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 1A).
	<ol>
		<li>Wash each construct with 5 mL of sterile, distilled water with magnetic agitation at room temperature for 15 min.</li>
		<li>Wash 1x with 5 mL of 70% ethanol with magnetic agitation at room temperature for 5 min.</li>
		<li>Wash 5x with 5 mL of sterile, distilled water with magnetic agitation at room temperature for 10 min per wash, to remove alcoholic residues.</li>
		<li>Dry the constructs on sterile paper towels inside the flow cabinet overnight.</li>
		<li>Store them in sterile 50 mL centrifuge tubes until use.</li>
	</ol>
	</li>
</ol>
2. Cell Seeding (Day -1)
<ol>
	<li>Before cell seeding, transfer the cleaned PDMS constructs to sterile plates and expose them to ultraviolet light for 5 min to ensure complete sterilization.</li>
	<li>Transfer each construct to a 35 mm cell culture plate, or 6-well plate,&#160;for immediate cell seeding.</li>
	<li>Trypsinize a confluent T75 flask of cardiac ATDPCs.
	<ol>
		<li>Wash the T75 flask with 5 mL of 1x phosphate-buffered saline (PBS).</li>
		<li>Add 1 mL of 0.05% trypsin-EDTA and incubate at 37 &#176;C for 5 min to detach the cells.</li>
		<li>Add 5 mL of complete medium to inactivate the trypsin-EDTA.</li>
		<li>Collect all cells in a 15 mL tube and wash the flask 2x with 5 mL of PBS to collect any remaining cells.</li>
		<li>Centrifuge at 230 x g for 5 min at 22 &#176;C, remove the supernatant, and resuspend the cells in 2 mL of complete medium to count them with the hemocytometer chamber.</li>
	</ol>
	</li>
	<li>Seed 200 &#181;L of cardiac ATDPCs (2.5 x 105 cells/mL) into the cell pool of the 12 PDMS constructs (Figure 1A,B) to have ~80% of the seeding surface covered by cells the day after, and incubate at 37 &#176;C and 5% CO2. 
	NOTE: After 2&#8211;4 h, the cells should be attached. Cell inoculum is prepared according to the cell size and growth. For smaller cells, the seeding density should be increased.</li>
	<li>Gently add 2 mL of prewarmed complete medium (&#945;-MEM supplemented with 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin-streptomycin) per plate.</li>
	<li>Incubate the constructs at culture conditions (usually 37 &#176;C and 5% CO2) overnight.</li>
</ol>
3. Electromechanical Stimulation Setup (Day 0)
<ol>
	<li>Before starting the procedure, take six constructs for electromechanical stimulation and six as nonstimulated controls. With fewer than six plates per stimulation, use empty constructs with the same medium volume to ensure proper electric field stimulation.</li>
	<li>Clean the stimulation unit with 70% ethanol and place it in the flow cabinet.</li>
	<li>Bring the sterile electrodes and tweezers inside the flow cabinet.</li>
	<li>Remove 90% of the media from the culture plate to easily manipulate the electrodes and constructs. First, place the PDMS constructs in the right position to ensure magnetic attraction (PDMS displacement within the culture plate toward the magnet) between both fixed and mobile magnets. Next, connect the platinum wire to the electrode connectors and the PTFE part in its designated space in the PDMS construct.</li>
	<li>Add 2.5 mL of fresh prewarmed complete medium to each construct. 
	NOTE: Maintain the sterility throughout the procedure and operate on one construct at a time. Maintain the rest of the constructs in the incubator at 37 &#176;C and 5% CO2 until use.</li>
	<li>Once all PDMS constructs are placed and electrically connected to the platform, bring the platform back into the incubator at 37 &#176;C and 5% CO2.</li>
	<li>Connect the electrical and mechanical source.</li>
	<li>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. Set the synchronism as follows.
	<ol>
		<li>Switch on the electrical stimulator. Wait for the main menu to appear in the display.
		<ol>
			<li>Then, select Option 2: Edit sequence + Enter.</li>
			<li>Edit the sequence Menu as follows.
			<ol>
				<li>Use the Mode tab to select either voltage or current. Select current by clicking + and press Enter.</li>
				<li>For the Amplitude tab, select 1 (mA) with +/- and press Enter.</li>
				<li>For the Period (T), select 1000 (ms) with +/- and press Enter.</li>
				<li>Set the Pulse duration (Tw) to 2 (ms) with +/- and press Enter.</li>
				<li>For the Trigger mode tab, select External by software and press Enter.</li>
				<li>Back in the main menu, select Option 4: Generate sequence and press Enter. 
				NOTE: The electrical stimulator rests in the standby until it receives a trigger command from the mechanical stimulator application through the serial port.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Perform the following steps in the mechanical stimulation section of the control application panel (Figure 2C).
		<ol>
			<li>Write 1,000 (ms) in the Pulse Period text control.</li>
			<li>Write 500 (ms) in the ON time (Tw) text control to set the mechanical pulse duration.</li>
			<li>Write 2,000 (AU) in the Excursion text control to deliver a 10% construct elongation. This is the number of steps in the linear control motor. 
			NOTE: The stimulation protocol applied here consists of alternating-current 2 ms monophasic square-wave pulses of 50 mV/cm at 1 Hz and 10% stretching for 7 days. The rise and fall times of the mechanical pulse are set at 100 ms, to roughly imitate the shape of the hearth pressure pulse. Also, the repetition mode is set to Continuous and there is a counter displaying the number of pulses.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Change the media 2x a week (Monday and Thursday afternoon). First, remove the old media; next, add the warm media on the sides of the PDMS support, never directly on the cell pool. 
	NOTE: If the cells have a high growth rate, the media should be changed 3x a week (e.g., Monday, Wednesday, and Friday). It is necessary to disconnect and reconnect all cables, but there is no need to remove the culture plates and electrodes from their place.</li>
	<li>Collect the samples after the experimentation is performed.</li>
</ol>
4. Sample Collection at the End of the Experimentation (Day 7)
<ol>
	<li>For RNA analyses
	<ol>
		<li>Wash the construct 2x with 3 mL of 1x PBS for 5 min at room temperature.</li>
		<li>Add 3 mL of 0.05% trypsin-EDTA to each plate (enough to cover the whole construct) and wait for 5 min at 37 &#176;C.</li>
		<li>After the cells are detached, add 2 mL of complete medium to inactivate the trypsin-EDTA.</li>
		<li>Collect all cells in a 15 mL tube and wash the construct 2x with 3 mL of PBS to collect any remaining cells.</li>
		<li>Centrifuge at 230 x g for 5 min at 22 &#176;C.</li>
		<li>Remove the supernatant and resuspend the pellet in 1 mL of PBS.</li>
		<li>Transfer the cell solution to a 1.5 mL tube and centrifuge at 230 x g for 5 min.</li>
		<li>Remove the supernatant and store the pellet at -80 &#176;C in 700 &#181;L of lysis reagent for further RNA isolation.</li>
		<li>Isolate RNA using a commercial kit, following the manufacturer&#39;s instructions.</li>
		<li>Reverse-transcribe the isolated RNA using the kit and random hexamers, according to the manufacturer&#39;s protocol.</li>
		<li>For small RNA concentrations, preamplify and, then, dilute 1:5 with RNase-free water before subsequent real-time reverse transcription polymerase chain reaction (RT-PCR) is performed. Continue with the standard protocol for real-time RT-PCR and check main cardiac markers. 
		NOTE: Typical cardiac markers comprise early and late markers from different categories, such as cardiac transcription factors (myocyte-specific enhancer factor 2A [MEF2A], GATA-binding protein 4 [GATA-4]) and structural (cardiac troponin I [cTnI], cardiac troponin T [cTnT], &#945;-actinin) and calcium regulation (Connexin43 [Cx43], sarco-/endoplasmic reticulum Ca2+-ATPase [SERCA2])30. Protein isolation can also be performed if needed. Simultaneous RNA and protein isolation can be performed with the same sample, using commercially available reagents and kits (Table of Materials) if the sample quantity is less.</li>
	</ol>
	</li>
	<li>For immunostainings 
	NOTE: This is performed directly on the cells attached to the cell pool of the PDMS construct. Therefore, we recommend placing, every time, 1 cm x 1 cm of paraffin film on the top of the cell pool, apart from the plate lid, to minimize the evaporation of the incubation solutions.
	<ol>
		<li>Wash the construct 2x with 3 mL of PBS for 5 min at room temperature.</li>
		<li>Fix the cells attached to the construct with 2 mL of 10% formalin for 15 min at room temperature.</li>
		<li>Wash the cells 3x with 3 mL of PBS for 5 min at room temperature. For long-term storage, leave the samples in PBS with 0.1% sodium azide at 4 &#176;C.</li>
		<li>Permeabilize the cells with 3 mL of PBS + 0.5% detergent (3x, each time 5&#8211;10 min, at room temperature).</li>
		<li>Incubate the cells with 100 &#181;L of PBS + 10% horse serum + 0.2% detergent + 1% bovine serum albumin at room temperature for 1 h, to block nonspecific antibody binding.</li>
		<li>Incubate the cells with 100 &#181;L of PBS + 10% horse serum + 0.2% detergent + 1% bovine serum albumin + primary antibody at room temperature for 1 h. For example, primary antibodies against Cx43 (1:100), sarcomeric &#945;-actinin (1:100), GATA-4 (1:50), MEF2 (1:25), and SERCA2 (1:50).</li>
		<li>Wash 3x with 3 mL of PBS at room temperature for 5 min.</li>
		<li>Incubate the cells with 100 &#181;L of PBS + secondary antibody at room temperature in the dark for 1 h. 
		NOTE: Secondary antibodies conjugated with different fluorophores and a counterstaining agent were used.</li>
		<li>Wash them 3x with 3 mL of PBS at room temperature in the dark for 5 min.</li>
		<li>Incubate the cells with 100 &#181;L of nuclear staining (0.1 &#181;g/mL) in PBS at room temperature in the dark for 15 min.</li>
		<li>Wash them 3x with 3 mL of PBS at room temperature in the dark for 5 min.</li>
		<li>Store the samples in 3 mL of PBS with 0.1% sodium azide at 4 &#176;C until the acquisition. 
		NOTE: Microscope acquisition is possible on inverted fluorescent and confocal microscopes with long-working distance objectives because the construct thickness is about 0.5 mm.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose, except that the stimulation device and protocol were previously patented (WO-2013185818-A1, WO-2017125159-A1).

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>
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			<td>Jackson ImmunoResearch</td>
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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 ...

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7.4K Views.  Health Science Research Institute Germans Trias i Pujol. 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.

Video: Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Simultaneous Synthesis of Single-walled Carbon Nanotubes and Graphene in a Magnetically-enhanced Arc Plasma

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very promising application for molecular sensors, field effect transistor and super thin and flexible electronic devices1-4. Anodic arc discharge supported by the erosion of the anode material is one of the most practical and efficient methods, which can provide specific non-equilibrium processes and a high influx of carbon material to the developing structures at relatively higher temperature, and consequently the as-synthesized products have few structural defects and better crystallinity.
 To further improve the controllability and flexibility of the synthesis of carbon nanostructures in arc discharge, magnetic fields can be applied during the synthesis process according to the strong magnetic responses of arc plasmas. It was demonstrated that the magnetically-enhanced arc discharge can increase the average length of SWCNT 5, narrow the diameter distribution of metallic catalyst particles and carbon nanotubes 6, and change the ratio of metallic and semiconducting carbon nanotubes 7, as well as lead to graphene synthesis 8. 
 Furthermore, it is worthwhile to remark that when we introduce a non-uniform magnetic field with the component normal to the current in arc, the Lorentz force along the J&times;B direction can generate the plasmas jet and make effective delivery of carbon ion particles and heat flux to samples. As a result, large-scale graphene flakes and high-purity single-walled carbon nanotubes were simultaneously generated by such new magnetically-enhanced anodic arc method. Arc imaging, scanning electron microscope (SEM), transmission electron microscope (TEM) and Raman spectroscopy were employed to analyze the characterization of carbon nanostructures. These findings indicate a wide spectrum of opportunities to manipulate with the properties of nanostructures produced in plasmas by means of controlling the arc conditions.

Anodic arc discharge is one of the most practical and efficient methods to synthesize various carbon nanostructures. To increase the arc controllability and flexibility, a non-uniform magnetic field was introduced to process the one-step synthesis of large-scale graphene flakes and high-purity single-walled carbon nanotubes.

Carbon nanostructures such as single-walled carbon nanotubes (SWCNT) and graphene attract a deluge of interest of scholars nowadays due to their very ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living components. Due to their inherent advantages, biorobots are gaining interest as alternatives to traditional fully artificial robots. Various studies have focused on harnessing the power of biological actuators, but only recently studies have quantitatively characterized the performance of biorobots and studied their geometry to enhance functionality and efficiency. Here, we demonstrate the development of a self-stabilizing swimming biorobot that can maintain its pitch, depth, and roll without external intervention. The design and fabrication of the PDMS scaffold for the biological actuator and biorobot followed by the functionalization with fibronectin is described in this first part. In the second part of this two-part article, we detail the incorporation of cardiomyocytes and characterize the biological actuator and biorobot function. Both incorporate a base and tail (cantilever) which produce fin-based propulsion. The tail is constructed with soft lithography techniques using PDMS and laser engraving. After incorporating the tail with the device base, it is functionalized with a cell adhesive protein and seeded confluently with cardiomyocytes. The base of the biological actuator consists of a solid PDMS block with a central glass bead (acts as a weight). The base of the biorobot consists of two composite PDMS materials, Ni-PDMS and microballoon-PDMS (MB-PDMS). The nickel powder (in Ni-PDMS) allows magnetic control of the biorobot during cells seeding and stability during locomotion. Microballoons (in MB-PDMS) decrease the density of MB-PDMS, and enable the biorobot to float and swim steadily. The use of these two materials with different mass densities, enabled precise control over the weight distribution to ensure a positive restoration force at any angle of the biorobot. This technique produces a magnetically controlled self-stabilizing swimming biorobot.

In this two-part study, a biological actuator was developed using highly flexible polydimethylsiloxane (PDMS) cantilevers and living muscle cells (cardiomyocytes), and characterized. The biological actuator was incorporated with a base made of modified PDMS materials to build a self-stabilizing, swimming biorobot.

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

Silicon Nanowires and Optical Stimulation for Investigations of Intra- and Intercellular Electrical Coupling

Myofibroblasts can spontaneously internalize silicon nanowires (SiNWs), making them an attractive target for bioelectronic applications. These cell-silicon hybrids offer leadless optical modulation capabilities with minimal perturbation to normal cell behavior. The optical capabilities are obtained by the photothermal and photoelectric properties of SiNWs. These hybrids can be harvested using standard tissue culture techniques and then applied to different biological scenarios. We demonstrate here how these hybrids can be used to study the electrical coupling of cardiac cells and compare how myofibroblasts couple to one another or to cardiomyocytes. This process can be accomplished without special equipment beyond a fluorescent microscope with coupled laser line. Also shown is the use of a custom-built MATLAB routine that allows the quantification of calcium propagation within and between the different cells in the culture. Myofibroblasts are shown to have a slower electrical response than that of cardiomyocytes. Moreover, the myofibroblast intercellular propagation shows slightly slower, though comparable velocities to their intracellular velocities, suggesting passive propagation through gap junctions or nanotubes. This technique is highly adaptable and can be easily applied to other cellular arenas, for in vitro as well as in vivo or ex vivo investigations.

This protocol describes the use of silicon nanowires for intracellular optical bio-modulation of cell in a simple and easy to perform method. The technique is highly adaptable to diverse cell types and can be used for in vitro as well as in vivo applications.