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>
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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. 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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. 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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) ...

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