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

Forfatterne anerkender følgende finansieringskilder: Magic That Matters Fund for Kardiovaskulær Forskning og Maryland Stem Cell Research Fund (2016-MSCRFI-2735).

1. Fremstilling af kardiomyocytter <ol><li> Generere og dyrke humane inducerede pluripotente stamceller (hiPSC&#39;er) på 6-brøndsplader overtrukket med basalmembranmatrix som beskrevet 10 . </li><li> Differentier hiPSC&#39;er i hiPSC-afledte cardiomyocytter (hiPSC-CM&#39;er) ved anvendelse af tidligere beskrevne fremgangsmåder 11 , 12 . </li><li> På dag 19 efter differentiering isolerer kardiomyocytterne ved anvendelse af 2 ml tryp...

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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...

Der findes mange forskellige metoder til 3D bioprinting 1 , 2 , 3 . 3D bioprinting klassificeres hyppigt af trykteknologi 1 , med eksempler som inkjet bioprinting, microextrusion bioprinting, laserassisteret bioprinting, en kombination af metoder eller nyere tilgange. 3D bioprinting kan også klassificeres i stilladsfrie eller stilladsafhængige metoder 4 . De fleste metoder til 3D bioprinting er stilladsafhængige, hvor der er behov for biomaterialer, fx bioinks 5 eller stilladser 6 . Stilladsafhængig 3D bioprinting står over for mange problemer og begrænsninger 4 , 7 , som f.eks. Immunogenicitet af stilladsmateriale, omkostninger til proprietære biobrændstoffer, langsom hastighed og toksicitet af nedbrydningsprodukter. SCAFFoldfri hjertevævsteknik ved anvendelse af sfæroider er blevet forsøgt 8 , med potentialet til at overvinde disse ulemper ved stilladsafhængig vævsteknik. Som det blev erkendt af forfatterne i det pågældende papir, havde det imidlertid været vanskeligt at håndtere og positionere sfæroider på faste steder under biofabricering. Den samtidige brug af 3D bioprinting og sfæroidbaseret vævsteknik har potentialet til at overvinde disse vanskeligheder. I denne protokol beskriver vi 3D bioprinting af hjertevæv uden andre biomaterialer ved kun at bruge celler i form af sfæroider. Stilladsfri sfæroid-baserede 3D-bioprintere 9 har evnen til at samle individuelle sfæroider ved hjælp af vakuumsugning og placere dem på en nålarray. Konceptet med positionering af sfæroider på en nålestik i 3D bioprinting er inspireret af brugen af nålarrayer (kendt som &quot; kenzan &quot;) i den gamle JapaNese kunst af blomster arrangement, ikebana. Dette system gør det muligt at placere spheroider præcist i enhver konfiguration og resulterer i, at de individuelle sfæroider smelter sammen i løbet af en kort periode for at skabe et 3D bioprintet væv. Denne metode gør det således muligt for spheroider at blive manipuleret med lethed, med potentielle konsekvenser for fremtiden for stilladsfri organbiobroduktion.

<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

Denne protokol beskriver 3D bioprinting af hjertevæv uden brug af biomaterialer ved anvendelse af kun celler. Kardiomyocytter, endotelceller og fibroblaster isoleres først, tælles og blandes ved ønskede celleforhold. De er samdyrket i individuelle brønde i 96-brønds plader med ultra-low attachment. Inden for 3 dage, formende sfæroider form. Disse sfæroider hentes derefter af en dyse ved hjælp af vakuumsugning og monteres på en nålopsætning ved hjælp af en 3D bioprinter. Sfærerne får lov til at smelte på nålen. Tre dage efter 3D bioprinting fjernes sfæroiderne som en intakt patch, som allerede spontant slår. 3D bioprintede hjertepletter udviser mekanisk integration af komponent sfæroider og er meget lovende i hjertevævregenerering og som 3D-modeller af hjertesygdomme.

Ved slutningen af ​​trin 4.4 (samkultur) bør cellerne i hver brønd aggregere i bunden af ​​de ultra-lave vedhæftede 96-brønds U-bundplader for at danne sfæroider med tyngdekraften. Disse sfæroider indeholder hiPSC-CM, HCF&#39;er og HUVEC&#39;er og kan inspiceres visuelt under lysmikroskopi, hvor de skal forekomme cirkulære ved todimensionelle fremspring ( Figur 1 ). I slutningen af ​​trin 6.3 skal det 3D bioprintede hjerteplaster indeholde...

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

Denne protokol beskriver 3D bioprinting af hjertevæv uden brug af biomaterialer. 3D bioprintede hjertepletter udviser mekanisk integration af komponent sfæroider og er meget lovende i hjertevævregenerering og som 3D-modeller af hjertesygdomme.

Oprettelse af hjertevæv, der udviser mekanisk integration af sfæroider ved hjælp af 3D bioprinting

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

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