Summary
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.
Abstract
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.
Introduction
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’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.
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Protocol
1. Construction of electrical stimulation cell culture chamber
- 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 0.5–1 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).
- 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.
- 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.
- Insert the longer (3 cm) bent end of one platinum wire (from inside to out) into each drilled hole, leaving 1–2 mm protruding from the outside of the lid, bend it using forceps. Secure the platinum wires in the lid holes with superconductive glue and leave it to dry for around 6 h.
- 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.
- 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).
- 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.
- 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.
- 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 3–5 mm layer of silicone adhesive and cover it with the other lid, allowing 12 h for the adhesive to dry.
- 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). - Adjust the dosage (voltage) and regimen of EStim delivered to the cells by regulating the DC power supply.
- Turn on the power supply by pressing the ON/OFF button on the front panel. Activate channel 1 by pressing button 1.
- 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.
- 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 =
; or
V = VEStim × d
Here, V = 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.
; or2. Mesenchymal stem cell culture in osteogenic medium
- 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.
- 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 °C.
- 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 °C, consisting of Dulbecco’s modified Eagle’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.
- In a laminar flow hood, remove the supernatant and carefully resuspend the cell pellet in 12 mL of NM prewarmed to 37 °C. Transfer the resuspended cells to a T-75 cell culture flask.
- Culture the cells at 37 °C, 5% CO2, 5% O2 until they reach an 80%–90% confluence (after approximately 3–5 days).
- Passage the cells.
NOTE: Perform all operations except centrifugation and incubation under sterile conditions in a laminar flow hood.- After reaching an 80%–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.
- 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.
- Discard the medium and resuspend cells in 1 mL of fresh normal medium. Assess the number of viable cells with trypan blue stain.
- Seed 1 x 106 cells in a new T-75 flask with 12 mL of prewarmed NM. Culture the cells at 37 °C, 5 % CO2, 5 % O2 until they reach an 80%–90% confluence.
NOTE: Cell passaging (steps 2.4.1–2.4.4) can be repeated a few times until the needed number of cells is obtained. Do not use cells older than passage 8.
- 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 °C, 5 % CO2, 5 % O2.
- 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 β-glycerophosphate, and 0.05 mM ascorbic acid-2-phosphate).
- Place the 6-well plate with cells in the incubator and incubate at 37 °C, 5% CO2, 5% O2 overnight.
3. Treating MSCs with EStim
- 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.
- 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.
- Set the power supply to 2.5 V load output and treat the cells with EStim for 1 h3,9.
- 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.
- 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.
- Repeat steps 3.1–3.5 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.
- After applying EStim for 7 consecutive days, maintain the cells in culture for an additional 7 days, exchanging the medium every 3–4 days.
4. Osteogenic differentiation measurements
- Analyze cell morphology changes under a microscope.
- 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.
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Representative Results
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 them using bright-field microscopy (calcium deposition analysis).
A detailed analysis of osteogenic marker gene expression changes was performed at days 3, 7, and 14 of culturing (Figure 3). This was done by measuring the relative expression of genes RunX2, Collagen I, Osteopontin, and Osterix by means of RT-qPCR and the comparative delta Ct (threshold cycle values) method10, where housekeeping genes Rplp1 and Ywhaz11 were used for normalization.
Exposing MSCs to 100 mV/mm of EStim for 3 days (1 h per day) had no effect; however, 7 days of exposure resulted in an increase in osteogenic differentiation, as determined by morphology changes (Figure 2A–C), calcium deposition (Figure 2E–G), and osteogenic marker gene expression changes (Figure 3) in comparison to a time-matched control without EStim. Prolonged EStim exposure (14 days) did not further enhance the osteogenic differentiation beyond that seen after 7 days of treatment (Figure 2D,H and Figure 3).
As shown in Figure 2, cells treated with EStim for 7 and 14 days appeared more condensed (Figure 2C,D) than those treated with EStim for 3 days or nontreated cells (Figure 2A,B) and showed an increased calcium deposition (Figure 2G,H) compared to those treated for only 3 days or nontreated controls (Figure 2E,F). Analysis of the osteogenic marker expression at 3, 7, and 14 days of culturing confirmed the enhanced osteogenic differentiation in cells treated with EStim for 7 and 14 days (Figure 3). The expression of osteogenic-differentiation-related marker genes12 RunX2, Collagen I, Osteopontin, and Osterix were the highest in cells treated with EStim for 7 days.
Figure 1: EStim cell culture chamber. (A) Electric circuit diagram of the EStim chamber showing the anodes (black), cathodes (red), and LEDs connected to the DC power supply. (B) Image of the marked 6-well plate lids and L-shaped platinum electrodes (bottom view) incorporated into the 6-well plate lid. (C) Assembled EStim chamber (top view) with wire connector, LEDs, and electrical insulation tape that shields the cells from the LED light (arrows). (D) EStim cell treatment setup with the EStim chamber in the incubator connected to the DC power supply, on the outside. Please click here to view a larger version of this figure.
Figure 2: Effect of EStim on MSC morphology and calcium deposition. Cells in osteogenic culture medium, exposed and not exposed (controls) to 100 mV/mm of EStim for 3, 7, and 14 days (1 h/day). (A–D) Morphology and (E–H) calcium deposition (alizarin red staining) on day 14 of culturing. Significant changes in cell morphology and calcium deposits were visible in cells treated with EStim for (C and G) 7 and (D and H) 14 days (10x magnification; the scale bar = 200 µm). This figure was modified from Eischen-Loges et al.9. Please click here to view a larger version of this figure.
Figure 3: Effect of EStim on MSC osteogenic marker gene expression. The osteogenic marker gene expression (measured with RT-qPCR at days 3, 7, and 14 of culturing) in cells treated with EStim for 3, 7, and 14 days, or nontreated. (A) At day 7 of culturing, the RunX2 expression was significantly higher in cells treated with EStim for 7 days. (B) The ColIa1 expression was significantly higher in cells treated for 7 days, measured at day 14 of culturing. (C) The expression of Osteopontin was significantly increased in EStim-treated cells at days 3, 7, and 14 of culturing. (D) The Osterix expression was absent in control cells at all time points and was seen only at 7 and 14 days of culture in cells exposed to EStim. Different letters on the bars indicate significant (p < 0.05) differences among groups at the same time point. The asterisk indicates significant (p < 0.05) differences between time points within the same group. This figure was modified from Eischen-Loges et al.9. Please click here to view a larger version of this figure.
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Discussion
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 as this metal is very malleable and delicate. While other metals, such as steel or tungsten, can be used, these are not recommended as they are prone to corrosion, which can be cytotoxic13. Also, cleaning/sterilizing the lid with the electrodes must be performed as precisely as described in the protocol since this method has been tested repeatedly and found to be effective in eliminating problems with contamination. Finally, while this chamber could be used to study other EStim-sensitive cell activities like migration14,15, proliferation, apoptosis16, cell membrane voltages17, and scaffold adhesion19, the EStim protocol we describe here (100 mV/mm, 1 h/day, 7 days) focused only on osteogenic differentiation in rat MSCs. Special attention should be given if higher voltages (≥150 mV/mm) and/or longer durations of EStim (>4–5 h) are applied since cytotoxic products resulting from electrolysis can accumulate in the medium. In this case, the medium must be carefully monitored and exchanged accordingly. To study other parameters and/or other cell types and conditions, we recommend that separate dosage (voltage) and regimen titration studies be conducted as these changes can affect how cells respond to EStim.
Possible malfunctioning of the EStim chamber can be due to breaks in the electrical circuitry after repeated use and can be monitored via the added LED lamps. In case of breakage (indicated by nonilluminated LED light[s]), the lid can be removed and easily disassembled to identify and repair the break. In addition, the simple design of the EStim chamber makes it easy to modify it according to the needs of different experimental setups and methods of the analysis. Examples include using different sizes of cell culture plates, by simply varying the length of and the distance between the electrodes or adding different (3D) culture conditions with ceramic scaffold material4 or conductive substrates18, by simply placing these materials seeded with cells between the electrodes.
As in all in vitro experiments, cell behaviors and functions observed/induced in the EStim chamber are not always directly transferable to in vivo models. Accordingly, when interpreting the EStim-induced cell behaviors/functions in the chamber, researchers must always take this into consideration.
The setup and method presented here have many advantages over other in vitro methods used to expose cells to EStim (systems with salt bridges5 and systems with electrodes incorporated in cell culture wells19). An important advantage of the system described here is that, since the cells are cultured in standard 6-well plates, they can be used after EStim treatment in other in vivo or in vitro protocols. In addition, the fact that electrodes are fixed on the lid of the 6-well plate makes it easy to clean and sterilize the device between experiments and to reuse it. Finally, the ability to simultaneously stimulate cells in six wells provides ample material for analysis and reproducibility.
To gain a better understanding of the mechanisms at the cellular membrane level by which EStim affects cell activities in ongoing studies using the chamber described here, we are exploring the relationship between externally delivered EStim and cell membrane potential (Vmem)17. In addition, based on previous findings9, in future studies, we will pretreat the cells with EStim in the chamber, alone and with different 3D scaffolds, and with conductive substrates, to stimulate specific cell functions; then, we will implant them into animal models to determine if they retain the observed enhanced functions in vivo. These studies will contribute to the growing body of information about the mechanisms by which EStim regulate cell function and, in doing so, could contribute to optimizing cell therapy approaches in regenerative medicine and tissue-engineering-based treatments.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
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.
Materials
| Name | Company | Catalog Number | Comments |
| Estim fabrication | |||
| Banana connector/Jack adaptor | Poppstars | 1008554 | 2 pieces |
| Cutting pliers | Knipex | 78 03 125 | |
| DC power supply (0-30V/0-3A) | B&K Precision | Model 9130B | Any simular model could be used |
| Insulated flexible wires (0.14 mm2) | Conrad Electronic International | 604794, 604093 | 2 pieces |
| Non-corrosive silicone rubber | Dow Corning | 3140 RTV | *could be purchased by many stores |
| Platinum Wire (999,5/1000; 1mm ø) | Junker Edelmetalle | 00D-3010 | 0.6 m needed for 1 Estim chamber |
| 70% Ethanol solution | any | Sterilisation of Estim chamber | |
| Silver coated copper wire (0.6 mm ø) | Conrad Electronic International | 409334 - 62 | ≈70 cm needed for 1 Estim device |
| Soldering iron Set | Conrad Electronic International | 1611410 - 62 | Any simular model could be used |
| TPP 6-well plate lid | Sigma-Aldrich | Z707759-126EA | 2 lids for Estim chamber |
| 2.2V wired circular LEDs | Conrad Electronic International | 599525 - 62 | 6 pieces |
| UHU Super glue | UHU GmbH & Co. KG | n/a | *could be purchased by many stores |
| MSC culture | |||
| β-Glycerophosphate disodium salt hydrate | Sigma-Aldrich | G9422 | osteogenic cell culture |
| DMEM, low glucose, GlutaMAX Supplement, pyruvate | Thermo-Fischer Scientific | 21885025 | cell culture |
| DPBS, no calcium, no magnesium | Thermo-Fischer Scientific | 14190144 | cell culture |
| Dexamethasone | Sigma-Aldrich | D4902 | osteogenic cell culture |
| Fetal Bovine Serum | Thermo-Fischer Scientific | 10500064 | cell culture |
| 50 ml Falcon tube | Sarstedt | 62,547,004 | cell culture |
| L-Ascorbic acid | Sigma-Aldrich | A4544 | osteogenic cell culture |
| Penicillin/Streptomycin | Thermo-Fischer Scientific | 15140122 | cell culture |
| Sprague-Dawley (SD) rat mesenchymal stem cells, bone marrow origin | Cyagen | RASMX-01001 | cell culture |
| Cell detachment solution | Thermo-Fischer Scientific | A1110501 | cell culture, cell detachment |
| TC Flask, T75 | Sarstedt | 833911302 | cell culture |
| TPP 6-well plates | Sigma-Aldrich | Z707759-126EA | cell culture |
| Trypan Blue Dye, 0.4% solution | Bio-Rad | 1450021 | cell count |
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