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

Os autores reconhecem as seguintes fontes de financiamento: Magic That Matters Fund for Cardiovascular Research e o Maryland Stem Cell Research Fund (2016-MSCRFI-2735).

1. Preparação de cardiomiócitos <ol><li> Gerar e cultivar células-tronco pluripotentes induzidas por humanos (hiPSCs) em placas de 6 poços revestidas com a matriz da membrana basal como descrito 10 . </li><li> Diferencie o HiPSCs em cardiomiócitos derivados de HiPSC (HiPSC-CMs) utilizando métodos previamente descritos 11 , 12 . </li><li> No dia 19 após a diferenciação, isolar os cardiomiócitos usando 2 mL de tripsina / EDTA 0...

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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...

Existem muitos métodos diferentes de bioprinção 3D 1 , 2 , 3 . A bioprinção em 3D é freqüentemente classificada pela tecnologia de impressão 1 , com exemplos como bioprintagem a jato de tinta, bioprinção de microextrusão, bioprinção assistida a laser, uma combinação de métodos ou abordagens mais recentes. A bioprinção 3D também pode ser classificada em métodos não dependentes de andaimes ou dependentes de andaimes 4 . A maioria dos métodos de bioprinção em 3D são dependentes de andaimes, onde há necessidade de biomateriais, por exemplo , bioinks 5 ou andaimes 6 . No entanto, a bioprinção 3D dependente do andaime enfrenta muitos problemas e limitações 4 , 7 , como a imunogenicidade do material de andaimes, o custo dos bioinks proprietários, a velocidade lenta e a toxicidade dos produtos de degradação. ScafA engenharia de tecido cardíaco sem dobra usando esferóides foi tentada 8 , com o potencial de superar essas desvantagens da engenharia de tecido dependente de andaimes. No entanto, como reconhecido pelos autores nesse artigo, foi difícil manipular e posicionar robustos esferoides em locais fixos, no processo de biofabricação. O uso concomitante de bioprinção 3D e engenharia de tecido à base de esferóides tem o potencial de superar essas dificuldades. Neste protocolo, descrevemos a bioprinção 3D do tecido cardíaco sem outros biomateriais, utilizando apenas células na forma de esferóides. As bioprinteiras 3D baseadas em esferóides sem cadastro 9 possuem a capacidade de retirar esferóides individuais usando aspiração a vácuo e colocá-los em uma disposição de agulhas. O conceito de posicionamento de esferóides em uma disposição de agulhas em bioprinção 3D, é inspirado no uso de matrizes de agulhas (conhecidas como &quot; kenzan &quot;) no antigo JapaNese arte de arranjo de flores, ikebana. Este sistema permite que os esferóides sejam posicionados com precisão em qualquer configuração e resulte em esferóides individuais fundindo em um curto período para criar um tecido 3D bioprinted. Este método permite que os esferóides sejam manipulados com facilidade, com implicações potenciais para o futuro da biofabricação de órgãos isentos de andaimes.

<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

Este protocolo descreve a bioprinção 3D do tecido cardíaco sem o uso de biomateriais, utilizando apenas células. Os cardiomiócitos, as células endotélias e os fibroblastos são primeiro isolados, contados e misturados às proporções de células desejadas. Eles são co-cultivados em poços individuais em placas de 96 poços de ultra-baixa fixação. Dentro de 3 dias, formando esferóides. Esses esferóides são então apanhados por um bico com aspiração a vácuo e montados em uma agulha com uma bioprantia 3D. Os esferóides são então autorizados a fundir na disposição da agulha. Três dias após a bioprinção em 3D, os esferóides são removidos como um remendo intacto, que já está espancando espontaneamente. Os remendos cardíacos bioprinted 3D apresentam integração mecânica de esferóides componentes e são altamente promissores na regeneração do tecido cardíaco e como modelos 3D de doenças cardíacas.

No final do passo 4.4 (co-cultura), as células em cada poço devem agregar no fundo das placas de fundo em U de 96 poços de montagem ultra baixa para formar esferóides por gravidade. Esses esferóides contêm hiPSC-CM, HCFs e HUVECs e podem ser vistos visualmente sob microscopia de luz, onde devem aparecer de forma circular por projeção bidimensional ( Figura 1 ). No final do passo 6.3, o remendo cardíaco bioprinted 3D deve conter vazios de tecido, dev...

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

Este protocolo descreve a bioprinção 3D do tecido cardíaco sem o uso de biomateriais. Os remendos cardíacos bioprinted 3D apresentam integração mecânica de esferóides componentes e são altamente promissores na regeneração do tecido cardíaco e como modelos 3D de doenças cardíacas.

Criação de tecido cardíaco exibindo integração mecânica de esferóides usando 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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