Name: construction and use of an electrical stimulation chamber for enhancing osteogenic differentiation in mesenchymal stem/stromal cells in vitro
Uploaded: 2019-01-31T15:00:00
Description: 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

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 elec...

<ol>
	<li>Oryan, A., Kamali, A., Moshiri, A., Baghaban Eslaminejad, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Mesenchymal+Stem+Cells+in+Bone+Regenerative+Medicine:+What+Is+the+Evidence?.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Adult+Mesenchymal+Stem+Cells+in+Bone+Tissue+Engineering+Applications:+Current+Status+and+Future+Prospects.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+effect+of+direct+current+electrical+stimulation+on+rat+mesenchymal+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+electrical+stimulation+and+tissue+engineering+to+treat+large+bone+defects+in+a+rat+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+direct+current+electric+fields+to+cells+and+tissues+in+vitro+and+modulation+of+wound+electric+field+in+vivo.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+responses+of+two-+and+three-dimensional+engineered+tissues+to+electric+fields.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+protocol+for+isolation+and+culture+of+mesenchymal+stem+cells+from+mouse+bone+marrow.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineered+vascularized+periosteal+flap+enriched+with+MSC/EPCs+for+the+treatment+of+large+bone+defects+in+rats.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pretreating+mesenchymal+stem+cells+with+electrical+stimulation+causes+sustained+long-lasting+pro-osteogenic+effects.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Curtis, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EF1alpha+and+RPL13a+represent+normalization+genes+suitable+for+RT-qPCR+analysis+of+bone+marrow+derived+mesenchymal+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+Expression+Profiles+of+Marker+Genes+in+Osteogenic+Differentiation+of+Human+Bone+Marrow-derived+Mesenchymal+Stem+Cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+corrosion+behaviors+and+surface+profiles+of+platinum-coated+electrodes+by+electrochemistry+and+complementary+microscopy:+biomedical+implications+for+anticancer+therapy.">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), 747-753 (2007).</li><li>Cho, Y., Son, M., Jeong, H., Shin, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+field-induced+migration+and+intercellular+stress+alignment+in+a+collective+epithelial+monolayer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrically+stimulated+cell+migration+and+its+contribution+to+wound+healing.">Electrically stimulated cell migration and its contribution to wound healing.</a> Burns &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+electrical+stimulation+on+cell+proliferation+and+apoptosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+principles+for+measuring+resting+membrane+potential+and+ion+concentration+using+fluorescent+bioelectricity+reporters.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+electrically+conductive+scaffold+as+the+skeleton+of+stem+cell+niche+in+regenerative+medicine.">The electrically conductive scaffold as the skeleton of stem cell niche in regenerative medicine.</a> Materials Science &amp; 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoblastic+differentiation+and+stress+response+of+human+mesenchymal+stem+cells+exposed+to+alternating+current+electric+fields.">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>

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>Estim fabrication</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&#38;K Precision</td>
			<td>Model 9130B</td>
			<td>Any simular model could be used</td>
		</tr>
		<tr>
			<td>Insulated flexible wires (0.14 mm2)</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 &#248;)</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 &#248;)</td>
			<td>Conrad Electronic International</td>
			<td>409334 - 62</td>
			<td>&#8776;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 &#38; Co. KG</td>
			<td>n/a</td>
			<td>*could be purchased by many stores</td>
		</tr>
		<tr>
			<td>MSC culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-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) ...

Here, we show how to build an electrical stimulation chamber using common six wall culture plates, and how to use it in order to stimulate osteogenic differentiation in mesenchymal stem cells. This chamber is easy and not expensive to build, and can be reused in multiple experiments. After being pre-treated with electrical stimulation in our chamber, mesenchymal stem cells can be used to improve outcomes in bone tissue engineering treatments.This chamber can also be used to investigate other cell types and electro-sensitive cell behaviors like migration, proliferation, changes in the membrane potential, apoptosis, and careful attachment. In a six well plate lid, mark and then drill two one-millimeter diameter holes, 25 millimeters apart, near the outer edge of each of the six wells. Next, cut a platinum wire into 12 five-centimeters long sections.Manually bend each five-centimeter section into an L"shape, leaving one end three centimeters long, and the other two centimeters long. Then, cut the silver-coated wire into two 35-centimeter lengths. Insert the longer bent end of a platinum wire into the drilled hole, leaving one to two millimeters protruding from the outside of the lid.Bend it using forceps. Afterward, secure the platinum wires in the lid holes with super-conductive glue and leave them to dry for approximately six hours. To prepare the cathodes, solder the tips of all six platinum wires protruding from the lid to one of the silver-coated wires.To prepare the anodes, solder the remaining six platinum wires to other silver-coated wires. Subsequently, add LEDs in the circuit between each of the six anode-cathode platinum-electrode pairs to confirm the functionality. Place a piece of black insulation tape under each LED to prevent exposing the cells in the culture plates to LED light.Next, glue the wire terminal block connector to the top left corner of the six-well plate lid, and connect both silver-coated wires to the input terminals. Then, cut out a 20-millimeter by 20-millimeter square section from the top left corner of a second six-well plate lid, 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.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. Turn on the power supply by pressing the ON-OFF button on the front panel. Activate channel one by pressing button one.Afterward, press button four to set the voltage. Set the load output at 2.5 volt. Then press Enter.On the day the cells are treated with E stem, sterilize the electrodes in 70%ethanol solution for 30 minutes. Then, dry them under UV light in a safety cabinet for an additional 30 minutes. In a laminar flow hood, cover the six-well plate containing the cultured MSCs with the lid equipped with the electrodes, making sure that the electrodes are completely submerged in the medium.Then, transfer the covered six-well plate with cells to the incubator and connect its wires to the power supply. Next, set the power supply to 2.5 volt load output and treat the cells with E stem for one hour. After stimulation, disconnect the power supply and remove the E stem chamber from the incubator.Under sterile conditions, replace the lid, equipped with electrodes, with a standard six-well plate lid. Subsequently, return the cells to the incubator, and leave them overnight. Clean the electrodes, first with PBS, and then with 70%ethanol solution.Then, clean the accumulated corrosion products from the electrode surface with fine sandpaper. Repeat the E stem treatment for six consecutive days. On day four, prior to applying electrical stimulation, and under sterile conditions, change the culture medium by aspirating 1.5 milliliters of medium and replacing it with 1.5 milliliters of pre-warmed, fresh osteogenic differentiation medium.After applying electrical stimulation for seven consecutive days, maintain the cells in culture for an additional seven days, exchanging the medium every three to four days. When the treatment is completed, analyze the cell morphology changes under a microscope. To assess the effect of electrical stimulation on MSC osteogenic differentiation, measure calcium deposition, alkaline phosphatase activity, and osteogenic marker gene expression, as described elsewhere.To evaluate the effect of 100 millivolts per millimeter of electrical stimulation on the MSC osteogenic differentiation, cells treated with electrical stimulation for three, seven, and 14 days and non-treated cells, were analyzed at day 14 of culture by assessing morphological changes and calcium deposition. Significant changes in cell morphology and calcium deposits were visible in cells treated with electrical stimulation for seven and 14 days. A detailed analysis of osteogenic marker gene expression changes was performed at days three, seven, and 14 of culture.At day seven of culture, RunX2 expression was significantly higher in cells treated with electrical stimulation for seven days. Whereas Colla1 expression was significantly higher in cells treated for seven days, measured at day 14 of culture. The expression of osteopontin was significantly increased in electrically stimulated cells at days three, seven, and 14 of culture.Osterix expression was absent in control cells at all time points, and was seen only at seven and 14 days of culture in cells exposed to electrical stimulation. The electrical stimulation chamber is relatively easy to build. However, special care must be taken when handling the platinum wires, and also when cleaning and sterilizing the electrodes.Cells pre-treated to electricity in our chamber, alone or seated on a scaffold material, could be implanted into animal models, to study how the positive effect of electrical stimulation persists in vitro. This technique provides a simple yet powerful tool to study the mechanism by which electrical stimulation regulates cell functions. This knowledge can be available in regenerative medicine cells.

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