Die Autoren danken Prof. Hendrik Tevaearai und die Abteilung für Gefäßchirurgie für die finanzielle Unterstützung für die erste Teil der Studie.

HINWEIS: Lewis männlichen und weiblichen Ratten wurden 200-220 g unter Standardlaborbedingungen (12 Stunden Licht und Dunkel-Zyklus, ad libitum Wasser und Nahrung, IVC-Käfig) untergebracht. Alle Tiere wurden in Übereinstimmung mit den Empfehlungen des FELASA und der Schweizer Tierschutzgesetzes behandelt. 1. Zell Zubereitung: mesenchymale Stammzelle Isolierung aus dem Knochenmark <ol><li> Betäuben die Ratten mit Isofluran 5% O 2 und 5 l / min in einer Induktionskammer für 5 min. Legen Sie das Tier die Schnauze in ein Nasenkegel auf die Anästhesie-System verbunden. Führen Sie eine Zehe oder Schwanz Prise zu Sedierung bestätigen. </li><li> Entfernen Sie die Haut von dem Bein mit der Schere, indem die Haut vom Knöchel bis zur Hüfte. Entfernen Sie die Muskeln und schneiden Sie die Oberschenkelarterie für Tier Ausbluten. Setzen Sie das Hüftgelenk und den Oberschenkelknochen verrenken Kopf. Es ist wichtig, den Oberschenkelkopf beschädigen. Zeigen die Knochen in sterilem PBS in ein 50 ml-Kunststoffröhrchen. </li><li> Entfernen Sie alle muscles und Bänder aus den Knochen, ohne zu brechen. HINWEIS: Es ist wichtig, dass der Knochen intakt bleibt, um die Verknüpfung von Spüllösung oder Kontamination mit Ethanol zu vermeiden. Spülen Sie die geschälten Knochen in 70% Ethanol für 5 min. </li><li> Unter der sterilen Laminarströmungshaube werden, beide Spitzen der Knochen mit einer Schere. Spülen Sie das Knochenmark durch die Injektion von sterilem PBS aus dem Knochen Extremität. Sammeln Mark in einen 15-ml-Tube. Zentrifuge 7 min bei 300 x g. </li><li> Entfernen Sie den Überstand. Hängen Sie das Pellet in 3 ml Lyse der roten Blutkörperchen Puffer. Inkubieren der Suspension für 1 min bei Raumtemperatur vor dem Verspinnen erneut für 7 Minuten bei 300 x g beträgt. In einem 150 ml-Kulturflasche, fügen sterile Zellkulturmedium (395 ml IMDM-Medium, 5 ml Penicillin / Streptomycin, 100 ml FBS) und Samen die Zellen. </li><li> Ändern Medium der zweite Tag zu nicht-adhärenten Zellen zu entfernen. <ol><li> Führen Mediumwechsel jeden zweiten Tag für 2-3 Wochen, bis die gewünschte Zellmenge erreicht ist. </li><li> Alternativ bereiten die biograft durch Samening der Zellen auf der festen Matrix nach Bedarf und an anderer Stelle beschrieben 3. </li></ol></li><li> Der Tag der Herzimplantation, bereiten Sie die Zellen oder ernten die biograft kurz vor epikardialen Anwendung. <ol><li> Sammeln Sie die Zellen mit Zellablösung Accutase Lösung. Zählen Sie die Zellen. Füllen einer 1,5 ml-Zentrifugenröhrchen mit dem Volumen der Zelle berechnet, um die gewünschte Menge zu erhalten Zellsuspension. Zentrifuge für 7 min bei 300 g und entfernen Sie den Überstand. </li><li> Alternativ ernten biograft von Zellkulturmedium gründlich in sterilem PBS und in frischem Kulturmedium ohne Serum zu halten. </li></ol></li></ol> 2. Erste Thorakotomie und LAD-Ligation <ol><li> Wiegen die Ratte. Schalten Sie das Heizkissen bei 37 ° C aufweist. Betäuben die Ratte mit 5% Isofluran und 5 l / min 100% O 2 in einer Induktionskammer für 5-7 min. Führen Sie eine Zehe oder Schwanz Prise zu Sedierung bestätigen. Legen Sie die Ratte auf dem Heizkissen. </li><li> Intubieren das Tier mit einem 14G IV-Katheter. Verbinden der Intubation Katheters zu einem 2,5 l / min Sauerstoff, 2,5% Isofluran, Atemvolumen von 2 ml, und einer Atemfrequenz von 90 Atemzügen / min programmiert Nagetier Ventilator. </li><li> Bereiten Sie ein Buprenorphin-Lösung bei 0,1 mg / kg. Injizieren subkutan ein Drittel der Lösung. Rasur den linken Teil des Brustkorbs. Desinfektion mit einer 1% Betadine Lösung. </li><li> Inzision der Haut senkrecht auf das Brustbein an der vierten Zwischenrippenraum. Trennen Sie die 3 Schichten des Brustmuskels (M. pectoralis major, aufsteigend Brustmuskel, und äußeren schrägen pectoralis). Öffnen Sie den vierten Zwischenrippenraum (zwischen den Rippen 4 und 5). </li><li> Verwenden Sie einen kleinen Aufroller, um die Rippen zu verbreiten und das Herz freizulegen. Öffnen Sie den Herzbeutel vorsichtig. Lokalisieren und zu ligieren den linken vorderen absteigenden Koronararterie (LAD) mit einem 7,0 Faden 4 mm unterhalb des Atrium. </li><li> Schließen Sie die Zwischenrippenraum mit zwei Maschen mit 3,0 Nähten. Positionieren Sie die beiden Fäden proximal und distal vom Brustbein. Erstens tighten das distale Naht. Spannen Sie den Ventilator Abluftschlauch für 2 Sekunden, um die Brust aufpumpen und vermeiden Sie jegliche Pneumothorax. Ziehen Sie die zweite Naht. Positionieren Sie die Muskeln Schichten wieder an Ort und Stelle (keine Nähte erforderlich sind). Schließen Sie die Haut mit einem 5,0 Faden. </li><li> Desinfizieren Sie die Naht mit 1% Betadine Lösung. Injizieren den Rest der Buprenorphin-Lösung. Schalten Sie das Anästhesiesystem. Intubation Katheter zu entfernen. </li><li> Halten Sie die Ratte im Käfig unter einer warmen Lampe für 1-2 Stunden. Halten ein Thermometer in den Käfig und Steuerung des Abstandes zwischen der warmen Lampe und dem Käfig, um ein Überhitzen zu vermeiden. Bringen Sie die Ratte in der IVC-Einheit. Injizieren subkutan Buprenorphin 0,05-0,1 mg / kg nach -operatively alle 6-12 h für 48 Stunden. </li></ol> 3. Epikardiale Verwaltung der Behandlung über eine zweite Thorakotomie <ol><li> Wiederholen Sie die Schritte 2.1-2.3. </li><li> Inzision der Haut senkrecht auf das Brustbein an der fünften Zwischenrippenraum. Trennen Sie die 3-Schichts der Brustmuskel (pectoralis major, aufsteigend pectoralis und äußeren schrägen pectoralis). Öffnen Sie den fünften Zwischenrippenraum (zwischen den Rippen 5 und 6). </li><li> Verwenden Sie einen kleinen Aufroller, um die Rippen zu verbreiten und das Herz freizulegen. Wenn es einige Festhalten, sorgfältig entfernen Sie sie mit einer feinen Pinzette. </li><li> Suchen Sie den Infarktbereich, der als hell Bereich unterhalb der Ligatur erscheint. HINWEIS: Wenn nötig, verwenden Sie ein 10 cm Stück Faden (7,0) an der Spitze eingesetzt, um den linken Ventrikel besser zu visualisieren. Ziehen Sie vorsichtig an der Naht lose durch eine Klammer gehalten, um das Herz freizulegen. </li><li> Tragen Sie eine der folgenden Behandlungen: <ol><li> Positionieren Sie den biograft an der Oberfläche des Herzens. Verwenden Sie die vorgefüllte Spritze Fibrinkleber bei Raumtemperatur für 10 min vorgewärmt. Geben Sie einen Tropfen (50-100 ul) Fibrinkleber unter der biograft mit einem 25 G Luer Nadel. Stellen Sie sicher, dass die biograft ist perfekt abgedichtet. </li><li> Alternativ gilt das Zellpellet mit einer Pipettenspitze an der Oberfläche gesammelt of Herzen auf dem blassen Bereich unterhalb der Ligatur. Geben Sie einen Tropfen (50-100 ul) Fibrinkleber auf dem Zellpellet. </li></ol></li><li> Entfernen Sie das Stück Faden von der Spitze. </li><li> Wiederholen Sie die Schritte 2.6-2.8. </li></ol>

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The authors have nothing to disclose.

Die dauerhafte Unterbindung der LAD irreversiblen Myokard-Schädigung. Die erste Tiermodell wurde im Jahre 1960 4 beschrieben. Seitdem es als Standard und geeignetes Modell für chronische MI berücksichtigt. Seine Stabilität und Reproduzierbarkeit erlaubt experimentelle Evaluation von Therapien für MI 5. Verbesserte Verfahren nach der ersten Beschreibung meldete einen operativen Mortalitätsrate von 35-13% 6. Wie erwartet, LAD-Ligation induzierte innerhalb...

Seit dem Ende des 19. Jahrhunderts hat sich Herz-Kreislauf-Krankheit die Nummer eins Serienmörder in den Industrieländern blieb. Unter ihnen, koronare Herzkrankheit stellt die Haupt Ätiologie. Die akute Phase Ergebnisse in Myokardinfarkt (MI) und wird durch maladaptive Umbau, die schrittweise entwickelt zu einer chronischen Phase und schwerer Herzinsuffizienz gefolgt. Trotz der jüngsten bedeutenden technologischen und therapeutische Fortschritte, die Morbidität und Mortalität aufgrund der Progression der Herzinsuffizienz noch wächst 1. In diesem Zusammenhang hat die Zelltherapie zunehmend Interesse als eine neue therapeutische Option, um die Progression der Krankheit zu Herzversagen zu stoppen und die kürzlich identifizierten Regenerationsfähigkeit des Herzmuskels stimulieren gewonnen. Experimentelle und klinische Untersuchungen haben überzeugende Beweise für die nach Herztransplantation von verschiedenen Zelltypen erhalten wohltuende Wirkung zur Verfügung gestellt. Wichtigsten Ergebnisse enthalten verbesserte Cardiac Kontraktionsfunktion, Abnahme der linksventrikulären Remodeling, reduzierte die Infarktgröße und erhöhte Gefäßdichte im Infarktgebiet. Die niedrige Zellzahl Retention nach Zellinjektion blieb jedoch ein wichtiger Nachteil. Verband der Zellen mit einer Biomatrix auf der Zellliefer 2 zu verbessern, haben kürzlich Forscher gefördert und klinische Interessen. Ligation der linken vorderen absteigenden Koronararterie (LAD) ist ein Referenzmethode für MI in der Kleintiermodell, das in transmuralen Infarkt und einer reifen Narbe führt. Zelltherapie in der chronischen Phase der MI angewendet erfordert einen zweiten chirurgischen Eingriff. Eine mediane Sternotomie wird üblicherweise durchgeführt, um intramyokardiale Injektion der Zellen oder epikardiale Implantation biografts ermöglichen. Solche invasive chirurgische Verfahren erhöhen die Sterberate, postoperative Erholungszeit, Schmerzen und Infektionsrisiko. Die minimal-invasive Ansatz hier präsentierten nicht nur verhindert, dass solche Voreingenommenheit, sondern bietet auch optimale Zugänglichkeit des Herzens zur Behandlung Anwendung. MI und epikardialen Implantation von Zellen, die mit einem gelartigen Biomatrix verbunden sind an einem schlagenden Herz über linke Rippenthorakotomien geführt.

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			<td height="21" style="height:21px;width:216px;">DUMONT #7 Forceps</td>
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			<td height="42" style="height:42px;width:216px;">Accutase cell detachment solution</td>
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cell-based therapy for heart failure in rat: double thoracotomy for myocardial infarction and epicardial implantation of cells and biomatrix

Herzzelltherapie wird zunehmend Interesse und die Implantation von Biomaterialien mit Zellen verbunden ist zu einem wichtigen Thema zu Herzmuskelzelle Lieferung optimieren. Nagetiermodell von Myokardinfarkt (MI), die aus linken vorderen absteigenden Arterie (LAD) Ligation üblicherweise über eine Thorakotomie durchgeführt worden ist; eine zweite Operation am offenen Herzen über eine Sternotomie traditionell für epikardiale Anwendung der Behandlung durchgeführt. Seit der Beschreibung der LAD-Ligation-Modell, hat nach der Operation Sterblichkeitsrate von 35 bis 13% gesunken, aber die zweite Operation hat kritische geblieben. Zur Verbesserung der postoperativen Erholung und verringern Schmerzen und Infektionen, sind minimal-invasive Operationsverfahren vorgestellt. Zwei Thorakotomien durchgeführt wurden, die die ursprüngliche für KOP-Ligation und die zweite für die Behandlung epikardialen Verwaltung. Biografts bestehend aus Zellen, die mit festen oder gelartigen Matrizen assoziiert wurden auf die Infarkt ar angewendetea. LAD-Ligation führte zu Verlust der Herzfunktion, wie durch Echokardiographie nach 2 und 6 Wochen durchgeführt bestätigt. Goldner Trichrom-Färbung auf Herz Abschnitten durchgeführt bestätigt transmurale Narbenbildung. Ersten und zweiten Operationen führten zu weniger als 10% der postoperativen Mortalität.

Alle Tiere innerhalb 1 Stunde nach Thorakotomien erholt. Die Wundheilung war schnell. Keine Infektion oder Ödeme beobachtet. Das Doppel Linksthorakotomie erlaubt optimalen Zugang zum Herzen (Abbildung 1). Schmerzen und postoperative Mortalität waren gering. Das Tier erholte sich schnell von der Operation und nahm an Gewicht zu (Abbildung 2). Kaplan Meir Überleben Prozent betrug 96% für die erste Thorakotomie. Vier Ratten über 104 starben innerhalb von 2...

Watch this Scientific Journal Video about Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix at JoVE.com

Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Implantation eines biograft Myokardinfarkt durch LAD-Ligation in einem Nagetiermodell induzierte Behandlung wurde üblicherweise erforderlichen zwei Operationen am offenen Herzen. Um die Sterblichkeit zu reduzieren und optimale Bedingungen für die Fixierung von festen und gallertartige Biomatrices mit Zellen verbunden sind, haben minimal-invasive Verfahren entwickelt worden.

Zell-basierte Therapie bei Herzinsuffizienz in Rat: Doppel Thorakotomie für Herzinfarkt und Epikardiale Implantation von Zellen und Biomatrix

Cardiac cell therapy has gained increasing interest and implantation of biomaterials associated with cells has become a major issue to optimize myocardial ...

cell-based-therapy-for-heart-failure-rat-double-thoracotomy-for

Research

JoVE Journal

Bioengineering

11.8K Aufrufe.  University of Fribourg.  Implantation eines biograft Myokardinfarkt durch LAD-Ligation in einem Nagetiermodell induzierte Behandlung wurde üblicherweise erforderlichen zwei Operationen am offenen Herzen. Um die Sterblichkeit zu reduzieren und optimale Bedingungen für die Fixierung von festen und gallertartige Biomatrices mit Zellen verbunden sind, haben minimal-invasive Verfahren entwickelt worden. 

Serie: Cell-based Therapy for Heart Failure in Rat: Double Thoracotomy for Myocardial Infarction and Epicardial Implantation of Cells and Biomatrix

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Biolumineszenz-Bildgebung für die Beurteilung von Immunantworten nach der Implantation von Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Forschung

Biotechnik

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Transplantation von induzierten pluripotenten Stammzellen-abgeleiteten mesoangioblast-ähnlichen myogenen Vorläufern in Mausmodellen der Muskelregeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

Analyse von Membranrohrnetze in Herzmuskelzellen von Vorhöfe und Kammern

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Aussaat und Implantation von einem biosynthetischen Tissue-Engineering trachealen Transplantat in einem Mausmodell

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Bewertung der Keratinozyten Verbreitung auf zwei- und dreidimensionale Typ I Kollagen Substrate

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Magnetic-, Acoustic-, and Optical-Triple-Responsive Microbubbles for Magnetic Hyperthermia and Pothotothermal Combination Cancer Therapy

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has been challenged. Here, we fabricate iron oxide nanoparticle shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform. Iron oxide nanoparticles serve as both magnetic and photothermal agents. Once intravenously injected, NSMs can be magnetically guided to the tumor site. Ultrasound triggers the release of iron oxide nanoparticles, facilitating the penetration of nanoparticles deep into the tumor due to the cavitation effect of microbubbles. Thereafter, magnetic hyperthermia and photothermal therapy can be performed on the tumor for combinational cancer therapy, a solution for cancer resistance due to the tumor heterogeneity. In this protocol, the synthesis and characterization of NSMs including structural, chemical, magnetic and acoustic properties were performed. In addition, the anti-cancer efficacy by thermal therapy was investigated using in vitro cell cultures. The proposed delivery strategy and combination therapy holds great promise in cancer treatment to improve both delivery and anticancer efficacies.

Presented here is a protocol for the fabrication of iron oxide nanoparticle-shelled microbubbles (NSMs) through self-assembly, synergizing magnetic, acoustic, and optical responsiveness in one nanotherapeutic platform for magnetic hyperthermia and photothermal combination cancer therapy.

Magnetische, akustische und optische dreifach ansprechende Mikroblasen für magnetische Hyperthermie und pothotothermische Kombinationskrebstherapie

The precision delivery of anti-cancer agents which aim for targeted and deep-penetrated delivery as well as a controlled release at the tumor site has ...

Preparation of Mesh-Shaped Engineered Cardiac Tissues Derived from Human iPS Cells for In Vivo Myocardial Repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from human induced pluripotent stem cells (hiPSCs), which are developed towards the goal of clinical use. HiPSC-derived cardiomyocytes, endothelial cells, and vascular mural cells are mixed with gel matrix and then poured into a polydimethylsiloxane (PDMS) tissue mold with rectangular internal staggered posts. By culture day 14 ECTs mature into a 1.5 cm x 1.5 cm mesh structure with 0.5 mm diameter myofiber bundles. Cardiomyocytes align to the long-axis of each bundle and spontaneously beat synchronously. This approach can be scaled up to a larger (3.0 cm x 3.0 cm) mesh ECT while preserving construct maturation and function. Thus, mesh-shaped ECTs generated from hiPSC-derived cardiac cells may be feasible for cardiac regeneration paradigms.

The present protocol generates mesh-shaped engineered cardiac tissues containing cardiovascular cells derived from human induced pluripotent stem cells to allow the investigation of cell implantation therapy for heart diseases.

Vorbereitung von Mesh-Shaped Engineered Cardiac Tissues derived from Human iPS Cells for In Vivo Myokard repair

The current protocol describes methods to generate scalable, mesh-shaped engineered cardiac tissues (ECTs) composed of cardiovascular cells derived from ...

Transplantation of a 3D Bioprinted Patch in a Murine Model of Myocardial Infarction

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ligation is a challenging procedure and has a high mortality rate due to its nature. We developed a method to consistently transplant bioprinted patches of cells and hydrogels onto the epicardium of an infarcted mouse heart to test their regenerative properties in a robust and feasible way. First, a deeply anesthetized mouse is carefully intubated and ventilated. Following left lateral thoracotomy (surgical opening of the chest), the exposed LAD is permanently ligated and the bioprinted patch transplanted onto the epicardium. The mouse quickly recovers from the procedure after chest closure. The advantages of this robust and quick approach include a predicted 28-day mortality rate of up to 30% (lower than the 44% reported by other studies using a similar model of permanent LAD ligation in mice). Moreover, the approach described in this protocol is versatile and could be adapted to test bioprinted patches using different cell types or hydrogels where high numbers of animals are needed to optimally power studies. Overall, we present this as an advantageous approach which may change preclinical testing in future studies for the field of cardiac regeneration and tissue engineering.

This protocol aims to transplant a 3D bioprinted patch onto the epicardium of infarcted mice modeling heart failure. It includes details regarding anesthesia, the surgical chest opening, permanent ligation of the left anterior descending (LAD) coronary artery and application of a bioprinted patch onto the infarcted area of the heart.

Transplantation eines biobedruckten 3D-Patches in einem Murine-Modell des Myokardinfarkts

Testing regenerative properties of 3D bioprinted cardiac patches in vivo using murine models of heart failure via permanent left anterior descending (LAD) ...

Quantification of Mouse Heart Left Ventricular Function, Myocardial Strain, and Hemodynamic Forces by Cardiovascular Magnetic Resonance Imaging

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations result in pathology, and how myocardial diseases may be treated. Cardiovascular magnetic resonance imaging (CMR) has become an indispensable tool for a comprehensive in vivo assessment of cardiac anatomy and function. This protocol shows detailed measurements of mouse heart left ventricular function, myocardial strain, and hemodynamic forces using 7-Tesla CMR. First, animal preparation and positioning in the scanner are demonstrated. Survey scans are performed for planning imaging slices in various short- and long-axis views. A series of prospective ECG-triggered short-axis (SA) movies (or CINE images) are acquired covering the heart from apex to base, capturing end-systolic and end-diastolic phases. Subsequently, single-slice, retrospectively gated CINE images are acquired in a midventricular SA view, and in 2-, 3-, and 4-chamber views, to be reconstructed into high-temporal resolution CINE images using custom-built and open-source software. CINE images are subsequently analyzed using dedicated CMR image analysis software.
Delineating endomyocardial and epicardial borders in SA end-systolic and end-diastolic CINE images allows for the calculation of end-systolic and end-diastolic volumes, ejection fraction, and cardiac output. The midventricular SA CINE images are delineated for all cardiac time frames to extract a detailed volume-time curve. Its time derivative allows for the calculation of the diastolic function as the ratio of the early filling and atrial contraction waves. Finally, left ventricular endocardial walls in the 2-, 3-, and 4-chamber views are delineated using feature-tracking, from which longitudinal myocardial strain parameters and left ventricular hemodynamic forces are calculated. In conclusion, this protocol provides detailed in vivo quantification of the mouse cardiac parameters, which can be used to study temporal alterations in cardiac function in various mouse models of heart disease.

This study describes a comprehensive cardiovascular magnetic resonance imaging (CMR) protocol to quantify the left ventricular functional parameters of the mouse heart. The protocol describes the acquisition, post-processing, and analysis of the CMR images as well as assessment of different cardiac functional parameters.

Quantifizierung der linksventrikulären Funktion des Mausherzens, des Myokardstamms und der hämodynamischen Kräfte durch kardiovaskuläre Magnetresonanztomographie

Mouse models have contributed significantly to understanding genetic and physiological factors involved in healthy cardiac function, how perturbations ...

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 ...