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

This work was supported by a grant-in-aid from the Heart and Stroke Foundation of Canada (G-14-0006265), operating grants from the Canadian Institutes of Health Research (137352 and 143066), and a J.P. Bickell foundation grant (1013821) to SSN.

1. Master Design and Fabrication
NOTE: Use soft lithography for device fabrication. Make a two-layer SU-8 master for polydimethylsiloxane (PDMS) molding.
<ol>
	<li>Design the device using a design and drafting software (Figure 1A, left). Draw each layer of the master separately. Print the device design on two photomasks at 20,000 dpi, corresponding to the two layers of the master15. Set the device pattern as transparent and the surrounding area as dar...

<ol>
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This manuscript describes the setup and implementation of the engineered platform, biowire, to improve the maturation of hPSC-CMs. The device can be made in standard microfabrication facilities, and biowires can be produced with common cell culture techniques and an electrical stimulator.
To our knowledge, there is no reported method similar to biowires. This strategy reveals that improved maturation properties were dependent on the electrical stimulation regimen, as evidenced by the greater m...

Cell-based therapy is one of the most promising and investigated strategies to achieve cardiac repair/regeneration. It has been aided by cardiac tissue engineering and the co-delivery of biomaterials1,2. Most available cell sources have been studied in animal models for their potentially beneficial effects on damaged, diseased, or aged hearts3. In particular, significant efforts have been made to use human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CM), a potentially unlimited autologous cell source for cardiac tissue engineering. hPSC-CMs can be produced using several established protocols4,5,6. However, the obtained cells display fetal-like phenotypes, with a range of immature characteristics compared to adult ventricular cardiomyocytes7,8. This can be an obstacle to the application of hPSC-CMs as models of adult heart tissue in drug discovery research and in the development of adult cardiac disease models9.
In order to overcome this limitation of phenotypic immaturity, new approaches have been actively investigated to promote cardiomyocyte maturation. Early studies revealed effective pro-maturation properties in neonatal rat cardiomyocytes via cyclic mechanical10 or electrical stimulation11. Gel compaction and cyclic mechanical stimulation were also shown to improve some aspects of hPSC-CM maturation12,13, with minimal enhancement of the electrophysiological and calcium handling properties. Therefore, a platform system called &#34;biological wire&#34; (biowire) was devised by providing both structural cues and electrical field stimulation to enhance the maturation of hPSC-CMs14. This system uses a microfabricated platform to create aligned cardiac tissue that is amenable to electrical field stimulation. This can be used to improve the structural and electrophysiological maturity of hPSC-CMs. Here, we describe the details of making such 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

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been a promising cell source and have thus encouraged the investigation of their potential applications in cardiac research, including drug discovery, disease modeling, tissue engineering, and regenerative medicine. However, cells produced by existing protocols display a range of immaturity compared with native adult ventricular cardiomyocytes. Many efforts have been made to mature hPSC-CMs, with only moderate maturation attained thus far. Therefore, an engineered system, called biowire, has been devised by providing both physical and electrical cues to lead hPSC-CMs to a more mature state in vitro. The system uses a microfabricated platform to seed hPSC-CMs in collagen type I gel along a rigid template suture to assemble into aligned cardiac tissue (biowire), which is subjected to electrical field stimulation with a progressively increasing frequency. Compared to nonstimulated controls, stimulated biowired cardiomyocytes exhibit an enhanced degree of structural and electrophysiological maturation. Such changes are dependent upon the stimulation rate. This manuscript describes in detail the design and creation of biowires.

The rational for the use of a suture in the biowires is to serve as a template for the formation of 3D constructs that align in one axis and mimic the shape of cardiac fibers. We show that after seven days of culture in the biowire, cells remodeled the gel around the suture (Figure 3A). The cells assembled along the axis of the suture to form aligned cardiac tissue (Figure 3). After 7 days of preculture, the biowires were subjected to 7 days of electrical...

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

The cardiac biowire platform is an in vitro method used to mature human embryonic and induced pluripotent stem cell-derived cardiomyocytes (hPSC-CM) by combining three-dimensional cell cultivation with electrical stimulation. This manuscript presents the detailed setup of the cardiac biowire platform.

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

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