Name: creation of cardiac tissue exhibiting mechanical integration of spheroids using 3d bioprinting
Uploaded: 2017-07-02T13:00:00
Description: Watch this Scientific Journal Video about Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting at JoVE.com

Gli autori riconoscono le seguenti fonti di finanziamento: Magic That Matters Fund per la ricerca cardiovascolare e il Maryland Stem Cell Research Fund (2016-MSCRFI-2735).

1. Preparazione dei cardiomiociti <ol><li> Generare e coltivare cellule staminali pluripotenti indotte da umano (hiPSCs) su piastre a 6 pozzetti rivestite con matrice a membrana basale come descritto 10 . </li><li> Differenziare i hiPSC nei cardiomiociti derivanti da hiPSC (hiPSC-CMs) utilizzando metodi descritti in precedenza 11 , 12 . </li><li> Il giorno 19 dopo la differenziazione, isolare i cardiomiociti usando 2 mL di Trypsin / EDT...

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T., Fontes, A., Vemuri, M. C., Macarthur, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+induced+pluripotent+stem+cells+with+CytoTune,+a+non-integrating+Sendai+virus.">Generation of induced pluripotent stem cells with CytoTune, a non-integrating Sendai virus.</a> Methods Mol Biol. 997, 45-56 (2013).</li><li>Li, S., Cheng, H., Tomaselli, G. F., Li, R. 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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+Human+Pluripotent+Stem+Cell+Surface+N-Glycoproteome+Resource+Reveals+Markers,+Extracellular+Epitopes,+and+Drug+Targets.">A Human Pluripotent Stem Cell Surface N-Glycoproteome Resource Reveals Markers, Extracellular Epitopes, and Drug Targets.</a> Stem Cell Rep. 3, 185-203 (2014).</li><li>ScienCell Research Laboratories. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Human Cardiac Fibroblasts (adult ventricular) Product Sheet. , (2017).</li><li>Lonza Walkersville, Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Endothelial Cell Systems - Technical Information &amp; Instructions.,. , (2015).</li><li>Thermo Fisher Scientific, Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunofluorescence Method for IHC Detection. , (2017).</li><li>Murata, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preliminary+study+of+osteochondral+regeneration+using+a+scaffold-free+three-dimensional+construct+of+porcine+adipose+tissue-derived+mesenchymal+stem+cells.">A preliminary study of osteochondral regeneration using a scaffold-free three-dimensional construct of porcine adipose tissue-derived mesenchymal stem cells.</a> J Orthop Surg Res. 10, (2015).</li><li>Mosadegh, B., Xiong, G., Dunham, S., Min, J. 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It is important to use beating, functional spheroids for 3D bioprinting. If spheroids are not beating, continuing to use them will invariably result in a non-functional 3D bioprinted patch.
One benefit of this approach is the ability to manipulate the cell content of the patch by varying the total number of cells and the percentage of cardiomyocytes, endothelial cells, and fibroblasts in the spheroids. This allows for many different types of cardiac patches to be printed, with varying histolog...

Ci sono molti metodi differenti di bioprinting 3D 1 , 2 , 3 . La bioprinting 3D è spesso classificata dalla tecnologia di stampa 1 , con esempi come bioprinting a getto d&#39;inchiostro, bioprinting a microestrusione, bioprinting assistito da laser, combinazione di metodi o approcci più recenti. La bioprinting 3D può essere classificata anche in metodi scaffold-dependent o scaffold-dependent 4 . La maggior parte dei metodi di bioprinting 3D sono dipendenti da scaffold, dove vi è la necessità di biomateriali, ad es. Biochip 5 o scaffold 6 . Tuttavia, il bioprinting 3D dipende da molte problematiche e limitazioni 4 , 7 , come l&#39;immunogenicità del materiale di ponteggio, il costo dei biochimici proprietari, la velocità lenta e la tossicità dei prodotti di degradazione. SCAFÈ stato tentato l&#39;ingegnerizzazione dei tessuti cardiaci senza piombo usando sferoidi 8 , con il potenziale per superare questi svantaggi dell&#39;ingegneria tissutale dipendente dal ponteggio. Tuttavia, come riconosciuto dagli autori in tale documento, era stato difficile gestire in modo robusto e posizionare sferoidi in posizioni fissi, nel processo di biofabbricazione. L&#39;uso concomitante di bioprinting 3D e di ingegneria tissutale a base di sferoidi ha il potenziale per superare queste difficoltà. In questo protocollo, descriviamo la bioprinting 3D del tessuto cardiaco senza altri biomateriali, usando solo le cellule sotto forma di sferoidi. I bioprinters 3D a base di sferoidi a base di scaffali 9 hanno la capacità di prelevare singoli sferoidi usando l&#39;aspirazione del vuoto e posizionarli su una matrice di ago. Il concetto di sferoidi di posizionamento su un array di ago in bioprinting 3D è ispirato dall&#39;utilizzo di array d&#39;ago (detto &quot; kenzan &quot;) nell&#39;antico JapaSe l&#39;arte della disposizione dei fiori, ikebana. Questo sistema permette che i sferoidali siano posizionati in modo preciso in qualsiasi configurazione e che i singoli sferoidi si fusero insieme per un breve periodo per creare un tessuto 3D bioprintato. Questo metodo consente quindi di manipolare con facilità le sferoidi, con potenziali implicazioni per il futuro della biofabbricazione degli organi senza scaffalature.

<table border="0" cellpadding="0" cellspacing="0" width="587">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Geltrex</td>
			<td style="width:157px;">Invitrogen&#160;</td>
			<td style="width:88px;">A1413202</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Trypsin/EDTA 0.05%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">15400054</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Defined Trypsin inhibitor 0.0125%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">R007100</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:176px;">RPMI Cell Media</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:88px;">11875-093</td>
			<td style="width:167px;">RPMI supplemented with B27 constitutes HIPSC-CM culture media</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:176px;">B-27 Supplement</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">17504044</td>
			<td style="width:167px;">RPMI supplemented with B27 constitutes HIPSC-CM culture media</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Countess Automated Cell Counter</td>
			<td style="width:157px;">Invitrogen</td>
			<td style="width:88px;">C10227</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:176px;">Human cardiac fibroblasts (adult ventricular type)</td>
			<td style="width:157px;">Sciencell</td>
			<td style="width:88px;">6310</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Human umbilical vein endothelial cells</td>
			<td style="width:157px;">Lonza</td>
			<td style="width:88px;">CC-2935</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:176px;">PrimeSurface ultra-low attachment 96-well U-bottom plates&#160;</td>
			<td style="width:157px;">Akita Sumitomo Bakelite Co.</td>
			<td style="width:88px;">MS-9096UZ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Regenova Bio 3D Printer</td>
			<td style="width:157px;">Cyfuse Biomedical K.K.</td>
			<td style="width:88px;">N/A</td>
			<td>www.cyfusebio.com/en/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:176px;">Troponin T Antibody</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">701620</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">Connexin 43 (Cx43) Antibody</td>
			<td style="width:157px;">Chemicon</td>
			<td>MAB3068</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:176px;">ProLong Gold Antifade Mountant with DAPI</td>
			<td style="width:157px;">Thermo Fisher</td>
			<td style="width:88px;">P36935</td>
			<td></td>
		</tr>
	</tbody>
</table>

creation of cardiac tissue exhibiting mechanical integration of spheroids using 3d bioprinting

Questo protocollo descrive il bioprinting 3D del tessuto cardiaco senza l&#39;uso di biomateriali, utilizzando solo le cellule. I cardiomiociti, le cellule endoteliali ei fibroblasti vengono prima isolati, contati e mescolati a rapporti cellulari desiderati. Sono co-coltivati ​​in singoli pozzetti in piastre a 96 pozzetti ad attacco ultra-basso. Entro 3 giorni, battendo forma sferoide. Questi sferoidi vengono quindi prelevati da un ugello usando l&#39;aspirazione del vuoto e assemblati su un array di ago utilizzando un bioprinter 3D. I sferoidi sono quindi autorizzati a fondere sull&#39;array dell&#39;ago. Tre giorni dopo la bioprinting 3D, i sferoidi vengono rimossi come una patch intatta, che sta già battendo spontaneamente. Le zone cardiache 3D bioprinted espongono l&#39;integrazione meccanica dei sferoidi dei componenti e sono altamente promettenti nella rigenerazione del tessuto cardiaco e come modelli 3D di malattie cardiache.

Alla fine del punto 4.4 (co-cultura), le cellule in ciascun pozzetto dovrebbero aggregarsi nella parte inferiore delle piastre a basso profilo a 96 pozzetti ad attacco ultra-basso per formare sferoidi per gravità. Questi sferoidi contengono HiPSC-CM, HCFs e HUVECs e possono essere sottoposti a ispezione visiva sotto microscopia leggera, dove devono apparire circolari per proiezione bidimensionale ( Figura 1 ). Alla fine del passo 6.3, la patch cardiaca 3D b...

Watch this Scientific Journal Video about Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting at JoVE.com

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

Questo protocollo descrive la bioprinting 3D del tessuto cardiaco senza l&#39;uso di biomateriali. Le zone cardiache 3D bioprinted espongono l&#39;integrazione meccanica dei sferoidi dei componenti e sono altamente promettenti nella rigenerazione del tessuto cardiaco e come modelli 3D di malattie cardiache.

Creazione di tessuto cardiaco che mostra l&#39;integrazione meccanica degli spheroidi usando la biopittura 3D

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

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