Die Autoren wollen Yabin Wu für die Kultivierung der Gewebe in Abbildung 2 dargestellt danken, wurde das Bild von Bart van Overbeeke genommen. Die Arbeit wurde finanziell unterstützt durch SenterNovem, Gewährung ISO 42022 unterstützt.

Ein. Kultur Murine Myoblast Vorläuferzellen oder C2C12 Zellen <ol><li> Isolat Zellen gemäß dem Protokoll anfänglich durch Shefer und Kollegen 10 veröffentlicht und später von Collins et al. 11 und Boonen et al. 12 angepasst und speichern diese in flüssigem Stickstoff. Dies erfordert Mäusen, zB C57Bl / 6. Alternative Verfahren sind in anderen Labors verwendet werden, zB ein Verfahren, in Journal of Visualized Experiments durch Li Y et al. 13. Für die Reagenzien und Ausrüstung, die Sie in der Tabelle auf Seite 7 und 8 bezeichnet. Von den Muskel aus einer Maus, die Sie in der Regel erhalten, genügend Zellen auf etwa 20 Ampullen in flüssigem Stickstoff Kryokonservierung. Als nächstes starten wir das Protokoll, um die Zellen aus dem flüssigen Stickstoff Speicher auftauen. </li><li> Platzieren der Zellen von 1 Fläschchen in einen 25 cm 2 Matrigel (1 mg / ml) beschichteten Gewebekulturplatten eingewogen und Wachstumsmedium (GM), die aus (335 ml DMEM fortgeschrittene, 100 ml fötalem bovine Serum (FBS), 50 ml HS, 5 ml Penicillin / Streptomycin, 5 ml L-Glutamin, 5 ml Hühnerembryo Extrakt (CEE)). </li></ol> Hinweis: Mantel für 2 Stunden bei Raumtemperatur und entfernen Sie die Matrigel durch Aspiration. <ol start="3"><li> Subkultur die Zellen etwa 1:3 alle 3 Tage. Das bedeutet: am Tag 3: Passage Zellen aus einem 25 cm 2 auf eine 75 cm 2-Kolben, am Tag 6: passage Zellen aus einem 75 cm 2 Kolben auf zwei 150 cm 2-Kolben (mit 30 min Vorabscheidungsbehandlung in unbeschichteten Kolben). Am Tag 9 Transfer ein 150 cm 2 Kolben 1 Dreibettzimmer 150 cm 2-Kolben oder falls nicht verfügbar zu drei 150 cm 2 Flaschen (falls erforderlich Vorbeschichtungszyklus wiederum abhängig vom Phänotyp der Zellen). Am Tag 12 die Zellen sind bereit für die Aussaat in einen Muskel Konstrukt. </li></ol> Hinweise: - Per Triple 150 cm 2 die Anzahl der Zellen wird etwa 4,5 x 10 6 Zellen. - Über Verwendungls aus unterschiedlichen Isolierungen zu mischen, um eine gemischte Population von Zellen zum Zeitpunkt der Aussaat zu erhalten. Hinweis: Wenn Sie nicht mit primären Zellen arbeiten wollen, sind C2C12 Myoblasten eine gute Alternative. 2. Engineering Skeletal Muscle Tissues <ol><li> Bereiten Silikonkleber durch Mischen des &quot;Elastomer&quot; mit dem &quot;Härter&quot; (10:1). Cut + / - 5 mm quadratische Segmente von Klett mit einem dreieckigen Seitenteilen (Haus-Form; Abbildung 1). Leim das Klettelement in die Wells einer 6-Well-Kulturplatte in einem Abstand von etwa 12 mm zwischen den Quadraten. </li></ol> Hinweise: - Verwenden Sie nur die weiche Seite des Velcro und das Gesicht dieser Seite nach oben. - Sicherstellen, dass die Dächer einander gegenüberliegen. - Nur decken den Klettverschluss mit Silikonkleber, nicht Kleber während der gut verteilt. - Für die spätere elektrische Stimulation ist es sinnvoll, die Konstrukte in vertikaler Richtung des Bohrlochs auszurichten plaß (entlang der langen Achse). <ol start="2"><li> Lassen Sie es über Nacht trocknen im Vakuumofen, nicht beheizt, vor allem um Luftblasen zu entfernen. Sterilisieren durch Zugabe von 70% igem EtOH zu den Vertiefungen, Inkubation für 15 min. Abspülen mit PBS 3x und legte unter UV für 15 min. Entfernen Sie alle PBS aus dem Brunnen und den Klettverschluss und Ort im Inkubator bis zur Verwendung. </li><li> Auftauen Matrigel-Lösung in den Kühlschrank und bereiten die Kollagenlösung auf die gewünschte Konzentration kurz bevor die 3D-Konstrukte. Verdünnt das Lager Kollagen mit steriler 0,02% Essigsäure (Endkonzentration 3,2 mg / ml). </li></ol> Hinweis: lassen Sie alles auf Eis. <ol start="4"><li> Trypsinieren die Zellen, in GM und zählen zu resuspendieren. Lassen Sie Zellen in Zentrifugenröhrchen im Inkubator. </li><li> Fügen Sie die gewünschte Menge an Collagen Stammlösung für die Zahlen von Konstrukten, die Sie an einem Rohr (gemäß Tabelle 1) machen wollen, fügen Sie 0,5 M NaOH zu dieser Kollagenlösung bis zu einem hellrosa color ist, die einen pH-Wert von 7,5. Vorsichtig mischen durch Auf-und Abpipettieren und vermeiden Blasenbildung. Dann fügen Sie die Matrigel und mischen sehr sanft, aber gründlich. Schließlich, fügen Sie die GM auf die Kollagen / Matrigel Mischung (für die entsprechenden Mengen siehe Tabelle 1). </li></ol> Hinweise: - Führen Sie jeden Schritt auf dem Eis! Matrigel und Kollagen wird leicht Gel bei steigender Temperatur. - Vorsicht, dass die Farbe der Tat ist rosa! Eine Erhöhung des pH-Wertes wird auch induzieren schnelle Gelierung. - Final Konzentration von Kollagen: 1,6 mg / ml. <ol start="6"><li> Zentrifuge die gewünschte Menge der Zellen bei 1000 rpm für 5 min und Überstand. </li></ol> Anmerkung: - Je nach Aktivität der Zellen die Anzahl der Zellen pro Konstrukt angepasst werden muss. Typischerweise liegt die Anzahl der Zellen, die zwischen 750.000 und 1.250.000 Zellen pro Konstrukt. <ol start="7"><li> Verwenden einige der Gelmischung, um das Zellpellet zu resuspendierenund Übertragung der Zellen in das verbleibende Gemisch und sorgfältig vermischt, jedoch ohne Einleiten von Luftblasen. </li><li> Nehmen die vorerhitzten Well-Platten aus dem Inkubator und Pipette 0,35 -0,4 ml der Zell-Gel-Gemisch in den ersten Klettverschluss. Dann beginnen, um die Mischung aus der Mitte zwischen den Befestigungsstellen pipettieren und reichen bis zum Klettverschluss. Schließlich verwenden den Rest des Gels zu füllen die Lücke zwischen den beiden Velcro Anker und Pipette um die Velcro Stücke. </li><li> Vorsichtig nach 5-10 min überprüfen, ob die Gele solide genug übertragen werden sollen, dh leicht abklopfen Gerichte und Sichtprüfung, wenn das Gel ist starr. Wenn ja, legen Sie sorgfältig die Gerichte in einem Inkubator. Normalerweise können sie nach zehn Minuten abgeholt. Dann wird nach 1-2 Stunden, sanft überlagern Gels mit 4 ml warmem GM. </li></ol> Hinweis:-Nehmen Sie keine heftigen Bewegungen beim Umgang mit den Platten. <ol start="10"><li> Ersetzen GM durch Differenzierung Medium (Wachstumsmediumohne Hühnchenembryoextrakt) nach 24 h und mit frischem Medium alle 2-3 Tage zu ersetzen. Am Tag 7 sollten Sie reifen orientierten Muskelfasern erhalten haben, wie sie mit Färbung für Querstreifung Entwicklung in den fusionierten Muskelfasern ausgewertet werden. </li></ol> 3. Elektrostimulation <ol><li> Sterilisieren Sie die Elektroden aus dem ionoptix Platte (siehe Tabelle von Reagenzien und Geräte) vor dem Gebrauch mit 70% Ethanol und anschließend trocken unter UV in einer Sicherheitswerkbank. Die Platte mit Elektroden auf die Kulturplatte mit den Konstrukten und decken mit dem Deckel von der Kulturplatte und Transfer in einem Inkubator. Schließen Sie das Ionoptix C-Schrittmacher mit den entsprechenden Kabeln. </li></ol> Anmerkung: sind Elektroden parallel neben den Muskel-Konstrukt (Abbildung 2) platziert. <ol start="2"><li> Übernehmen Sie die Stimulationsprotokoll wie erwartet. </li></ol> Hinweis: Wir verwenden in der Regel 4 V / CM,6ms Impulse mit einer Frequenz von 2 Hz. Ändern Kulturmedium alle 24 Stunden während der Stimulation.

<ol>
	<li>Langelaan, M. L. P., Boonen, K. J. M., Polak, R. B., 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=Meet+the+new+meat:+tissue+engineered+skeletal+muscle.">Meet the new meat: tissue engineered skeletal muscle.</a> Trends Food Sci. Tech. 21 (2), 59-66 (2010).</li><li>Shansky, J., Chromiak, J., Tatto, M., 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=A+simplified+method+for+tissue+engineering+skeletal+muscle+organoids+in+vitro.">A simplified method for tissue engineering skeletal muscle organoids in vitro.</a> In Vitro Cell Dev. Biol. Animal. 33 (9), 659-661 (1997).</li><li>Vandenburgh, H., Del Tatto, M., Shansky, J., 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+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>Yaffe, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retention+of+differentiation+potentialities+during+prolonged+cultivation+of+myogenic+cells.">Retention of differentiation potentialities during prolonged cultivation of myogenic cells.</a> Proc. Natl. Acad. Sci. U.S.A. 61 (2), 477-483 (1968).</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>Koning, M., Werker, P. M. N., vander Schaft, D. W. J., Bank, R. A., Harmsen, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-1+and+MicroRNA-206+Improve+Differentiation+Potential+of+Human+Satellite+Cells:+A+Novel+Approach+for+Tissue+Engineering+of+Skeletal+Muscle.">MicroRNA-1 and MicroRNA-206 Improve Differentiation Potential of Human Satellite Cells: A Novel Approach for Tissue Engineering of Skeletal Muscle.</a> Tissue Eng. Part A. , (2011).</li><li>Darabi, R., Pan, W., Bosnakovski, 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=Functional+myogenic+engraftment+from+mouse+iPS+cells.">Functional myogenic engraftment from mouse iPS cells.</a> Stem Cell Rev. 7 (4), 948-957 (2011).</li><li>Langelaan, M. L. P., Boonen, K. J. M., Rosaria-Chak, K. Y., 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=Advanced+maturation+by+electrical+stimulation:+Differences+in+response+between+C2C12+and+primary+muscle+progenitor+cells.">Advanced maturation by electrical stimulation: Differences in response between C2C12 and primary muscle progenitor cells.</a> J. Tissue Eng. Regen. Med. 5 (7), 529-539 (2011).</li><li>van der Schaft, D., van Spreeuwel, A. C., van Assen, H. C., Baaijens, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanoregulation+of+vascularization+in+aligned+tissue+engineered+muscle;+a+role+for+VEGF.">Mechanoregulation of vascularization in aligned tissue engineered muscle; a role for VEGF.</a> Tissue Eng. Part A. , (2011).</li><li>Shefer, G., Wleklinski-Lee, M., Yablonka-Reuveni, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+satellite+cells+can+spontaneously+enter+an+alternative+mesenchymal+pathway.">Skeletal muscle satellite cells can spontaneously enter an alternative mesenchymal pathway.</a> J. Cell. Sci. 117 (Pt. 22), 5393-5404 (2004).</li><li>Collins, C. A., Olsen, I., Zammit, P. 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=Stem+cell+function,+self-renewal,+and+behavioral+heterogeneity+of+cells+from+the+adult+muscle+satellite+cell+niche.">Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche.</a> Cell. 122 (2), 289-301 (2005).</li><li>Boonen, K. J. M., Rosaria-Chak, K. Y., Baaijens, F. P. T., van der Schaft, D. W. J., Post, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+environmental+cues+from+the+satellite+cell+niche:+optimizing+proliferation+and+differentiation.">Essential environmental cues from the satellite cell niche: optimizing proliferation and differentiation.</a> Am. J. Physiol. Cell Physiol. 296 (6), C1338-C1345 (2009).</li><li>Li, Y., Pan, H., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolating+Stem+Cells+from+Soft+Musculoskeletal+Tissues.">Isolating Stem Cells from Soft Musculoskeletal Tissues.</a> J. Vis. Exp. (41), e2011 (2010).</li><li>Boonen, K. J. M., Langelaan, M. L. P., Polak, R. B., 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=Effects+of+a+combined+mechanical+stimulation+protocol:+Value+for+skeletal+muscle+tissue+engineering.">Effects of a combined mechanical stimulation protocol: Value for skeletal muscle tissue engineering.</a> J. Biomech. 43 (8), 1514-1521 (2010).</li><li>Gawlitta, D., Boonen, K. J. M., Oomens, C. W. J., Baaijens, F. P. T., Bouten, C. V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+serum-free+culture+conditions+on+skeletal+muscle+differentiation+in+a+tissue-engineered+model.">The influence of serum-free culture conditions on skeletal muscle differentiation in a tissue-engineered model.</a> Tissue Eng. Part A. 14 (1), 161-171 (2008).</li><li>Koning, M., van Luijn, M., van der Schaft, D. W. J., 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=Human+skeletal+muscle+formation+and+engraftment+In+vivo+is+independent+of+preconditioning+In+vitro+with+HUVEC.">Human skeletal muscle formation and engraftment In vivo is independent of preconditioning In vitro with HUVEC.</a> , (2013).</li><li>Levenberg, S., Rouwkema, J., Macdonald, 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=Engineering+vascularized+skeletal+muscle+tissue.">Engineering vascularized skeletal muscle tissue.</a> Nat. Biotechnol. 23 (7), 879-884 (2005).</li><li>Koffler, J., Kaufman-Francis, K., Yulia, 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=Improved+vascular+organization+enhances+functional+integration+of+engineered+skeletal+muscle+grafts.">Improved vascular organization enhances functional integration of engineered skeletal muscle grafts.</a> Proc. Natl. Acad. Sci. U.S.A. 108 (36), 14789-14794 (2011).</li></ol>

Die Autoren haben nichts zu offenbaren.

Das Engineering von Muskelgewebe hat ein großes Potenzial für den Einsatz als eine Krankheit Modell für Wirkstoff-Screening, in der regenerativen Medizin und für die Fleischproduktion. Allerdings unterscheiden sich die Anforderungen für diese Anwendungen. Wir entschieden uns, mit einer Kombination aus Collagen und Matrigel arbeiten, weil Kollagen ermöglicht Zellausrichtung und weil die Myoblasten Vorläuferzellen erfordern das Vorhandensein von Basalmembran abgeleitete Proteine ​​wie in früheren Studien 2D <s...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Matrigel-Wachstumsfaktor reduziert </td><td> Beckton und Dickinson </td><td></td><td></td></tr><tr><td> DMEM (hoher Glucose) * </td><td> Gibco </td><td> 42430 </td><td></td></tr><tr><td> Erweiterte DMEM </td><td> Gibco </td><td> 12491 </td><td></td></tr><tr><td> Pferdeserum </td><td> Gibco </td><td> 65050-122 </td><td></td></tr><tr><td> Fötales Rinderserum </td><td> Greiner </td><td> 758075 </td><td></td></tr><tr><td> 0,45 und 0,22 um Spritzenfilter * </td><td> Whatmann (Schleicher und Scheull) </td><td> 10462100 </td><td></td></tr><tr><td> L-Glutamin </td><td> Gibco </td><td> 25030024 </td><td></td></tr><tr&gt; <td> Penicillin / Streptomycin </td><td> Gibco </td><td> 10378016 </td><td></td></tr><tr><td> Amphotericin </td><td> Gibco </td><td> 15290-018 </td><td></td></tr><tr><td> Kultur Kunststoff </td><td> Greiner </td><td></td><td> Inklusive Kulturflaschen und Pipetten </td></tr><tr><td> Hühnchenembryoextrakt </td><td> United States Biological </td><td> C3999 </td><td></td></tr><tr><td> Pasteurpipette * </td><td> Hilgenberg </td><td></td><td> Pasteurpipetten mit Verengung, mit Baumwolle, offene Spitze L: 230 mm mit Spitzendurchmesser von 0,9 - 1,1 mm </td></tr><tr><td> Pasteurpipette * </td><td> Hilgenberg </td><td></td><td> Pasteur Pipetten, mit Verengung, mit Baumwolle, offene Spitze L: 230 mm mit Spitze Durchmesser von 1,4 - 1,6 mm </td></tr><tr><td> Pasteurpipette </td><td> VWR </td><td> 612-1702 </td><td></td></tr><tr><td> Collagenase Typ I* </td><td> Sigma </td><td> C0130-16 </td><td></td></tr><tr><td> 40 um Zellsieb * </td><td> BD Falcon </td><td> 352340 </td><td></td></tr><tr><td> 19G Nadel </td><td></td><td></td><td></td></tr><tr><td> Elastomer </td><td> Dow Corning Corporation </td><td> 3097358-1004 </td><td> Silastic MDX 4-4210 # </td></tr><tr><td> Härter </td><td> Dow Corning Corporation </td><td></td><td> Silastic MDX 4-4210 # </td></tr><tr><td> Klettband </td><td> Regelmäßige Store </td><td></td><td> Sie können dies an einem regulären Geschäft zu kaufen, verwenden Sie nur die weiche Seite </td></tr><tr><td> Kollagen Typ I, Rattenschwanz </td><td> BD Biosciences </td><td> 3544236 </td><td></td></tr><tr><td> C-Pace EP Kultur Pacer </td><td> Ionoptix </td><td></td><td></td></tr><tr><td> 6-Well-Kulturschalen zur elektrischen Stimulation </td><td> Beckton Dickinson-Falcon </td><td> BD Falcon # 353846 </td><td></td></tr><tr><td> C-Dish Kulturschale Elektroden </td><td> Ionoptix </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> * Für die Isolierung von Zellen (Punkt 1.1) benötigt # Together in one kit </td></tr></tbody></table>

engineering skeletal muscle tissues from murine myoblast progenitor cells and application of electrical stimulation

Engineered Muskelgewebe kann für verschiedene Zwecke verwendet werden, die die Produktion von Geweben für den Einsatz als eine Krankheit Modell in vitro, zB um Druckgeschwüre zu studieren, für die regenerative Medizin und als Fleisch Alternative 1. Die erste gemeldete 3D Muskel-Konstrukte wurden vor vielen Jahren und machte Pioniere auf dem Gebiet sind Vandenburgh und Kollegen 2,3. Advances in Muskelgewebe Technik gemacht sind nicht nur das Ergebnis aus dem großen Gewinn an Wissen über biochemische Faktoren, Stammzellen und Vorläuferzellen, sondern sind insbesondere auf der Basis von Erkenntnissen von Forschern gewonnen, dass physische Faktoren eine essentielle Rolle bei der Kontrolle von Verhalten der Zelle spielen und Gewebe Entwicklung. State-of-the-art engineered Muskel baut derzeit von Zell-besiedelten Hydrogel Konstrukten bestehen. In unserem Labor diese in der Regel aus murine Myoblasten Vorläuferzellen, aus murinen Hinterbein Muskeln isoliert oder einem murinen Myoblasten-Zelllinie C2C12, mixed mit einer Mischung aus Kollagen / Matrigel und ausplattiert zwischen zwei Verankerungspunkten, imitiert die Muskel Bänder. Andere Zellen können ebenfalls berücksichtigt werden, z. B. alternative Zelllinien wie L6 Ratte Myoblasten 4, neonataler Muskel abgeleitet Vorläuferzellen 5, aus adulten Muskelgewebe aus anderen Spezies, wie Mensch 6 oder sogar induzierte pluripotente Stammzellen (iPS) 7 abgeleitete . Zellkontraktilität bewirkt Ausrichtung der Zellen entlang der langen Achse des Konstrukts 8,9 und Differenzierung der Muskel-Vorläuferzellen nach etwa einer Woche der Kultur. Außerdem kann die Anwendung der elektrischen Stimulation des Prozesses der Differenzierung teilweise 8 verbessern. Aufgrund seiner geringen Größe (8 x 2 x 0,5 mm) das komplette Gewebe analysiert mit Hilfe der konfokalen Mikroskopie zB Rentabilität, Differenzierung und Ausrichtung der Zellen zu überwachen. Abhängig von der spezifischen Anwendung der Bedingungen für die engineered Muskelgewebe variieren, z. B. Einsatz für die regenerative Medizin erfordert die Upscaling von Gewebe Größe und Vaskularisation, während als ein Fleisch alternative Übersetzung in andere Arten ist notwendig, zu dienen.

Das Endprodukt wird Muskel-Konstrukte wie in 3 angedeutet ist. Die Größe des Gewebes ca. 8 mm lang, 2 mm breit und 0,5 mm dick sein. Elektrische Stimulation während der Differenzierung ändert die Expression von Myosin schwere Kette-Isoformen, aber nicht wesentlich bessere Differenzierung wie durch den Differenzierungsmedium 8 induziert wird, sondern eine elektrische Stimulation kann auch am Ende des Verfahrens angewendet werden, um für die Funktionalität des Muskels zu überprüfen Da ein Muskel m...

Watch this Scientific Journal Video about Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation at JoVE.com

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

Engineered Muskelgewebe hat ein großes Potenzial in der regenerativen Medizin, als Krankheit Modell und auch als alternative Quelle für Fleisch. Hier beschreiben wir die Konstruktion von einem Muskel-Konstrukt, in diesem Fall von der Maus Myoblasten Vorläuferzellen, und die Stimulation durch elektrische Impulse.

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 ...

engineering-skeletal-muscle-tissues-from-murine-myoblast-progenitor

Research

JoVE Journal

Bioengineering

20.8K Aufrufe.  Eindhoven University of Technology, The Netherlands. Engineered Muskelgewebe hat ein großes Potenzial in der regenerativen Medizin, als Krankheit Modell und auch als alternative Quelle für Fleisch. Hier beschreiben wir die Konstruktion von einem Muskel-Konstrukt, in diesem Fall von der Maus Myoblasten Vorläuferzellen, und die Stimulation durch elektrische Impulse.

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

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

Herstellung von Micro-Gewebe mit Modulen von Collagen Gel enthaltenden Zellen

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Forschung

Biotechnik

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

Ingenieurwesen Fibrin-basierten Tissue Konstrukte von Myofibroblasten und Anwendung von Constraints und abseihen to Cell und Collagen (Re) Organisation Induce

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

Electrospun Fibrous Scaffolds of Poly(glycerol-dodecanedioate) for Engineering Neural Tissues From Mouse Embryonic Stem Cells

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among the various fabrication methods, electrospinning is the most attractive one due to its simplicity and versatility. Additionally, electrospun nanofibers mimic the size of natural extracellular matrix ensuring additional support for cell survival and growth. This study showed the viability of the fabrication of long fibers spanning a larger deposit area for a novel biodegradable and biocompatible polymer named&#160;poly(glycerol-dodecanoate) (PGD)1 by using a newly designed collector for electrospinning. PGD exhibits unique elastic properties with similar mechanical properties to nerve tissues, thus it is suitable for neural tissue engineering applications. The synthesis and fabrication set-up for making fibrous scaffolding materials was simple, highly reproducible, and inexpensive. In biocompatibility testing, cells derived from mouse embryonic stem cells could adhere to and grow on the electrospun PGD fibers. In summary, this protocol provided a versatile fabrication method for making PGD electrospun fibers to support the growth of mouse embryonic stem cell derived neural lineage cells.

Electrospun Fibrous Scaffolds of Polyglycerol-dodecanedioate for Engineering Neural Tissues From Mouse Embryonic Stem Cells

Synthesis and fabrication of electrospun long fibers spanning a larger deposit area via a newly designed collector from a novel biodegradable polymer named poly(glycerol-dodecanoate) (PGD) was reported. The fibers were able to support the growth of cells derived from mouse pluripotent stem cells.

Elektro Faser-Gerüste von Poly (Glycerin-dodecandioat) für Ingenieurnervengewebe aus embryonalen Stammzellen der Maus

For tissue engineering applications, the preparation of biodegradable and biocompatible scaffolds is the most desirable but challenging task. &#160;Among ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Die Messung der isometrischen Maximalkraft von permeabilisierten Skelettmuskelfasern generiert

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Technik Dreidimensionale Epithelgewebe Eingebettet in extrazellulären Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living components. Due to their inherent advantages, biorobots are gaining interest as alternatives to traditional fully artificial robots. Various studies have focused on harnessing the power of biological actuators, but only recently studies have quantitatively characterized the performance of biorobots and studied their geometry to enhance functionality and efficiency. Here, we demonstrate the development of a self-stabilizing swimming biorobot that can maintain its pitch, depth, and roll without external intervention. The design and fabrication of the PDMS scaffold for the biological actuator and biorobot followed by the functionalization with fibronectin is described in this first part. In the second part of this two-part article, we detail the incorporation of cardiomyocytes and characterize the biological actuator and biorobot function. Both incorporate a base and tail (cantilever) which produce fin-based propulsion. The tail is constructed with soft lithography techniques using PDMS and laser engraving. After incorporating the tail with the device base, it is functionalized with a cell adhesive protein and seeded confluently with cardiomyocytes. The base of the biological actuator consists of a solid PDMS block with a central glass bead (acts as a weight). The base of the biorobot consists of two composite PDMS materials, Ni-PDMS and microballoon-PDMS (MB-PDMS). The nickel powder (in Ni-PDMS) allows magnetic control of the biorobot during cells seeding and stability during locomotion. Microballoons (in MB-PDMS) decrease the density of MB-PDMS, and enable the biorobot to float and swim steadily. The use of these two materials with different mass densities, enabled precise control over the weight distribution to ensure a positive restoration force at any angle of the biorobot. This technique produces a magnetically controlled self-stabilizing swimming biorobot.

In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot.

Herzmuskelzellbasierter Aktuator und selbststabilisierender Biorobot - TEIL 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living ...

Cardiac Muscle Cell-based Actuator and Self-stabilizing Biorobot - Part 2

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. These devices, called biorobots, are powered solely by the force generated from the contractile activity of the living component and, due to their many inherent advantages, could be an alternative to conventional fully artificial robots. Here, we describe the methods to seed and characterize a biological actuator and a biorobot that was designed, fabricated, and functionalized in the first part of this two-part article. Fabricated biological actuator and biorobot devices composed of a polydimethylsiloxane (PDMS) base and a thin film cantilever were functionalized for cell attachment with fibronectin. Following functionalization, neonatal rat cardiomyocytes were seeded onto the PDMS cantilever arm at a high density, resulting in a confluent cell sheet. The devices were imaged every day and the movement of the cantilever arms was analyzed. On the second day after seeding, we observed the bending of the cantilever arms due to the forces exerted by the cells during spontaneous contractions. Upon quantitative analysis of the cantilever bending, a gradual increase in the surface stress exerted by the cells as they matured over time was observed. Likewise, we observed movement of the biorobot due to the actuation of the PDMS cantilever arm, which acted as a fin. Upon quantification of the swimming profiles of the devices, various propulsion modes were observed, which were influenced by the resting angle of the fin. The direction of motion and the beating frequency were also determined by the resting angle of the fin, and a maximum swim velocity of 142 &#181;m/s was observed. In this manuscript, we describe the procedure for populating the fabricated devices with cardiomyocytes, as well as for the assessment of the biological actuator and biorobot activity.

In this study, a biological actuator and a self-stabilizing, swimming biorobot with functionalized elastomeric cantilever arms are seeded with cardiomyocytes, cultured, and characterized for their biochemical and biomechanical properties over time.

Herzmuskelzell-basierter Aktuator und selbststabilisierender Biorobot - Teil 2

In recent years, hybrid devices that consist of a living cell or tissue component integrated with a synthetic mechanical backbone have been developed. ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Mikrofluidische Bioprinting für Engineering durchblutet, Gewebe und Organellen

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Two Methods for Decellularization of Plant Tissues for Tissue Engineering Applications

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor biocompatibility, and cost. Plant tissues have favorable characteristics that make them uniquely suited for use as scaffolds, such as high surface area, excellent water transport and retention, interconnected porosity, preexisting vascular networks, and a wide range of mechanical properties. Two successful methods of plant decellularization for tissue engineering applications are described here. The first method is based on detergent baths to remove cellular matter, which is similar to previously established methods used to clear mammalian tissues. The second is a detergent-free method adapted from a protocol that isolates leaf vasculature and involves the use of a heated bleach and salt bath to clear the leaves and stems. Both methods yield scaffolds with comparable mechanical properties and low cellular metabolic impact, thus allowing the user to select the protocol which better suits their intended application.

Here we present, and contrast two protocols used to decellularize plant tissues: a detergent-based approach and a detergent-free approach. Both methods leave behind the extracellular matrix of the plant tissues used, which can then be utilized as scaffolds for tissue engineering applications.

Zwei Methoden zur Decellularization von Pflanzengewebe für Tissue-Engineering-Anwendungen

The autologous, synthetic, and animal-derived grafts currently used as scaffolds for tissue replacement have limitations due to low availability, poor ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells&#039; Cardiomyogenic Potential

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Gleichzeitige elektrische und mechanische Stimulation, Cells Cardiomyogenic Potenzial zu steigern

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...