Effects of Electrical Fields on Neuroblastoma (N2A) Cell

Differentiation: Preliminary Results

Daniel Martin Fernández

, Pablo Pérez García

, María E. Martín

, Paula Daza

Juan Alfonso Serrano-Viseas

, Gloria Huertas

and Alberto Yúfera

Instituto de Microelectrónica de Sevilla (IMSE), Universidad de Sevilla, Av. Americo Vespuccio, S/N Sevilla, Spain

Departamento de Biología Celular, Facultad de Biología, Universidad de Sevilla, Av. Reina Mercedes, S/N, Sevilla, Spain

Keywords: Biomedical Circuits, Electrical Pulse Stimulation (EPS), Microelectrodes, Neuroblastoma (N2A), Stem Cell

Differentiation.

Abstract: This work describes Electrical Stimulations (ES) assays on stem cells. The neuroblastoma (N2A) cell linage

was submitted to several electrical fields to enable and enhance its differentiation toward neurons. Both Direct

Current (DC) and Alternated Current (AC) time dependent electric field protocols were applied to N2A cell

culture under differentiation conditions, obtaining different responses. Control and electrically excited

samples’ number of differentiated cells and neurite lengths were measure after differentiation. Results showed

that DC fields have a strong influence on N2A differentiation since the percentage of differentiated cells and

the neurites lengths were the highest. In addition, a significant alignment of neurites measured with the applied

electrical field has been detected, which demonstrates the high sensitivity of differentiation processes to

electrical field polarity.

1 INTRODUCTION

In the most advanced organisms, each cell has a

specific function, being its morphology and

physiology adapted for greater efficiency carrying out

that function. This process is known as

differentiation. Cellular differentiation, therefore, is

the process by which a cell pauses its division process

and changes its function and phenotype; that is, it

expresses parts of its DNA that were previously

suppressed and vice versa. In our specific case, N2A

cells differentiate into neurons. Undifferentiated N2A

cells are small and round; when differentiation

begins, the cytoplasm extends in one or more

directions, forming threads called neurites, also the

cell flattens (Echalier, 2018).

N2A can be cultivated in suspension or with

substrate. Differentiation occurs either way;

although, in suspension cultures, the morphology

https://orcid.org/0000-0001-7283-7254

https://orcid.org/0000-0002-3204-1726

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https://orcid.org/0000-0001-9881-0148

https://orcid.org/0000-0001-5851-2576

https://orcid.org/0000-0002-1814-6089

remains round and the number of differentiated cells

decreases; these cells, once transferred to a culture

with substrate, reach a normal degree of

differentiation and a flat morphology. Cells cultured

with substrate differentiate with a normal rate and flat

morphology (Figure 1) (Ross, 1975).

Figure 1: N2A cells in the process of differentiation:

Suspension culture (left) and substrate culture (right) (Ross,

1975).

152

Fernández, D., García, P., Martín, M., Daza, P., Serrano-Viseas, J., Huertas, G. and Yúfera, A.

Effects of Electrical Fields on Neuroblastoma (N2A) Cell Differentiation: Preliminary Results.

DOI: 10.5220/0010320101520159

In Proceedings of the 14th International Joint Conference on Biomedical Engineering Systems and Technologies (BIOSTEC 2021) - Volume 1: BIODEVICES, pages 152-159

ISBN: 978-989-758-490-9

An Electric Field (EF) plays, under the adequate

electrostimulation (ES) protocol, a relevant role in the

reorganization of the cell cytoskeleton (McCaig,

2005) (Villanueva, 2019). Normally, cytoskeletal

reorganization is mediated, inter alia, by extracellular

signaling kinase dependent (ERK) pathways. A

signaling pathway is a common process at the cellular

level. It starts from a chemical reactant that initiates a

series of reactions within the cell; this ultimately

produces a result. Signaling pathways are the primary

method most cells use to transmit and translate

chemical signals. The ERK pathway produces

cytoskeletal reorganization and, ultimately, cell

differentiation. One of the steps in this signaling

pathway is dependent on cyclic adenosine

monophosphate (cAMP) (Liu, 2001). Several studies

have demonstrated the efficacy of cAMP (Chang,

1976), or of factors that increase cAMP activity

(Chatterjee, 1992), (Tremblay, 2009), inducing cell

differentiation in N2A, as well as the efficacy of β -

Hydroxy-β-methylbutyrate (HMB). HMB, in turn,

activates the ERK signaling cascade (Salto, 2015).

There are also authors that claim that cyclic AMP acts

similarly, when it is activated by an electric field and

it is activated by chemical factors (Pullar, 2005),

when it comes to migrating keratinocytes. This could

prove that the application of an electric field produces

the activation of cAMP. With this information, it is

possible to link electrostimulation and cell

differentiation. There could be a relationship between

the application of electrostimulation and the ERK

signaling cascade; possibly involving the activation

of cyclic AMP. Therefore, an electric field could use

the ERK signaling pathway to reorganize the

cytoskeleton and ultimately lead to cell

differentiation, by activating cAMP.

The main objective of this work is to promote the

differentiation and growth of neurites in mouse N2A

cells, applying electric fields during the

differentiation process. To achieve that, two

experiments will be carried out with different

electrostimulation protocols, keeping the same cell

incubation protocol. Experiments are planned to

compare the efficacy of both excitation protocols

enabling the differentiation of N2A cells into

neurons. The proposed electrostimulation system

main blocks are shown in Figure 2. The functional

blocks are: the electrodes for cell culture; the circuits

for electrical stimulation, generating the proposed

signals, and the electrical system encapsulation for

insulation inside the incubator. Two electrical signals

(protocols) are applied: DC, with several values, and

squared signals at the same frequency and duty

cycles, modifying its amplitude.

Figure 2: Main blocks in proposed electrostimulation

system.

2 MATERIALS AND METHODS

This section will describe the experimental procedure

followed on electrostimulation assays with N2A.

2.1 N2A Cell Culture

Initially, 25,000 cells were seeded for the first

experiment (DC) and 12,500 cells for the second

(AC), both in 600ml, located on each of the 8 wells of

the plate, that will be explained in the next section.

The DC culture grew for 72 hours and the AC for 96

hours. For sample preparation, DNEM (Dulbecco's

Modified Eagle Medium) was added initially.

Subsequently, they were placed in an incubator at

37ºC with 5% CO2 for 24 hours. This medium was

then removed and serum-free medium (Opti-MEM,

Reduced Serum Medium) was added for the cells to

differentiate, and returned to the incubator for 24

hours more. After that time, electrostimulation was

carried out for 6 hours. Once 18 hours more of

incubation had passed, the experiment was finished

(Figure 3) and the results obtained were evaluated.

Figure 3: AC incubation protocol.

2.2 Electrodes

The choice of electrodes is important. The results will

depend on the material from which they are made of,

Effects of Electrical Fields on Neuroblastoma (N2A) Cell Differentiation: Preliminary Results

153

their geometry, and layout. In many studies, the

culture is made in Petri dishes and, therefore, there is

total freedom in the choice of electrodes and their

configuration. The best electrode arrangement is

calculated then, as well as some way to place them in

that arrangement. In our case, the culture took place

in wells on a plate with electrodes, so all these

drawbacks were eliminated. The chosen plate is

specifically designed for Electrical Cell Impedance

Sensing (ECIS) techniques, being applicable directly

to electrostimulation assays. As advantages: it can be

used with large numbers of cells, it reduces

impedance fluctuations, its electrodes are uniformly

arranged in space, and all wells hold the same

volume. The cells were cultured in wells located in a

commercial plate from Applied Biophysics (Applied

Biophysics, 2020) using the model 8W10E+ from the

ECIS Cultureware line shown in Figure 4.

Figure 4: AB Electrodes (Applied Biophysics, 2020).

Each of the eight wells has two sets of 20 circular

electrodes with a diameter of 250µm. The electrodes

are arranged in an interlocking fingers (interdigit)

configuration, where sets of five electrodes face each

other; one set with the signal and one with the

reference, as it is illustrated in Figure 5.

The electrostimulation signal, therefore, will be

applied from the working to the reference electrodes

and vice versa. It was necessary to calculate the

separation between both electrodes to apply the

electric field with the correct amplitude, in terms of

volts by centimeter [V/cm]. For this end, the distance

tool within Matlab image tools, was employed.

Knowing that the diameter of the electrode is 250 µm,

we could extrapolate the distance between the

electrodes. We calculate the distance between

electrodes, in pixels, as the average of the

measurements taken, and we applied the conversion

factor calculated previously. The result is 0.947mm,

approximately. With this data, we were able to

transform the amplitudes defined in V/cm to specific

voltages for our experimental setup.

Figure 5: Electrode configuration inside a well. There are

20 gold electrodes in parallel (working electrode) vs 20

gold electrodes in parallel (reference electrode). Each

circular electrode has a 250µm diameter. Model 8W10E+.

2.3 Stimulation Circuits. ES Signals

2.3.1 DC ES Signals

In the first experiment, four direct current signals

were used, one of them being ground. The

characteristics of the signals were chosen according

to the bibliography. The main work contributions to

DC electrostimulation of N2A cells and similar are in

Table 1. The chosen amplitude values are consistent

with the values that other authors have used in their

studies, and specifically with this cell type.

Table 1: Summary of the voltage amplitudes reported for

DC electrostimulation.

Cell line Amplitude

(V/cm)

Ref.

N2A 1.1-10 (Jain, 2013)

SH-SY5Y 1.5 (Xiong, 2015)

Xenopus Neurons 0.1 - 10 (Jaffe, 1977)

Considering the values in Table I, and the distance

between electrodes of 1mm, the V/cm values are

calculated for our electrode setup. The final DC

amplitudes in our assay are listed in Table 2.

Table 2: Amplitude selected for the DC assay.

Amplitude 1 2 3 4 (GND)

(mV)

125 250 500 0

(V/cm) 1.25 2.5 5 0

For the electrostimulation circuit, an inverter

amplifier was used, built with an operational

amplifier and two resistances, with its gain defined by

the resistance’s ratio.

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2.3.2 AC ES Signals

In the case of alternating current, more parameters

had to be decided, not only the amplitude, but also the

waveform, frequency and pulse width (Table 3).

Three different signals were applied. To make

comparisons between setups and to facilitate the

assembly of the circuit, it was decided to apply the

same signal with three different amplitudes, as in the

case of direct current. Ours, as well as other possible

proposals are listed in Table 3. The squared signal

proposed in this work is illustrated in Figure 6.

Table 3: ES parameters for AC assays.

Signal Freq

[Hz]

Pulse

width

Amplitu

Ref.

Squa.

Bipha.

100 2.5ms pos

2.5ms neg

125,250,

500

mV/mm

This work

Squa.

Bipha

n.a. 200µs–1ms 100mV-

(Braeken,

2009)

Squa.

Bipha

100 50-200µs 4 -

32µA/cm

(Chang,

2011)

Sin 1,10,50 ---- 1V/cm (Lim, 2013)

Squa. 100 5ms 300mV/m

(Chang,

2016)

Squa. 1 2ms 5V (Xiong,

2015)

Squa. 1,3,5 0.25–10ms 1 –

6V/cm

(Tandon,

2011)

Figure 6: Proposed waveform for the AC signal. Three

amplitudes are considered. Frequency is 100Hz, and the

selected amplitudes are 100mV, 200mV and 425mV.

The timer circuit generates square pulses of

frequency 100Hz and pulse width 2.5ms; in addition

to a circuit that varied its amplitude. It was decided to

use assemblies based on the 555 integrated circuit

(IC) for the timer and differential amplifiers based

opamps for the amplitude variation. The timing

circuit needed a square pulse generator and two

circuits to regulate the width of the pulse, one for the

positive pulse and one for the negative pulse; both

pulses had to happen one immediately after the other.

With two IC 555s in monostable mode we would be

able to do that. Later we would use a differential

amplifier to invert the negative pulse and combine it

with the positive one, in addition to regulating the

amplitude of both. In this from, we would have the

complete circuit to generate one of the three AC

signals. Each, DC and AC signal is applied to two

wells. Figure 7 displays the plate layout of the DC

signals applied. AC is similar.

Figure 7: Proposed DC signals layout. Each DC signal is

applied to two wells. Wells 7 and 8 are the controls.

2.4 System Encapsulation

The whole electrostimulation system, circuits plus

electrodes, was put inside an incubator during

experiments. Cells in the plate were set over the cage,

while the circuits were placed inside it, to insulate

them from the incubators conditions (Figure 8).

Figure 8: Cage system: electrode wells are put over the cage

and the electrostimulation circuits inside.

2.5 Cell Measurements

Several measurements were performed, to evaluate

the performance assay.

 Number of cells

 Number of differentiated cells

 Number of neurites

 Neurite lengths

 Neurite polarization

Effects of Electrical Fields on Neuroblastoma (N2A) Cell Differentiation: Preliminary Results

155

3 EXPERIMENTAL RESULTS

The experimentation process consists of several parts:

the first is the culture of N2A cells, then

electrostimulation is carried out and, finally,

photographs are taken under the microscope of each

electrode. This procedure lasted three days, during

which the direct current and alternating current

experiments were carried out in an overlapping

manner. The cultures had been prepared previously,

according to the procedure detailed in the Material

and Methods section.

After the stimulation process, an optical

microscope and a Leica family camera were used to

take the snapshots, communicating to a computer

using the LAS EZ software (Leica, 2020). Using this

procedure, we were able to take pictures of each

electrode, whilst adjusting parameters such as

exposition, gain, gamma, etc. to highlight neurites on

differentiated cells. Only images that provided

information were taken, that is, electrodes with

multiple layers of cells or damaged electrodes would

not be photographed. The photographs were stored in

folders according to their well.

From the pictures taken, the several paramezters

proposed to characterize the experiment were measure:

number of cells, number of differentiated cells, number

of neurites, length of neurites and neurite polarization.

To distinguish between differentiated and

undifferentiated cells, a criterion based on morphology

was followed: cells with extensions (neurites) are

differentiated cells (Figure 9). On the other hand, it was

necessary to distinguish between living and dead cells.

Dead cells are normally found in “spongy”-looking

aggregates (Figure 10).

With clear criteria, the cells were counted first. A

Matlab script was programmed for this task (Figure

11). The script opens the images found in the folder

(corresponding to a well) one by one, that is, it opens

one and, when given the order, closes it and opens the

next one. Once an image is opened, the view is

zoomed in, in order to visualize the electrode better;

click where a cell is to count it, and when all have

been counted, proceed with the differentiated cells.

After that, the image will be closed and the next one

will be opened. Once finished, the script will generate

a .csv file with the number of total cells in each image,

another with the differentiated cells and another with

the

coordinates of the points where the differentiated

cells are (Figure 12).

Figure 9: Example: differentiated and non-differentiated

cell.

Figure 10: Example: dead cells.

Figure 11: Cell counting Matlab script block diagram.

Figure 12: Matlab display when cells are being counted.

BIODEVICES 2021 - 14th International Conference on Biomedical Electronics and Devices

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The next step was to count and measure the

neurites of the differentiated cells. For this, another

script has been developed in Matlab (Figure 13). This

script opens the images in a similar way to the

previous one, in this case the coordinates of the points

calculated in the last step are read in order to mark the

differentiated cells with a red cross. In turn, a ruler

with two moving points is shown. The ruler must be

placed at the beginning and end of the neurite and

when given the order, the length of the neurite, its

starting point and its end are recorded. When the last

neurite of an image is measured, the order is given

and the image changes to the next. Subsequently, two

.csv files are generated, one with the distances of the

neurites per electrode and the other with the starting

and ending points of the neurites (Figure 14).

Figure 13: Neurite length measuring Matlab script block

diagram.

Figure 14: Example measuring the neurite length with

Matlab.

As additional information, the orientation of the

neurites has also been obtained, that is, the angle they

form in the image, respect a given coordinate origin.

This was possible thanks to the nature of the

information extraction process from the images. By

knowing the start and end points of the neurite (Figure

15), it was possible to obtain the angle it forms, using

the inverse of the tangent; as shown in Equation (1).

This approximation, although not perfect, allows us

to recognize some type of pattern in the angles of the

neurites, if it exists.

tan







(1)

Where y = (y1-y2), x = (x1-x2) and ϴ is the angle

that the neurite forms (Figure 15).

An extensive analysis was performed to obtain the

expected measurements from the experiment. In the

following are summarized some of the more relevant,

illustrating the sensitivity of N2A cells to DC and AC

electric fields. Figures 16 and 17 show the mean value

of the number of differentiated cells in DC and AC

conditions, for the several values of amplitudes

applied.

Figure 15: Parameters used in the calculus of the neurite

angle. The center of the electrode is taken as reference axis.

Figure 16: Number of differentiate cells vs DC level.

Figure 17: Number of differentiate cells vs AC level.

The percentage of differentiate cell, with respect

to the total number of counted cells is displayed in

Figures 18 and 19, for DC and AC conditions

respectively. The efficiency on differentiation seems

to be higher for DC electrical fields, being highest at

125mV (1.25V/cm).

Effects of Electrical Fields on Neuroblastoma (N2A) Cell Differentiation: Preliminary Results

157

Figure 18: Differentiation coefficient for DC.

Figure 19: Differentiation coefficient for AC.

Finally, the phase angle tested over the

photographs for the two assays (DC and AC) are

displayed on Figures 20 and 21, together with their

respective controls. In both cases (DC and AC), it can

be appreciated a high polarization around (25º- 40º)

and (130º-150º) intervals, validating the fact that one

of the neurite answers to electrical field is its biasing

along the applied ES electric field (Patel, 1982).

The presented results show how N2A cells can be

electro-stimulated using lateral electric fields defined

from the bottom electrodes, and how their neurites

were biased along the direction defined by the electric

fields applied.

Figure 20: Neurites polarization (DC): Number of neurites

measure for each angle between 0º and 180º. The red line

represents an approximated fitting curve.

Figure 21: Neurites polarization (AC): Number of neurites

measure for each angle between 0º and 180º. The red line

represents an approximated fitting curve.

4 CONCLUSIONS

An experimental procedure and the results of

electrostimulation assays on neuroblastoma cells, in

the process of differentiation towards neurons, have

been presented. Two types of signals were utilized on

the experiments: the first one consisting of a direct

current voltage signal and the second one a biphasic

square voltage signals at 100 Hz, both with three

different amplitudes. The results obtained

demonstrate that, for the selected amplitude values,

the differentiation process is more sensitive to DC

than to AC signals, with similar amplitudes. This

could be due to the biphasic nature of the AC signal

applied. A biphasic signal polarizes cells in the two

directions of the electrical field, whilst a DC signal

only polarizes towards the cathode. Another factor to

keep in mind is the rms value of the AC, this being,

approximately, 0.71 times the amplitude of the signal.

Therefore, the AC signals applied have less effective

amplitude than the DC ones. This could also explain

the tendency seen in Figure 19, where the coefficient

increases as the amplitude does. The optimum lateral

ES field values, 100mV/mm, is coincident with the

reported by other authors (Jain, 2013), using vertical

electrical fields over its setup. This result supported

the validity of the assays. A remarkable alignment of

the neurites was observed on the maximum electric

field directions expected (30º – 150º), which means a

polarization response of N2A cells to external

stimulus, as stated in (Patel, 1982). More in-depth

experiments must be done in AC ES, for wider

frequency ranges and duty cycles, to fully

characterize ES signals effects on N2A

differentiation. In addition, higher/smaller ES periods

of 6h should be tested, for the same reason. Finally,

as future work, it is programmed to incorporated

some biomarkers, as cAMP, in parallel with

electrostimulation, for biological validation of the

assays.

BIODEVICES 2021 - 14th International Conference on Biomedical Electronics and Devices

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ACKNOWLEDGEMENTS

This work was supported partially by the Spanish

founded Project: System for measure and

electrostimulation applied to differentiation and

motility of cells. P18-FR-2308. PAIDI 2020 Project.

Funded by: Junta de Andalucía, Consejería de

Economía y Conocimiento, and also by the Spanish

Government’s Ministerio de Ciencia, Innovación y

Universidades, Plan Estatal 2017-2020 Retos-

Proyectos I+D+I: Real time monitoring of

hemodynamic variables using intelligent stents

(iSTENT) with capacitive sensors and bioimpedance,

under the project RTI2018-093512-B-C21, co-

financed with FEDER.

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