Experimental setup
The in vitro experimental setup included several blocks: the signal generation circuit, the cell culture plate with electrodes, and the 3D printed bed that holds the connections to the circuit and the culture chambers.
The circuit designed consisted of a USB oscilloscope working as a DC power supply (Analog Discovery 2, Digilent, Washington, USA), that allowed us to obtain a low noise DC signal, and an amplification stage. The amplification stage was powered at +-5V and employed OPA4228 operational amplifiers (Texas Instruments, Texas, USA) at inverter amplifier configuration to generate the desired DC signals from the negative supply voltage. The inverter amplifier gains were set by resistance ratios that ultimately define the DC voltage levels outputted to the cell culture. The amplification stage was soldered on a perforated board, all circuitry was encapsulated in a hermetic enclosure to protect it from the environmental conditions of the cell incubator (Figure 1A).
In this study, 8W10E+ cell culture plates from Applied Biophysics (Troy, USA) were used (Figure 1B). These plates are made of a clear polycarbonate substrate and have 8 wells, each with two sets of 20 round interdigitated transparent gold electrodes: one set positive and the other set to ground. The electrodes have a diameter of 250 μm and a separation of approximately 1 mm between the closest ones; this means that all applied voltages can be directly expressed in mV/mm. These plates allow us to be completely sure about the EF suffered by the cells, since there are 20 positive (and 20 ground) EF application points on the culture substrate and cells rest on top of them, electrically stimulated cells will sense the same or similar EF. The configuration of the electrodes in space imposes the weakest field lines at 90º, while the strongest ones are located around 45º and 135º, in reference to the electrode (Figure 1D).
The 3D printed bed was designed to hold both the cell culture plate and a printed circuit board (PCB) that connects it with the signal generation circuit. The PCB has 9 connectors that make contact with the gold fingers on the 8W10E+ plate and a pin header connector to interface with the signal generation circuit (Figure 1C).
Cell and culture conditions
N2a
N2a cells were generously provided by Dr. Diego Ruano (Institute of Biomedicine of Sevilla, Sevilla, Spain). Cells were cultured in medium consisting of 50% DMEM High glucose (Biowest, Nuaillé, France) and 50% Opti-MEM (Gibco, Alcobendas, Spain) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, Alcobendas, Spain), 2mM L-glutamine, 50μg/mL streptomycin, and 50U/mL penicillin (Sigma-Aldrich, Madrid, Spain). 25.000 N2a cells were seeded on 500μL of completed medium in each of the 8 wells of an 8W10E+ plate; this number of cells ensures the optimal density: plenty of cells to form a layer and with enough space for cells to differentiate properly. The day before electrical stimulation, serum-free medium was added, replacing the completed medium. Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2 and they were routinely subcultured in order to be in exponential growth phase when used for experiments. Each experiment was independently performed at least in triplicate.
MC3T3
MC3T3E1, a murine pre-osteoblast cell line (CRL-2593, from ATCC, Manassas, VA, USA) was used to evaluate the effects of ES on proliferation, viability, and differentiation process. Cells were cultured in Minimum Essential Medium (MEM), containing 10% fetal bovine serum plus antibiotics (100 U/mL penicillin and 100 mg/mL streptomycin) (Invitrogen) and antifungal (amphotericin B 100mg/mL (BioWittaker Lonza). Osteoblasts cells were seeded at a cellular density of 175,000 cells per condition. Plates were kept at 37 ◦C in a humidified 5% CO2 atmosphere.
At 72h of osteoblast culture, the cell medium was changed to osteogenic media (α-MEM medium) supplemented with 10 mM ascorbic acid (Merck, Germany), 10 mM of β-glycerophosphate (StemCell Technologies, Canada) and 10nM of dexamethasone (Sigma). In-vitro experiments were carried out at 4, 7 and 10 days of cell incubation, in which the supernatants were transferred to vials to be stored at -80ºC until the last day of the experiment, to evaluate cell differentiation.
Electrical Stimulation Protocol
N2a cells were stimulated for 6 hours and left in serum-free medium for another 18 hours. MC3T3 cells were stimulated 3 hours every day for 11 days; the medium was refreshed every other day before stimulation. The stimulation signals consisted of DC voltages of amplitudes: 0 (control), 125, 250 and 500mV/mm for both cell lines; conditions were duplicated in 2 wells of the culture plate, thus using the 8 wells of the 8W10E+ plate. For MC3T3 cells, two identical plates were seeded and stimulated, a higher cell number was required in order to perform the gene study. This stimulation protocol was designed based on previous works (Table 1). Works aimed at neural differentiation and neurite outgrowth apply DC EF for 1 to 8 hours, most often in a single day, with amplitudes lower than 1 V/mm. For osteogenesis, the DC amplitudes used are similar, but the number of days of ES is larger, ranging from 3 to 14 days.
Table 1: Several references of Electrical Stimulation conditions reported using DC signals, on neuroblastoma and osteoblast cell lines.
Cell Line
|
Amplitudes (mV/mm)
|
ES duration
|
Effect
|
Ref
|
Hours/day
|
Days
|
N2a
|
110, 250, 500, 750, 1000
|
6
|
1
|
Differentiation and neurite outgrowth
|
[2]
|
Dorsal Root Ganglia Neurons
|
50
|
8
|
1
|
Neurite outgrowth
|
[39]
|
PC12
|
3000
|
1
|
1
|
Neurite polarization and outgrowth
|
[40]
|
Neural precursor cells (NPC)
|
115
|
2
|
2
|
Migration
|
[41]
|
Immortalized keratinocytes HaCaT
|
150
|
1
|
1
|
Proliferation and migration
|
[42]
|
MC3T3
|
100
|
1, 2
|
3
|
Differentiation and Osteogenesis
|
[26]
|
Rat bone marrow stromal cells
|
3.5, 35, 350
|
2, 4, 12
|
14
|
Osteogenesis
|
[22]
|
hMSC
|
100
|
0.167 (10min)
|
21
|
Differentiation and osteogenesis
|
[23]
|
hMSC
|
100, 200
|
1
|
21
|
Proliferation, differentiation and osteogenesis
|
[24]
|
Rat mesenchymal stem cells
|
100
|
1
|
14
|
Osteogenesis
|
[28]
|
N2a Counting method and differentiation analysis
After the stimulation protocol was performed, images of each electrode were taken in an inverted microscope (Leica, Wetzlar, Germany) with a 20X objective. The images were then analyzed: undifferentiated and differentiated cells were counted, neurites were measured, and their orientation was also assessed. In order to measure neurite orientation, images were stabilized taking the electrode as reference, and angles were extracted from the coordinates of the neurites ends. For this purpose, eq. (1) was used, where θ represents the orientation of the neurite and (x1, y1) and (x2, y2) are the coordinates of the ends of the neurite (Figure 2A).
Only cells on top of the electrode surface were considered (Figure 2B); in this way we ensure that the counted cells had fully experienced the electrical stimulation delivered. A MATLAB program was developed in order to analyze these images [43], the cited program allows one to click on cells to count them and displays a ruler tool with moving ends to measure the length of neurites (Figure 2A). In the whole process, four steps were necessary to obtain the results: a) firstly, all cells were counted; b) secondly, only differentiated cells were selected; c) thirdly, the images were stabilized in order to do the last pass d) fourthly, neurite length and orientation were measured with the ruler tool.
N2a Inmunofluorescence assay
An additional experiment was carried out for the immunofluorescence assay on N2a cells. Cells were stimulated for 6 hours at 500mV/mm, they were then incubated for 18h in serum-free medium, after which they were fixed with 4% paraformaldehyde (Fluka, Honeywell International) in PBS at room temperature for 15 minutes and permeabilized with 0.5% Triton X-100 (Merck KGaA, Darmstadt, Germany) in PBS at room temperature for 5 minutes. Samples were blocked in PBS with 0.1% Tween20 (Sigma-Aldrich, Madrid, Spain) and 1% bovine serum albumin (Sigma-Aldrich, Madrid, Spain). A primary monoclonal Anti-a-Tubulin (Sigma-Aldrich, Madrid, Spain) was diluted 1:3000 in the solution used for blocking, cells were kept in this solution for 45 minutes at room temperature. Goat Polyclonal secondary antibody to mouse IgG (H&L) - Alexa Fluor 488 was diluted 1:500 in the same blocking solution and incubated for 30 minutes at room temperature. Epifluorescence microscopy was performed using an inverted Leica microscope with a 10X objective (Leica, Wetzlar, Germany).
MC3T3 Cell Viability and Proliferation Assay
In order to quantify the proliferation of MC3T3 an AlamarBlue assay was carried out the day after the last stimulation protocol (day 10). Briefly, 300μL AlamarBlue mixture containing 10%AlamarBlueTM stock solution (ThermoFisherScientific, UK) and 90% cell culture medium was added directly to the samples in a culture plate with electrodes, then incubated at 37°C for 120min. Following incubation, three 50μL replicates of the aliquots were transferred from each sample into a clear 96 well plate. A sample control (media plus AlamarBlueTM reagent) was required on this assay. Fluorescence readings were taken using a TECAN, Infinity 200 Pro microplate reader at 570nm excitation.
MC3T3 Differentiation by Alkaline Phosphatase (ALP)
MC3T3 differentiation levels were evaluated through alkaline phosphatase (ALP) activity, using the Alkaline Phosphatase Assay Kit Colorimetric (Abcam ab83369, Abcam, Cambridge, UK). MC3T3 medium was refreshed every other day before electrical stimulation. The used medium was stored at -80ºC until the last day of stimulation. The assay was performed at 3, 4, 5, 6, 7, 10 and 14 days, by triplicate, according to manufacturer’s protocol. The absorbance at 405 nm of 4-nitrophenol was measured in a 96-well microplate reader. Data were expressed as U/mL of pNPP (para-Nitrophenylphosphate).
Real-time PCR analysis on MC3T3
RNA was isolated from MC3T3E1 cells by using Trizol, following manufacturer’s instructions (Invitrogen, CA, USA). To carry out the Real-time PCR, total RNA was reverse-transcribed with RT First strand kit (SABioscience, USA) according to manufacturer’s instructions.
To validate the PCR assay, Real-time PCR was done with cDNA from each condition, and was repeated three times for each gene using SYBRGreen (Roche,) and the primers of Runx2, osteoprotegerin (OPG), Ostx and 18S rRNA (housekeeping gene) were provided by QuantiTectPrimer Assay (Qiagen, USA). Real-time PCR were performed in Step-One system (Applied Biosystems, USA).
Statistical analysis
All statistical analyses were performed using one-way analysis of variance (ANOVA) test, where p values <0.05 were considered statistically significant. All tests have been performed with reference to the control condition.