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

De auteurs erkennen de volgende financieringsbronnen: Magic That Matters Fund voor Cardiovascular Research en het Maryland Stamcellen Research Fund (2016-MSCRFI-2735).

1. Bereiding van cardiomyocyten <ol><li> Genereer en kweek menselijke geïnduceerde pluripotente stamcellen (hiPSCs) op 6-putjes platen die zijn bekleed met basismembraanmatrix zoals beschreven 10 . </li><li> Differentieer hiPSCs in hiPSC afgeleide cardiomyocyten (hiPSC-CMs) met behulp van eerder beschreven methoden 11 , 12 . </li><li> Op dag 19 na differentiatie isoleren de cardiomyocyten met behulp van 2 ml trypsine / EDTA 0,05% in el...

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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...

Er zijn veel verschillende methoden van 3D bioprinting 1 , 2 , 3 . 3D bioprinting wordt vaak geclassificeerd door printtechnologie 1 , met voorbeelden zoals inkjet bioprinting, microextrusion bioprinting, laser assisted bioprinting, een combinatie van methoden of nieuwere benaderingen. 3D bioprinting kan ook worden ingedeeld in steigervrije of steigerafhankelijke methoden 4 . De meeste methoden van 3D bioprinting zijn steigerafhankelijk, waar biomaterialen nodig zijn, bijv. Bioinks 5 of steigers 6 . Echter, steigerafhankelijke 3D bioprinting ondervindt veel problemen en beperkingen 4 , 7 , zoals immunogeniciteit van steigermateriaal, kosten van eigen bio-verbindingen, trage snelheid en toxiciteit van afbraakproducten. SCAFVouwvrije hartweefseltechniek met behulp van sferoïden is geprobeerd 8 , met het potentieel om deze nadelen van steigerafhankelijke weefseltechniek te overwinnen. Zoals echter door de auteurs in dat papier werd erkend, was het moeilijk om robuuste handgreep en positionering van spheroïden op vaste locaties, in het proces van biofabricatie. Het gelijktijdige gebruik van 3D bioprinting en spiraal gebaseerde weefseltechniek heeft de mogelijkheid om deze moeilijkheden te overwinnen. In dit protocol beschrijven we 3D bioprinting van hartweefsels zonder andere biomaterialen, waarbij alleen cellen in de vorm van sferoïden worden gebruikt. Steigervrije spheroïde-gebaseerde 3D bioprinters 9 hebben de mogelijkheid om individuele spheroïden op te halen met behulp van vacuümzuigen en positioneren op een naald array. Het concept van positionering sferoïden op een naald array in 3D bioprinting, is geïnspireerd op het gebruik van naald arrays (bekend als &quot; kenzan &quot;) in de oude JapaNese kunst van bloemstuk, ikebana. Met dit systeem kunnen spheroïden precies in elke configuratie gepositioneerd worden, waardoor de individuele spheroïden over een korte periode samenvoegen om een ​​3D bioprintend weefsel te creëren. Met deze methode kunnen spheroïden gemakkelijk worden gemanipuleerd, met mogelijke implicaties voor de toekomst van de stellingvrije organische biofabricatie.

<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

Dit protocol beschrijft 3D bioprinting van hartweefsel zonder gebruik van biomaterialen, waarbij alleen cellen worden gebruikt. Cardiomyocyten, endotheelcellen en fibroblasten worden eerst geïsoleerd, geteld en gemengd bij gewenste celverhoudingen. Ze worden gecultiveerd in individuele putten in ultra-low attachment 96-wells platen. Binnen 3 dagen slaat er spheroidsvorm. Deze sferoïden worden dan opgevangen door een mondstuk met vacuümzuigkracht en gemonteerd op een naald array met behulp van een 3D bioprinter. De sferoïden mogen dan op de naald array zitten. Drie dagen na 3D bioprinting worden de sferoïden verwijderd als een intacte patch, die al spontaan slaat. 3D bioprinted cardiac patches vertonen mechanische integratie van component sferoïden en zijn zeer veelbelovend in hartweefselregeneratie en als 3D-modellen van hart-en vaatziekten.

Aan het einde van stap 4.4 (co-cultuur) moeten de cellen in elke put bij de onderkant van de ultra-lage bevestiging 96-putjes U-bodemplaten samenvoegen om de spheroïden door zwaartekracht te vormen. Deze sferoïden bevatten hiPSC-CM, HCF&#39;s en HUVECs, en kunnen visueel geïnspecteerd worden onder lichtmicroscopie, waar ze circulair door tweedimensionale projectie moeten worden weergegeven ( Figuur 1 ). Aan het einde van stap 6.3, moet de 3D bioprinted ca...

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

Dit protocol beschrijft 3D bioprinting van hartweefsel zonder het gebruik van biomaterialen. 3D bioprinted cardiac patches vertonen mechanische integratie van component sferoïden en zijn zeer veelbelovend in hartweefselregeneratie en als 3D-modellen van hart-en vaatziekten.

Het creëren van cardiale weefsels die mechanische integratie van sferoïden tonen met behulp van 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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