This work was supported in part by an AO Foundation Start-Up Grant (S-14-03H) and the Friedrichsheim Foundation (Stiftung Friedrichsheim) based in Frankfurt/Main, Germany.

1. Construction of electrical stimulation cell culture chamber
<ol>
	<li>To build the EStim chamber, collect two lids of standard 6-well cell culture plates; 99.99% platinum wire, 60 cm in length with a diameter of 0.5&#8211;1 mm; silver-coated copper wire, 70 cm in length with a diameter of 0.6 mm; cutting pliers; soldering iron kit; one tube of superconductive glue; one wire terminal block connector, six small 2.2 V LEDs (optional); one tube of noncorrosive silicone adhesive coating (optional); one roll of black electrical insulation tape; standard, flexible, insulated copper electric wire (0.14 mm2), 2 m in length (Table of materials).</li>
	<li>In a 6-well plate lid, mark and then drill two holes (with a diameter of ~1 mm), 25 mm apart, near the outer edge of each of the six wells (12 holes in total), as shown in Figure 1A,B.</li>
	<li>Cut twelve 5 cm lengths of platinum wire with cutting pliers. Bend each of the wires manually into an L-shape, leaving one end 3 cm long and the other 2 cm. Cut the silver-coated wire into two 35 cm lengths.</li>
	<li>Insert the longer (3 cm) bent end of one platinum wire (from inside to out) into each drilled hole, leaving 1&#8211;2 mm protruding from the outside of the lid,&#160;bend it using forceps. Secure the platinum wires in the lid holes with superconductive glue and leave it to dry for around 6 h.</li>
	<li>Solder the tips of all six platinum wires (that will later serve as cathodes, see Figure 1A) protruding from the lids to one of the silver-coated wires. Repeat the same procedure, soldering the remaining six platinum wires (that will later serve as anodes) to the other silver-coated wire.
	<ol>
		<li>Add LEDs in the circuit between each of the six anode-cathode platinum electrode pairs, to confirm functionality during the experiments (optional). Place a piece of black insulation tape under each LED to prevent exposing the cells in the culture plates to the LED light (Figure 1C).</li>
	</ol>
	</li>
	<li>Glue the wire terminal block connector to the top left corner of the 6-well plate lid and connect both silver-coated wires to the input terminals, as shown in Figure 1C.</li>
	<li>Cut out a 20 mm x 20 mm section from the top left corner of a second 6-well plate lid (Figure 1B, Lid Nr. 2) to accommodate the terminal block connector on the first lid. Cover the first lid, equipped with the electrodes, with the second lid and tape them together with adhesive tape.
	<ol>
		<li>To improve the bonding of the two lids, use silicone adhesive coating (optional). To do so, cover the lid with the silver electrodes with a 3&#8211;5 mm layer of silicone adhesive and cover it with the other lid, allowing 12 h for the adhesive to dry.</li>
	</ol>
	</li>
	<li>Connect one end of the two standard insulated copper wires to the output terminals of the wire connector and the other ends to banana male connectors (4 mm). 
	NOTE: The length of these wires depends on the distance from the shelf in the incubator, where the cells will be kept, to the power supply, outside the incubator (Figure 1D).</li>
	<li>Adjust the dosage (voltage) and regimen of EStim delivered to the cells by regulating the DC power supply.
	<ol>
		<li>Turn on the power supply by pressing the ON/OFF button on the front panel. Activate channel 1 by pressing button 1.</li>
		<li>Press button Nr. 4 (V-set) to set the voltage. Press buttons 2, . and 5 to set the load output at 2.5 V. Press Enter.</li>
		<li>Ensure that the load output of 2.5 V (2,500 mV) corresponds to an EStim of 100 mV/mm, according to the following, simplified equation. 
		VEStim = <img alt="Equation" src="/files/ftp_upload/59127/59127eq1.jpg" />; or 
		V&#160;=&#160;VEStim &#215; d 
		Here, V&#160;= the power supply voltage output in millivolts; d = the distance between electrodes in millimeters; VEStim = the stimulation voltage in millivolts per millimeter. 
		NOTE: This simplification applies only in case of constant DC voltage. The resistance between the medium and the cells is negligible as electrodes are not in direct contact with the cells; instead, EStim is delivered to the cells through the medium.</li>
	</ol>
	</li>
</ol>
2. Mesenchymal stem cell culture in osteogenic medium
<ol>
	<li>Purchase and store commercially available rat MSCs (see Table of Materials) in liquid nitrogen until the day of the experiment. Alternatively, isolate MSCs from other animals according to protocols published elsewhere7,8 in accordance with local institutional regulations for the use of experimental animals.</li>
	<li>On the day of the experiment, remove one vial (1 x 106 cells) of MSCs from the liquid nitrogen storage, and quickly (within 1 min) thaw the cells in a water bath preheated to 37 &#176;C.
	<ol>
		<li>Under sterile conditions in a laminar flow hood, pipette the vial content into a 50 mL falcon tube and add 9 mL of normal medium (NM) prewarmed to 37 &#176;C, consisting of Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM; 1x) with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin solution. Pellet the cells for 5 min by centrifugation at 300 x g.</li>
		<li>In a laminar flow hood, remove the supernatant and carefully resuspend the cell pellet in 12 mL of NM prewarmed to 37 &#176;C. Transfer the resuspended cells to a T-75 cell culture flask.</li>
	</ol>
	</li>
	<li>Culture the cells at 37 &#176;C, 5% CO2, 5% O2 until they reach an 80%&#8211;90% confluence (after approximately 3&#8211;5 days).</li>
	<li>Passage the cells. 
	NOTE: Perform all operations except centrifugation and incubation under sterile conditions in a laminar flow hood.
	<ol>
		<li>After reaching an 80%&#8211;90% confluence, retrieve the cells with cell detachment solution. Aspirate the cell culture medium, wash 2x with 1x phosphate-buffered saline (PBS), add 5 mL of 1x cell detachment solution, and return the cells to the incubator for 5 min.</li>
		<li>Once the cells are detached, add an equal amount of culture medium to inactivate the detachment solution. Collect the cells in a 50 mL falcon tube and spin them at 300 x g for 5 min.</li>
		<li>Discard the medium and resuspend cells in 1 mL of fresh normal medium. Assess the number of viable cells with trypan blue stain.</li>
		<li>Seed 1 x 106 cells in a new T-75 flask with 12 mL of prewarmed NM. Culture the cells at 37 &#176;C, 5 % CO2, 5 % O2 until they reach an 80%&#8211;90% confluence. 
		NOTE: Cell passaging (steps 2.4.1&#8211;2.4.4) can be repeated a few times until the needed number of cells is obtained. Do not use cells older than passage 8.</li>
	</ol>
	</li>
	<li>Seed 9 x 104 cells in 3 mL of NM (10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, DMEM) in each well of a 6-well culture plate. Incubate the cells for 1 day at 37 &#176;C, 5 % CO2, 5 % O2.</li>
	<li>The next day, aspirate culture medium and apply 3 mL of osteogenic differentiation medium (OM; normal medium supplemented with 10-7 M dexamethasone, 10 mM &#946;-glycerophosphate, and 0.05 mM ascorbic acid-2-phosphate).</li>
	<li>Place the 6-well plate with cells in the incubator and incubate at 37 &#176;C, 5% CO2, 5% O2 overnight.</li>
</ol>
3. Treating MSCs with EStim
<ol>
	<li>On the day the cells are treated with EStim, sterilize the electrodes in 70% ethanol solution for 30 min; then, dry them under UV light in a safety cabinet for an additional 30 min.</li>
	<li>In a laminar flow hood, cover the 6-well plate containing the cultured MSCs with the lid equipped with the electrodes, making sure that the electrodes are completely submerged in medium (if necessary, add medium). Transfer the covered 6-well plate (EStim chamber) with the cells to the incubator and connect its wires to the power supply.</li>
	<li>Set the power supply to 2.5 V load output and treat the cells with EStim for 1 h3,9.</li>
	<li>After stimulation, disconnect the power supply and remove the EStim chamber from the incubator. Under sterile conditions, exchange the lid equipped with electrodes with a standard 6-well plate lid.</li>
	<li>Return the cells to the incubator and leave them overnight. Clean the electrodes, first with PBS and then with 70% ethanol solution. Clean the accumulated corrosion products from the electrode surface with fine sandpaper.</li>
	<li>Repeat steps 3.1&#8211;3.5 for 6 consecutive days. On day 4, prior to applying EStim and under sterile conditions, change the culture medium by aspirating 1.5 mL of medium and replacing it with 1.5 mL of prewarmed fresh OM.</li>
	<li>After applying EStim for 7 consecutive days, maintain the cells in culture for an additional 7 days, exchanging the medium every 3&#8211;4 days.</li>
</ol>
4. Osteogenic differentiation measurements
<ol>
	<li>Analyze cell morphology changes under a microscope.</li>
	<li>To assess the effect of EStim on MSC osteogenic differentiation, measure calcium deposition, alkaline phosphatase activity, and osteogenic marker gene expression, as described elsewhere3,9.</li>
</ol>

<ol><li>Oryan, A., Kamali, A., Moshiri, A., Baghaban Eslaminejad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+Mesenchymal+Stem+Cells+in+Bone+Regenerative+Medicine%3A+What+Is+the+Evidence%3F%5BTitle%5D)+AND+%22Cells%2C+Tissues%2C+Organs%22%5BJournal%5D)">Role of Mesenchymal Stem Cells in Bone Regenerative Medicine: What Is the Evidence?</a> Cells, Tissues, Organs. 204 (2), 59-83 (2017).</li>
<li>Mauney, J. R., Volloch, V., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+Adult+Mesenchymal+Stem+Cells+in+Bone+Tissue+Engineering+Applications%3A+Current+Status+and+Future+Prospects%5BTitle%5D)+AND+%22Tissue+Engineering%22%5BJournal%5D)">Role of Adult Mesenchymal Stem Cells in Bone Tissue Engineering Applications: Current Status and Future Prospects.</a> Tissue Engineering. 11 (5-6), 787-802 (2005).</li>
<li>Mobini, S., Leppik, L., Thottakkattumana Parameswaran, V., Barker, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+effect+of+direct+current+electrical+stimulation+on+rat+mesenchymal+stem+cells%5BTitle%5D)+AND+%22PeerJ%22%5BJournal%5D)">In vitro effect of direct current electrical stimulation on rat mesenchymal stem cells.</a> PeerJ. 5, e2821(2017).</li>
<li>Leppik, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combining+electrical+stimulation+and+tissue+engineering+to+treat+large+bone+defects+in+a+rat+model%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Combining electrical stimulation and tissue engineering to treat large bone defects in a rat model.</a> Scientific Reports. 8 (1), S1(2018).</li>
<li>Song, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+direct+current+electric+fields+to+cells+and+tissues+in+vitro+and+modulation+of+wound+electric+field+in+vivo%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo.</a> Nature Protocols. 2 (6), 1479-1489 (2007).</li>
<li>Hronik-Tupaj, M., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+the+responses+of+two-+and+three-dimensional+engineered+tissues+to+electric+fields%5BTitle%5D)+AND+%22Tissue+Engineering.+Part+B%2C+Reviews%22%5BJournal%5D)">A review of the responses of two- and three-dimensional engineered tissues to electric fields.</a> Tissue Engineering. Part B, Reviews. 18 (3), 167-180 (2012).</li>
<li>Huang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+protocol+for+isolation+and+culture+of+mesenchymal+stem+cells+from+mouse+bone+marrow%5BTitle%5D)+AND+%22Journal+of+Orthopaedic+Translation%22%5BJournal%5D)">An improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow.</a> Journal of Orthopaedic Translation. 3 (1), 26-33 (2015).</li>
<li>Nau, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+engineered+vascularized+periosteal+flap+enriched+with+MSC%2FEPCs+for+the+treatment+of+large+bone+defects+in+rats%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Medicine%22%5BJournal%5D)">Tissue engineered vascularized periosteal flap enriched with MSC/EPCs for the treatment of large bone defects in rats.</a> International Journal of Molecular Medicine. 39 (4), 907-917 (2017).</li>
<li>Eischen-Loges, M., Oliveira, K. M. C., Bhavsar, M. B., Barker, J. H., Leppik, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pretreating+mesenchymal+stem+cells+with+electrical+stimulation+causes+sustained+long-lasting+pro-osteogenic+effects%5BTitle%5D)+AND+%22PeerJ%22%5BJournal%5D)">Pretreating mesenchymal stem cells with electrical stimulation causes sustained long-lasting pro-osteogenic effects.</a> PeerJ. 6, 4959(2018).</li>
<li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2%28-Delta+Delta+C%28T%29%29+Method%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), San Diego, CA. 402-408 (2001).</li>
<li>Curtis, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EF1alpha+and+RPL13a+represent+normalization+genes+suitable+for+RT-qPCR+analysis+of+bone+marrow+derived+mesenchymal+stem+cells%5BTitle%5D)+AND+%22BMC+Molecular+Biology%22%5BJournal%5D)">EF1alpha and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells.</a> BMC Molecular Biology. 11, 61(2010).</li>
<li>Wang, L., Li, Z. -y, Wang, Y. -p, Wu, Z. -h, Yu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+Expression+Profiles+of+Marker+Genes+in+Osteogenic+Differentiation+of+Human+Bone+Marrow-derived+Mesenchymal+Stem+Cells%5BTitle%5D)+AND+%22Chinese+Medical+Sciences+Journal+%28Chung-kuo+i+hsueh+k%27o+hsueh+tsa+chih%29%22%5BJournal%5D)">Dynamic Expression Profiles of Marker Genes in Osteogenic Differentiation of Human Bone Marrow-derived Mesenchymal Stem Cells.</a> Chinese Medical Sciences Journal (Chung-kuo i hsueh k'o hsueh tsa chih). 30 (2), 108-113 (2015).</li>
<li>Kim, H. B., Ahn, S., Jang, H. J., Sim, S. B., Kim, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+corrosion+behaviors+and+surface+profiles+of+platinum-coated+electrodes+by+electrochemistry+and+complementary+microscopy%3A+biomedical+implications+for+anticancer+therapy%5BTitle%5D)+AND+%22Micron%22%5BJournal%5D)">Evaluation of corrosion behaviors and surface profiles of platinum-coated electrodes by electrochemistry and complementary microscopy: biomedical implications for anticancer therapy.</a> Micron. 38 (7), Oxford, UK. 747-753 (2007).</li>
<li>Cho, Y., Son, M., Jeong, H., Shin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electric+field-induced+migration+and+intercellular+stress+alignment+in+a+collective+epithelial+monolayer%5BTitle%5D)+AND+%22Molecular+Biology+of+the+Cell%22%5BJournal%5D)">Electric field-induced migration and intercellular stress alignment in a collective epithelial monolayer.</a> Molecular Biology of the Cell. , mbcE18010077(2018).</li>
<li>Tai, G., Tai, M., Zhao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrically+stimulated+cell+migration+and+its+contribution+to+wound+healing%5BTitle%5D)+AND+%22Burns+%26+Trauma%22%5BJournal%5D)">Electrically stimulated cell migration and its contribution to wound healing.</a> Burns & Trauma. 6, 20(2018).</li>
<li>Love, M. R., Palee, S., Chattipakorn, S. C., Chattipakorn, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+electrical+stimulation+on+cell+proliferation+and+apoptosis%5BTitle%5D)+AND+%22Journal+of+Cellular+Physiology%22%5BJournal%5D)">Effects of electrical stimulation on cell proliferation and apoptosis.</a> Journal of Cellular Physiology. 233 (3), 1860-1876 (2018).</li>
<li>Adams, D. S., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((General+principles+for+measuring+resting+membrane+potential+and+ion+concentration+using+fluorescent+bioelectricity+reporters%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">General principles for measuring resting membrane potential and ion concentration using fluorescent bioelectricity reporters.</a> Cold Spring Harbor Protocols. 2012 (4), 385-397 (2012).</li>
<li>Jin, G., Li, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+electrically+conductive+scaffold+as+the+skeleton+of+stem+cell+niche+in+regenerative+medicine%5BTitle%5D)+AND+%22Materials+Science+%26+Engineering.+C%2C+Materials+for+Biological+Applications%22%5BJournal%5D)">The electrically conductive scaffold as the skeleton of stem cell niche in regenerative medicine.</a> Materials Science & Engineering. C, Materials for Biological Applications. 45, 671-681 (2014).</li>
<li>Hronik-Tupaj, M., Rice, W. L., Cronin-Golomb, M., Kaplan, D. L., Georgakoudi, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Osteoblastic+differentiation+and+stress+response+of+human+mesenchymal+stem+cells+exposed+to+alternating+current+electric+fields%5BTitle%5D)+AND+%22Biomedical+Engineering+Online%22%5BJournal%5D)">Osteoblastic differentiation and stress response of human mesenchymal stem cells exposed to alternating current electric fields.</a> Biomedical Engineering Online. 10, 9(2011).</li>
</ol>

The authors have nothing to disclose.

Here we describe the construction of a chamber and a method for treating mesenchymal stem cells with EStim that results in enhanced osteogenic differentiation.
The EStim setup presented does not require special equipment/knowledge and can be performed in a standard stem cell biology/biochemistry laboratory by junior researchers. However, when building and using the EStim chamber, special care must be taken in a few critical steps. When handling the platinum electrodes, extra care must be taken...

An increase in trauma and/or disease-induced bone defects are being treated using different combinations of cell therapy and regenerative medicine technologies. MSCs are the cell of choice in such treatments, due to their relatively high osteogenic activity, isolation and expansion efficiency, and safety1. To maximize their osteogenic activity and, thus, optimize their therapeutic effectiveness, several methods have been introduced to manipulate MSCs prior to their use in these treatments (as reviewed by Mauney et al.2). One such method is EStim, which has been shown to enhance MSC osteogenic differentiation in vitro3 and promote bone healing in vivo4. Despite the growing number of studies focusing on treating MSCs with EStim, an optimal regimen for maximizing EStim&#8217;s pro-osteogenic effect has yet to be defined.
Other in vitro methods using EStim utilize salt bridges submerged in the culture medium, which separates cells from metallic electrodes5. The advantage of this is that delivering EStim through salt bridges eliminates the introduction of chemical byproducts (e.g., corrosion of metallic electrodes) that may be cytotoxic. Despite this advantage, salt bridges are cumbersome to work with, and the EStim they deliver differs from that delivered in in vivo models, making it difficult to correlate results obtained when using the two systems. Setups that deliver EStim via metallic or carbon electrodes fixed inside the cell culture wells (as reviewed by Hronik-Tupaj and Kaplan6) better simulate devices used in vivo; however, these devices are difficult to clean/sterilize between uses and the number of cells that can be studied per experiment is limited. We designed the EStim chamber presented here specifically to address the limitations of these other setups. While most of our experience using this EStim chamber has been with 2D and 3D cultures containing bone-marrow- and adipose-tissue-derived MSCs3,4, a major benefit of this chamber is that it is versatile and, with relatively minor changes, can be adapted to study other cell types under a variety of different conditions.
Here we describe the construction of an EStim cell culture chamber; then, we demonstrate its use by treating MSCs with different regimens of EStim and measuring the resulting effect on osteogenic differentiation. MSC osteogenic differentiation is assessed via calcium deposition, alkaline phosphatase activity, and osteogenic marker gene expression. Importantly, in past experiments that used this setup, we observed that these pro-osteogenic effects persist long after the EStim treatment was discontinued.

<table><tbody><tr><td>&lt;strong&gt;Estim fabrication&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Banana connector/Jack adaptor</td><td>Poppstars</td><td>1008554</td><td>2 pieces</td></tr><tr><td>Cutting pliers</td><td>Knipex</td><td>78 03 125</td><td></td></tr><tr><td>DC power supply (0-30V/0-3A)</td><td>B&amp;amp;K Precision</td><td>Model 9130B</td><td>Any simular model could be used</td></tr><tr><td>Insulated flexible wires (0.14 mm&lt;sup&gt;2&lt;/sup&gt;)</td><td>Conrad Electronic International</td><td>604794, 604093</td><td>2 pieces</td></tr><tr><td>Non-corrosive silicone rubber</td><td>Dow Corning</td><td>3140 RTV</td><td>*could be purchased by many stores</td></tr><tr><td>Platinum Wire (999,5/1000; 1mm &amp;oslash;)</td><td>Junker Edelmetalle</td><td>00D-3010</td><td>0.6 m needed for 1 Estim chamber</td></tr><tr><td>70% Ethanol solution</td><td>any</td><td></td><td>Sterilisation of Estim chamber</td></tr><tr><td>Silver coated copper wire (0.6 mm &amp;oslash;)</td><td>Conrad Electronic International</td><td>409334 - 62</td><td>&amp;asymp;70 cm needed for 1 Estim device</td></tr><tr><td>Soldering iron Set</td><td>Conrad Electronic International</td><td>1611410 - 62</td><td>Any simular model could be used</td></tr><tr><td>TPP 6-well plate lid</td><td>Sigma-Aldrich</td><td>Z707759-126EA</td><td>2 lids for Estim chamber</td></tr><tr><td>2.2V wired circular LEDs</td><td>Conrad Electronic International</td><td>599525 - 62</td><td>6 pieces</td></tr><tr><td>UHU Super glue</td><td>UHU GmbH &amp;amp; Co. KG</td><td>n/a</td><td>*could be purchased by many stores</td></tr><tr><td>&lt;strong&gt;MSC culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;beta;-Glycerophosphate disodium salt hydrate</td><td>Sigma-Aldrich</td><td>G9422</td><td>osteogenic cell culture</td></tr><tr><td>DMEM, low glucose, GlutaMAX Supplement, pyruvate</td><td>Thermo-Fischer Scientific</td><td>21885025</td><td>cell culture</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>Thermo-Fischer Scientific</td><td>14190144</td><td>cell culture</td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td>osteogenic cell culture</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo-Fischer Scientific</td><td>10500064</td><td>cell culture</td></tr><tr><td>50 ml Falcon tube</td><td>Sarstedt</td><td>62,547,004</td><td>cell culture</td></tr><tr><td>L-Ascorbic acid</td><td>Sigma-Aldrich</td><td>A4544</td><td>osteogenic cell culture</td></tr><tr><td>Penicillin/Streptomycin</td><td>Thermo-Fischer Scientific</td><td>15140122</td><td>cell culture</td></tr><tr><td>Sprague-Dawley (SD) rat mesenchymal stem cells, bone marrow origin</td><td>Cyagen</td><td>RASMX-01001</td><td>cell culture</td></tr><tr><td>Cell detachment solution</td><td>Thermo-Fischer Scientific</td><td>A1110501</td><td>cell culture, cell detachment</td></tr><tr><td>TC Flask, T75</td><td>Sarstedt</td><td>833911302</td><td>cell culture</td></tr><tr><td>TPP 6-well plates</td><td>Sigma-Aldrich</td><td>Z707759-126EA</td><td>cell culture</td></tr><tr><td>Trypan Blue Dye, 0.4% solution</td><td>Bio-Rad</td><td>1450021</td><td>cell count</td></tr></tbody></table>

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.

To evaluate the effect of 100 mV/mm of EStim on the osteogenic differentiation of MSCs, cells treated with EStim for 3, 7, and 14 days or nontreated (control) were analyzed at day 14 of culturing by assessing morphological changes and calcium deposition (Figure 2). This was done by imaging cells using bright-field microscopy (morphology changes) or by fixing cells in 4% paraformaldehyde solution, staining them with 0.02% alizarin red solution and then imaging...

Watch this Scientific Journal Video about Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro at JoVE.com

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

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) ...

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11.1K Views.  Johann Wolfgang Goethe-University. Hier laten we zien hoe je een elektrische stimulatiekamer bouwt met behulp van gemeenschappelijke zes wandkweekplaten, en hoe je deze gebruiken om osteogene differentiatie in mesenchymale stamcellen te stimuleren. Deze kamer is eenvoudig en niet duur om te bouwen, en kan worden hergebruikt in meerdere experimenten. Na voorbehandeling met elektrische stimulatie in onze kamer, kunnen mesenchymale stamcellen worden gebruikt om de resultaten in botweefsel engineering behandelingen te verbeteren.

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

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated phenomena such as bone remodeling, muscular hypertrophy, and smooth muscle cell plasticity. However, the forces involved are often extremely complex and difficult to monitor and control in vivo. To better investigate the effects of mechanical forces on cells, we have developed an in vitro method for applying uniaxial cyclic tensile strain to adherent cells cultured on elastic membranes. This method utilizes a custom-designed bioreactor with a motorized cam-rotor system to apply the desired force. Here we present a step-by-step video protocol demonstrating how to assemble the various components of each "stretch chamber", including, in this case, a silicone membrane with micropatterned topography to orient the cells with the direction of the strain. We also describe procedures for sterilizing the chambers, seeding cells onto the membrane, latching the chamber into the bioreactor, and adjusting the mechanical parameters (i.e. magnitude and rate of strain). The procedures outlined in this particular protocol are specific for seeding human mesenchymal stem cells onto silicone membranes with 10 &micro;m wide channels oriented parallel to the direction of strain. However, the methods and materials presented in this system are flexible enough to accommodate a number of variations on this theme: strain rate, magnitude, duration, cell type, membrane topography, membrane coating, etc. can all be tailored to the desired application or outcome. This is a robust method for investigating the effects of uniaxial tensile strain applied to cells in vitro.

It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates.

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated ...

Biology

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Electrically Conductive Scaffold to Modulate and Deliver Stem Cells

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection has been used for decades to deliver stem cells in stroke models, this technique is limited by the lack of ability to manipulate the stem cells prior to injection. This paper details a method of using an electrically conductive polymer scaffold for stem cell delivery. Electrical stimulation of stem cells using a conductive polymer scaffold alters the stem cell's genes involved in cell survival, inflammatory response, and synaptic remodeling. After electrical preconditioning, the stem cells on the scaffold are transplanted intracranially in a distal middle cerebral artery occlusion rat model. This protocol describes a powerful technique to manipulate stem cells via a conductive polymer scaffold and creates a new tool to further develop stem cell-based therapy.

This protocol describes fabrication of a cell culture system to allow seeding of stem cells on a conductive polymer scaffold for in vitro electrical stimulation and subsequent in vivo implantation of the stem cell-seeded scaffold using a minimally invasive technique.

Stem cell therapy has emerged as an exciting stroke therapeutic, but the optimal delivery method remains unclear. While the technique of microinjection ...

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.

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 ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients&#8217; life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited ...

Medicine

Artificial Cartilage Implants for Bone Regeneration via Endochondral Ossification

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it does not have a mechanism to induce angiogenesis. Implanting artificial cartilage tissue fabricated from MSCs induces angiogenesis and bone formation in vivo via endochondral ossification (ECO). Therefore, this ECO-mediated approach may be a promising bone regeneration therapy in the future. An important aspect of the clinical application of this ECO-mediated approach is establishing a protocol for preparing enough cartilage to be implanted to repair the bone defect. It is especially not practical to design a single mass of grafted cartilage of a size that conforms to the shape of the actual bone defect. Therefore, the cartilage to be transplanted must have the property of forming bone integrally when multiple pieces are implanted. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification to meet clinical requirements. Although many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo, the optimal scaffold material to meet the needs of clinical applications has yet to be determined. Hyaluronic acid (HA) is a crucial component of the cartilage extracellular matrix and is a biodegradable and biocompatible polysaccharide. Here, we show that HA hydrogels have excellent properties to support in vitro differentiation of MSC-based cartilage tissue and promote endochondral bone formation in vivo.

Author Spotlight: Enhancing Bone Regeneration with Vascularized Artificial Cartilage Integration 

Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it ...

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

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