Name: maturation of human stem cell-derived cardiomyocytes in biowires using electrical stimulation
Uploaded: 2017-05-06T13:00:00
Description: Watch this Scientific Journal Video about Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation at JoVE.com

Questo lavoro è stato sostenuto da una sovvenzione-in-aiuto del Heart and Stroke Foundation of Canada (G-14-0.006.265), sovvenzioni di funzionamento dal Canadian Institutes of Health Research (137.352 e 143.066), e una borsa di studio fondazione JP Bickell (1.013.821 ) al SSN.

1. master e Fabrication Nota: utilizzare litografia soft per la fabbricazione di dispositivi. Fare un doppio strato SU-8 master per polidimetilsilossano (PDMS) stampaggio. <ol><li> Progettare il dispositivo utilizzando un design e redazione software (Figura 1A, sinistra). Disegnare ogni strato del maestro separatamente. Stampa il design del dispositivo su due fotomaschere a 20.000 dpi, corrispondenti ai due strati del maestro 15. Impostare il modell...

<ol>
	<li>Sun, X., Nunes, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+hydrogel-based+strategies+for+application+in+cardiac+tissue+regeneration.">Overview of hydrogel-based strategies for application in cardiac tissue regeneration.</a> Biomed Mater. 10 (3), 034005 (2015).</li><li>Sun, X., Altalhi, W., Nunes, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascularization+strategies+of+engineered+tissues+and+their+application+in+cardiac+regeneration.">Vascularization strategies of engineered tissues and their application in cardiac regeneration.</a> Adv Drug Deliv Rev. 96, 183-194 (2016).</li><li>Hastings, C. 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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=Biowire:+a+platform+for+maturation+of+human+pluripotent+stem+cell-derived+cardiomyocytes.">Biowire: a platform for maturation of human pluripotent stem cell-derived cardiomyocytes.</a> Nat Methods. 10 (8), 781-787 (2013).</li><li>Lake, 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=Microfluidic+device+design,+fabrication,+and+testing+protocols.">Microfluidic device design, fabrication, and testing protocols.</a> Protocol Exchange. , (2015).</li><li>Shiba, Y., Hauch, K. D., Laflamme, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+applications+for+human+pluripotent+stem+cells.">Cardiac applications for human pluripotent stem cells.</a> Curr Pharm Des. 15 (24), 2791-2806 (2009).</li><li>Yang, X., 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=Tri-iodo-l-thyronine+promotes+the+maturation+of+human+cardiomyocytes-derived+from+induced+pluripotent+stem+cells.">Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes-derived from induced pluripotent stem cells.</a> J Mol Cell Cardiol. 72, 296-304 (2014).</li><li>Zhang, 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=Tissue-engineered+cardiac+patch+for+advanced+functional+maturation+of+human+ESC-derived+cardiomyocytes.">Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.</a> Biomaterials. 34 (23), 5813-5820 (2013).</li><li>Radisic, 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=Oxygen+gradients+correlate+with+cell+density+and+cell+viability+in+engineered+cardiac+tissue.">Oxygen gradients correlate with cell density and cell viability in engineered cardiac tissue.</a> Biotechnol Bioeng. 93 (2), 332-343 (2006).</li><li>Reubinoff, B. 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Questo manoscritto descrive la configurazione e implementazione della piattaforma progettata, BIOWIRE, per migliorare la maturazione di HPSC-CM. Il dispositivo può essere realizzato in impianti di microfabbricazione standard e biowires può essere prodotto con tecniche di coltura cellulare comuni e uno stimolatore elettrico. A nostra conoscenza, non esiste un metodo simile a quello riportato biowires. Questa strategia rivela che il miglioramento delle proprietà di maturazione sono dipenden...

La terapia a base di cellule è una delle strategie più promettenti e studiati per raggiungere cardiaca riparazione / rigenerazione. E &#39;stato aiutato da ingegneria dei tessuti cardiaco e il co-fornitura di biomateriali 1, 2. La maggior parte delle fonti di cellule disponibili sono stati studiati in modelli animali per i loro effetti potenzialmente benefici sulla danneggiati, malati, o cuori di età compresa tra 3. In particolare, notevoli sforzi sono stati fatti per usare cellule staminali pluripotenti umane (HPSC) -derived cardiomiociti (HPSC-CM), una fonte di cellule autologhe potenzialmente illimitato per l&#39;ingegneria tissutale cardiaco. HPSC-CM può essere prodotto utilizzando diversi protocolli stabiliti 4, 5, 6. Tuttavia, le cellule ottenute mostrano fenotipi fetali simile, con una serie di caratteristiche immaturi rispetto al cardiomiociti ventricolari adulti 7, </sup&gt; 8. Questo può essere un ostacolo per l&#39;applicazione di HPSC-CMS come modelli di tessuto cardiaco degli adulti nella ricerca scoperta di nuovi farmaci e lo sviluppo di modelli di malattia cardiaca per adulti 9. Per superare questa limitazione di immaturità fenotipica, nuovi approcci sono stati attivamente studiati per promuovere la maturazione dei cardiomiociti. I primi studi hanno rivelato efficaci proprietà pro-maturazione in cardiomiociti neonatali di ratto tramite ciclico 10 meccanica o stimolazione elettrica 11. Compattazione gel e ciclica stimolazione meccanica stati mostrati anche di migliorare alcuni aspetti della HPSC-CM maturazione 12, 13, con un minimo aumento delle proprietà di manipolazione elettrofisiologiche e di calcio. Pertanto, un sistema piattaforma chiamata &quot;wire biologica&quot; (BIOWIRE) è stato ideato fornendo sia segnali strutturali e campo elettrico stimulation per migliorare la maturazione di HPSC-CM 14. Questo sistema utilizza una piattaforma microfabbricazione per creare tessuto cardiaco allineato che è suscettibile di stimolazione campo elettrico. Questo può essere usato per migliorare la maturità strutturale ed elettrofisiologico di HPSC-CM. Qui, descriviamo i dettagli di tale biowires.

<table border="0" cellpadding="0" cellspacing="0" width="582">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">L-Ascorbic acid</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">A-4544</td>
			<td style="width:167px;">hPSC-CM culture media componet</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">AutoCAD</td>
			<td style="width:80px;">Autodesk, Inc</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Software to design device</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Carbon rods, &#216; 3 mm</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Electrical stimulator chamber component</td>
		</tr>
		<tr height="206">
			<td height="206" style="height:206px;width:216px;">Collagen, type 1, rat tail</td>
			<td style="width:80px;">BD Biosciences</td>
			<td style="width:119px;">354249</td>
			<td style="width:167px;">Collagen gel: 2.1 mg/ml of rat tail collagen type I in 24.9 mM glucose, 23.8 mM NaHCO3, 14.3 mM NaOH, 10 mM HEPES, in 1xM199 media with 10 % of growth factor-reduced Matrigel.</td>
		</tr>
		<tr height="164">
			<td height="164" style="height:164px;width:216px;">Collagenase type I&#160;</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">C0130</td>
			<td style="width:167px;">0.2% collagenase type I (w/v) and 20% FBS (v/v) in PBS with Ca2+ and Mg2+. Sterilize with 0.22 &#956;m filter and make 12 ml aliquots. Store at -20 &#176;C.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:216px;">Deoxyribonuclease I (DNase I)</td>
			<td style="width:80px;">EMD Millipore</td>
			<td style="width:119px;">260913-25MU</td>
			<td style="width:167px;">Make 1 mg/ml DNase I stock solution in water. Filter sterile and store 0.5 ml aliquots at &#8722;20 &#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Drill &#38; drill bits (&#216; 1mm and 2 mm)</td>
			<td style="width:80px;">Dremel</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Drill holes in polycarbonate frames</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Electrical stimulator</td>
			<td style="width:80px;">Grass</td>
			<td style="width:119px;">s88x</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Fetal bovine serum (FBS)</td>
			<td style="width:80px;">WISENT Inc.</td>
			<td style="width:119px;">080-450</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">D-(+)-Glucose&#160;</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">G5767</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">L-Glutamine</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25030081</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">H2O</td>
			<td style="width:80px;">MilliQ</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">18.2 M&#937;&#183;cm at 25 &#176;C, ultrapure, to make all solutions</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">HEPES</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">H4034</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Hot plate</td>
			<td style="width:80px;">Torrey Pines</td>
			<td style="width:119px;">HS40</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Iscove&#39;s Modified Dulbecco&#39;s Medium(IMDM)</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">12440053</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Mask aligner</td>
			<td style="width:80px;">EVG&#160;</td>
			<td style="width:119px;">EVG 620</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Matrigel, growth factor reduced&#160;</td>
			<td style="width:80px;">Corning</td>
			<td style="width:119px;">354230</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Medium 199 (M199)</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">11150059</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Monothioglycerol (MTG)</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">M-6145</td>
			<td style="width:167px;">hPSC-CM culture media componet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Orbital shaker</td>
			<td style="width:80px;">VWR</td>
			<td style="width:119px;">89032-088</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Penicillin/Streptomycin (P/S)</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">15070063</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Phosphate-buffered saline (PBS) with Ca2+ and Mg2+&#160;</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">14040133</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Plate (6-well)</td>
			<td style="width:80px;">Corning</td>
			<td style="width:119px;">353046</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Plate (6-well), low attachment</td>
			<td style="width:80px;">Corning</td>
			<td style="width:119px;">3471</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Platinum wires, 0.2 mm</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Electrical stimulator chamber component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:80px;">Dow Corning</td>
			<td style="width:119px;">Sylgard 184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Propylene glycol monomethyl ether acetate (PGMEA)</td>
			<td style="width:80px;">Doe &#38; Ingalls Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">To develop the wafer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Pouch, peel-open</td>
			<td style="width:80px;">Convertors</td>
			<td style="width:119px;">92308</td>
			<td style="width:167px;">For steam sterilization</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Silicon wafer, 4-inch</td>
			<td style="width:80px;">UniversityWafer Inc.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">Sodium bicarbonate (NaHCO3)</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Sodium hydroxide</td>
			<td style="width:80px;">Sigma</td>
			<td style="width:119px;">S8045</td>
			<td style="width:167px;">Collagen gel component</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Sprin coater</td>
			<td style="width:80px;">Specialty Coating Systems</td>
			<td style="width:119px;">G3P-8</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="280">
			<td height="280" style="height:280px;width:216px;">StemPro-34 culture medium</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">10639011</td>
			<td style="width:167px;">hPSC-CM culture medium. To make 50 ml, add 1.3 ml supplement, 500 &#956;l of 100&#215; L-Glutamine, 250 &#956;l of 30 mg/ml transferrin, 500 &#956;l of 5 mg/ml ascorbic acid, 150 &#956;l of 26 &#956;l /2 ml monothioglycerol (MTG), and 500 &#956;l (1 %) penicillin/streptomycin.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Stop media</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">&#160;Wash medium:FBS (1:1)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">SU-8 50&#160;</td>
			<td style="width:80px;">MicroChem Corp.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">photoresist, master component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">SU-8 2050&#160;</td>
			<td style="width:80px;">MicroChem Corp.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">photoresist, master component</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Transferrin</td>
			<td style="width:80px;">Roche</td>
			<td style="width:119px;">10-652-202</td>
			<td style="width:167px;">hPSC-CM culture media componet</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Trypsin/EDTA, 0.25%</td>
			<td style="width:80px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">25200056</td>
			<td style="width:167px;">hPSC-CM culture media componet</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:216px;">Wash medium</td>
			<td style="width:80px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">IMDM containing 1% Penicillin/Streptomycin</td>
		</tr>
	</tbody>
</table>

maturation of human stem cell-derived cardiomyocytes in biowires using electrical stimulation

cardiomiociti derivati ​​dalle cellule staminali umane pluripotenti (HPSC-CMS) sono stati una fonte di cellule promettente e hanno così favorito l&#39;esame delle loro potenziali applicazioni nel campo della ricerca cardiaca, tra cui la scoperta di farmaci, la modellazione della malattia, l&#39;ingegneria dei tessuti e della medicina rigenerativa. Tuttavia, le cellule prodotte da protocolli esistenti mostrano una serie di immaturità rispetto ai cardiomiociti adulti ventricolare nativi. Molti sforzi sono stati fatti maturare HPSC-CM, con solo la maturazione moderata raggiunto finora. Pertanto, un sistema ingegnerizzato, chiamato BIOWIRE, è stato ideato prevedendo stimoli fisici ed elettrici di condurre HPSC-CM ad uno stato più maturo in vitro. Il sistema utilizza una piattaforma microfabbricazione per seminare HPSC-CM in collagene tipo I gel lungo un modello di sutura rigido da montare nel tessuto cardiaco allineata (BIOWIRE), sottoposto alla stimolazione campo elettrico con una frequenza progressivamente crescente. Rispetto ai controlli non stimolate,stimolati biowired cardiomiociti presentano un maggiore grado di maturazione strutturale ed elettrofisiologico. Tali cambiamenti dipendono dalla frequenza di stimolazione. Questo manoscritto descrive in dettaglio la progettazione e realizzazione di biowires.

Il razionale per l&#39;uso di una sutura nelle biowires è servire come stampo per la formazione di costrutti 3D che si allineano in un asse e imitano la forma di fibre cardiache. Abbiamo dimostrato che dopo sette giorni di coltura in BIOWIRE, cellule rimodellato il gel intorno la sutura (Figura 3A). Le celle assemblate lungo l&#39;asse della sutura per formare in linea di tessuto cardiaco (Figura 3). Dopo 7 giorni di precoltura, i biowires sono stati so...

Watch this Scientific Journal Video about Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation at JoVE.com

Maturation of Human Stem Cell-derived Cardiomyocytes in Biowires Using Electrical Stimulation

La piattaforma BIOWIRE cardiaco è un metodo in vitro utilizzato per maturare embrionale umano e cardiomiociti derivati dalle cellule staminali pluripotenti indotte (HPSC-CM) combinando coltura cellulare tridimensionale con stimolazione elettrica. Questo manoscritto presenta la configurazione dettagliata della piattaforma BIOWIRE cardiaco.

Maturazione di staminali umane cardiomiociti derivati ​​dalle cellule in Biowires Utilizzando Stimolazione Elettrica

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their ...

The overall goal of this procedure is to utilize controlled three dimensional cell cultivation methods in electrical stimulation to promote the maturation of human embryonic and induced pluripotent stem cells derived cardiomyocytes. This method can help answer key questions in the cardiac tissue engineering field including how different electrical stimulation parameters or length of time affect cardiac cell function and maturation state. The end results are engineered human cardiac tissues of improved maturity.The implications of this technique extend toward diagnosis applications such as testing the safety and efficacy of new drugs because cardiomyocyte with mature characteristics such as electrical progress are most likely to serve as more accurate models of human cardiac cells in drug screening studies. Combine the PDMS monomer and curing agent at a 10:1 ratio and mix well. Let it set for five minutes to degas.Then load a Petri dish containing the master for the biowire template with the PDMS mixture. Try to minimize air bubble formation. Next cure the PDMS at 70 degree Celsius for two hours.Then recover the solidified mold by first using a sharp blade around the edges and then peeling it away. Then trim the excess PDMS and collect all the templates in a sterilization pouch. Steam auto clave the templates at 121 degree Celsius for 20 minutes.And then load each template into a sterile Petri dish under the ventilation of a biosafety cabinet. Now for each template use tweezers to mount a piece of sterile 6.0 silk suture into the groove spanning the entire channel. The templates are then ready for cell seeding.The suture will serve as a template for the formation of 3D constructs that align along the suture axis mimicking the shape of cardiac fibers. For each chamber, make a pair of rectangular polycarbonate pieces to serve as the frame for the electrical stimulation chamber. Along the center line of each piece, drill two three millimeter wide holes, two centimeters apart.For each chamber prepare two 1.5 centimeter long carbon rods. About 3 millimeters from the end of each rod drill a one millimeter wide hole. Then thread each hole with a platinum wire and wrap one end of the wire around the rod.Next, for each chamber insert two rods between two polycarbonate pieces making a frame. Then pour about two milliliters of PDMS into a well of a six well plate and place the frame into the well with PDMS, submerging the bottom of the frames. Cure the PDMS at 70 degree Celsius for two hours so the PDMS secures the frame.Then remove the completed chamber from the plate with the PDMS attached. To test the function of an assembled chamber connect the platinum wires to a battery and use a voltmeter to check that electricity is being passed through the rods. Now transfer the functional chambers into a sterilization pouch and use 20 minutes of steam sterilization at 121 degree celsius to prepare them for cell culture.To prepare the gel, precool all the gel components on ice before mixing them in a sterile tube. Make sure to add the collagen and extra cellular matrix last and mix the components using pipetting. For the cells have cardiomyocytes differentiated from human pluripotent stem cells.Collect the cells and for each biowire preparation plant prepare 3.5 microliters of collagen gel containing half a million cells. Now use a P10 pipette tip to load approximately four milliliters of suspension into the 0.5 centimeter long channel template in each dish. Adjust the position of the suture using sterile forceps as needed so it is fully stretched.Once seeding the cells, make sure to always resuspend the gel cell mixture before taking autoclave in order to have consistent results. After loading the channels, cover the bottom of the dish with sterile PBS making sure not to touch the gel. This will ensure that the biowires do not dehydrate during polymerization.After incubating the biowires at 37 degree celsius for 30 minutes, replace the PBS with enough culture medium to cover the cells. 12 milliliters is usually enough. Then culture the dish for a week replacing the medium every other day.In a bio safety cabinet add two milliliters of PBS to each well of a six well plate that will house an electrical stimulator chamber. Then use sterile tweezers to gently place the autoclave devices into the wells. Once in position, discard the PBS and replace it with five milliliters of warm medium which should completely cover the carbon rods.Before culturing the cells for 24 hours, observe the cardiomyocytes contractility in response to electrical stimulation to ensure that the cells are capturing the electrical stimulus and responding with contractions. Next, place the biowires into the electrical stimulator chamber and orient the channels perpendicularly to the carbon rods. Extend the platinum wires away from the plate and then cover the plates.Connect the wires to an electrical stimulator. Set the stimulator to deliver biphasic one millisecond pulses at one hertz and at three volts per centimeter. Then transfer the preparations in the electrical stimulator to an incubator at 37 degree celsius.Increase stimulation rate incrementally every 24 hours from one hertz at day zero to six hertz at day six. During this week of culture, change the medium every other day. And observe the cardiomyocytes contractility on a daily basis.After seven days of preculture in the biowire cells remolded the gel around the suture. The cells assembled along the axis of the suture to form aligned cardiac tissue. The cells expressed cardiac contractile proteins showed sarcomeric banding in the contractile apparatus and their myofibrils were aligned along the axis of the suture.Cells in the biowire exhibited lower proliferation rates than cells in embryoid bodies. Calcium handling was significantly improved in the cardiomyocytes cultivated in biowires. Treatment with caffeine induced an abrupt release of calcium from the sacroplasmic reticulum.Such transients were found only in stimulated cells. There were also significantly higher stimulation frequency depending calcium release amplitudes. In addition, cardiomyocytes cultured in biowires showed improved hERG and inward rectifier current densities compared to cultivation in embryoid bodies which was further enhanced by electrical stimulation.The improved inward rectifier current was consistent with increased expression of the potassium inwardly rectifying channel jean, KCN J2.Automaticity the ability to beat spontaneously was significantly higher in embryoid body derived cardiomyocytes compared to controlled biowires. Control showed automaticity comparable to biowire preparation subjected to the six hertz regimen. Once mastered the setup of the biowires can be done in half a day.The setup of electrical stimulation will take a couple of hours and the final results can be reached in two weeks. While attempting this procedure, it is important to remember that cardiomyocytes differentiated from different cell lines or by different protocols can vary slightly in their maturity and purity. Therefore optimizing the experimental parameters such as the stimulation rate to your cells might be required.Following this procedure, other conditions including variation of the cellular composition and the utilization of mutant cardiomyocytes can be optimized to answer a bevy of additional questions.

Research

JoVE Journal

Developmental Biology

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Derivazione di Cardiomyocytes altamente purificato da cellule staminali umane pluripotenti indotte utilizzando piccoli Differenziazione Molecola-modulato e successiva glucosio Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Ricerca

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Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

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Maturazione di staminali umane cardiomiociti derivati ​​dalle cellule in Biowires Utilizzando Stimolazione Elettrica

Maturazione di staminali umane cardiomiociti derivati dalle cellule in Biowires Utilizzando Stimolazione Elettrica