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

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 MSCs^3,4, 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.

Protocol

1. Construction of electrical stimulation cell culture chamber

1. 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).
2. 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.
3. 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.
4. 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.
5. 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.
   1. 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).
6. 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.
7. 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.
   1. 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.
8. 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).
9. Adjust the dosage (voltage) and regimen of EStim delivered to the cells by regulating the DC power supply.
   1. Turn on the power supply by pressing the ON/OFF button on the front panel. Activate channel 1 by pressing button 1.
   2. 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.
   3. 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.
      V_EStim = ; or
      V = V_EStim × d
      Here, V = the power supply voltage output in millivolts; d = the distance between electrodes in millimeters; V_EStim = 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.

2. Mesenchymal stem cell culture in osteogenic medium

1. 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 elsewhere^7,8 in accordance with local institutional regulations for the use of experimental animals.
2. 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.
   1. 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.
   2. 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.
3. Culture the cells at 37 °C, 5% CO₂, 5% O₂ until they reach an 80%–90% confluence (after approximately 3–5 days).
4. Passage the cells.
   NOTE: Perform all operations except centrifugation and incubation under sterile conditions in a laminar flow hood.
   1. 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.
   2. 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.
   3. Discard the medium and resuspend cells in 1 mL of fresh normal medium. Assess the number of viable cells with trypan blue stain.
   4. 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.
5. 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.
6. 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).
7. 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

1. 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.
2. 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.
3. Set the power supply to 2.5 V load output and treat the cells with EStim for 1 h^3,9.
4. 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.
5. 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.
6. 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.
7. 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

1. Analyze cell morphology changes under a microscope.
2. To assess the effect of EStim on MSC osteogenic differentiation, measure calcium deposition, alkaline phosphatase activity, and osteogenic marker gene expression, as described elsewhere^3,9.

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...

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...

Materials

List of materials used in this article

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