, Ein New Researcher Award vom Thrasher Research Fund, und Beiträge an die Reizleitung Fund an der Kinderklinik Boston; Diese Arbeit wird durch Forschungsmittel von den National Institutes of Health (HL088206 HL068915) unterstützt.

Teil 1: Montieren Sie bauen Gussformen <ol><li> Verwenden Sie eine Rasierklinge Silikonschlauch (VWR) zu halbieren und schneiden Sie es in 3 cm lange Stücke schneiden. </li><li> Geben Sie einen Tropfen von Implantat-grade RTV Silikonkleber (Rhodia) auf der Innenseite von jedem Ende des Schlauchs. </li><li> Schnell Ort ein kleines Stück (1 cm x 1 cm) von Polyester-Mesh (McMaster-Carr) auf dem Silikonkleber drop und richten Sie sie mit dem Ende des Schlauchs. Dies wird eine leicht erhöhte und ebene Fläche für Bau Anhang. Wiederholen Sie für das andere Ende. </li><li> Lassen Sie die Form bei Raumtemperatur für 3 Tage trocknen lassen. Es ist hilfreich, 20 bis 30 Formen in einer Zeit zu machen, da diese im Wesentlichen zu entsorgen sind. </li><li> Sobald der Kleber vollständig trocken ist, speichern diese Formen in ein Becherglas mit 70% Ethanol gefüllt, bis sie gebraucht werden. Dieser Schritt wird dazu beitragen, sterilisieren die Formen und das fertige Produkt wird in Abbildung 1 dargestellt. </li></ol> Teil 2: Vorbereitung für die Myoblasten Zellisolation <ol><li> Verdünnen Sie 1 mg Laminin (Sigma) in 250 ml 0,22 um Filter-sterilisierte Phosphat-gepufferter Salzlösung (PBS) mit 1% Penicillin / Streptomycin (Invitrogen) und 1% Fungizone (Invitrogen). </li><li> Coat 150 mm Zellkulturplatten (BD Falcon) mit 4 ml der verdünnten Laminin-Lösung aus Schritt 1,1 und bei 37 º C für mindestens 4 Stunden vor dem Ausplattieren der primären Skelettmyoblasten. </li><li> Machen Sie den Myoblasten Medium durch Mischen Hams F-10 Nutrient Mixture (Sigma) mit 20% FBS (Atlanta Biologicals), 5 ng / L basischen Fibroblasten-Wachstumsfaktor (Promega), 1% Penicillin / Streptomycin (Invitrogen) und 1% Fungizone ( Invitrogen). </li><li> Bereiten Sie die Skelettmuskulatur Verdauung durch Auflösen von 1 g ~ 295 U / mg Collagenase 2 (Worthington) in 100 ml 2,4 U / ml Dispase-2 (Roche). Add 0,037 g CaCl 2 und gut mischen. Filter sterilisieren die Verdauung Puffer durch einen 0,2 um Filter Festplatte auf eine 10 ml Spritze und speichern Aliquots bei -20 ° C eingebaut Setzen Sie den Betrag für die Gewebe-Verdau bei 37 ° C zusammen mit den Myoblasten Medien benötigt. </li></ol> Teil 3: Myoblasten isoliert von der Skelettmuskulatur <ol><li> Mit einer Pinzette und eine kleine Schere, zu entfernen paraspinalen Muskeln von toten Neugeborenen Lewis Ratten durch Aufschneiden den Rücken hinunter entlang des Rückenmarks, Abziehen der Haut und Faszie Schichten und dann Ausschneiden der Muskeln. </li><li> In einer Gewebekultur Kapuze, platzieren Sie den ausgeschnittenen Muskeln in einem 50 ml konischen Röhrchen (BD Falcon) mit 40 ml 1X Hanks Balanced Salt Solution (HBSS) (Invitrogen) auf Eis gefüllt. Lassen Sie den Muskel-Streifen auf den Boden des Röhrchens absetzen und entfernen Sie vorsichtig die meisten der Flüssigkeit und Blut Überreste von Vakuumsauger mit einer sterilen Pasteurpipette in eine Kultur Haube (Baker). </li><li> Gießen Sie die geernteten Muskel Stücke auf einer 150 mm Kulturschale und entfernen Sie so viel von der restlichen Flüssigkeit wie möglich abgesaugt. Mit 2 einseitig geschliffene Klingen zerkleinern das Gewebe zu einer dicken Paste. Gießen vorgewärmten Verdauung Lösung auf der Paste und die Masse in eine frische 50 ml Tube mit einem 10-ml-Pipette. Während der enzymatischen Verdauung, wird das Gewebe gelegentlich verrieben (ohne Blasenbildung) mit einer 10 mL Pipette auf ein schnurloses Pipette-Aid (Drummond) ausgestattet. Auch hier ermöglichen das Gewebe ansiedeln und entfernen Sie die überschüssige Flüssigkeit. </li><li> Das Röhrchen auf einem Schaukelstuhl Plattform und verdauen bei 37 º C für ca. 30 min. </li><li> Sobald der Muskel wird verflüssigt, übergeben Sie die Lösung durch einen 70 um Zellsieb (BD Falcon) und Zentrifuge bei Raumtemperatur für 10 min bei 600 xg (Beckman GP). Mit einer Pasteurpipette und Absaugung, entfernen Sie so viel von der Flüssigkeit wie möglich und das Pellet mit 10 ml warmem Myoblasten Medium. </li><li> Pre-Platte die Zellen auf einer unbeschichteten 150 mm Gewebekulturplatte für 15 Minuten zur Beseitigung von Fibroblasten. </li><li> Übertragen Sie die Zellen, die noch nicht auf die Laminin-beschichtete cultureware befestigt und verdünnen, so dass es eine Platte für jede von 1 bis 2 Rattenbabys. Die Platte wird in einem befeuchteten Inkubator eingestellt bei 37 ° C mit 5% CO 2. </li></ol> Teil 4: Casting gezüchteten Gewebe-Konstrukten (5 mL) <ol><li> Nach 1 bis 2 Tagen, entfernen 10 der 150 mm Myoblasten Zellkulturplatten aus dem Inkubator, spülen Sie sie zweimal mit 37 ° C 1X PBS, und fügen Sie 4 ml warmes 0,05% Trypsin-EDTA auf jeder Platte. Bringen Sie die Platten in den Inkubator für 5 min. </li><li> Ernte der abgelösten Myoblasten aus den Platten in der Kultur Kapuze und sammeln die Flüssigkeit in einer 50 ml Tube (BD Falcon). Verwenden Sie 10 ml Myoblasten mittel-bis alle Platten Spül-und kombinieren diese mit den Zellen in die Zentrifugenröhrchen. Pellet die Zellen bei Raumtemperatur für 10 min bei 600 xg (Beckman GP) und entfernen Sie überschüssige Flüssigkeit wie in Schritt 3.5 beschrieben. </li><li> Resuspendieren Myoblasten Zellen in 1,6 ml Myoblasten Medium und auf Eis stellen. </li><li> Mit einer Pinzette vorsichtig Entfernen 4 konstruieren Formen aus Schritt 1.5 und spülen Sie sie zweimal mit 1X PBS. Vakuum-Sauggreifer aus alle Spuren von PBS mit einer Pasteurpipette, bevor jeder Form in einer 6-well Gewebe culture Platte (BD Falcon). </li><li> Sammeln Sie die anderen Reagenzien für Bau Vorbereitung nötig (10X Hams F10, Antibiotika, Typ-1-Kollagen [3,9 mg / mL] [6] und Matrigel ™) auf Eis und halten Myoblasten bei 37 º C. Kombinieren Sie 500 ul 10x Ham F10, 100 ul Penicillin / Streptomycin, 100 ul Fungizone, 1,6 mL Kollagen, und 750 ul Matrigel ™ in ein konisches 15 ml Tube. Mischen Sie die Lösung für 10 sec mit einem Mini-Vortexer (VWR) auf 10 und auf Eis stellen. </li><li> Anschließend fügen Sie 80 ul von NaHCO 3 und die Myoblasten Suspension aus Schritt 3,3 bis die Mischung aus Schritt 3.5. Vortex die gesamte Mischung für 10 sec und kehren der Röhre in den Eiskübel für ein paar Minuten, um die Lösung von Bläschen klar. </li><li> Vorsichtig warf das Gemisch in die Formen, bevor sie sich verfestigen mit einer 10 ml Pipette beginnt. Jede Form hält ca. 1 mL der viskosen Flüssigkeit. Versuchen Sie zu vermeiden Einführung von Luftblasen während dieses Vorgangs. </li><li> Sorgfältig Übertragung der Teller mit der cast Konstrukte in den Inkubator. Nach ca. 30 min, entfernen Sie die erstarrten Konstrukte aus dem Inkubator und vorsichtig Myoblasten Medium in jede Vertiefung zur Deckung des Gewebes zu konstruieren. Bringen Sie die Platte in den Inkubator (Abbildung 2). </li></ol> Teil 5: Repräsentative Ergebnisse Wenn dieses Protokoll korrekt ausgeführt wird, wird die Myoblasten-haltigen Gewebe konstruieren bereit für in vivo-Implantation (dh, wenn aus der Form entfernt) oder für weitere in vitro-Analysen nach 2 Tagen. <img src="/files/ftp_upload/1137/Figure1.jpg" alt="" /> Abbildung 1. Beispiele für abgeschlossen zu konstruieren Formen (siehe Schritt 1.5). <img src="/files/ftp_upload/1137/Figure2.jpg" alt="" width="400" height="321" /> Abbildung 2. A verfestigt Myoblasten-haltigen Gewebe konstruieren [1]. <img src="/files/ftp_upload/1137/Figure3.jpg" alt="" /> Abbildung 3. H &amp; E gefärbten Schnitten eines Myoblasten-haltigen Gewebe zu konstruieren. &quot;A&quot; zeigt einen Längsschnitt und &quot;B&quot; zeigt einen Querschnitt.

<ol>
	<li>Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+conduction+through+engineered+tissue.">Cardiac conduction through engineered tissue.</a> Am J Pathol. 169 (1), 72-85 (2006).</li><li>Rando, T. A., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+mouse+myoblast+purification,+characterization,+and+transplantation+for+cell-mediated+gene+therapy.">Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy.</a> J Cell Biol. 125 (6), 1275-1287 (1994).</li><li>Blau, H. M., Webster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+human+muscle+cells.">Isolation and characterization of human muscle cells.</a> Proc Natl Acad Sci U S A. 78 (9), 5623-5627 (1981).</li><li>Powell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+human+bioartificial+muscles+expressing+a+foreign+recombinant+protein+for+gene+therapy.">Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy.</a> Hum Gene Ther. 10 (4), 565-577 (1999).</li><li>Vandenburgh, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+skeletal+muscle+organoids+for+reversible+gene+therapy.">Tissue-engineered skeletal muscle organoids for reversible gene therapy.</a> Hum Gene Ther. 7 (17), 2195-2200 (1996).</li><li>Gallop, P. M., Seifter, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Properties+of+Soluble+Collagens.">Preparation and Properties of Soluble Collagens.</a> Methods in Enzymology. 6, 635-641 (1963).</li></ol>

Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her durch die Institutional Animal Care und Verwenden Ausschusses im Kinderkrankenhaus Boston gesetzt durchgeführt.

Die Formen, in denen das Gewebe konstruieren gegossen werden kann in jeder Form und Größe hergestellt werden, allerdings muss es mindestens zwei Befestigungspunkte werden. Ansonsten bilden die Matrix und Zellen eine kugelförmige Struktur und die Zellen sterben. In der vorliegenden Protokoll, beschreiben wir die Verwendung eines Polyester-Mesh für diesen Zweck, aber wir haben auch erfolgreich Edelstahlgewebe verwendet. Offensichtlich werden größere Formen erfordern mehr Zellen und ein größeres Volumen der sonstig...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Silicone tubing</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>60985-724</td>
<td>&nbsp;</td>
<tr>
<td>Silicone adhesive</td>
<td>&nbsp;</td>
<td>Rhodia Silicones</td>
<td>MED ADH 4300 RTV</td>
<td>&nbsp;</td>
<tr>
<td>Polyester Mesh</td>
<td>&nbsp;</td>
<td>McMaster-Carr</td>
<td>93185T17</td>
<td>&nbsp;</td>
<tr>
<td>Laminin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L2020</td>
<td>&nbsp;</td>
<tr>
<td>Nutrient Mixture F-10 HAM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>N6908</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Atlanta Biologicals</td>
<td>S11550</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Fungizone</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15290-018</td>
<td>&nbsp;</td>
<tr>
<td>Dispase-2</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10295825001</td>
<td>&nbsp;</td>
<tr>
<td>Collagenase 2</td>
<td>&nbsp;</td>
<td>Worthington</td>
<td>46H8863</td>
<td>&nbsp;</td>
<tr>
<td>Basic Fibroblast Growth Factor</td>
<td>&nbsp;</td>
<td>Promega</td>
<td>G5071</td>
<td>&nbsp;</td>
<tr>
<td>150 mm tissue culture dishes</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353025</td>
<td>&nbsp;</td>
<tr>
<td>0.05% (1X) Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25300</td>
<td>&nbsp;</td>
<tr>
<td>1X Hanks Balanced Salt Solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14170-112</td>
<td>&nbsp;</td>
<tr>
<td>7.5% NaHCO3</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25080-094</td>
<td>&nbsp;</td>
<tr>
<td>70 &mu;m cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>6-well plates</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>353046</td>
<td>&nbsp;</td>
<tr>
<td>50 mL Conical Vial</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352098</td>
<td>&nbsp;</td>
<tr>
<td>15 mL Conical Vial</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>352099</td>
<td>&nbsp;</td>
<tr>
<td>0.2 &mu;m filter</td>
<td>&nbsp;</td>
<td>Nalgene</td>
<td>194-2520</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

fabrication of myogenic engineered tissue constructs

Trotz der Tatsache, dass die elektronische Herzschrittmacher sind lebensrettende medizinische Geräte, deren langfristige Performance bei pädiatrischen Patienten kann problematisch wegen der Einschränkungen durch ein Kind seiner geringen Größe und ihrer unvermeidlichen Wachstum auferlegt werden. Folglich gibt es einen echten Bedarf an innovativen Therapien speziell für pädiatrische Patienten mit Herzrhythmusstörungen entwickelt. Wir schlagen vor, dass eine leitende biologische Alternative, bestehend aus einer Kollagen-basierten Matrix mit autologen-abgeleiteten Zellen könnte besser zum Wachstum anzupassen, reduzieren die Notwendigkeit für wiederkehrende Operationen, und verbessern Sie die Qualität des Lebens für diese Patienten. In der vorliegenden Studie beschreiben wir ein Verfahren zur Einbindung primären Skelett Myoblasten Zellkulturen in einem Hydrogel-Matrix der Mode eine chirurgisch-implantierbaren Gewebe zu konstruieren, das als elektrische Leitung zwischen dem oberen und unteren Kammern des Herzens dienen. Letztlich erwarten wir mit dieser Art von gezüchteten Gewebe zu atrioventrikulären elektrische Leitfähigkeit bei Kindern mit komplettem Herzblock wiederherzustellen. In Anbetracht dieser, isolieren wir Myoblasten aus der Skelettmuskulatur von neonatalen Lewis Ratten und Platte sie auf Laminin-beschichtete Gewebe Kulturschalen mit einer modifizierten Version des etablierten Protokollen<sup&gt; [2, 3]</sup&gt;. Nach 1-2 Tagen werden kultivierten Zellen gesammelt und mit Antibiotika versetzt, Typ-1-Kollagen, Matrigel ™ und NaHCO<sub&gt; 3</sub&gt;. Das Ergebnis ist eine zähflüssige, einheitliche Lösung, die in eine Form von nahezu beliebiger Größe und Form gegossen werden kann<sup&gt; [1, 4, 5]</sup&gt;. Für unsere Gewebe Konstrukte beschäftigen wir Typ-1-Kollagen aus fetalen Lamm Haut unter Verwendung von Standardverfahren isoliert<sup&gt; [6]</sup&gt;. Sobald das Gewebe bei 37 ° C verfestigt, Kulturmedien ist sorgfältig auf die Platte gegeben, bis das Konstrukt ist untergetaucht. Die gezüchteten Gewebe wird dann weiter zu verdichten durch Dehydratisierung für 2 weitere Tage erlaubt, an welcher Stelle es reif ist<em&gt; In-vitro-</em&gt; Beurteilung oder chirurgische Implantation.

Watch this Scientific Journal Video about Fabrication of Myogenic Engineered Tissue Constructs at JoVE.com

Fabrication of Myogenic Engineered Tissue Constructs

Hier zeigen wir die Herstellung von Kollagen-basierten, baut Gewebe mit Skelettmyoblasten. Diese 3-D konstruiert Konstrukte verwendet zu ersetzen oder zu reparieren Gewebe werden<em&gt; In vivo</em&gt;. Für unsere Zwecke haben wir diese als AV-elektrische Leitung für die Reparatur von kompletten AV-Block entwickelt<sup&gt; [1]</sup&gt;.

Herstellung von Myogenic Engineered Tissue Konstrukte

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

fabrication-of-myogenic-engineered-tissue-constructs

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

10.4K Aufrufe.  Children's Hospital Boston and Harvard Medical School. Hier zeigen wir die Herstellung von Kollagen-basierten, baut Gewebe mit Skelettmyoblasten. Diese 3-D konstruiert Konstrukte verwendet zu ersetzen oder zu reparieren Gewebe werden In vivo. Für unsere Zwecke haben wir diese als AV-elektrische Leitung für die Reparatur von kompletten AV-Block entwickelt [1].

Serie: Fabrication of Myogenic Engineered Tissue Constructs

PDMS Device Fabrication and Surface Modification

PDMS Bauelementherstellung und Oberflächenmodifizierung

Forschung

Biologie

Fabrication of the Thermoplastic Microfluidic Channels

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using polymer/nanoparticle composite microscale lysis and solid phase extraction columns. The recovered samples are concentrated, small volume samples that are PCRable, without any additional cleanup. Here, we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then, we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns. We demonstrate grafting and polymerization of a carbon nanotube/polymer composite column for bacterial cell lysis. We then show the lysis process followed by solid phase extraction of nucleic acids from the sample on chip using a silica/polymer composite column. The attached protocols contain detailed instructions on how to make both lysis and solid phase extraction columns.

Here we demonstrate how to fabricate thermoplastic microfluidic chips using hot embossing and heat sealing. Then we demonstrate how to use in situ light directed surface grafting and polymerization through the sealed chip to form the composite solid phase columns.

Die Herstellung der thermoplastischen Mikrofluidikkanälen

In our lab, we have successfully isolated nucleic acids directly from microliter and submicroliter volumes of human blood, urine and stool using ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

Aktin Co-Sedimentation Assay; für die Analyse von Protein-Bindung an F-Aktin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Fabrication of Amperometric Electrodes

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we describe the techniques needed to generate low noise (&lt;0.5 pA) electrodes.  The techniques have been modified from published descriptions by previous researchers (1,2).  Electrodes are made by preparing carbon fibers and threading them individually into each capillary tube by using a vacuum with a filter to aspirate the fiber.  Next, the capillary tube with fiber is pulled by an electrode puller, creating two halves, each with a fine-pointed tip.  The electrodes are dipped in hot, liquid epoxy mixed with hardener to create an epoxy-glass seal.  Lastly, the electrodes are placed in an oven to cure the epoxy.  Careful handling of the electrodes is critical to ensure that they are made consistently and without damage.  This protocol shows how to fabricate and cut amperometric electrodes for recording from single cells.

This protocol describes how to generate carbon fiber electrodes. The electrodes are subsequently used to detect catecholamine release from vesicles with carbon fiber amperometry.

Herstellung von amperometrische Elektroden

Carbon fiber electrodes are crucial for the detection of catecholamine release from vesicles in single cells for amperometry measurements.  Here, we ...

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.

Here, we describe a cardiac surgical procedure to implant engineered tissue in the atrioventricular (AV)-groove of an adult Lewis rat.

Die Implantation von Engineered Tissue in der Ratte Herz

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Ein System zur Kultivierung Iris Pigmentepithelzellen nach Lens Regeneration in Newt Study

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism&prime;s program for development1, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics. 
Reporter transgenes provide a powerful method to study the in vivo function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE&prime;s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7 has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster to insert transgenes into specific genome landing sites8-10. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13 feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols. 
Here, we present a general protocol to quantitatively evaluate a CRE&prime;s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE&prime;s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.

Quantitative Comparison of cis-Regulatory Element CRE Activities in Transgenic Drosophila melanogaster

Phenotypic variation for traits can result from mutations in cis-regulatory element (CRE) sequences that control gene expression patterns. Methods derived for use in Drosophila melanogaster can quantitatively compare the levels of spatial and temporal patterns of gene expression mediated by modified or naturally occurring CRE variants.

Quantitativen Vergleich der Cis-Regulatory Element (CRE) Aktivitäten in transgenen Drosophila melanogaster</em

Gene expression patterns are specified by cis-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

Maus Sperma Kryokonservierung und Recovery mit der I · Cryo-Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

Herstellung und Verwendung von Mikroumgebung Microarrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering homologen Rekombination Konstrukte in<em&gt; Drosophila</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Herstellung von Myogenic Engineered Tissue Konstrukte

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