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

Este trabalho foi apoiado por uma bolsa-in-aid from the Heart and Stroke Foundation of Canada (G-14-0006265), subvenções de funcionamento dos Institutos Canadenses de Pesquisa em Saúde (137352 e 143066), e um subsídio da Fundação JP Bickell (1013821 ) à SSN.

1. Projeto mestre e fabricação NOTA: Use litografia suave para a fabricação do dispositivo. Faça um mestre SU-8 de duas camadas para a moldagem de polidimetilsiloxano (PDMS). <ol><li> Desenhe o dispositivo usando um software de desenho e desenho ( Figura 1A , à esquerda). Desenhe cada camada do mestre separadamente. Imprima o desenho do dispositivo em duas fotomassagens a 20.000 dpi, correspondendo às duas camadas do mestre 15 . Definir o pad...

<ol>
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Este manuscrito descreve a configuração e a implementação da plataforma de engenharia, biowire, para melhorar a maturao de HPSC-CMs. O dispositivo pode ser feito em instalações de microfabricação padrão, e biowires pode ser produzido com técnicas de cultura de células comuns e um estimulador elétrico. Para nosso conhecimento, não há nenhum método relatado semelhante ao biowires. Esta estratégia revela que a melhoria das propriedades de maturação foram dependentes do regime ...

terapia baseada em celular é uma das mais promissoras e investigados para alcançar cardíaca reparação / regeneração. Ele foi auxiliado por engenharia de tecidos cardíaco e o co-entrega de biomateriais 1, 2. A maioria das fontes celulares disponíveis têm sido estudados em modelos animais para os seus efeitos potencialmente benéficos sobre danificados, doentes, ou corações com idades entre 3. Em particular, os esforços significativos foram feitos para usar células-tronco pluripotentes humanas (HPSC) derivada de cardiomiócitos (HPSC-CM), uma fonte de células autólogas potencialmente ilimitado para a engenharia de tecido cardíaco. HPSC-CMs pode ser produzido usando vários protocolos estabelecidos 4, 5, 6. No entanto, as células obtidas a exibir os fenótipos fetais semelhante, com uma gama de características imaturas em comparação com cardiomiócitos ventriculares do adulto 7, </sup&gt; 8. Isso pode ser um obstáculo para a aplicação de HPSC-CMs como modelos de tecido cardíaco de adultos na pesquisa de descoberta de drogas e no desenvolvimento de adultos modelos de doenças cardíacas 9. De modo a ultrapassar esta limitação de imaturidade fenotípica, novas abordagens têm sido investigado activamente para promover a maturação de cardiomiócitos. Os primeiros estudos revelaram propriedades pró-maturação eficazes em cardiomiócitos de ratos neonatais através cíclico mecânico 10 ou estimulação eléctrica 11. Compactação do gel e estimulação mecânica cíclico também foram exibidas para melhorar alguns aspectos da HPSC-CM maturação 12, 13, com realce mínimo das propriedades de manuseamento electrofisiológicos e de cálcio. Portanto, um sistema de plataforma chamado &quot;fio biológica&quot; (biowire) foi concebido, fornecendo ambas as pistas estruturais e estimulação do campo eléctricon para melhorar a maturação de HPSC-CMs 14. Este sistema utiliza uma plataforma microfabricado para criar tecido cardíaco alinhados que é passível de estimulação do campo eléctrico. Isso pode ser usado para melhorar a maturidade estrutural e eletrofisiológica da HPSC-CMS. Aqui, descrevemos os detalhes de fazer tais 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

cardiomiócitos derivados de células-tronco pluripotentes humanas (HPSC-CMS) têm sido uma fonte de células promissor e, assim, encorajados a investigação de suas possíveis aplicações na pesquisa cardíaca, incluindo a descoberta de medicamentos, modelagem de doença, a engenharia de tecidos e medicina regenerativa. No entanto, as células produzidas por protocolos existentes exibir uma gama de imaturidade comparado com cardiomiócitos ventriculares adulto nativas. Muitos esforços têm sido feitos para amadurecer HPSC-CMs, com apenas maturação moderada alcançado até agora. Portanto, um sistema de engenharia, chamado biowire, foi concebida, fornecendo pistas tanto físicas e eléctricas para levar HPSC-MC para um estado mais maduro in vitro. O sistema utiliza uma plataforma microfabricado para semear HPSC-CMs em colagénio do tipo I gel ao longo de um fio de sutura molde rígido para montar em tecido cardíaco alinhados (biowire), que é submetido a estimulação do campo eléctrico com uma frequência progressivamente crescente. Em comparação com os controlos não estimulados,estimulada biowired cardiomiócitos exibem um maior grau de maturação estrutural e electrofisiológica. Tais alterações são dependentes da taxa de estimulação. Este manuscrito descreve em detalhe a concepção e criação de biowires.

O racional para o uso de uma sutura no biowires é servir como um molde para a formação de construções 3D que se alinham em um eixo e imitam a forma de fibras cardíacas. Mostramos que após sete dias de cultura no biowire, as células remodelaram o gel ao redor da sutura ( Figura 3A ). As células foram montadas ao longo do eixo da sutura para formar o tecido cardíaco alinhado ( Figura 3 ). Após 7 dias de pré-cultura, os biowires foram submetidos...

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

A plataforma biowire cardíaca é um método in vitro utilizado para amadurecer embrionário humano e cardiomiócitos derivadas de células estaminais pluripotentes induzidas (HPSC-CM) através da combinação de cultura de células tridimensional com estimulação eléctrica. Este manuscrito apresenta a configuração detalhada da plataforma biowire cardíaca.

Maturação de cardiomiócitos estaminais humanas derivadas de células em Biowires Usando Electrical Stimulation

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

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