Name: simultaneous electrical and mechanical stimulation to enhance cells' cardiomyogenic potential
Uploaded: 2019-01-18T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential at JoVE.com

The authors want to thank the members of the ICREC Research Program (IGTP, Badalona) and the Electronic and Biomedical Instrumentation Group (UPC, Barcelona), especially Prof. J. Rosell-Ferrer. In addition, the authors acknowledge STEM CELLS Translational Medicine journal and AlphaMed Press for permitting the adaptation of previously published figures (Lluci&#224;-Valldeperas, et al.30). The development of this prototype and the design of the protocol were supported by Ministerio de Educaci&#243;n y Ciencia (SAF 2008-05144), Ministerio de Econom&#237;a y Competitividad (SAF 2014-59892), the European Commission 7th Framework Programme (RECATABI, NMP3-SL-2009-229239), Fundaci&#243; La Marat&#243; de TV3 (080330, 201516, 201502), and Fundaci&#243;n para la Innovaci&#243;n y la Prospectiva en Salud en Espa&#241;a (FIPSE; 06-00001396-15).

This study uses human cardiac ATDPCs from patient samples. Their use has been approved by the local ethics committee, and all patients gave informed consent. The study protocol conforms to the principles outlined in the Declaration of Helsinki.
1. Preparations
<ol>
	<li>Autoclave two tweezers, 12 platinum PTFE electrodes for electrical stimulation, and some paper towels, at 121 &#176;C for 20 min.</li>
	<li>Sterilize 12 PDMS custom-made constructs (Figure 1</stro...

Electromechanical stimulation appears to be a safe alternative for preparing cells for a hostile cardiac environment and enhancing their cardiac commitment. Here, a protocol described for cardiac progenitor cells increased the expression of main cardiac markers and was reported to be beneficial for their next implantation on infarcted murine myocardium30. In general, electromechanically stimulated cardiac ATDPCs increased the expression of genes related to early, structural, and calcium regulation...

Heart function is based on the coupling of electrical excitation and mechanical contraction. Briefly, cardiomyocyte intercellular junctions permit electrical signal propagation to produce almost synchronous contractions of the heart that pump blood systemically and through the pulmonary system. Cardiac cells, thus, undergo both electrical and mechanical forces that regulate gene expression and cellular function. Accordingly, many groups have attempted to develop culture platforms that mimic the cardiac physiological environment to understand the role of mechanical and electrical stimulation on cardiac development, function, and maturation. In vitro electrical and mechanical stimulations individually have been applied extensively in cardiac tissue engineering to enhance functional properties, increase cell maturation, or improve cell-cell coupling and calcium handling1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21. Nevertheless, synchronous electromechanical conditioning remains unexploited because of the challenge of developing a stimulator and protocol, and because of the mandatory optimization22.
Preliminary work addressed electromechanical stimulation as a combination of electrical stimulation and media perfusion; however, the flow does not involve the strain-based deformation typical of ventricular filling23,24,25. Later, more physiological approaches combined electrical stimuli with physical deformation or stretch to mimic the isovolumetric contraction26,27,28,29,30,31. Feng et al. described the first demonstration of electromechanical stimulation in 2005, reporting enhanced cardiomyocyte size and contractile properties26. Wang et al. pretreated mesenchymal stem cells with 5-azacytidine and applied simultaneous electrical and mechanical conditioning, improving recellularization, cell viability, cardiac differentiation, and tissue remodeling27. Since those publications, more groups have reported on electromechanical stimulation of cell monolayers or engineered tissues (e.g., Black28, Vunjak-Novakovic29,31, and our group30) with the first conditioned cells tested in vivo30. Briefly, Morgan and Black tested several combinations of electrical and mechanical stimuli, reporting that the timing between stimulations was crucial because delayed combined electromechanical stimulation yielded the best results28. Next, Godier-Furn&#233;mont and collaborators optimized an electromechanical stimulation protocol for engineered heart muscle constructs from neonatal rat heart cells and achieved, for the first time, a positive force-frequency relationship29. Afterward, our group reported that electromechanically preconditioned cells increased the expression of main cardiac markers in vitro and broad beneficial effects in vivo, such as improved cardiac function or increased vessel density in the infarct border region30. The most recent publication demonstrated that cardiac tissues from stem-cell-derived cardiomyocytes subjected to electromechanical conditioning reached a maturation level closer to human adult cardiac structure and function31. Additionally, alternative three-dimensional stimulation platforms comprise electroactive scaffolds that provide electrical, mechanical, and topographical cues to the cells attached32. Moreover, mechanical deformation (cell monolayer stretching and compression) can also be induced with stretchable electrodes mimicking normal physiological conditions, as well as extreme conditions33.
Therefore, the rationale is that in vitro electromechanical stimuli based on physiological conditions could enhance the cardiomyogenic potential of a cell. Indeed, this stimulation could benefit further integrations of therapeutic cells into the myocardium in a clinical scenario or increase tissue maturation for drug-screening applications.
In addition, we isolated and characterized a population of human adipose tissue-derived progenitor cells of cardiac origin (cardiac ATDPCs)34. These cells are located in the epicardial fat. These cells display beneficial histopathological and functional effects in the treatment of myocardial infarction and also maintain cardiac and endothelial differentiation potential.30,35. We hypothesized that these benefits would increase after biophysical stimulation.
Consequently, we developed a device and a stimulation regime for the cell population of interest and investigated the effects. This electromechanical protocol is a new strategy to induce active cell stretching in a sterile manner and noninvasively compared to previous publications36, in combination with electric field stimulation. The technique reported here explains in detail the device and method used for the electrical, mechanical, and electromechanical stimulation of cells.
This device can provide both electrical and mechanical stimulation, independently or simultaneously. The stimulation is performed with a noninvasive and aseptic novel approach that includes presterilized cell support, electrodes placed inside a standard culture plate, and a platform that induces the mechanical and electrical forces (Figure 1).
The platform can hold up to six culture plates and consists of a sandwich structure of laser-cut poly(methyl methacrylate) and printed circuit-board pieces. The platform prototype relies on a combination of a monophasic programmable computer-controlled electrical stimulator, a printed circuit board for the robust connection of the electrodes, and six 10 mm x 10 mm x 5 mm nickel-plated neodymium-fixed magnets placed near one side of the culture plates. There is also an aluminum bar with six driving magnets (same model) placed in front of the other side of the culture plates and moved with a linear servomotor. The motor is driven by a motor controller, operated through an RS-232 port by commercial software (see the Table of Materials). Through the user interface and programmable stimulator, it is possible to program the electrical intensity, the pulse duration and frequency, the frequency of mechanical stimulation, its duty cycle, the number of pulses, the pulse amplitude (magnet excursion), and the slope.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58934/58934fig1.jpg" /> 
Figure 1: Electromechanical stimulator. (A) PDMS construct used for the cell conditioning. (B) Drawing of the PDMS construct, including electrodes and magnets. (C) Detail of the printed circuit board (platform) used to perform the electromechanical conditioning. This panel has been modified from Lluci&#224;-Valldeperas et al.30. (D) Picture of the electromechanical stimulation platform and user interface (computer). <a href="https://www.jove.com/files/ftp_upload/58934/58934fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Both the stimulator and the method for electromechanical conditioning are fully described in two international patents, WO-2013185818-A137 and WO-2017125159-A138.
The biocompatible silicone constructs designed to provide structural support to cells, electrodes, and magnets have been described previously10,21. Briefly, they consist of polydimethylsiloxane (PDMS), molded and cured at room temperature, with a Young&#39;s modulus of 1.3 MPa, close to physiological levels. The construct contains a cell culture pool in a flexible area (10 mm x 10 mm x 2 mm), two inner transverse slots to hold the electrodes, and two embedded 6 mm x 2 mm x 4 mm nickel-plated neodymium magnets. The electrodes are built with 0.2 mm platinum wire twisted around a 2 mm x 3 mm x 12 mm polytetrafluoroethylene (PTFE) core bar (21 cm per electrode, approximately 23 turns) and placed at opposite sides of the flexible area to create an electric field for inducing electrical stimulation. Mechanical stretching is achieved through magnetic attraction between magnets embedded in the support and external magnets placed next to the culture plate and on the moving aluminum arm. In this way, the cell support can be extended without breaking the sterile barrier. This approach is suitable for a cell monolayer but could be adapted to three-dimensional constructs, as well.
In addition, a regular pattern could be imprinted where the cells are seeded, using a ruled diffraction grating (1,250 grooves/mm). The direct visualization of cells cultured on the PDMS construct under brightfield and fluorescent microscopes is possible because of its transparency and 0.5 mm thickness. In the current case, the PDMS culture pool has a vertical surface pattern, perpendicular to the stretching force, to align the cells perpendicularly to the electric field, which minimizes the electric field gradient across the cell.
Figure 1 shows a detailed description of the construct and device used for the stimulation. The PDMS construct and characteristics are optimized for cell stretching (Figure 1A,B). The stimulator is developed and validated for the effective application of the desired electrical and mechanical stimulation to cells attached to the PDMS construct. This process includes ensuring good connectivity and user operability through the software interface (Figure 1C,D).
The procedure for cell stimulation using this custom-made device is described in the protocol section.

<table >
	<tbody>
		<tr >
			<td >Stimulator</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >nickel plated neodymium magnets</td>
			<td >Supermagnete</td>
			<td >Q-10-10-05-N</td>
			<td></td>
		</tr>
		<tr >
			<td >nickel-plated neodymium magnets</td>
			<td >Supermagnete</td>
			<td >Q-06-04-02-HN</td>
			<td></td>
		</tr>
		<tr >
			<td >polydimethylsiloxane (PDMS) SYLGAR 184 Silicone Elastomer Kit</td>
			<td >Dow Corning Corp</td>
			<td >184</td>
			<td></td>
		</tr>
		<tr >
			<td >ruled diffraction grating (1250 grooves/mm)</td>
			<td >Newport</td>
			<td >05RG150-1250-2</td>
			<td></td>
		</tr>
		<tr >
			<td >Motor controller</td>
			<td >Faulhaber</td>
			<td>MCLM-3006-S</td>
			<td></td>
		</tr>
		<tr >
			<td >Labview</td>
			<td >National Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Cell culture</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >phosphate-buffered saline (PBS)</td>
			<td >Gibco</td>
			<td >70013-065</td>
			<td></td>
		</tr>
		<tr >
			<td >0.05% trypsin-EDTA</td>
			<td >Gibco</td>
			<td >25300-120</td>
			<td></td>
		</tr>
		<tr >
			<td >35 mm cell culture dish</td>
			<td>BD Falcon</td>
			<td>45353001</td>
			<td></td>
		</tr>
		<tr >
			<td >fetal bovine serum (FBS)</td>
			<td >Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr >
			<td >L-Glutamine 200 mM, 100x</td>
			<td >Gibco</td>
			<td >25030-024</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicilina/Streptomicine, 10.000 U/mL</td>
			<td >Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr >
			<td >Minimum essential medium eagle (alfa-MEM)</td>
			<td >Sigma</td>
			<td>M4526-24x500ML</td>
			<td></td>
		</tr>
		<tr >
			<td >Protein &amp; RNA analyses</td>
			<td ></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >protease inhibitor cocktail</td>
			<td >Sigma</td>
			<td >P8340</td>
			<td></td>
		</tr>
		<tr >
			<td >QIAzol Lysis Reagent</td>
			<td >Qiagen</td>
			<td>79306</td>
			<td></td>
		</tr>
		<tr >
			<td >AllPrep RNA/Protein Kit</td>
			<td >Qiagen</td>
			<td>50980404</td>
			<td></td>
		</tr>
		<tr >
			<td >Rneasy mini kit</td>
			<td >Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr >
			<td >iTaq Universal Probes One-Step Kit</td>
			<td>Bio-Rad Laboratories</td>
			<td >172-5140</td>
			<td></td>
		</tr>
		<tr >
			<td >Random hexamers</td>
			<td >Qiagen</td>
			<td >79236</td>
			<td></td>
		</tr>
		<tr >
			<td >TaqMan PreAmp MasterMix 2X</td>
			<td>Applied Biosystems</td>
			<td >4391128</td>
			<td></td>
		</tr>
		<tr >
			<td >TaqMan Universal PCR MasterMix</td>
			<td>Applied Biosystems</td>
			<td >4324018</td>
			<td></td>
		</tr>
		<tr >
			<td >Immunostaining</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >10% formalin</td>
			<td >Sigma</td>
			<td >HT-501128-4L</td>
			<td></td>
		</tr>
		<tr >
			<td >horse serum</td>
			<td >Sigma</td>
			<td >H1138</td>
			<td></td>
		</tr>
		<tr >
			<td >Triton X-100</td>
			<td >Sigma</td>
			<td >X100-500ML</td>
			<td></td>
		</tr>
		<tr >
			<td >Bovine Serum Albumina (BSA)</td>
			<td >Sigma</td>
			<td >A7906-100G</td>
			<td></td>
		</tr>
		<tr >
			<td >PARAFILM</td>
			<td >Sigma</td>
			<td >P6543</td>
			<td></td>
		</tr>
		<tr >
			<td >4',6-diamidino-2-phenylindole (DAPI)</td>
			<td >Sigma</td>
			<td >D9542</td>
			<td></td>
		</tr>
		<tr >
			<td >Phalloidin Alexa 568</td>
			<td >Invitrogen</td>
			<td >A12380</td>
			<td></td>
		</tr>
		<tr >
			<td >sodium azide</td>
			<td >Sigma</td>
			<td >S8032-100g</td>
			<td></td>
		</tr>
		<tr >
			<td >Hoechst 33342</td>
			<td >Sigma</td>
			<td >14533</td>
			<td></td>
		</tr>
		<tr >
			<td >Connexin-43 rabbit primary antibody</td>
			<td >Sigma</td>
			<td>C6219 lot#061M4823</td>
			<td></td>
		</tr>
		<tr >
			<td >sarcomeric &#945;-actinin mouse primary antibody</td>
			<td >Sigma</td>
			<td>A7811 lot#080M4864</td>
			<td></td>
		</tr>
		<tr >
			<td >GATA-4 goat primary antibody</td>
			<td>R&amp;D</td>
			<td>AF2606 VAZ0515101</td>
			<td></td>
		</tr>
		<tr >
			<td >MEF2 rabbit primary antibody</td>
			<td>Santa Cruz</td>
			<td>sc-313 lot#E0611</td>
			<td></td>
		</tr>
		<tr >
			<td >SERCA2 goat primary antibody</td>
			<td>Santa Cruz</td>
			<td>sc-8095 lot#D2709</td>
			<td></td>
		</tr>
		<tr >
			<td >Cy3 secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td >711-165-152</td>
			<td></td>
		</tr>
		<tr >
			<td >Cy3 secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td >715-165-151</td>
			<td></td>
		</tr>
		<tr >
			<td >Cy3 secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td >712-165-150</td>
			<td></td>
		</tr>
		<tr >
			<td >Cy2 secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td >715-225-150</td>
			<td></td>
		</tr>
		<tr >
			<td >Cy2 secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td >711-225-152</td>
			<td></td>
		</tr>
		<tr >
			<td >Cy2 secondary antibody</td>
			<td>Jackson ImmunoResearch</td>
			<td >705-225-147</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous electrical and mechanical stimulation to enhance cells' cardiomyogenic potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Figure 2 represents the general schema followed for the cell stimulation. Briefly, cells were seeded on the PDMS construct and subjected to electromechanical stimulation, with a media change performed twice a week. Nonstimulated cells were used as a control for the electromechanical conditioning. Additionally, we added an extra control to the experiment, and subcutaneous ATDPCs were used as a control for cardiac ATDPCs. Subcutaneous ATDPCs are obtained from s...

Watch this Scientific Journal Video about Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential at JoVE.com

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

This is a method to apply electromechanical stimulation on cells. It has multiple applications to study its effects on a cell population, pre-training for in vivo delivery, and cell maturation. The advantage is that electrical and mechanical stimuli can be applied with the same device individually or simultaneously while keeping sterile viral intact.This technique is an indirect approach to our therapy. Cell therapy is considered electromechanically stimulated cells may be an interesting cell population to treat intro-myocardium. This method generally belongs to the cardiovascular field, but it could also be applied to the nervous system.This is an easy technique that requires patience. The most difficult part will be working with the small pieces, and keep the sterility throughout. There are little details of manipulation which are difficult to explain, so the visual demonstration is really helpful.Begin by transferring 12 cleaned PDMS constructs to sterile 10 centimeter plates. To insure complete sterilization, expose the plates to UV light for five minutes. Then transfer each construct to a separate 35 millimeter cell culture plate, or 6-well plates for immediate cell seeding.To start cell seeding, first wash a confluent T75 flask of cardiac ATDPCs with five milliliters of 1x PBS. To detach the cells, add one milliliter of 0.05%trypsin-EDTA. And incubate at 37 degrees Celsius for five minutes.Then, add five milliliters of complete medium to inactivate the trypsin-EDTA. Collect all detached cells in a 15 milliliter tube. Wash the cell glass twice with five milliliters of PBS to collect any remaining cells, and add them to the 15 milliliter tube.Centrifuge at 230 g for five minutes at 22 degrees Celsius. Remove the supernatant, and re-suspend the cells in two milliliters of complete medium, and count them with the hemocytometer. Seed 200 microliters of cardiac ATDPCs into each plate with the PDMS constructs to obtain about 80%of the seeding surface covered by cells the following day.Incubate at 37 degrees Celsius, and 5%CO2. Gently add 2 milliliters of pre-warmed complete medium per plate. Incubate the cells with the constructs at 37 degrees Celsius, and 5%CO2 overnight.Before starting this procedure, assign six out of 12 constructs for electromechanical stimulation, and keep six for non-stimulated controls. Use 70%ethanol to clean the stimulation unit. Place the stimulation unit, sterile electrodes, and tweezers inside the flow cabinet.In order to easily manipulate the electrodes and constructs, remove 90%of the medium from each cell culture plate. Place the PDMS constructs in the right position toward the magnet to insure magnetic attraction between both fixed and mobile magnets. These two previous steps in which you manipulate the cell's seeded PDMS constructs and the electrodes while keeping the sterility throughout the process are the most critical.Then, connect the platinum wire to the electrode connectors. Orient the PTFE part of the electrodes in their designated spaces in the PDMS constructs. Add 2.5 milliliters of fresh, pre-warmed complete medium to each construct.Once all PDMS constructs are placed and electrically connected to the platform, place the platform back into the 37 degrees Celsius, and 5%CO2 incubator. Then connect the electrical and mechanical source. To configure the stimulation program, specify electrical and mechanical stimulation regimes through the user interfaces of the electrical stimulator and the application, which controls the mechanical stimulation.To set the synchronism, switch on the electrical stimulator, and wait for the main menu to appear on the display. Then select option two, edit sequence, plus Enter. To edit the sequence menu, use the mode tab to select current"by clicking plus"and pressing Enter"For the period, select 1000"with plus/minus and press Enter"And for trigger mode tab, select external by software and press Enter"For the amplitude tab, select 1"with plus/minus and press Enter"Then, back in the main menu, select option 4, and then generate sequence, and press Enter"In the mechanical stimulation section of the control application panel, first write 1000"in the plus period text control.Then write 500"in the ON time text control, to set the mechanical pulse duration. Finally, write 2000"in the excursion text control to deliver a 10%construct elongation. Change the cell media twice a week, by first removing the old media, and then adding warm, fresh media on the sides of the PDMS support.On day seven, at the end of the experiment, collect the samples as described in the manuscript. Electromechanically stimulated cardiac ATDPCs enhanced their cardiomyogenic potential. This was indicated by real-time PCR showing increased expression of early and late cardiac genes specifically the cardiac transcription factor GATA-4, the structural marker beta-myosin heavy chain, and the calcium related gene Connexin43.Phalloidin staining against actin fibers showed that the majority of cells aligned according to the vertical pattern. Connexin43 distribution was mostly in the cytoplasm, and at the plasma membrane, contributing to intracellular communication through gap junctions. MEF2 and GATA-4 transcription factors were located at the nuclei in cardiac ATDPCs.But GATA-4 was not detected in subcutaneous ATDPCs. The cytoplasmic markers SERCA2, and sarcomeric alpha-actinin, did not show a mature sarcomere organization typical for cardiomyocytes. And beating was not observed in control and stimulated cell populations.A healthy cell monolayer at the beginning of this protocol and keeping the sterility throughout the procedure are crucial. After sample collection, the standard genome protein expression techniques can be performed. This technique helps to better understand the effect of cell electrical and/or mechanical stimuli.Until recently, it was impossible to use the same device for both stimulation, which made results harder to compare.

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