td><td> Kendall </td><td> 170060 </td><td></td></tr><tr><td> Ti Rohr </td><td> Tico Titanium </td><td> N / A </td><td> Angegeben als ½ &quot;OD, .065&quot; Wand, .370 &quot;ID, 0,1737 £ / ft </td></tr><tr><td> Trypsin </td><td> Lonza </td><td> CC-5012 </td><td></td></tr><tr><td> Trypsin neutralisierende Lösung (TNS) </td><td> Lonza </td><td> CC-5002 </td><td> 0,03% </td></tr><tr><td> Vetropolycin </td><td> PharmaDerm Tiergesundheit </td><td></td><td> NAC #: 12920110 </td></tr><tr><td> Vicryl Naht (3-0) </td><td> Ethicon </td><td> J808T </td><td></td></tr><tr><td> Wasserbad Sonicator </td><td> Branson </td><td> B200 </td><td></td></tr></tbody></table>

autologous endothelial progenitor cell-seeding technology and biocompatibility testing for cardiovascular devices in large animal model

Implantierbare Herz-Kreislauf-Geräte werden aus künstlichen Materialien (zB Titan (Ti), expandiertem Polytetrafluorethylen), die das Risiko von Thromboembolien Bildung 1,2,3 Pose hergestellt. Wir haben eine Methode, um die innere Oberfläche des Ti Rohre mit Eigenblut-derived Mensch oder Schwein endothelialen Vorläuferzellen (EPC) 4-Linie entwickelt. Durch die Implantation Ti Röhrchen eine konfluente Schicht von Schweine-EPCs in die untere Hohlvene (IVC) von Schweinen, testeten wir die verbesserte Biokompatibilität der Zell-Oberfläche ausgesät in der prothrombotischen Umfeld eines großen Tiermodell und im Vergleich zu unmodifizierten blanken Metalloberflächen 5,6,7 (Abbildung 1). Diese Methode kann verwendet werden, um Geräte innerhalb von Minuten nach der Implantation endothelialize und testen ihre antithrombotischen Funktion in vivo werden. Periphere Blut wurde von 50 kg Yorkshire Schweinen und ihren mononukleäre Zellfraktion kultiviert, um die EPCs 4,8 isolieren erhalten. Ti Rohre (9,4 mm ID) wurden in drei 4,5 cm Längsschnitten vorgestanzten und wieder mit Schrumpfschlauch. Eine Aussaat Gerät gebaut wurde, das kann für langsame Rotation des Ti Röhren. Wir führten eine Laparotomie auf die Schweine und externalisiert den Darm und Harnblase. Scharfe und stumpfe Präparation wurde die IVC von der Bifurkation distal der rechten Nierenarterie proximalen Skelettierung. Die Ti Röhrchen wurden anschließend mit autologen EPC Federung und drehen bei 10 RPH x 30 min fluoreszenzmarkierten einheitlichen Zell-Beschichtung 9 erreichen gefüllt. Nach Verabreichung von 100 USP / kg Heparin wurden beide Enden der IVC und eine lumbale Vene festgeklemmt. A 4 cm veinotomy wurde durchgeführt und das Gerät eingelegt und mit Phosphat-gepufferter Kochsalzlösung. Als veinotomy mit einem 4:0-Prolene fortlaufenden Naht verschlossen war, wurde eine Klammer zu de-air der IVC entfernt. Am Ende des Verfahrens, der Faszie mit 0-PDS (Polydioxanon Naht) wurde angenähert, schloss die subkutane Raum mit 2-0 Vicryl und die Haut geheftet geschlossen. Nach 3 bis 21 Tage, Schweine wurden getötet, das Gerät explantiert en-Block und fixiert. Die Ti Rohre wurden demontiert und die inneren Oberflächen abgebildet mit einem Fluoreszenzmikroskop. Wir fanden, dass das blanke Metall Ti Rohre komplett verschlossen, während die EPC-seeded Röhren blieb Patent. Darüber hinaus konnten wir eine konfluente Schicht von EPCs auf der Innenseite mit Blut in Kontakt Oberfläche zu demonstrieren. Abschließend kann unsere Technologie verwendet, um Ti Rohre innerhalb von Minuten nach der Implantation endothelialize mit autologen EPCs zu Thrombosen des Geräts zu verhindern. Unsere OP-Methode ermöglicht die Prüfung der verbesserten Biokompatibilität von solchen modifizierten Geräte mit minimalem Blutverlust und EPC-Oberfläche ausgesät Störung.

Watch this Scientific Journal Video about Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model at JoVE.com

Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Ein Verfahren zur Aussaat Titan mit Blut in Kontakt Biomaterialien mit autologen Zellen und Prüfung Biokompatibilität beschrieben. Diese Methode verwendet endothelialen Vorläuferzellen und Titan Rohre, innerhalb von Minuten von chirurgischen Implantation in porcine Hohlvenen ausgesät. Diese Technik ist geeignet für viele andere implantierbare biomedizinische Geräte.

Autologe endotheliale Vorläuferzellen Cell-Sätechnik und Biokompatibilitätstestung Für Herz-Kreislauf-Geräte in Großtiermodell

Implantable cardiovascular devices are manufactured from artificial materials (e.g. titanium (Ti), expanded polytetrafluoroethylene), which pose the risk ...

autologous-endothelial-progenitor-cell-seeding-technology

Research

JoVE Journal

Bioengineering

20.3K Aufrufe.  Duke University. Ein Verfahren zur Aussaat Titan mit Blut in Kontakt Biomaterialien mit autologen Zellen und Prüfung Biokompatibilität beschrieben. Diese Methode verwendet endothelialen Vorläuferzellen und Titan Rohre, innerhalb von Minuten von chirurgischen Implantation in porcine Hohlvenen ausgesät. Diese Technik ist geeignet für viele andere implantierbare biomedizinische Geräte.

Serie: Autologous Endothelial Progenitor Cell-Seeding Technology and Biocompatibility Testing For Cardiovascular Devices in Large Animal Model

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Elektro-Zell-Substrat-Impedance Sensing zur Quantifizierung der endothelialen Proliferation, Barrierefunktion und Beweglichkeit

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Forschung

Biotechnik

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

In-vitro-Zellkulturmodell für Toxic Inhalative Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Schicht-für-Schicht der Kollagenabscheidung in Mikrofluidik-Vorrichtungen zum Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Ferromagnetic Bare Metal Stent for Endothelial Cell Capture and Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. The feasibility of using magnetic forces to capture and retain endothelial outgrowth cells (EOC) labeled with super paramagnetic iron oxide nanoparticles (SPION) has been shown previously. But this technique requires the development of a mechanically functional stent from a magnetic and biocompatible material followed by in-vitro and in-vivo testing to prove rapid endothelialization. We developed a weakly ferromagnetic stent from 2205 duplex stainless steel using computer aided design (CAD) and its design was further refined using finite element analysis (FEA). The final design of the stent exhibited a principal strain below the fracture limit of the material during mechanical crimping and expansion. One hundred stents were manufactured and a subset of them was used for mechanical testing, retained magnetic field measurements, in-vitro cell capture studies, and in-vivo implantation studies. Ten stents were tested for deployment to verify if they sustained crimping and expansion cycle without failure. Another 10 stents were magnetized using a strong neodymium magnet and their retained magnetic field was measured. The stents showed that the retained magnetism was sufficient to capture SPION-labeled EOC in our in-vitro studies. SPION-labeled EOC capture and retention was verified in large animal models by implanting 1 magnetized stent and 1 non-magnetized control stent in each of 4 pigs. The stented arteries were explanted after 7 days and analyzed histologically. The weakly magnetic stents developed in this study were capable of attracting and retaining SPION-labeled endothelial cells which can promote rapid healing.

Our goals were to design, manufacture and test ferromagnetic stents for endothelial cell capture. Ten stents were tested for fracture and 10 more stents were tested for retained magnetism. Finally, 10 stents were tested in-vitro and 8 more stents were implanted in 4 pigs to show cell capture and retention.

Ferromagnetische Bare Metal Stent für Endothelzellen-Abscheidung und Retention

Rapid endothelialization of cardiovascular stents is needed to reduce stent thrombosis and to avoid anti-platelet therapy which can reduce bleeding risk. ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Herstellung von benutzerdefinierten Agarose Brunnen für die Aussaat von Zelle und Gewebe Ring Selbstmontage mit 3D-Druck Formen

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

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, ...

A Large Animal Model for Pulmonary Hypertension and Right Ventricular Failure: Left Pulmonary Artery Ligation and Progressive Main Pulmonary Artery Banding in Sheep

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing novel therapeutics for PH requires a clinically relevant large animal model of increased pulmonary vascular resistance and RVF. This manuscript discusses the latest development of the previously published ovine PH-RVF model that utilizes left pulmonary artery (PA) ligation and main PA occlusion. This model of PH-RVF is a versatile platform to control not only the disease severity but also the RV&#39;s phenotypic response.
Adult sheep (60-80 kg) underwent left PA (LPA) ligation, placement of main PA cuff, and insertion of RV pressure monitor. PA cuff and RV pressure monitor were connected to subcutaneous ports. Subjects underwent progressive PA banding twice per week for 9 weeks with sequential measures of RV pressure, PA cuff pressures, and mixed venous blood gas (SvO2). At the initiation and endpoint of this model, ventricular function and dimensions were assessed using echocardiography. In a representative group of 12 animal subjects, RV mean and systolic pressure increased from 28 &#177; 5 and 57 &#177; 7 mmHg at week 1, respectively, to 44 &#177; 7 and 93 &#177; 18 mmHg (mean &#177; standard deviation) by week 9. Echocardiography demonstrated characteristic findings of PH-RVF, notably RV dilation, increased wall thickness, and septal bowing. The longitudinal trend of SvO2 and PA cuff pressure demonstrates that the rate of PA banding can be titrated to elicit varying RV phenotypes. A faster PA banding strategy led to a precipitous decline in SvO2 &#60; 65%, indicating RV decompensation, whereas a slower, more paced strategy led to the maintenance of physiologic SvO2 at 70%-80%. One animal that experienced the accelerated strategy developed several liters of pleural effusion and ascites by week 9. This chronic PH-RVF model provides a valuable tool for studying molecular mechanisms, developing diagnostic biomarkers, and enabling therapeutic innovation to manage RV adaptation and maladaptation from PH.

This manuscript describes the surgical technique and experimental approach to develop severe right ventricular pressure overload to model their adaptive and maladaptive phenotypes.

Ein großes Tiermodell für pulmonale Hypertonie und rechtsventrikuläres Versagen: Ligatur der linken Lungenarterie und progressive Hauptbelarterienbandierung bei Schafen

Decompensated right ventricular failure (RVF) in pulmonary hypertension (PH) is fatal, with limited medical treatment options. Developing and testing ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Großflächige, automatisierte Produktion von aus Fett gewonnenen Stammzellsphäroiden für den 3D-Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

In Silico Clinical Trials for Cardiovascular Disease

The SILICOFCM project mainly aims to develop a computational platform for in silico clinical trials of familial cardiomyopathies (FCMs). The unique characteristic of the platform is the integration of patient-specific biological, genetic, and clinical imaging data. The platform allows the testing and optimization of medical treatment to maximize positive therapeutic outcomes. Thus, adverse effects and drug interactions can be avoided, sudden cardiac death can be prevented, and the time between the commencement of drug treatment and the desired result can be shortened. This article presents a parametric model of the left ventricle automatically generated from patient-specific ultrasound images by applying an electromechanical model of the heart. Drug effects were prescribed through specific boundary conditions for inlet and outlet flow, ECG measurements, and calcium function for heart muscle properties. Genetic data from patients were incorporated through the material property of the ventricle wall. Apical view analysis involves segmenting the left ventricle using a previously trained U-net framework and calculating the bordering rectangle based on the length of the left ventricle in the diastolic and systolic cycle. M-mode view analysis includes bordering of the characteristic areas of the left ventricle in the M-mode view. After extracting the dimensions of the left ventricle, a finite elements mesh was generated based on mesh options, and a finite element analysis simulation was run with user-provided inlet and outlet velocities. Users can directly visualize on the platform various simulation results such as pressure-volume, pressure-strain, and myocardial work-time diagrams, as well as animations of different fields such as displacements, pressures, velocity, and shear stresses.

In Silico Clinical Trials for Cardiovascular Disease

This protocol demonstrates the workflow of the SILICOFCM platform for automatically generating a parametric model of the left ventricle from patient-specific ultrasound images by applying a multi-scale electromechanical model of the heart. This platform enables in silico clinical trials intended to reduce real clinical trials and maximize positive therapeutic outcomes.

In Silico Klinische Studien für Herz-Kreislauf-Erkrankungen

The SILICOFCM project mainly aims to develop a computational platform for in silico clinical trials of familial cardiomyopathies (FCMs). The unique ...

Bewertung der hydrodynamischen Eigenschaften in montierten Porcine Aortenklappen

An Ex Vivo Porcine Model for Hydrodynamic Testing of Experimental Aortic Valve Procedures and Novel Medical Devices

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a method for mounting a porcine aortic valve in a pulse duplicator to evaluate its hydrodynamic properties. These properties can then be evaluated before and after the procedure under investigation is performed and/or the investigative medical device is applied. Securing the inflow segment presents some difficulty owing to the lack of circumferential myocardium in the left ventricular outflow tract. This method addresses that issue by securing the inflow segment using the anterior leaflet of the mitral valve and then suturing the left ventricular free wall around the inflow fixture. The outflow segment is secured simply by inserting the fixture into an incision in the superior aspect of the aortic arch. We found that specimens had significantly different hydrodynamic properties before and after tissue fixation. This finding induced us to use fresh specimens in our testing and should be considered when using this method. In our work, we used this method to test novel intracardiac patch materials for use in the valvular position by performing an aortic valve neocuspidization procedure (Ozaki procedure) on the mounted porcine aortic valves. These valves were tested before and after the procedure to assess the change in hydrodynamic properties in comparison to the native valve. Herein, we report a platform for hydrodynamic testing of experimental aortic valve procedures that enables comparison with the native valve and between different devices and techniques used for the procedure under investigation.

Autor im Rampenlicht: Weiterentwicklung von Tests kardialer Verfahren vor Beginn von Großtierstudien 

We present a method for mounting a porcine aortic valve on a pulse duplicator to test its hydrodynamic properties. This method can be used to determine the change in hydrodynamics after the application of an experimental procedure or novel medical device prior to use in a large animal model.

Ein ex vivo Schweinemodell für die hydrodynamische Erprobung experimenteller Aortenklappenverfahren und neuartiger Medizinprodukte

The options for testing new cardiac procedures and investigative medical devices prior to use in an animal model are limited. In this study, we present a ...