Name: automated robotic dispensing technique for surface guidance and bioprinting of cells
Uploaded: 2016-11-18T13:00:00.000+00:00
Duration: 10 min 14 s
Description: Watch this Scientific Journal Video about Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells at JoVE.com

The work presented here is supported by the Singapore National Research Foundation under CREATE program (NRF-Technion): The Regenerative Medicine Initiative in Cardiac Restoration Therapy Research Program and by the Public Sector Funding (PSF) 2012 from the Science and Engineering Research Council (SERC) under the Agency for Science, Technology and Research (A*STAR).

Hinweis: Dieses Protokoll beschreibt die Verwendung eines Rückdruck unterstützt Roboter - Dispensorsystem (1A) als Oberflächenätzung (1B) und Extrusionsbasis bioprinter (1C) 10. 1. Änderung eines Polystyrol-Oberfläche <ol><li> Verwenden Sie 1 mm Polystyrolplatten, da Polystyrol-Gewebekulturplatten neigen dazu, nach oben in der Mitte zu beugen, ruinieren die Höhe Konsistenz beider Ätzen und Drucken. Hinweis: Da die Polystyrolplatten nicht für Zelladhäsion modifiziert sind, eine Plasmabehandlung durchgeführt wird. </li><li> Sauerstoffplasmabehandlung. <ol><li> Wählen Sie zunächst einen Druck von 2 bar auf den Regulator des Sauerstoffflasche auf dem Plasma-Maschine angeschlossen. Dann schalten Sie den Plasma-Maschine auf und geben Sie die folgenden Bedingungen in das Bedienfeld der Maschine: 150 W, 30 SSCM Sauerstoff, 10 min (für Strom, Gasströmung und Zeit beziehungsweise). </li><li> Legen Sie das Polystyrol substrate in die Plasmakammer, die Tür verschließen und den Startknopf auf dem Bedienfeld drücken. Einweichen des Sauerstoffplasma behandelt Polystyrolsubstrat in fötalem Rinderserum und inkubieren bei 37 ° C für 2 h vor dreimal 1x phosphatgepufferter Kochsalzlösung gewaschen (PBS). </li></ol></li></ol> 2. Programmierung Print Patterns <ol><li> Verwenden Sie das Programm zunächst Etch die Oberfläche mit einem Stylus oder Skalpell. <ol><li> Wenn Sie einen Stift verwenden, legen Sie einen Durchmesser von 1,5 mm Textil-Nadel (von innen mit großer Sorgfalt) in die Düse einer Abgabespritze (entweder 5 oder 10 ml), bis er eingeklemmt und gesichert wird. Bei Verwendung eines Skalpells, wählen Sie eine Runde behandelt Skalpell so dass er in den Klemmmechanismus des Druckarmes befestigt werden kann. HINWEIS: Der Stift gekrümmte Muster besser als die Skalpellklinge ätzt. </li><li> Wenn zuerst eine bioprinted Anordnung zu schaffen versucht, skizzieren das gewünschte Muster auf Millimeterpapier mit nummerierten Achsen der XY-Koordinaten zu erzeugen. Dann input die Koordinaten des geätzten / bioprinted Muster in das Tabellenkalkulationsprogramm (Abbildung 2). HINWEIS: Die &quot;gewünschte Muster&quot; kann in vielen verschiedenen Formen sein, wie beispielsweise linear, S-förmig oder kreisförmig. Die Wahl hängt von dem experimentellen Modell erforderlich ist, wie beispielsweise parallele lineare Linien für die Differenzierung von MSCs zu Cardiomyozyten wie hier demonstriert. Die Streudiagramm-Funktion ermöglicht die Visualisierung der vorgeschlagenen Handlung Muster. </li><li> Öffnen Sie die Druck / Ausgabe-Software. Wählen Sie &quot;Programm&gt; Programm hinzufügen&quot;, gefolgt von &quot;Bearbeiten&gt; Punkt hinzufügen&quot;, um das Programm einzurichten. Exportieren Sie die x- und y-Koordinatenwerte aus der Tabelle in den erhaltenen Druck / Spende Software die Verwendung von &quot;Copy and Paste&quot; Funktion. </li><li> Kalibrieren Sie die &quot;z&quot; Höhe des Roboters vor jedem Lauf, um den Stift / print Düse auf die Oberfläche zu platzieren. <ol><li> In der Druck- / Spende Software, wählen Sie die &quot;Robot&quot; Option, klicken Sie auf &quot;Ändern Mode &amp;# 34; und aktivieren Sie die &quot;Lernmodus&quot; -Option. Einmal ausgewählt, ermöglicht die Software die Funktion &quot;Tippen&quot; des Roboters. </li><li> Joggen, initialisieren zuerst den Roboter in seine Standardposition, indem Sie die folgenden Befehle in der Menüleiste ausgewählt wird; &quot;Robot&gt; Meca initialisieren&quot;, und wählen Sie &quot;Robot&gt; Jog&quot;. Eingabe der numerischen Werte (in mm) in den &quot;X und Y Slots&quot; erforderlich, um den Stift genau auf den Ursprung des Musters zu platzieren. </li><li> Als nächstes Eingang in der &quot;Z-Slot&quot; ein Zahlenwert (in mm) mit der Oberfläche mit dem Stift oder Druckdüse in Kontakt zu bringen, aber nicht biegen oder die Oberfläche einrücken. Dieser Punkt wird als &quot;Z&quot; Startwert bezeichnet. HINWEIS: Die Tiefe jeder Rille kann leicht die Z-Höhe des Systems unter Verwendung variiert werden. Nuten 40, 80 und 170 &amp; mgr; m gezeigt , in die Oberfläche von 1 mm dicke Polystyrolplatten (4 und Tabelle 1) zu schneiden. </li></ol></li><li> Wählen Sie die prucken Anweisung für jede der Koordinatenpunkte, das heißt &quot;Start of Line Dispense&quot; den ersten Punkt und Druck Einleitung zu definieren &quot;Linie , die&quot; die Zwischenpunkte zu bestimmen und &quot;End of Line Dispense&quot; an den Roboter zu signalisieren , das zu beenden Druckauflage. </li><li> Kommunizieren Sie das Programm an den Roboter durch die Folgebefehle in der oberen Menüleiste auswählen: &quot;Robot&gt; Senden C &amp; T Data&quot;. </li><li> Starten Sie die Radierung / Druckauflage durch den Roboter in die Betriebsart &quot;Run&quot; zu ändern. Tun Sie dies, indem Sie &quot;Robot&gt; Ändern Modus&gt; Run-Modus&quot; in der oberen Menüleiste. </li><li> Starten Sie den Druckvorgang mit der grünen &quot;Start-Taste&quot; auf dem Roboter Spender Konsole drücken. </li></ol></li></ol> 3. Herstellung und Druck von zellhaltigen Gelatine Bioink <ol><li> Löse 2% Gelatine in Minimum Essential Medium Alpha Medium (&amp; agr; MEM) (mit 10% FBS ergänzt und 2% Antibiotikum / antimycotic) bei 60 ° C für 2 Stunden die bioink Lösungen herzustellen. </li><li> Vorkultur der Red Fluorescent Protein exprimieren mesenchymalen Stammzellen (RFP-MSCs) bis 70% Konfluenz in 10 cm Gewebekulturschalen mit &amp; agr; MEM / 10% FBS. Lösen der anhaftenden Zellen in Suspension durch Entfernen des Mediums und Beschichtung mit 1x Trypsin-EDTA-Lösung für 5 min bei 37 ° C. </li><li> Pellet die Zellen durch Zentrifugation bei 1000 × g für 5 min und entfernen Sie den Überstand. Zellpellet in 0,5 ml 1x PBS und die Zelldichte zählen eine Zählkammer. </li><li> Nachdem man die bioink auf Raumtemperatur abkühlen, sanft in einem ausreichenden Volumen , um die Aussetzung der RFP-MSCs in den bioink mischen , um eine Endkonzentration von 5 x 10 6 Zellen ml -1 zu erreichen. </li><li> Gießen Sie die Zelle Lager bioink in eine Druckspritze mit der Düse verschlossen. Kühlen Sie das geladene Spritze auf 4 ° C, um eine druckbare Viskosität zu erreichen. </li><li> Legen Sie die geladene Spritze auf dem automatisiertenRoboter-Abgabesystem und befestigen Sie die Luftdruckleitungen. Entfernen Sie die Spritze Luer Dichtung und befestigen Sie die Druckdüsen. </li><li> Auspressen cellularized bioinks in dünne Linien unter Verwendung von 0,05 MPa Rückdruck, bei 5 mm / sec Schreibgeschwindigkeit von 10 ml Spritze über eine 27 G-Nadel / Düse (konisch über zylindrische empfohlen) auf eine Polystyrolfolienoberfläche nach dem vorprogrammierten Ablagerung Muster in Schritt 2.1 beschrieben, um die cellularized bioink auf die vorgeätzten Rillen zu platzieren. </li><li> Nach 1 Stunde Inkubation bei Raumtemperatur, mit 10 ml Wachstumsmedium (mit 10% FBS ergänzt und Antibiotika) und Inkubieren der Zellen für 24 h (die Zellen zu erlauben, auf die geätzten Merkmale zu erfassen und reagieren) vor der Anzeige unter Verwendung eines invertierten Fluoreszenzmikroskop bei 10-facher Vergrößerung. </li></ol>

<ol>
	<li>Ma, Z., Ringeisen , R. B., Spargo, J. B., Wu, K. P., 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> Cell and Organ Printing. , 137-159 (2010).</li><li>Kaji, H., Camci-Unal, G., Langer, R., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+systems+for+the+generation+of+patterned+co-cultures+for+controlling+cell-cell+interactions.">Engineering systems for the generation of patterned co-cultures for controlling cell-cell interactions.</a> Biochim Biophys Acta. 1810 (3), 239-250 (2011).</li><li>Goubko, C. A., Cao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+multiple+cell+types+in+co-cultures:+A+review.">Patterning multiple cell types in co-cultures: A review.</a> Materials Science and Engineering: C. 29 (6), 1855-1868 (2009).</li><li>Pati, F., Jang, J., Lee, J. W., Cho, D. W., Yoo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of 3D Biofabrication and Translation. , 123-152 (2015).</li><li>Derby, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printing+and+prototyping+of+tissues+and+scaffolds.">Printing and prototyping of tissues and scaffolds.</a> Science. 338 (6109), 921-926 (2012).</li><li>Malda, 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=25th+anniversary+article:+Engineering+hydrogels+for+biofabrication.">25th anniversary article: Engineering hydrogels for biofabrication.</a> Adv Mater. 25 (36), 5011-5028 (2013).</li><li>Billiet, T., Vandenhaute, M., Schelfhout, J., Van Vlierberghe, S., Dubruel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+trends+and+limitations+in+hydrogel-rapid+prototyping+for+tissue+engineering.">A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering.</a> Biomaterials. 33 (26), 6020-6041 (2012).</li><li>Skardal, A., Yoo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of 3D Biofabrication and Translation. , 1-17 (2015).</li><li>Irvine, S. 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=Printing+cell-laden+gelatin+constructs+by+free-form+fabrication+and+enzymatic+protein+crosslinking.">Printing cell-laden gelatin constructs by free-form fabrication and enzymatic protein crosslinking.</a> Biomed Microdevices. 17 (1), 16 (2015).</li><li>Bhuthalingam, 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=A+novel+3D+printing+method+for+cell+alignment+and+differentiation.">A novel 3D printing method for cell alignment and differentiation.</a> International Journal of Bioprinting. 1, 57-65 (2015).</li><li>Curtis, A., Wilkinson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topographical+control+of+cells.">Topographical control of cells.</a> Biomaterials. 18 (24), 1573-1583 (1997).</li><li>Tay, C. 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=Micropatterned+matrix+directs+differentiation+of+human+mesenchymal+stem+cells+towards+myocardial+lineage.">Micropatterned matrix directs differentiation of human mesenchymal stem cells towards myocardial lineage.</a> Exp Cell Res. 316 (7), 1159-1168 (2010).</li><li>Tay, C. 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=Bio-inspired+micropatterned+platform+to+steer+stem+cell+differentiation.">Bio-inspired micropatterned platform to steer stem cell differentiation.</a> Small. 7 (10), 1416-1421 (2011).</li><li>Li, H., 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=Direct+laser+machining-induced+topographic+pattern+promotes+up-regulation+of+myogenic+markers+in+human+mesenchymal+stem+cells.">Direct laser machining-induced topographic pattern promotes up-regulation of myogenic markers in human mesenchymal stem cells.</a> Acta Biomater. 8 (2), 531-539 (2012).</li><li>Kolind, K., Leong, K. W., Besenbacher, F., Foss, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidance+of+stem+cell+fate+on+2D+patterned+surfaces.">Guidance of stem cell fate on 2D patterned surfaces.</a> Biomaterials. 33 (28), 6626-6633 (2012).</li><li>Agrawal, 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=Smooth+Muscle+Cell+Alignment+and+Phenotype+Control+by+Melt+Spun+Polycaprolactone+Fibers+for+Seeding+of+Tissue+Engineered+Blood+Vessels.">Smooth Muscle Cell Alignment and Phenotype Control by Melt Spun Polycaprolactone Fibers for Seeding of Tissue Engineered Blood Vessels.</a> International Journal of Biomaterials. 2015, (2015).</li><li>Chang, 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=Phenotypic+modulation+of+primary+vascular+smooth+muscle+cells+by+short-term+culture+on+micropatterned+substrate.">Phenotypic modulation of primary vascular smooth muscle cells by short-term culture on micropatterned substrate.</a> PLoS One. 9 (2), e88089 (2014).</li><li>Li, 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=Engineering+cell+alignment+in+vitro.">Engineering cell alignment in vitro.</a> Biotechnol Adv. 32 (2), 347-365 (2014).</li><li>Blaeser, 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=Controlling+Shear+Stress+in+3D+Bioprinting+is+a+Key+Factor+to+Balance+Printing+Resolution+and+Stem+Cell+Integrity.">Controlling Shear Stress in 3D Bioprinting is a Key Factor to Balance Printing Resolution and Stem Cell Integrity.</a> Adv Healthc Mater. 5 (3), 326-333 (2016).</li><li>Nery, A. 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=Human+mesenchymal+stem+cells:+From+immunophenotyping+by+flow+cytometry+to+clinical+applications.">Human mesenchymal stem cells: From immunophenotyping by flow cytometry to clinical applications.</a> Cytometry Part A. 83A (1), 48-61 (2013).</li><li>Guvendiren, M., Lu, H. D., Burdick, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear-thinning+hydrogels+for+biomedical+applications.">Shear-thinning hydrogels for biomedical applications.</a> Soft Matter. 8 (2), 260-272 (2012).</li><li>Markstedt, K., 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=3D+Bioprinting+Human+Chondrocytes+with+Nanocellulose-Alginate+Bioink+for+Cartilage+Tissue+Engineering+Applications.">3D Bioprinting Human Chondrocytes with Nanocellulose-Alginate Bioink for Cartilage Tissue Engineering Applications.</a> Biomacromolecules. 16 (5), 1489-1496 (2015).</li><li>Kolesky, D. 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=3D+bioprinting+of+vascularized,+heterogeneous+cell-laden+tissue+constructs.">3D bioprinting of vascularized, heterogeneous cell-laden tissue constructs.</a> Adv Mater. 26 (19), 3124-3130 (2014).</li><li>Merceron, T. K., Murphy, S. V., Yoo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of 3D Biofabrication and Translation. , 249-270 (2015).</li><li>Carrow, J. K., Kerativitayanan, P., Jaiswal, M. K., Lokhande, G., Gaharwar, A. K., Yoo, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of 3D Biofabrication and Translation. , 229-248 (2015).</li><li>Lee, K. V., Turksen, K., 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> Bioprinting in Regenerative Medicine. , 33-66 (2015).</li><li>Ozbolat, I. T., Hospodiuk, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+advances+and+future+perspectives+in+extrusion-based+bioprinting.">Current advances and future perspectives in extrusion-based bioprinting.</a> Biomaterials. 76, 321-343 (2016).</li><li>Li, M., Tian, X., Schreyer, D. J., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+needle+geometry+on+flow+rate+and+cell+damage+in+the+dispensing-based+biofabrication+process.">Effect of needle geometry on flow rate and cell damage in the dispensing-based biofabrication process.</a> Biotechnol Prog. 27 (6), 1777-1784 (2011).</li><li>Nikkhah, M., Edalat, F., Manoucheri, S., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+microscale+topographies+to+control+the+cell-substrate+interface.">Engineering microscale topographies to control the cell-substrate interface.</a> Biomaterials. 33 (21), 5230-5246 (2012).</li><li>Khademhosseini, 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=Microfluidic+patterning+for+fabrication+of+contractile+cardiac+organoids.">Microfluidic patterning for fabrication of contractile cardiac organoids.</a> Biomed Microdevices. 9 (2), 149-157 (2007).</li><li>Chelli, 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=Neural+cell+alignment+by+patterning+gradients+of+the+extracellular+matrix+protein+laminin.">Neural cell alignment by patterning gradients of the extracellular matrix protein laminin.</a> Interface Focus. 4 (1), 20130041 (2014).</li><li>Aubin, H., 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=Directed+3D+cell+alignment+and+elongation+in+microengineered+hydrogels.">Directed 3D cell alignment and elongation in microengineered hydrogels.</a> Biomaterials. 31 (27), 6941-6951 (2010).</li><li>B&#233;duer, 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=Engineering+of+adult+human+neural+stem+cells+differentiation+through+surface+micropatterning.">Engineering of adult human neural stem cells differentiation through surface micropatterning.</a> Biomaterials. 33 (2), 504-514 (2012).</li><li>Bian, W., Jackman, C. P., Bursac, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+structural+and+functional+anisotropy+of+engineered+cardiac+tissues.">Controlling the structural and functional anisotropy of engineered cardiac tissues.</a> Biofabrication. 6 (2), 024109 (2014).</li><li>Chen, P. 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=Fabrication+of+large+perfusable+macroporous+cell-laden+hydrogel+scaffolds+using+microbial+transglutaminase.">Fabrication of large perfusable macroporous cell-laden hydrogel scaffolds using microbial transglutaminase.</a> Acta Biomater. 10 (2), 912-920 (2014).</li><li>Bertassoni, L. E., 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=Direct-write+bioprinting+of+cell-laden+methacrylated+gelatin+hydrogels.">Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels.</a> Biofabrication. 6 (2), 024105 (2014).</li><li>Zhao, X., 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=3D+patterned+substrates+for+bioartificial+blood+vessels+-+The+effect+of+hydrogels+on+aligned+cells+on+a+biomaterial+surface.">3D patterned substrates for bioartificial blood vessels - The effect of hydrogels on aligned cells on a biomaterial surface.</a> Acta Biomater. 26, 159-168 (2015).</li></ol>

The authors declare they have no competing financial interest.

Der entscheidende Schritt dieses Verfahrens ist die tatsächliche Bioprinting Lieferung der Stammzellen, da der Prozess zu den Merkmalen Zellsedimentation erlauben müssen, drucken, ohne bioink Verbreitung / Blutungen, liefern Zellen, ohne den Tod Schubspannung Zelle und nicht die Differenzierung gegenüber unerwünschten Linie auslösen. Wenn die erwartete Zellenausrichtung ausbleibt, dann sollte die bioink Viskosität für ihre Eignung für den Druck beurteilt werden. Es ist wichtig, dass ...

Die gezielte Strukturierung von Zell Platzierung ermöglicht die Bildung von Kulturen , die 1 in vivo zellulären Organisation zu imitieren. Tatsächlich Erforschung der Interaktionen zwischen mehreren Zelltypen können durch die Organisation ihrer räumlichen Anordnung 2,3- unterstützt. Die meisten Strukturierungssysteme beruhen auf Oberflächenmodifikation Verfahren zur Förderung oder Zelladhäsion mit anschließender passiver Zellaufbringeinrichtung zu verhindern. Bioprinting bietet räumliche und zeitliche Kontrolle über Zellverteilungen 1. Zusätzlich zu diesen Funktionen, Bioprinting wurde zur Erzeugung von geometrisch komplexen Gerüsten 4 als eine technisch einfache, schnelle und kostengünstige Methode beschrieben. Es nutzt Design - Software - Computer und ermöglicht die Einführung von Zellen in dem Herstellungsverfahren 4. Bioprinting Systeme kategorisiert auf der Grundlage ihrer Arbeitsprinzipien als laserbasierte, Inkjet-basierten oder Extrusionsbasis 4. Extrusion Bioprinting wurde als der vielversprechendste beschrieben , da sie die Herstellung von Konstrukten organisiert klinisch relevanter Größen innerhalb eines realistischen Zeitrahmen 4-6 ermöglicht. Es wird entweder durch mechanische oder Gegendruck unterstützt Extrusion einer Zelle Lager Hydrogel bioink durchgeführt. In dem hier vorgestellten Verfahren wurde Gegendruck eingesetzt. Wie erwähnt, werden die Zellen in einem cytocompatible bioink geliefert. Eine solche bioink sollte die Lieferung von Zellen ohne Erzeugung schädlicher Scherspannung unterstützen, und sein einer ausreichenden Viskosität , die Integrität der gedruckten Bahn zu halten, ohne zu kollabieren oder Spreiz (bezeichnet als &quot;Ausbluten&quot;) , 7-10. Die Wechselwirkung von Zellen mit ihrer Haftfläche ist bekannt, Zellverhalten zu beeinflussen. Die Topographie Oberfläche kann steuern die Zellform, Orientierung 11 und auch den Phänotyp. Insbesondere haben die Herstellung von Nuten und Kanälen gezeigt, zu induziereneine gestreckte, längliche Morphologie auf mehreren Zelltypen. Die Annahme dieser Morphologie wurde mit dem Phänotyp von multi- und pluripotenten Zellen zu beeinflussen gefunden. Wenn zum Beispiel auf Rillen ausgerichtet sind , zeigen mesenchymalen Stammzellen (MSC) den Nachweis der Differenzierung zu Kardiomyozyten 12,13 und glatten Gefäßmuskelzellen übernehmen die Kontraktions Phänotyp über den synthetischen 10,14-17. Die Zellkanäle oder Nuten ausrichten können über eine Anzahl von Verfahren auf einer Polymeroberfläche erzeugt werden, beispielsweise tiefes reaktives Ionenätzen, Elektronenstrahllithographie, Laserdirektdruck, Femtosekundenlaser, Photolithographie und Trockenätzen im Plasma 18. Diese Ansätze sind oft zeitaufwendig, erfordern eine komplexe Vorrichtung und kann in der Form des Musters erzeugt werden, zu begrenzen. Darüber hinaus synchronisiert sie nicht Musterung mit Bioprinting und nicht für den sofortigen Zellularisierung ermöglichen. Die koordinativ kontrollierte Bewegung eines automatisiertenAbgabesystem kann für die Abscheidung von Lösungen komplexer Muster folgen. Hier zeigen wir, wie die mikroskaligen gesteuerte Bewegung genutzt werden können Kanäle für die Zell Orientierung zu schaffen. Ein geschärfte Stift oder Skalpell wird an den Druckkopf anstelle der Extrusions Spritze befestigt und das Gerät kann dann die Polymeroberfläche unter der Anleitung der Software zur grafischen Darstellung ätzen. Das Verfahren bietet Vielseitigkeit in Musterentwurf und ist anwendbar auf polymere Materialien, die üblicherweise in Bioengineering verwendet, wie Polystyrol, PTFE, und Polycaprolacton. Als weiteren Schritt zum Ätzen können die Zellen auf die zerkratzte Nuten bioprinted direkt. Die Gelatine bioink hier verwendete konnte sowohl die Spur halten und die abgeschiedenen Zellen ermöglichen, die geätzten Strukturen zu erfassen. Mesenchymalen Stammzellen auf die geätzten Nuten bioprinted wurden an ihnen entlang in verschiedene Linien zu verlängern demonstriert.

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Robotic Dispensing System</td><td>Janome</td><td>2300N</td><td></td></tr><tr><td>Plasma Machine</td><td>Femto Science</td><td>Covance</td><td></td></tr><tr><td>USB Microscope</td><td></td><td></td><td></td></tr><tr><td>Optical Microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spreadsheet</td><td>Excel</td><td>Excel</td><td></td></tr><tr><td>Printing Co-ordinate Software</td><td>Janome</td><td>JR C-Points</td><td></td></tr><tr><td>Imaging Software</td><td>National Institutes of Health (NIH)</td><td>ImageJ</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Stylus (Blade)</td><td>OLFA</td><td>AK-5</td><td></td></tr><tr><td>5 ml printing syringe</td><td>San-ei Tech</td><td>SH10LL-B</td><td></td></tr><tr><td>30 G printing needle</td><td>San-ei Tech</td><td>SH30-0.25-B</td><td></td></tr><tr><td>1 mm polystyrene sheets</td><td></td><td>Purchased locally</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Invitrogen&amp;nbsp;</td><td>10270-098</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Gelatin from porcine skin, Gel strength 300, Type A</td><td>Sigma Aldrich</td><td>9000-70-8</td><td></td></tr><tr><td>&amp;alpha;MEM</td><td>Invitrogen</td><td>41061-029</td><td></td></tr><tr><td>Antibiotc antimycotic</td><td>Sigma Aldrich</td><td>A5955-100ML</td><td></td></tr><tr><td>Red Fluorescent Protein Mesenchymal Stem Cells (RFP-MSCs)</td><td>Cyagen Biosciences Incorporation</td><td>RASMX-01201</td><td></td></tr></tbody></table>

automated robotic dispensing technique for surface guidance and bioprinting of cells

Dieses Manuskript beschreibt die Einführung von Zell Führung Merkmale durch die direkte Lieferung von Zellen auf diese Funktionen in einem Hydrogel bioink unter Verwendung eines automatisierten Roboter-Abgabesystem verfolgt wird. Die besondere bioink ausgewählt wurde, wie es Zellen erlaubt, in Richtung zu sedimentieren und die Merkmale erfassen. Das Abgabesystem bioprints lebenden Zellen in Hydrogel bioinks einen Gegendruck unterstützt Druckkopf verwendet wird. Doch durch den Druckkopf mit einem geschärften Stift oder Skalpell ersetzt wurde, kann das Abgabesystem auch topographische Hinweise durch Anätzen zu erstellen verwendet werden. Der Taststift Bewegung kann in Schritten von 10 &amp; mgr; m in der X, Y und Z-Richtung programmiert werden. Die gemusterte Nuten konnten mesenchymalen Stammzellen zu orientieren, beeinflussen sie eine längliche Morphologie in Ausrichtung mit den Nuten &#39;Richtung zu übernehmen. Die Strukturierung könnte entworfen werden unter Verwendung von Software zur grafischen Darstellung in geraden Linien, konzentrischen Kreisen und Sinuswellen. In einem nachfolgenden Verfahren, FibroBlasten und mesenchymalen Stammzellen wurden in einer 2% igen Gelatine bioink suspendiert, in einem Gegendruck angetrieben Extrusionsdruckkopf Bioprinting. Die Zellenlager bioink wurde dann gedruckt die gleichen programmierten Koordinaten für das Ätzen verwendet. Die bioprinted Zellen waren in der Lage zu den geätzten Merkmalen zu erfassen und darauf zu reagieren, wie durch ihre gedehnten Orientierung entlang der Richtung der geätzten Rillen gezeigt.

Die repräsentativen Ergebnisse zeigen , dass der Gegendruck unterstützt Roboterabgabesystem kann sowohl Oberfläche als Extrusionsbasis bioprinter verwendet werden Ätzen für die Durchführung und bioink Druck (Abbildung 1 A). Es kann für die Erzeugung von geätzten Rillen in Polymeroberflächen verwendet werden, und anschließend zu den Merkmalen (1 B und C) eine Zelle Lager bioink direkt zu drucken. <p class="jove_content" fo:keep-together.with...

Watch this Scientific Journal Video about Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells at JoVE.com

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Automatische Robotic Dosiertechnik für Oberflächen Ausrichtungs- und Bioprinting von Zellen

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

automated-robotic-dispensing-technique-for-surface-guidance

Research

JoVE Journal

Bioengineering

6.5K Aufrufe.  Nanyang Technological University. This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

Serie: Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

Prescribed 3-D Direct Writing of Suspended Micron/Sub-micron Scale Fiber Structures via a Robotic Dispensing System

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer fiber length and orientation is defined by the spatial positioning of the dispensing system 3-axis stages. The fiber diameter is defined by the prescribed dispense time of the dispensing system valve, the feed rate (the speed at which the stage traverses from an initiating to a terminating position), the gauge diameter of the dispensing tip, the viscosity and surface tension of the polymer solution, and the programmed drawing length. The stage feed rate affects the polymer solution&#8217;s evaporation rate and capillary breakup of the filaments. The dispensing system consists of a pneumatic valve controller, a droplet-dispensing valve and a dispensing tip. Characterization of the direct write process to determine the optimum combination of factors leads to repeatedly acquiring the desired range of fiber diameters. The advantage of this robotic dispensing system is the ease of obtaining a precise range of micron/sub-micron fibers onto a desired, programmed location via automated process control. Here, the discussed self-assembled micron/sub-micron scale 3D structures have been employed to fabricate suspended structures to create micron/sub-micron fluidic devices and bioengineered scaffolds.

Here, we present a protocol to fabricate freely-suspended, micron/sub-micron scale polymer fibers and &#8220;web-like&#8221; structures generated via automated direct writing procedure by means of a 3-axis dispensing system.

Vorgeschriebene 3-D direkte Schreiben von Hänge Micron / Sub-Mikrometer-Maßstab Faserstrukturen über eine Robotic Dosiersystem

A 3-axis dispensing system is utilized to control the initiating and terminating fiber positions and trajectory via the dispensing software. The polymer ...

Forschung

Ingenieurwesen

The Submerged Printing of Cells onto a Modified Surface Using a Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell phenotype after printing, poor organization and distribution of desired cells, and the inability to deliver drugs and/or nutrients to targeted areas in the array. Our 3D microfluidic printing technology is uniquely capable of sealing and printing arrays of cells onto submerged surfaces in an automated and multiplexed manner. The design of the microfluidic cell array (MFCA) 3D fluidics enables the printhead tip to be lowered into a liquid-filled well or dish and compressed against a surface to form a seal. The soft silicone tip of the printhead behaves like a gasket and is able to form a reversible seal by applying pressure or backing away. Other cells printing technologies such as pin or ink-jet printers are unable to print in submerged applications. Submerged surface printing is essential to maintain phenotypes of cells and to monitor these cells on a surface without disturbing the material surface characteristics. By printing onto submerged surfaces, cell microarrays are produced that allow for drug screening and cytotoxicity assessment in a multitude of areas including cancer, diabetes, inflammation, infections, and cardiovascular disease.

This 3D microfluidic printing technology prints arrays of cells onto submerged surfaces. We describe how arrays of cells are delivered microfluidically in 3D flow cells onto submerged surfaces. By printing onto submerged surfaces, cell microarrays were produced that allow for drug screening and cytotoxicity assessment in a multitude of areas.

Die Tauchdruck der Zellen auf eine modifizierte Oberfläche mit einem Continuous Flow Microspotter

The printing of cells for microarray applications possesses significant challenges including the problem of maintaining physiologically relevant cell ...

Biotechnik

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

Roboter-Produktion von Cancer Cell Sphäroide mit einer wässrigen Zwei-Phasen-System für Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

Viability of Bioprinted Cellular Constructs Using a Three Dispenser Cartesian Printer

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of three-dimensional bioprinting in tissue engineering has generated new methods for the printing of cells and matrix to fabricate biomimetic tissue constructs. The solid freeform fabrication (SFF) method developed for three-dimensional bioprinting uses an additive manufacturing approach by depositing droplets of cells and hydrogels in a layer-by-layer fashion. Bioprinting fabrication is dependent on the specific placement of biological materials into three-dimensional architectures, and the printed constructs should closely mimic the complex organization of cells and extracellular matrices in native tissue. This paper highlights the use of the Palmetto Printer, a Cartesian bioprinter, as well as the process of producing spatially organized, viable constructs while simultaneously allowing control of environmental factors. This methodology utilizes computer-aided design and computer-aided manufacturing to produce these specific and complex geometries. Finally, this approach allows for the reproducible production of fabricated constructs optimized by controllable printing parameters.

A Cartesian bioprinter was designed and fabricated to allow multi-material deposition in precise, reproducible geometries, while also allowing control of environmental factors. Utilizing the three-dimensional bioprinter, complex and viable constructs may be printed and easily reproduced.

Lebensfähigkeit Bioprinted Cellular Konstrukte unter Verwendung einer Drei Dispenser kartesischen Drucker

Tissue engineering has centralized its focus on the construction of replacements for non-functional or damaged tissue. The utilization of ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

Schaffung von Herzgewebe, die mechanische Integration von Sphäroiden mit 3D-Bioprinting ermöglicht

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

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 ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting von Knorpel und Haut-Gewebe-Analoga nutzen eine neuartige Passive Einheit Mischtechnik für Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Affordable Oxygen Microscopy-Assisted Biofabrication of Multicellular Spheroids

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue assembling units for biofabrication. While the main power of the spheroid model is in mimicking physical-chemical gradients at the tissue microscale, the real physiological environment (including dynamics of metabolic activity, oxygenation, cell death, and proliferation) inside the spheroids is generally ignored. At the same time, the effects of the growth medium composition and the formation method on the resulting spheroid phenotype are well documented. Thus, characterization and standardization of spheroid phenotype are required to ensure the reproducibility and transparency of the research results. The analysis of average spheroid oxygenation and the value of O2 gradients in three dimensions (3D) can be a simple and universal way for spheroid phenotype characterization, pointing at their metabolic activity, overall viability, and potential to recapitulate in vivo tissue microenvironment. The visualization of 3D oxygenation can be easily combined with multiparametric analysis of additional physiological parameters (such as cell death, proliferation, and cell composition) and applied for continuous oxygenation monitoring and/or end-point measurements. The loading of the O2 probe is performed during the stage of spheroid formation and is compatible with various protocols of spheroid generation. The protocol includes a high-throughput method of spheroid generation with introduced red and near-infrared emitting ratiometric fluorescent O2 nanosensors and the description of multi-parameter assessment of spheroid oxygenation and cell death before and after bioprinting. The experimental examples show comparative O2 gradients analysis in homo- and hetero-cellular spheroids as well as spheroid-based bioprinted constructs. The protocol is compatible with a conventional fluorescence microscope having multiple fluorescence filters and a light-emitting diode as a light source.

The protocol describes high-throughput spheroid generation for bioprinting using the multi-parametric analysis of their oxygenation and cell death on a standard fluorescence microscope. This approach can be applied to control the spheroids viability and perform standardization, which is important in modeling 3D tissue, tumor microenvironment, and successful (micro)tissue biofabrication.

Erschwingliche Sauerstoffmikroskopie-unterstützte Biofabrikation von mehrzelligen Sphäroiden

Multicellular spheroids are important tools for studying tissue and cancer physiology in 3D and are frequently used in tissue engineering as tissue ...

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

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

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

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

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

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. 
On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 &#176;C and melting temperature of ~90 &#176;C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Shear processing during hydrogel formation results in the production of microgel suspensions that shear-thin but rapidly restructure following the removal of shear forces. Such materials have been used as a supporting matrix for bioprinting complex, cell-laden structures. Here, methods used to manufacture the supporting bed and compatible bioinks are described.

Agarose-Flüssiggele, die durch Scherverarbeitung während der Gelierung für suspendierten 3D-Biodruck gebildet werden

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several ...