Name: a net mold-based method of scaffold-free three-dimensional cardiac tissue creation
Uploaded: 2018-08-05T13:00:00
Description: Watch this Scientific Journal Video about A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation at JoVE.com

Forfatterne anerkender den følgende finansieringskilde: magi at sager fonden for hjerte-kar-forskning.

1. forberedelse af Cardiomyocytes
<ol><li>Pels 6-godt plader med basalmembranen matrix og kultur menneskelige-induceret pluripotente stamceller (hiPSCs) som tidligere beskrevet17.</li>
	<li>Differentiere hiPSCs i hiPSC-CMs ved hjælp af tidligere beskrevne metoder18.</li>
	<li>På 16-18 d efter differentiering, suspendere cardiomyocytes ved at skylle hver brønd med 2 mL 1 x fosfatbufferet saltopløsning (PBS) uden calcium eller magnesium, efterfulgt af inkubation med 1 m...

<ol>
	<li>Gao, L., 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=Myocardial+Tissue+Engineering+With+Cells+Derived+From+Human-Induced+Pluripotent+Stem+Cells+and+a+Native-Like,+High-Resolution,+3-Dimensionally+Printed+Scaffold.">Myocardial Tissue Engineering With Cells Derived From Human-Induced Pluripotent Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold.</a> Circulation Research. 120 (8), 1318-1325 (2017).</li><li>Borovjagin, A. V., Ogle, B. M., Berry, J. L., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Microscale+Devices+to+3D+Printing:+Advances+in+Fabrication+of+3D+Cardiovascular+Tissues.">From Microscale Devices to 3D Printing: Advances in Fabrication of 3D Cardiovascular Tissues.</a> Circulation Research. 120 (1), 150-165 (2017).</li><li>Tandon, N., 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=Electrical+stimulation+systems+for+cardiac+tissue+engineering.">Electrical stimulation systems for cardiac tissue engineering.</a> Nature Protocols. 4 (2), 155-173 (2009).</li><li>Duran, A. G., 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=Regenerative+Medicine/Cardiac+Cell+Therapy:+Pluripotent+Stem+Cells.">Regenerative Medicine/Cardiac Cell Therapy: Pluripotent Stem Cells.</a> The Journal of Thoracic and Cardiovascular Surgery. 66 (1), 53-62 (2018).</li><li>Lux, M., 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=In+vitro+maturation+of+large-scale+cardiac+patches+based+on+a+perfusable+starter+matrix+by+cyclic+mechanical+stimulation.">In vitro maturation of large-scale cardiac patches based on a perfusable starter matrix by cyclic mechanical stimulation.</a> Acta Biomaterialia. 30, 177-187 (2016).</li><li>Eschenhagen, T., 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=Three-dimensional+reconstitution+of+embryonic+cardiomyocytes+in+a+collagen+matrix:+a+new+heart+muscle+model+system.">Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system.</a> The FASEB Journal. 11 (8), 683-694 (1997).</li><li>Moldovan, N. I., Hibino, N., Nakayama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+the+Kenzan+Method+for+Robotic+Cell+Spheroid-Based+Three-Dimensional+Bioprinting.">Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting.</a> Tissue Engineering Part B: Reviews. 23 (3), 237-244 (2017).</li><li>Sugiura, T., Hibino, N., Breuer, C. K., Shinoka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+cardiac+patch+seeded+with+human+induced+pluripotent+stem+cell+derived+cardiomyocytes+promoted+the+regeneration+of+host+cardiomyocytes+in+a+rat+model.">Tissue-engineered cardiac patch seeded with human induced pluripotent stem cell derived cardiomyocytes promoted the regeneration of host cardiomyocytes in a rat model.</a> Journal of Cardiothoracic Surgery. 11 (1), 163 (2016).</li><li>Caspi, O., 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=Tissue+engineering+of+vascularized+cardiac+muscle+from+human+embryonic+stem+cells.">Tissue engineering of vascularized cardiac muscle from human embryonic stem cells.</a> Circulation Research. 100 (2), 263-272 (2007).</li><li>Ong, C. 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=Biomaterial-Free+Three-Dimensional+Bioprinting+of+Cardiac+Tissue+using+Human+Induced+Pluripotent+Stem+Cell+Derived+Cardiomyocytes.">Biomaterial-Free Three-Dimensional Bioprinting of Cardiac Tissue using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes.</a> Scientific Reports. 7 (1), 4566 (2017).</li><li>Kelm, J. M., 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+concept+for+scaffold-free+vessel+tissue+engineering:+self-assembly+of+microtissue+building+blocks.">A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks.</a> Journal of Biotechnology. 148 (1), 46-55 (2010).</li><li>Noguchi, 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=Development+of+a+three-dimensional+pre-vascularized+scaffold-free+contractile+cardiac+patch+for+treating+heart+disease.">Development of a three-dimensional pre-vascularized scaffold-free contractile cardiac patch for treating heart disease.</a> The Journal of Heart and Lung Transplantation. 35 (1), 137-145 (2016).</li><li>Zuppinger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+culture+for+cardiac+cells.">3D culture for cardiac cells.</a> Biochimica et Biophysica Acta. 1863 (7 Pt B), 1873-1881 (2016).</li><li>Langenbach, F., 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=Generation+and+differentiation+of+microtissues+from+multipotent+precursor+cells+for+use+in+tissue+engineering.">Generation and differentiation of microtissues from multipotent precursor cells for use in tissue engineering.</a> Nature Protocols. 6 (11), 1726-1735 (2011).</li><li>Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C., de Boer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+culture+as+a+tool+for+creating+3D+complex+tissues.">Spheroid culture as a tool for creating 3D complex tissues.</a> Trends in Biotechnology. 31 (2), 108-115 (2013).</li><li>Agocha, A., Sigel, A. V., Eghbali-Webb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+adult+human+heart+fibroblasts+in+culture:+a+comparative+study+of+growth,+proliferation+and+collagen+production+in+human+and+rabbit+cardiac+fibroblasts+and+their+response+to+transforming+growth+factor-beta1.">Characterization of adult human heart fibroblasts in culture: a comparative study of growth, proliferation and collagen production in human and rabbit cardiac fibroblasts and their response to transforming growth factor-beta1.</a> Cell Tissue Research. 288 (1), 87-93 (1997).</li><li>Baudin, B., Bruneel, A., Bosselut, N., Vaubourdolle, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+isolation+and+culture+of+human+umbilical+vein+endothelial+cells.">A protocol for isolation and culture of human umbilical vein endothelial cells.</a> Nature Protocols. 2 (3), 481-485 (2007).</li><li>Pimentel, C. 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=Three-dimensional+fabrication+of+thick+and+densely+populated+soft+constructs+with+complex+and+actively+perfused+channel+network.">Three-dimensional fabrication of thick and densely populated soft constructs with complex and actively perfused channel network.</a> Acta Biomaterialia. 65, 174-184 (2018).</li><li>Shimizu, T., 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=Polysurgery+of+cell+sheet+grafts+overcomes+diffusion+limits+to+produce+thick,+vascularized+myocardial+tissues.">Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues.</a> The FASEB Journal. 20 (6), 708-710 (2006).</li><li>Sakaguchi, K., Shimizu, T., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+three-dimensional+vascularized+cardiac+tissue+with+cell+sheet+engineering.">Construction of three-dimensional vascularized cardiac tissue with cell sheet engineering.</a> Journal of Controlled Release. 205, 83-88 (2015).</li><li>Ong, C. 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=Creation+of+Cardiac+Tissue+Exhibiting+Mechanical+Integration+of+Spheroids+Using+3D+Bioprinting.">Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting.</a> Journal of Visualized Experiments. 125 (e55438), (2017).</li><li>Tan, 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=Cell+number+per+spheroid+and+electrical+conductivity+of+nanowires+influence+the+function+of+silicon+nanowired+human+cardiac+spheroids.">Cell number per spheroid and electrical conductivity of nanowires influence the function of silicon nanowired human cardiac spheroids.</a> Acta Biomaterialia. 51, 495-504 (2017).</li><li>Karra, R., Poss, K. 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Betydningen af denne metode ligger i dens reproducerbarhed og effektiviteten af den resulterende flerlaget hjerte væv. I feltet i hjertets vævsmanipulering er en af de nuværende mål at identificere en metode til at konstruere slå, flerlaget og funktionelle 3D-hjerte patches. Vi rapporterer en effektiv og reproducerbar metode til oprettelse af flerlaget hjerte væv af direkte manuel såning af spheroids sammensat af cardiomyocytes, endotel celler og fibroblaster i en roman netto støber. Netto formen anvendes i denne...

Målet med aktuelle hjerte vævsmanipulering er at udvikle en behandling for at udskifte eller reparere struktur og funktion af tilskadekomne Myokardie væv1. Metoder til at skabe 3D-hjerte væv modeller udstiller de vigtige kontraktile og elektrofysiologiske egenskaber af indfødte hjerte væv har hastigt ekspanderende2,3. En række forskellige strategier er blevet undersøgt og anvendes i undersøgelser4,5. Disse metoder spænder fra brugen af specifikke syntetiske og naturlige bioaktive hydrogels, såsom gelatine, kollagen, fibrin og peptider6, at bio-blæk deposition teknologier2 og bioprinting teknologier7.
Det har vist sig at stillads-gratis metoder kan producere sammenlignelige væv som biomateriale-baserede metoder, uden ulemperne at integrere udenlandske stilladser materiale8. Oren Caspi et al. demonstreret, at indarbejdelsen af forskellige typer af celler giver mulighed for generation af stærkt vaskulariserede human engineering hjerte væv9. Hagen et al. udviklet en 3-D udskrivningsmetoden for kardiale patch skabelse fra spheroids. Resulterende patches er sammensat af cardiomyocytes, fibroblaster og endotelceller i en 70:15:15 forholdet10. Spheroids har vist sig at være effektiv "byggesten" af stillads-fri hjerte væv skabelse, som de er resistente mod hypoxi og besidder tilstrækkelig mekanisk integritet for implantation11,12. Tidligere undersøgelser har påvist flere fabrikation metoder til oprettelse af sfæroide, herunder brug af hængende drop metode, spinner kolber13, mikrofluid systemer14og ikke-tilhænger kultur overflader ubestrøget eller bestrøget med Agarosen mikro-forme15. I denne protokol, vi bruger hængende drop enhed, som indeholder micropores til kondenserende hundredvis af spheroids ad gangen.
Denne undersøgelse præsenterer en roman og effektiv stillads-fri metode til oprettelse af hjertets væv, som omfatter manuelt såning af spheroids i en firkantet støbeform hulrum og inkubere væv på en shaker til modning. Under sædvanlige statisk kultur er ilt diffusion begrænset til de ydre aspekter af væv konstruktion, hvilket resulterer i central nekrose. Dog med den netto mug, er alle spheroids seedede i formen nedsænket i medierne med en konstant fluidic bevægelse, giver mulighed for øget udbredelse af næringsstoffer og ilt. Derudover denne skimmel-baseret metode giver mulighed for samtidig oprettelse af forskellige størrelser væv pletter med minimal manuelle indsats og den deraf følgende væv let kan fjernes fra formen. Denne nye metode giver mulighed for effektiv og reproducerbare oprettelsen af stillads-fri, flerlaget hjerte pletter.

<table border="0" cellpadding="0" cellspacing="0" style="width:708px;" width="708">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Human Cardiac fibroblasts (HCF)</td>
			<td style="width:139px;">Sciencell</td>
			<td style="width:119px;">6310</td>
			<td style="width:220px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">FM-2 Consists of Basal Medium</td>
			<td style="width:139px;">Sciencell</td>
			<td style="width:119px;">2331</td>
			<td style="width:220px;">HCF culture medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:231px;">Human umbilical vein endothelial cells (HUVEC)</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:119px;">CC-2935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EGM+Bullet Kit&#160;</td>
			<td style="width:139px;">Lonza</td>
			<td style="width:119px;">CC5035</td>
			<td>HUVEC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">E8 media&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A1517001</td>
			<td>HiPSC culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Geltrex&#160;</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">A1413202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">TrypLE Express Enzyme (1X)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">12604013</td>
			<td>Trypsin and Cell dissociation reagent</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">RPMI media</td>
			<td style="width:139px;">Invitrogen</td>
			<td style="width:119px;">11875093</td>
			<td style="width:220px;">RPMI media with B-27 supplement is hiPSC-CM culture medium</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:231px;">B-27 supplement (50x)</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">17504044</td>
			<td style="width:220px;">RPMI media with B-27 supplement is hiPSC-CM culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trypan Blue Solution, 0.4%</td>
			<td style="width:139px;">Thermo Fisher</td>
			<td style="width:119px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Novel net mold&#160;</td>
			<td style="width:139px;">TissueByNet Co.,Ltd</td>
			<td style="width:119px;">NM25-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hanging drop plate</td>
			<td style="width:139px;">Kuraray Co.,Ltd</td>
			<td style="width:119px;">MPc350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">6 well plates&#160;</td>
			<td style="width:139px;">Sigma-Aldrich</td>
			<td style="width:119px;">CLS-3516</td>
			<td></td>
		</tr>
	</tbody>
</table>

a net mold-based method of scaffold-free three-dimensional cardiac tissue creation

Denne protokol beskriver en roman og let netto skimmel-baseret metode til at oprette tre-dimensionelle (3-D) hjerte væv uden yderligere stillads materiale. Human-induceret pluripotente stamceller-celle-afledte cardiomyocytes (iPSC-CMs), menneskelige hjerte fibroblaster (HCFs) og menneskelige umbilical vene endothelial celler (HUVECs) er isoleret og bruges til at generere en cellesuspension med 70% iPSC-CMs, 15% HCFs og 15% HUVECs. De er co kulturperler i et ultra-lavt vedhæftet fil "hængende drop" system, som indeholder micropores til kondenserende hundredvis af spheroids ad gangen. Cellerne samlede og danner spontant slå spheroids efter 3 dage med fælles kultur. Spheroids er høstet, seedet i en roman støbeform hulrum og kulturperler på en shaker i rugemaskinen. Spheroids blevet en moden funktionelle væv ca. 7 dage efter såning. De deraf følgende lag væv består af sammenvoksede spheroids med tilfredsstillende strukturelle integritet og synkron slå adfærd. Denne nye metode har et lovende potentiale som en reproducerbar og omkostningseffektiv metode til at oprette manipuleret væv til behandling af hjertesvigt i fremtiden.

I vores forsøg udnyttet vi en cellesuspension af 70% iPSC-CMs, 15% HCFs og 15% HUVECs i RPMI/B-27 celle medier på en koncentration af 2,475,000 celler pr. mL. Når du har oprettet cellesuspension, udleveret vi 4 mL cellesuspension til hver brønd af udlægget ultra-lavt hængende drop system, som beskrevet i trin 4.3 i protokollen. Brugen af hængende drop system resulterede i spontan dannelsen af hundredvis af slå spheroids efter 3 dages kultur ved 37 ° C, 5% CO2og 95% luf...

Watch this Scientific Journal Video about A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation at JoVE.com

A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation

En netto skimmel-baseret metode til stillads-gratis tre-dimensionelle hjerte væv skabelse

Denne protokol beskriver en netto skimmel-baseret metode for at oprette tre-dimensionelle stillads-fri hjerte væv med tilfredsstillende strukturelle integritet og synkron slå adfærd.

This protocol describes a novel and easy net mold-based method to create three-dimensional (3-D) cardiac tissues without additional scaffold material. ...

Research

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Bioengineering

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A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

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