Tracy C. Grikscheit, Erik R. Barthel, und Frédéric G. Sala von der California Institute for Regenerative Medicine (CIRM), Gewährung Zahlen RN2-00946-1 (TCG) und TG2-01.168 (ERB, FGS) unterstützt. Allison L. Speer ist eine Gesellschaft der Universität Surgeons Ethicon Gelehrter. Yasuhiro Torashima wird von einem Kinderkrankenhaus in Los Angeles Saban Institut Forschung Career Development Fellowship gefördert.

Ein. Organoid Units Vorbereitung <ol><li> Instrumente geeignet für Maus Dissektion (Schere und Pinzette) sollte im Autoklaven sterilisiert werden. </li><li> Menschlich einschläfern den Spender Maus nach lokalen IACUC Protokolle. Stellen Sie sicher, dass das Tier tot ist, bevor Sie fortfahren. </li><li> Einen Mittellinieneinschnitt um Zugang zu dem Peritonealraum gewinnen. Hautlappen können reflektiert je nach Bedarf, um die Exposition zu verbessern. </li><li> Ausweiden des Dünndarms und teilen sie knapp distal des Lig. Treitz. Trennen Sie den Dünndarm von seinem Mesenterium mit scharfen und sanften stumpf. Identifizieren Sie die ileocecal Kreuzung und unterteilen den Dünndarm 5 mm proximal dazu. </li><li> Mit Schere, öffnen Sie den Darm längs der Kraterbildung Grenze in einer Petrischale mit 10 ml 4 ° C, sterile Hanks &#39;Salzlösung (HBSS, Invitrogen, Carlsbad CA) / 1X Antibiotika-Antimykotika (Anti-Anti, Invitrogen) Lösung. Löschen Fäkalien aus dem geöffneten Darm mitvorsichtiges Schütteln dann übertragen eröffnet Darm zu einem 15ml Zentrifugenröhrchen mit 10 ml 4 ° C, sterile HBSS / 1X Anti-Anti. </li><li> Waschen Sie die geöffneten Darm dreimal in 10 ml 4 ° C, sterile HBSS / 1X anti-anti in einem Reagenzglas. Jede Wäsche kann mit mildem Schütteln der 15 ml Tube für 30 sec durchgeführt werden. Nach Schütteln, sinkt das Darmgewebe dem Boden des Röhrchens. Entsorgen schwimmenden Material, das mesenchymalen Fremdkörpern ist. Entfernen Sie die Waschlösung vorsichtig mit einer Pipette. </li><li> Mince des gewaschenen Darm in einer Petrischale mit 10 ml 4 ° C, sterile HBSS / 1X anti-anti auf weniger als 1 mm quadratische Stücke mit einer Schere. Sammeln Sie die gehackte Material mit einer automatischen Pipette und legen Sie sie in ein Reagenzglas. </li><li> Zentrifugieren bei 500 Upm für 8 min. Überstand verwerfen, die Fett-und Bindegewebes enthält. </li><li> Verdauen zerkleinertem, gewaschenem Material mit 10 ml sterilem HBSS / 1X anti-anti plus 0,125 mg / ml Dispase (Invitrogen) und 800 Einheiten / ml Collagenase Typ 1 (Worthington, Lakewood NJ). Bis 40 ml der Aufschlusslösung vorzubereiten, abwiegen 5 mg Dispase, 142 mg Kollagenase, und fügen Sie sterile HBSS bis zu einem Volumen von 40 ml. Diese Lösung jeweils frisch organoide Einheiten vorbereitet sind, und halten bei 4 ° C bis zum Gebrauch. Fügen Sie die Aufschlusslösung direkt an den Pellet aus Schritt 1.8. </li><li> Inkubieren der Test-Röhrchen mit dem zerkleinerten, gewaschenen Materials mit der Aufschlußlösung bei 37 ° C für 20 min. </li><li> Rufen Sie das Reagenzglas und weiter stören die verdaute Gewebe durch Verreiben mit einer 10 ml Pipette. Wiederholen zwischen 20 bis 50 mal, bis ein einheitliches Erscheinungsbild entsteht. </li><li> Zentrifuge das Reagenzglas 5 min bei 800 UpM. Überstand verwerfen, die einzelne Zellen enthält. </li><li> Stoppen des Verdaureaktion mit 10 ml 4 ° C, steril Dulbeccos modifiziertem Eagle Medium (DMEM, Invitrogen) plus 10% v / v hitzeinaktiviertem fötalem Rinderserum (FBS HALLO-, Invitrogen). Resuspendieren pellet und schütteln Sie die Röhre. </li><li> Zentrifuge das Reagenzglas 5 min bei 800 UpM. Entfernen Sie den Überstand vorsichtig mit einer automatischen Pipette bis zu den letzten paar Tropfen. Verwenden Sie eine Einweg-Kunststoff-Pipette für den letzten Tropfen zu vermeiden Resuspendieren des Pellets. </li></ol> 2. Laden von Polyglykolsäure Scaffold <ol><li> Form 2-mm lang, 5-mm Außendurchmesser zylindrischen Gerüste aus Vlies Polyglykolsäure (2-mm Blechdicke, 60 mg cm-3 Schüttdichte; Porosität&gt; 95%, Concordia Fibers, Coventry RI) wie in Lit. beschrieben. 4. </li><li> Schneiden Sie die distalen 2 mm eines Einweg 1.000 Mikroliter Pipette mit einer Schere mit 70% Ethanol in destilliertem Wasser zubereitet kippen. </li><li> Legen Sie das Gerüst in ein 4-Loch-Kulturplatte. Laden der organoiden Einheiten auf das Stützgerüst mit der 1000 Mikroliter-Pipette, zuerst in das Lumen und dann auf die äußere Oberfläche. Verwenden Sie eine Pinzette Beschichtung des Lumen zu gewährleisten. Nicht stören oder Aufbrechen der zylindrischen Form des Polymers. </li&gt; </ol> 3. Implantation in Wirt Maus <ol><li> Verwenden Sie einen syngenen Host Maus auf dem gleichen Hintergrund wie der Spender, falls verfügbar. Ansonsten beschäftigen einen immungeschwächten nonobese diabetischen / schwere kombinierte Immunschwäche oder NOD / SCID Tier (Jackson Laboratories, Sacramento CA). </li><li> Erbrechen Vollnarkose mit Isofluran. Rasur, prep und drapieren der Maus den Bauch. </li><li> Eine 5 mm Mittellinieneinschnitt zum Eingang in die Bauchhöhle zu gewinnen. Identifizieren und sorgfältig ausweiden das Omentum. Setzen Sie den geladenen Polymers auf das Omentum und wickeln Sie es mit dem Gewebe. Nicht reißen die Omentum. </li><li> Sichern Sie das Polymer auf die Omentum mit einem 5-0 Monocryl Naht. Gently ersetzen Omentum mit dem gewickelt Polymer in seine anatomische Position. </li><li> Schließen Sie den Bauchschnitt in Schichten mit 4-0 Vicryl Nähte. Führen Sie den Muskel-Verschluss und kümmern sich nicht um die Bauchorgane unterhalb des Einschnitts zu verletzen. Verwenden Knopfnähte für die Haut. </li><li> Verwalten postoperative Analgesie mit 2 mg / kg Ketoprofen (Ketofen, Fort Dodge Animal Health) in sterilem Wasser als subkutane Quaddel neben dem Einschnitt. Die Tiere sollten täglich ausgewertet werden, und wenn das Tier zeigt Anzeichen von Schmerzen oder Leiden, eine zusätzliche Dosis von Ketoprofen können auf postoperativen Tag 2 verabreicht werden. Mit dem dritten postoperativen Tag, sollte das Tier komplett ohne Anzeichen von Schmerzen oder Leiden wiederhergestellt werden. Wenn Schmerzen oder Leiden weiter auf postoperativen Tag 3, wird dies als abnormal und sollte in Übereinstimmung mit den IACUC und Tier Betreuungseinrichtung Protokollen angesprochen werden. </li><li> Lassen Sie die Maustaste zu erholen und das Tissue-Engineering-Darm für vier Wochen wachsen. Geben Sie das Tier ad libitum Zugang zu Nagetierfutter (Lab Diet 5001, PMI Nutrition, St. Louis MO) und Wasser mit Septra 200 mg / 40 mg pro 5 ml (Hallo-Tech Pharmacal, Amityville NY) bei einer Verdünnung von 1:100. </li></ol> 4. Ernte <ol><li> Humanely euthanize das Wirtstier vier Wochen nach der Implantation. </li><li> Erneuten Öffnen der ursprünglichen Einschnitt und spiegeln die Haut kopfwärts Zugang zur Bauchhöhle erleichtern. </li><li> Öffnen Sie die Muskulatur und Identifizierung der Tissue-Engineering-Konstrukt als eine Kugel von Gewebe. </li><li> Nehmen Sie sich Verwachsungen der Konstrukt aus intraabdominellen Eingeweide mit scharfen Dissektion. </li><li> Befestigen Sie das Konstrukt in Formalin für spätere Paraffin Montage oder Verwendung das Gewebe frisch biochemischen Assays wie real-time PCR-oder Protein-Isolation. </li></ol>

<ol>
	<li>Grikscheit, T. C., Siddique, A., Ochoa, E. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+small+intestine+improves+recovery+after+massive+small+bowel+resection.">Tissue-engineered small intestine improves recovery after massive small bowel resection.</a> Ann. Surg. 240, 748-754 (2004).</li><li>Wales, P. W., Christison-Lagay, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+bowel+syndrome:+epidemiology+and+etiology.">Short bowel syndrome: epidemiology and etiology.</a> Sem. Ped. Surg. 19, 3-9 (2010).</li><li>Evans, G. S., Flint, N., Somers, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+a+method+for+the+preparation+of+rat+intestinal+epithelial+cell+primary+cultures.">The development of a method for the preparation of rat intestinal epithelial cell primary cultures.</a> J. Cell Sci. 101, 219-231 (1992).</li><li>Sala, F. G., Matthews, J. A., Speer, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicellular+approach+forms+a+significant+amount+of+tissue-engineered+small+intestine+in+the+mouse.">A multicellular approach forms a significant amount of tissue-engineered small intestine in the mouse.</a> Tiss. Eng. Part A. 17, 1841-1850 (2011).</li><li>Speer, A. L., Sala, F. G., Matthews, J. A., Grikscheit, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+tissue-engineered+stomach+demonstrates+epithelial+differentiation.">Murine tissue-engineered stomach demonstrates epithelial differentiation.</a> J. Surg. Res. 171, 6-14 (2011).</li><li>Haxhija, E. Q., Yang, H., Spencer, A. U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+epithelial+cell+proliferation+is+dependent+on+the+site+of+massive+small+bowel+resection.">Intestinal epithelial cell proliferation is dependent on the site of massive small bowel resection.</a> Pediatr. Surg. Int. 23, 379-390 (2007).</li><li>Zhao, L., Cheng, Z., Dhall, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+corrective+pullthrough+surgery+in+a+mouse+model+of+Hirschsprung's+disease.">A novel corrective pullthrough surgery in a mouse model of Hirschsprung's disease.</a> J. Pediatr. Surg. 44, 759-766 (2009).</li><li>Petrosyan, M., Guner, Y. S., Williams, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+regarding+the+pathogenesis+of+necrotizing+enterocolitis.">Current concepts regarding the pathogenesis of necrotizing enterocolitis.</a> Ped. Surg. Int. 25, 309-318 (2009).</li><li>Shew, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+concerns+in+malrotation+and+midgut+volvulus.">Surgical concerns in malrotation and midgut volvulus.</a> Ped. Radiol. 39, S167-S171 (2009).</li><li>Sampietro, G. M., Corsi, F., Maconi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospective+study+of+long-term+results+and+prognostic+factors+after+conservative+surgery+for+small+bowel+Crohn's+disease.">Prospective study of long-term results and prognostic factors after conservative surgery for small bowel Crohn's disease.</a> Clin. Gastroenterol. Hepatol. 7, 183-191 (2009).</li><li>Klempnauer, J., Grothues, F., Bektas, H., Pichlmayr, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+results+after+surgery+for+acute+mesenteric+ischemia.">Long-term results after surgery for acute mesenteric ischemia.</a> Surgery. , 121-239 (1997).</li><li>Fitzgibbons, S. C., Jones, B. A., Hull, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+biopsy-proven+parenteral+nutrition-associated+liver+fibrosis+and+biochemical+cholestasis+in+children+with+short+bowel+syndrome.">Relationship between biopsy-proven parenteral nutrition-associated liver fibrosis and biochemical cholestasis in children with short bowel syndrome.</a> J. Ped. Surg. 45, 95-99 (2010).</li><li>Spencer, A. U., Kovacevich, D., McKinney-Barnett, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pediatric+short+bowel+syndrome:+the+cost+of+comprehensive+care.">Pediatric short bowel syndrome: the cost of comprehensive care.</a> Am. J. Clin. Nutr. 88, 1552-1559 (2008).</li><li>Kato, T., Tzakis, A. G., Selvaggi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+and+multivisceral+transplantation+in+children.">Intestinal and multivisceral transplantation in children.</a> Ann. Surg. 243, 756-766 (2006).</li><li>Reyes, J., Bueno, J., Kocoshis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+intestinal+transplantation+in+children.">Current status of intestinal transplantation in children.</a> J. Ped. Surg. 33, 243-254 (1998).</li></ol>

Keine Interessenskonflikte erklärt.

Wir präsentieren ein Protokoll zur Herstellung Tissue-Engineering-Darm der Maus über ein organoides Einheiten-on-Gerüst Ansatz. Die wichtigsten Schritte sind solche der organoiden Einheiten Zubereitung. Es ist darauf zu reinigen und ausreichend mechanisch verarbeiten das Gewebe, aber gleich ist darauf nicht overdigest oder overtriturate die organoiden Einheiten werden nach dem Verdau durchgeführt (Schritt 1.11). Wenn dies erledigt ist, die organoiden Einheiten zu Einzelzellen, die in dem Überstand von Schritt 1,12 ...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> HBSS </td><td> Gibco </td><td> 114170-112 </td><td></td></tr><tr><td> Antibiotikum-Antimykotikum 100X </td><td> Invitrogen </td><td> 15240-062 </td><td></td></tr><tr><td> Dispase </td><td> Gibco </td><td> 17105-041 </td><td></td></tr><tr><td> Collagenase Typ 1 </td><td> Worthington </td><td> LS004194 </td><td></td></tr><tr><td> DMEM high glucose 1X </td><td> Gibco </td><td> 11995-065 </td><td></td></tr><tr><td> Hitzeinaktiviertem FBS </td><td> Invitrogen </td><td> 16140-071 </td><td></td></tr><tr><td> Biofelt 100% PGA </td><td> Concordia Medical </td><td> FELT01-1005 </td><td> Für Polymer Zubereitung nach Ref.4 </td></tr><tr><td> Poly-L-Milchsäure </td><td> Durect </td><td> B6002-1 </td><td> Für Polymer Zubereitung nach Ref. 4 </td></tr><tr><td> Typ-I-Kollagen, Rattenschwanz </td><td> Sigma-Aldrich </td><td> C3867-1VL </td><td> Für Polymer Zubereitung nach Ref. 4 </td></tr><tr><td> Ketoprofen 100 mg / ml </td><td> Fort Dodge Animal Health </td><td> 71-KETOI-100-50 </td><td></td></tr><tr><td> LabDiet 5001 Nagetierfutter </td><td> LabDiet </td><td> 5001 </td><td></td></tr><tr><td> Septra 200 mg / 40 mg pro 5 ml, USP </td><td> Hallo-Tech Pharmacal </td><td> 50383-824-16 </td><td></td></tr><tr><td> Isofluran, USP </td><td> Phoenix Pharmaceuticals </td><td> 57319-507-06 </td><td></td></tr></tbody></table>

tissue engineering of the intestine in a murine model

Tissue-Engineering Dünndarm (TESI) erfolgreich verwendet worden, um Lewis Ratten nach massiven Dünndarmresektion zu retten, was im Gegenzug die präoperative Gewichte innerhalb von 40 Tagen. 1 Bei Menschen, massive Dünndarmresektion in Kurzdarmsyndrom, eine funktionelle malabsorptive führen Staat, der erheblichen Morbidität, Mortalität und Kosten im Gesundheitswesen einschließlich der parenteralen Ernährung abhängig, Leberversagen und Zirrhose, und die Notwendigkeit für multiviszerale Organtransplantation. 2 In diesem Papier verleiht, beschreiben und dokumentieren wir unser Protokoll für die Erstellung von Tissue-Engineering-Darm in einem Mausmodell mit einem vielzelligen organoide Einheiten-on-Gerüst Ansatz. Organoiden Einheiten sind mehrzellige Aggregate aus dem Darm abgeleitet, die sowohl mukosale und mesenchymalen Elemente enthalten, 3 die Beziehung zwischen denen die intestinale Stammzellnische bewahrt. 4 In laufende und zukünftige Forschung, der Übergang der Technik in derMaus zur Untersuchung der Vorgänge während TESI Bildung durch die Nutzung der transgenen Instrumenten in dieser Art beteiligt zu erlauben. 5 Die Verfügbarkeit von immungeschwächten Mausstämmen auch erlauben uns, die Technik für die menschliche Darmgewebe anwenden und optimieren die Bildung des menschlichen TESI als Maus-Xenograft vor dessen Übergang in den Menschen. Unsere Methode beschäftigt Good Manufacturing Practice (GMP)-Reagenzien und Materialien, die bereits für die Verwendung bei menschlichen Patienten zugelassen wurden, und bietet somit einen deutlichen Vorteil gegenüber Ansätzen, die auf dezellularisierten tierischen Geweben verlassen. Das ultimative Ziel dieser Methode ist die Übersetzung für den Menschen als regenerative Medizin therapeutische Strategie für Kurzdarmsyndrom.

Abbildung 1 zeigt eine Gesamtansicht Schema für das Protokoll dokumentiert. Das Endergebnis dieses Protokolls ist eine Kugel oder kugelförmige Struktur des Tissue-Engineering murinen Darm mit einem Lumen, Schleimhaut, submucosa, Muscularis und Umgebung. 2A zeigt eine typische Globus im Vergleich zum Ausgangsmaterial Polymergerüst. 2B zeigt dasselbe Konstrukt zweiklappigen scharf zu sein Lumen offenbaren. Abbildung 3 zeigt eine Hämatoxylin / Eosin-ge...

Watch this Scientific Journal Video about Tissue Engineering of the Intestine in a Murine Model at JoVE.com

Tissue Engineering of the Intestine in a Murine Model

Dieser Artikel und das dazugehörige Video präsentieren unser Protokoll zur Erzeugung von Tissue-Engineering-Darm der Maus, mit einem organoiden Einheiten-on-Gerüst Ansatz.

Tissue Engineering des Darms in einem Mausmodell

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

tissue-engineering-of-the-intestine-in-a-murine-model

Research

JoVE Journal

Bioengineering

14.8K Aufrufe.  Keck School of Medicine of the University of Southern California. Dieser Artikel und das dazugehörige Video präsentieren unser Protokoll zur Erzeugung von Tissue-Engineering-Darm der Maus, mit einem organoiden Einheiten-on-Gerüst Ansatz.

Serie: Tissue Engineering of the Intestine in a Murine Model

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Verkapselung von Kardiomyozyten in einer Fibrin-Hydrogel für Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Forschung

Biotechnik

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Postproduction Verarbeitung von Elektrogesponnene Fasern für Tissue Engineering

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Engineering Skeletal Muscle Gewebe von Murine Myoblast Progenitor Cells und Anwendung von Elektrostimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Entwurf eines Biaxiale Mechanische Belastung Bioreaktor für Tissue Engineering

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Tissue Engineering eines Menschen 3D<em&gt; In-vitro-</em&gt; Tumor-Testsystem

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Erzeugung eines dreidimensionalen Vollhaut Equivalent und automatisierte Verwundung

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Engineering 3D cellularized Kollagengele für Gefäßgeweberegeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Tissue Engineering von Intrinsic Vaskularisation in ein<em&gt; In Vivo</em&gt; Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

The Arteriovenous (AV) Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in pathological situations such as cancer, vascularization plays a leading role in disease progression. Consequently, there is a strong need for a standardized and well-characterized in vivo model in order to elucidate the mechanisms of neovascularization and develop different vascularization approaches for tissue engineering and regenerative medicine.
We describe a microsurgical approach for a small animal model for induction of a vascular axis consisting of a vein and artery that are anastomosed to an arteriovenous (AV) loop. The AV loop is transferred to an enclosed implantation chamber to create an isolated microenvironment in vivo, which is connected to the living organism only by means of the vascular axis. Using 3D imaging (MRI, micro-CT) and immunohistology, the growing vasculature can be visualized over time. By implanting different cells, growth factors and matrices, their function in blood vessel network formation can be analyzed without any disturbing influences from the surroundings in a well controllable environment. 
In addition to angiogenesis and antiangiogenesis studies, the AV loop model is also perfectly suited for engineering vascularized tissues. After a certain prevascularization time, the generated tissues can be transplanted into the defect site and microsurgically connected to the local vessels, thereby ensuring immediate blood supply and integration of the engineered tissue. By varying the matrices, cells, growth factors and chamber architecture, it is possible to generate various tissues, which can then be tailored to the individual patient's needs.

The Arteriovenous AV Loop in a Small Animal Model to Study Angiogenesis and Vascularized Tissue Engineering

We describe a microsurgical approach for the generation of an arteriovenous (AV) loop as a model for analyzing vascularization in vivo in an isolated and well-characterized environment. This model is not only useful for the investigation of angiogenesis, but is also optimally suited for engineering axially vascularized and transplantable tissues.

Die arteriovenöse (AV) Loop in einem Kleintiermodell zur Untersuchung Angiogenesis und Vaskularisierte Tissue Engineering

A functional blood vessel network is a prerequisite for the survival and growth of almost all tissues and organs in the human body. Moreover, in ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Behandlung von Ligamentenkonstrukten mit ausüberschüssigem Serum: Ein Translations-Gewebe-Engineering-Modell

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...