Bioelectromagnetics 37:264^278 (2016)

Long-Term Electromagnetic Exposure of

Developing Neuronal Networks: A Flexible
Experimental Setup
Stefan Oster,1 AndreasW. Daus,1 Christian Erbes,1 Michael Goldhammer,1,2
Ulrich Bochtler,2 and ChristianeThielemann1*
1
BioMEMS Lab, Aschaffenburg University of Applied Sciences, Aschaffenburg, Germany
2
Laboratory for Electromagnetic Compatibility (EMC), Aschaffenburg University of
Applied Sciences, Aschaffenburg, Germany
Neuronal networks in vitro are considered one of the most promising targets of research to assess
potential electromagnetic field induced effects on neuronal functionality. A few exposure studies
revealed there is currently no evidence of any adverse health effects caused by weak
electromagnetic fields. Nevertheless, some published results are inconsistent. Particularly, doubts
have been raised regarding possible athermal biological effects in the young brain during neuronal
development. Therefore, we developed and characterized a flexible experimental setup based on a
transverse electromagnetic waveguide, allowing controlled, reproducible exposure of developing
neuronal networks in vitro. Measurement of S-parameters confirmed very good performance of the
Stripline in the band of 800–1000 MHz. Simulations suggested a flexible positioning of cell culture
dishes throughout a large exposure area, as specific absorption rate values were quite independent
of their position (361.7
11.4 mW/kg) at 1 W, 900 MHz. During exposure, thermal drift inside
cellular medium did not exceed 0.1 K. Embryonic rat cortical neurons were cultivated on
microelectrode array chips to non-invasively assess electrophysiological properties of electrogenic
networks. Measurements were taken for several weeks, which attest to the experimental setup being
a reliable system for long-term studies on developing neuronal tissue. Bioelectromagnetics.
37:264–278, 2016. © 2016 Wiley Periodicals, Inc.

Key words: RF exposure setup; mobile communication frequencies; SAR dosimetry;

microelectrode array; electrophysiology

INTRODUCTION
It is well known that ionizing radiation can
induce serious damage to living cells. A general
indicator for likely effects and their impact is total
energy deposition and absorbed dose of tissue. Major
bio-damage within tissue is caused by radicals and
secondary electrons related to high energy deposition.
Radiobiological implications like chromosomal aber-
rations, gene mutation, or genomic instability of
DNA, all of which may result in cancer, are likely to
emerge. In contrast, electromagnetic fields (EMF) at
radiofrequency (RF) according to mobile communica-
tion standards are clearly assigned to non-ionizing
radiation. They only introduce weak energy levels
into living tissue and are considered incapable of
inducing DNA damage or influencing physiological
functions [Adey, 1993]. Nevertheless, interaction of
RF-EMFs and the human body is of serious concern,
indicated by a remarkable number of recent publica-
tions addressing this issue. In a cohort study, Poulsen
et al. [2013] evaluated possible correlation between
mobile phone use and risk of cancer and found little
evidence of increased cancer risk among mobile
phone users. Limitations of cohort studies are obvi-
ously the long period of time until data is generated,
nevertheless epidemiological research is ongoing,
which is approved by the cohort study of mobile
phone use and health (COSMOS), a recently started
Grant sponsor: Bundesministerium f€ur Bildung und Forschung
(BMBF, engl. Federal Ministry of Education and Research);
grant numbers: 17N2208 PT-AIF, 03FH026I3.
Conflicts of interest: None.
*Correspondence to: Christiane Thielemann, Wuerzburger Straße
45, D-63743 Aschaffenburg, Germany.
E-mail: christiane.thielemann@h-ab.de
Received for review 17 July 2014; Accepted 14 March 2016
DOI: 10.1002/bem.21974
Published online in Wiley Online Library
(wileyonlinelibrary.com).

2016 Wiley Periodicals, Inc.


international study monitoring mobile phone use and
possible human health effects [COSMOS, 2013].
Epidemiologic studies have been supported by
in vivo and in vitro experiments that benefit from
more specific exposure conditions. They are widely
employed if an understanding of basic mechanisms is
foregrounded. Also, long-term assays can be executed
for up to several weeks within appropriate exposure
setups. Kumlin et al. [2007] exposed male Wistar rats
with pulsed Global System for Mobile Communica-
tions (GSM) phone radiation by radial transmission
lines. Whole-body averaged specific absorption rate
(SAR) related to RF input power at 900 MHz was
applied up to 3 W/kg and rats were subjected to
radiation for 10 h per week over a period of 5 weeks.
At all times, a point of criticism is movement of rats
in their cages. Due to rats’ motion, SAR could alter
temporarily from 0.7 to 12 W/kg. In vitro exposure
experiments help to overcome this problem. They are
a favorable approach to studying possible effects on
a cellular level, regardless of whether cell cultures
are seeded as monolayers [Schuderer et al., 2004a;
O’Connor et al., 2010; Moretti et al., 2013], in
suspension [Sch€ onborn et al., 2000; Schuderer et al.,
2004a] or as three-dimensional (3-D) reaggregates
[Daus et al., 2011].
A focus of research is possible impact of mobile
phone frequencies on the human brain. Although there
is only little evidence of major health risks for the
brain, a few published results are inconsistent and
their findings have led to contentious interpretations.
For instance, Zeni et al. [2012] asserted that cellular
endpoints of neuron-like cells were not affected by
RF radiation, whereas Buttiglione et al. [2007]
opposed the suggestion that RF radiation impairs cell
cycle progression of neuroblasts. Tattersall et al.
[2001] found that low intensity RF-EMF affected
neuronal activity in rat hippocampal slices; exposure
to continuous wave RF fields led to potentiation of the
amplitude of the population spike by up to 20%
in Cornu Ammonis (CA) 1 or reduced or abolished
epileptiform bursting in 36% of tested slices. Other in
vitro studies revealed acute electrophysiological
effects like modulated excitability or cellular signals,
which synchronized with modulation frequency
[Caddemi et al., 1986; Tamburello et al., 1991;
Abbate et al., 1996]. In this field, also a number of
human electroencephalogram (EEG) studies are avail-
able. The group of Achermann disclosed signal-
depended changes in human EEG, as pulse-modulated
RF-fields affected the EEG compared to continuous
wave signals [Huber et al., 2002; Regel et al., 2007].
Findings of Schmid et al. [2012] supported those
studies as they discovered pulse-modulated effects
Exposure of Developing Neuronal Networks 265
and found short-term alterations on sleep EEG of
humans. But in contrast, a recent study showed that
the alpha band activity of healthy volunteers was
altered by either continuous wave or pulsed modu-
lated RF EMFs [Perentos et al., 2013]. With equal
exposure conditions as in Schmid’s study [2012],
Loughran et al. [2013] were not able to demonstrate
exposure-related effects on cognition and EEG of
adolescents. Additionally, they did not find enhanced
sensitivity of adolescents to RF-EMFs. Scoring epide-
miological data, Divan et al. [2008] showed a possible
correlation between behavioral abnormalities of chil-
dren and cell phone exposure. Adolescents who were
prenatally or postnatally subjected to EMFs were
more likely to show hyperactivity or be prone to
headache-related symptoms [Sudan et al., 2012].
To provide reliable experimental conditions for
in vitro studies, various exposure setups have been
developed. Paffi et al. [2010a] reviewed such systems
in terms of SAR homogeneity, number of sample
holders, and operating frequencies. Schuderer et al.
[2004a] developed a rectangular waveguide setup for
exposure of cell monolayers and cells in suspension.
Exposure setup was designed for up to eight mono-
layer sample holders (35 mm) exposed to local SAR
level of 1.3 (W/kg)/W in the H-field maximum. The
resonant system was optimized accordingly to GSM
900 at 900 MHz. As this setup was based on standing
waves, a sole frequency and accurate placement of
sample holders is essential for reproducible field
parameters. These conditions limit its application in
terms of frequency variability and usability. An open
RF exposure setup based on transverse electromag-
netic (TEM) waves was presented by Merla et al.
[2011] and Moretti et al. [2013]. To avoid external
disturbances in the open structured setup, the system
is located in an electromagnetic compatibility test
chamber. Broad frequency range (900 and 2450 MHz
are presented) and good accessibility of samples are
advantages of this approach. Koester et al. [2007]
presented a rectangular wave-guide operating in the
propagating and standing wave mode, designed for
high frequency signals as continuous wave or as
generic signals known from Universal Mobile Tele-
communication System (UMTS). A resonant exposure
setup developed from a small planar antenna structure,
the so-called wire patch cell, has been introduced by
Laval et al. [2000]. Two 15  15 cm2 metallic plates
separated by four 2.9 cm high probes furnish space for
eight 35 mm standard Petri dishes. The setup is
coaxially fed with the RF signal and establishes a
highly uniform field inside the antenna cavity, which
is optimized for 900 MHz. An advantage of the
system is its high electric field strength (1300 Vm1)
Bioelectromagnetics
266 Oster et al.
at an input power level of 1 W. However, an obvious
disadvantage is narrow bandwidth, caused by limita-
tions of a resonant field.
The German Federal Office for Radiation
Protection states in the summary of the large German
Mobile Telecommunication Research Program that
two questions could not yet be answered satisfactorily:
questions about health risks from long-term use of
mobile telephones and whether children could be
more sensitive to high frequency EMFs than adults
[GCRP, 2011]. We addressed these open issues by
designing an in vitro experiment that allowed for
long-term RF-EMF exposure of developing embry-
onic cortical networks in vitro. The overall challenge
here was full control of exposure-related temperature
increase—certainly a disadvantage of the in vitro
situation—and exact dosimetry. Electrophysiological
recordings provide functional parameters that may
help interpret possible impacts on mammalian neuro-
nal communication. Being aware of the fact that in
vitro experiments with cultures of embryonic neurons
are not feasible to model full complexity of a
developing young brain, we focused in this project
on possible impact of RF-EMFs on developing
electrophysiological activity and connectivity of corti-
cal networks during the first weeks in vitro.
For this purpose, we established and character-
ized a flexible experimental setup based on an open
TEM-cell as defined in Weston [2001], ready for
long-term experiments with biological samples. Up to
eight 35 mm Petri dishes or six Microelectrode Array
(MEA) chips can be placed in the exposure domain,
providing a uniform EM field. The latter are favored
tools for assessment of electrophysiological properties
of cellular networks in a non-invasive way and were
chosen in this study to evaluate possible effects of
EMF exposure on developing and mature activity of
neuronal networks. MEAs are a common approach to
achieve a basic understanding of electrophysiological
properties of neuronal networks [Egert et al., 2002;
Stett et al., 2003; Daus et al., 2012]. A flexible
frequency range can be provided by the system,
covering mobile communication standards as there
are Terrestrial Trunked Radio (TETRA, 400 MHz) or
GSM (900 and 1800 MHz). Numerical simulations
and experimental field measurements were performed
to guarantee accurate dosimetry. For long-term
experiments, cells were exposed inside an incubator
equipped with absorber materials to minimize un-
avoidable reflections of incubator walls.
For the first time, we showed here that long-
term exposure of neuronal networks during the phase
of developing electrophysiological activity and
connectivity including, for example, axon growth and
Bioelectromagnetics
synaptogenesis, is possible at highly controlled con-
ditions. A number of experiments were performed
with embryonic cortical networks of rats exposed to
EMF for up to 60 days in vitro (div) and cellular
signals were analyzed in terms of network activity
and communication patterns.
MATERIALS AND METHODS
Cell Culture
Cryopreserved rat cortical neurons from embry-
onic rats (E18 or E19) were purchased from Lonza,
Basel, Switzerland. Cell cultivation followed a modi-
fied protocol of Otto et al. [2003]. Briefly, vials
containing 4  106 cells were stored in liquid nitrogen
at 196 8C. After thawing, cells were diluted drop-
wise with pre-warmed serum-free cell culture medium
and seeded at density of 100–300 cells/mm2 onto
Poly-D-Lysine- and Laminin-coated (Sigma–Aldrich,
Taufkirchen, Germany) MEAs. Twice a week, half of
the medium was replaced with fresh, pre-warmed
medium.
MEA Recording
Neuronal signals were recorded extracellularly
at a sampling rate of 10 kHz and stored for offline
analysis with a custom-made software tool [Bestel
et al., 2012; Nick et al., 2013] covering signal
detection as well as spike-rate and burst-rate determi-
nation. Signal acquisition was conducted daily starting
from 14 div until 62 div. Recordings were performed
outside exposure setup. To maintain sample tempera-
ture at 37 8C, a temperature-controller (Multi Channel
Systems, Reutlingen, Germany) was employed during
recordings.
Exposure Setup
Exposure setup used in this study is based on an
open TEM-cell [Weston, 2001]. In detail, the asym-
metric stripline antenna is defined by two stacked,
metallic plates (septum and ground plate), whereas
the septum has two bent, tapered ends soldered to the
inner conductor of an RF connector (Figs. 1 and 2).
Characteristic impedance of the system was calculated
following Hammerstadt’s equation [Janssen, 1992]
and determined to 50 V for the given width-to-height
ratio of septum and ground plate. As a result, a widely
uniform EM-field is generated between upper and
ground plate. For simplification, we refer to our
exposure setup in the following text as Stripline.
Although the setup is generally not based on
resonances, a broad flexible frequency range can be
applied at varying input powers without changes in
 Exposure of Developing Neuronal Networks 267

Fig. 1. Top and side view of Stripline geometry.

geometry. However, constraints due to parasitic
effects, like standing waves, can limit the application
and have to be kept in mind. Wave signals can either
be continuous or pulsed with respect to mobile
communication standards. Interplate field strength
E can easily be estimated by
pffiffiffiffiffiffiffiffiffiffiffiffi
PZ
E¼
h
where P is Stripline input power, Z is Stripline’s
characteristic impedance, and h is interplate distance.
Stable environmental conditions are a prerequi-
site for long-term exposure of in vitro cell cultures.
Consequently, the Stripline was integrated into a
CO2-incubator (BC 170, SalvisLab, Rotkreuz,
Switzerland) providing optimal temperature and gas
composition for the neuronal cell culture. However,
undesired reflections from the inner metallic incubator
walls can occur during the exposition procedure. As a
countermeasure, we implemented absorber structures
(C-RAM FLX-2.0, emc-technik, Stuttgart, Germany)
on the inner walls of the incubator.
ð1Þ
RESULTS
Computational Simulation and Measurement of
Field Distribution in Empty Stripline
COMSOL Multiphysics (RF Module, version
3.5a, Comsol Multiphysics, Goettingen, Germany)
was used to rebuild our exposure setup as a numerical

Fig. 2. For exposure experiments, we defined (a) an utilizable exposure domain provided
with several Petri dishes, and (b) an exact exposure domain for more delicate experiments.
The feeding and matching transitions of septum and inner conductor of the coaxial connec-
tions are enlarged. See also Figure 1 for exact geometry.
Bioelectromagnetics
268 Oster et al.
equivalent, based on partial differential equations
(PDE), and to calculate field distribution using finite
element method (FEM). COMSOL allowed analysis
of the entire modeling domain for the electric field
vector following the PDE
   
1 s
r r  E  k 0 er  j
2
E¼0
mr ve0
where mr is relative permeability, k0 is wave number
in free space, ɛr is relative permittivity, s is electric
conductivity, ɛ0 is permittivity of vacuum, and v is
angular frequency.
The numerical model of the empty Stripline is a
geometrically appropriate imitation of the real expo-
sure setup. Metallic structures were considered as
Perfect Electric Conductors (PEC) and the system’s
impedance was assumed to be exactly 50 V. Incubator
walls were simulated as PEC too, whereas the surface
of the absorber structure was integrated as scattering
boundary, which absorbed incident waves. er and mr
were set by the default values of air to 1 and PECs are
considered as lossless, so resistivity r is zero every-
where. Simulation of electrical field distribution was
realized with approximately 4 mesh elements/cm3. As
the Stripline allows a broad frequency range to be
applied, simulations were made in the range from DC
to 2200 MHz graded in steps of 100 MHz (Fig. 3, see
also Fig. 5 for detailed results with error bars). With
regard to the subsequent cell culture experiments, we
defined two distinct subareas inside the Stripline. One
is the so-called “utilizable exposure domain,” which
covers a large area of 144  104 mm2, sufficient for
several standard Petri dishes (Fig. 2a). In a second
Fig. 3. Simulation results of mean electric field strength at the central plane of the empty
Stripline averaged over the utilizable (dashed line, triangles) and exact (solid line, circles)
exposure domain as a function of frequency. Markers indicate frequencies currently used in
wireless communication.
Bioelectromagnetics
“exact exposure domain,” which covers a smaller area
of 54  54 mm2, single MEA chip exposures will be
performed (Fig. 2b).
For 900 MHz and 1 W, the numerically derived
mean electrical field strength for “utilizable” and
“exact” exposure domain was almost equal to the
value of 282.8 Vm1, calculated with Equation 1. For
ð2Þ
frequencies above 1700 MHz, results indicate a de-
crease in mean field strength compared to low
frequencies with some deviation between the two
exposure domains (Fig. 3). In general, lower frequen-
cies featured excellent field homogeneity, while
uniformity diminished with increasing frequency.
Table 1 gives a concise overview concerning frequen-
cies currently used in wireless communication. Sum-
marizing, we found that “utilizable exposure domain”
allows for moderate to low field strength deviation,
while field homogeneity in the “exact exposure
domain” was very low and suitable for more delicate
studies.
To quantify the system’s matching and cutoff
frequency, the reflection coefficient S11 and the
transmission coefficient S21 were measured with a
network analyzer (8753 ES, Hewlett Packard, Palo
Alto, CA) at frequencies between 800 and 1000 MHz.
Inside the incubator, we found a well-balanced system
as the maximal return loss was 26 mW (16 dB), for
an input power of 1 W. With the front door open,
corresponding to the situation for E-field probe
measurements, the S11 reflection coefficient generally
decreased as the return loss was at most 14 mW
(18.5 dB); see Figure 4. Note that for relevant
measurement frequency of 900 MHz the situation is
especially good as the S11 coefficients are very close
 Exposure of Developing Neuronal Networks 269

TABLE 1. Numerical Results for the Electric Field Strength (Averaged) and Inhomogeneity in the Empty Stripline at
Common Wireless Communication Frequencies

Utilizable exposure domain Exact exposure domain

Frequency/MHz Electric field/V/m Inhomogeneity/dB Electric field/V/m Inhomogeneity/dB

400 (TETRA) 271.4
0.33 275.6
0.12
900 (GSM) 266.9
0.16 270.5
0.03
1800 (GSM) 250.0
0.78 236.0
0.64
2000 (UMTS) 221.3
1.01 180.1
0.85

(20 dB). Measurement of the coefficient S21 at
900 MHz (Fig. 4) revealed transmission losses of
1.5 dB in the empty Stripline and 1.3 dB with the
front door open. We conclude that for field measure-
ments (as described below) with the incubator door
unavoidably open, conditions in the Stripline are only
slightly modified as compared to the door closed.
Therefore experimentally and simulated results at
900 MHz can be promptly compared. Furthermore,
we extended the frequency range and investigated
S-parameters from DC to 2200 MHz. Not quite
unexpected, we found for numeric (data not shown) as
well as experimental results at frequencies above
1.0 GHz parasitic effects, like standing waves presum-
ably caused by the incubator walls and the Stripline.
Therefore, we claim that frequencies between 1.0 and
1.7 GHz are only applicable with some limitations as
the input reflection coefficient S11 was not more than
10 dB and the transmission coefficient S21 in the
range of 3 dB. The cutoff frequency was measured
to be 1.7 GHz.
Finally for the measurement of field distribution,
a signal generator (SML03, Rohde & Schwartz,
Munich, Germany) supplied electromagnetic power,
which was amplified (ZHL-10W-2Gþ, Mini-Circuits,
Camberley, England), continuously monitored (R&S
NRT, Rohde & Schwartz) in terms of power and
SWR level and fed into the Stripline via a RF
connector (1-1337418-0, Tyco Electronics, Schaff-
hausen, Switzerland). At the end of the Stripline,
the signal was decoupled by an identical connector
and matched, inside the incubator, with a coaxial
cable connected to a 50 V termination impedance
(MOD 374BNF, Globes Elektronik, Heilbronn,
Germany). The Stripline was integrated into a CO2-
incubator. Reflections from inner metallic incubator
walls, caused by the open waveguide structure, are
minimized by plane silicon rubber absorber patterns
fixed on a slide-in shelf above the Stripline and at
the front door.
Experimental field measurements were per-
formed with the Dosimetric Assessment System
(DASY). DASY is a six-axis robot-based (RX 90
robot, St€aubli, Horgen, Switzerland) scanning system,
equipped with high sensitive E-field probes
(ET3DV4, Schmid & Partner Engineering, Zurich,
Switzerland or EFS-105, enprobe, Berlin, Germany),
which is commonly used for SAR-measurements of

Fig. 4. Measurement of reflection coefficient S11 and transmission coefficient S21 of the Stri-
pline as a function of frequency. S-parameters were determined for the empty Stripline placed
inside incubator.
Bioelectromagnetics
270 Oster et al.
cellular phones and other wireless communication
devices [EN 62209, 2006]. The probes allow for
E-field determination with
0.3 dB deviation from
isotropy and a resolution of 10 mV/m at 900 MHz,
with six DoF, the robot can move toward any random
point in a region of interest with a positional deviation
of
20 mm. Overall measurement uncertainty was
estimated to be 14% and hence in good agreement
with literature [Paffi et al., 2012]. Measurements were
performed inside the incubator, unavoidably with
open door. We defined discrete measurement posi-
tions in steps of 5 mm across the “utilizable exposure
domain.” As we analyzed the frequency band
GSM 900, our emphasis was placed on the field
distribution in a nearby frequency range of 840–
920 MHz, with 20 MHz-steps (Fig. 5). Average
field strength measured at an input power of
1 W for a continuous wave signal was 244
16 Vm1
at 880 MHz, 235
17 Vm1 at 900 MHz, and 257

11 Vm1 at 920 MHz. Field strength inside the
incubator but outside the Stripline was also recorded
to study conditions for sham experiments. An attenua-
tion of 25 dB was found above and 30 dB below
the Stripline setup.
Comparison of Simulated and Measured Field
Distribution in Empty Stripline
A detailed comparison of computational simula-
tions and experimental field measurements of the
uplink and downlink band of GSM 900 is shown for
the empty Stripline in Figure 5. Mean and standard
deviation of the E-field were calculated for the
“utilizable exposure domain” at the central plane of
300
250
electric field / V/m
200
150
100
simulation
50 probe measurement
0
820 840 860 880 900 920 940
frequency / MHz
Fig. 5. Mean of the simulated (triangles) and measured
(circles) electrical field strength is shown as a function of the
uplink and downlink band frequencies of GSM 900. Mean and
standard deviation of the E-field were calculated for the utiliz-
able exposure domain at the central plane of the empty
Stripline. A continuous wave signal of 1W was applied for sim-
ulations and measurements.
Bioelectromagnetics
the Stripline. Computational analysis (triangles)
revealed as expected low dependency of the simulated
mean electric field on the frequency. A maximal
variance of only 0.3 dB was calculated for the given
frequency range. In contrast, experimental results
(circles) varied stronger with frequency. Their mean
electric field values strayed by a factor of 0.8 dB
(E900 MHz: 235
17 Vm1, E920 MHz: 257
11 Vm1)
in the given frequency range. More importantly, there
is a difference of only 1.1
0.6 dB at 900 MHz
between averaged E-field strength of numeric and
experimental results. Summarizing, results for the
empty Stripline support the assumption of good but
not excellent consistency between simulated and
experimental findings, so that reliable conclusions can
be drawn from simulations when restrictions are kept
in mind.
Computational Simulations and Measurement
of SAR in Cell Culture Dishes
Exposure conditions for cell culture experiments
were characterized for two kinds of cell culture
dishes: coverslips (H873.2, Carl Roth, Karlsruhe,
Germany) placed in common Petri dishes (XH88.1,
Carl Roth, Karlsruhe, Germany) and MEA chips
(60MEA200/30iR-Ti-gr, Multi Channel Systems,
Reutlingen, Germany). Coverslips are easy to handle
and widely used for growing microorganisms or cell
cultures in vitro. MEAs incorporate a number of
titanium nitride electrodes in a glass substrate and
allow extracellular recordings of electrogenic tissue
[Taketani and Baudry, 2006]. MEAs used provided a
glass chamber for cell culture medium with inner
diameter of 20 mm containing recording electrodes
(Fig. 6a). In the center of the MEA chip, there was a
substrate-embedded 8  8 electrode array (Fig. 6b) of
60 electrodes (30 mm in diameter, 1 mm in height,
interspace of 200 mm). We refer to this 4 mm2 area as
the “recording domain” (Fig. 6a and b). A numerical
equivalent of a coverslip (20  20 mm2) in a solution-
filled (Fig. 7a) Petri dish (35  10 mm2) was aligned
in the center of the Stripline’s ground plate (“exact
exposure domain”). Geometric dimensions and
material parameters of coverslip and Petri dish, as
well as dielectric properties of cellular medium
960
are summarized in Table 2. Furthermore, we imple-
mented a model simulating eight coverslip-equipped
Petri dishes, as described above, located in the
“utilizable exposure domain,” see Figure 2a. To
consider possible medium losses due to evaporation,
we set up three scenarios with different medium
volumes, respectively level heights. We titled these
conditions “replaced,” “evaporation loss,” and “re-
placement needed,” reflecting the situation instantly
 Exposure of Developing Neuronal Networks 271

Fig. 6. Microelectrode array for non-invasive extracellular recordings. Shown is the top view
(a) of the MEA chip, the enlarged recording domain (b) with layout of microelectrode array,
and a cross-section (c) of the MEA chip. Referring to Egert et al. [1998], aspect ratio (1:30) of
metal electrodes is given by the TiN-layer thickness of 1 mm and the electrodes diameter of
30 mm.

after medium renewal, during ongoing cultivation, and
when medium replacement is needed to guarantee
survival of cells (Fig. 7 and Table 3). Simulation
results were achieved with an extremely fine mesh,
corresponding to 3 mesh elements/mm3 in the medium
of each Petri dish.
Additionally, a MEA chip placed in the center of
the ground plate (“exact exposure domain”) was
modeled (Fig. 8a). Its material properties and dimen-
sions [Multi Channel Systems MCS, 2016] and proper-
ties of the cell culture medium are summarized in
Table 2. As it was possible to place six MEA chips in
the “utilizable exposure domain” we set up a model for
that condition as well (Fig. 8b). Simulation results were
achieved with an extremely fine mesh, corresponding
to approximately 4 mesh elements/mm3 in the medium
of each MEA chip.
To improve model accuracy of the MEA chip
including micro-sized electrodes, we employed a two-
step simulation strategy allowing for more detailed
computation. In the first step, we simulated the
incubator including the Stripline with the MEA chip

Fig. 7. Vertical cross-sections (a-c) through the center of a Petri dish. Shown is effect of the
medium volume/height on SAR values (for quantitative filling levels compare Table 3). (d) Hori-
zontal cross-section in 1 mm height above the coverslip bottom. Shown is SAR distribution
(medium level: replaced) at input power of 1W and a frequency of 900 MHz.
Bioelectromagnetics
272 Oster et al.

TABLE 2. Dimensions and Material Parameters Employed in Simulations at 900 MHz Frequency and 1 W Input Power

Item Detail l/mm w/mm h/mm ID/mm OD/mm er s/S/m r/kg/m3

Petri dish Dish 10 35 36 4.2
Lid 6 36.5 37.5 4.2
Coverslip 20 20 0.16 4.6
MEA Substrate 49 49 1 4.2
Container 6 20 24 4.2
Electrode 0.001 0.03 1 58  106
Cell culture medium 71 2.1 1000
l, length; w, width; h, height; ID, inner diameter; OD, outer diameter; er, relative permittivity; s, electric conductivity; r, mass density.

situated in the “exact exposure domain.” The level of
cell culture medium was in the state “replaced.” Based
on the SAR value calculated for the first model, we
set up a second model, placing more emphasis on
delicate electrodes in the “recording domain”
(Fig. 9e and f). A subarea—slightly larger than the
“recording domain” of the entire MEA chip—was
implemented enclosing the subtle microelectrode
array. An array of 60 metallic electrodes with the
realistic electrode interspace of 200 mm was inte-
grated into the MEA chip’s substrate model. Meshing
of electrodes was done with about 3 mesh elements/
mm3. Metallic connection lines were also modeled
and simulated with a “floating” potential. For better
clarity, connection lines were not depicted in
Figure 6b. Their impact on the overall result was
much smaller than field distortions due to electrodes
and therefore neglected.
For all simulation hereafter, SAR values were
calculated for 1 Watt input power and 900 MHz
operating frequency following
s 2
SAR ¼ E ð3Þ
r condition “replaced” was 361.7
6.9 mW/kg and for
where s is specific conductivity, r is density of cell
culture medium, and E is rms-value of the electric
field within the medium [EN 62209, 2006].
Vertical and horizontal SAR distribution in a
coverslip-equipped Petri dish, located in the “exact
exposure domain,” is visualized in Figure 7. The
distinct impact of the medium level on SAR
value is illustrated in Figure 7a–c. Generally, the
higher the medium level, the larger the all-over
inhomogeneity along the vertical axis. For the medium
condition “replaced,” deviation between the SAR
value (0.15 W/kg) at the center and the SAR value
(0.60 W/kg) at the medium-air boundary was calcu-
lated to be 6 dB. For decreasing medium levels like
“evaporation loss” or “replacement needed” smaller
vertical SAR gradients of 4 and 2 dB, respectively,
were observed. In Figure 7d, a horizontal cut of the
Petri dish is shown 1 mm above the coverslip bottom,
which corresponds to the location of the adhering cell
monolayer. The medium condition was “replaced.”
SAR values deviate across the coverslip by at most
1 dB (135.7
31.5 mW/kg), thus expressing good ho-
mogeneity (Table 4). In the “usable exposure domain”
space for up to eight Petri dishes (with coverslips) is
present. Looking at the SAR homogeneity throughout
all coverslips the deviation is still good as no value
differs more than 1 dB (121.7
28.4 mW/kg).
In Figure 9a–c, SAR values for MEA chips at
different filling levels are presented. The picture
resembles the one for Petri dishes. Averaged SAR
values and corresponding inhomogeneities increased
with medium level. The value for the medium
“replacement needed” only 100.9
5.6 mW/kg across
the “recording domain” at 1 mm height over the MEA
container bottom (Table 4). As shown in Figure 9d–f,
SAR homogeneity for the “recording domain” of an
MEA chip placed in the “exact exposure domain” was
excellent. An average value of 361.7
6.9 mW/kg
(<0.1 dB) was prevalent in a horizontal slice 1 mm
above the electrode array. It should be emphasized
that we found a field enhancement of 0.75 dB
(429.9 mW/kg) compared to the average value of

TABLE 3. Medium Conditions and Corresponding Filling Levels in Liquid Containers While Ongoing Cultivation

Petri dish medium condition Volume/ml hmedium/mm MEA medium condition Volume/ml hmedium/mm
“Replaced” 3.6 3.7 “Replaced” 1.6 5.1
“Evaporation loss” 2.7 2.8 “Evaporation loss” 1.2 3.8
“Replacement needed” 1.8 1.9 “Replacement needed” 0.8 2.5

Bioelectromagnetics
 Exposure of Developing Neuronal Networks 273

To quantify the matching of the Stripline loaded

Fig. 8. Grayscale of simulated SAR distribution for (a) a sin-
gle-MEA-equipped Stripline and (b) a six-MEA-equipped Stri-
pline. SAR values are given in a horizontal slice 1 mm above
the recording domain where monolayer cell cultures typically
adhere. The scale bar depicts absolute SAR values.
361.7 mW/kg on top of every single microelectrode,
whereas no deviation was observed for interspaces.
Hence, for “recording domain,” no considerable effect
of metallic electrodes was declared, as they represent
only a small fraction (1%) of the area. Concerning the
“utilizable exposure domain” equipped with six MEA
chips the overall homogeneity (0.13 dB) is likewise
very good as the SAR
standard deviation (sd) was
calculated to be 362.5
11.4 mW/kg across all chips.
These results reinforce the choice of the biosensor for
the post-exposure electrophysiology of cell culture
monolayers.
with cell culture dishes, the S11 input reflection
coefficient and S21 transmission coefficient were
measured for frequencies from 800 to 1000 MHz.
Inside the incubator, we found for the Stripline
equipped with six MEA chips, a well-balanced system
as the maximal return loss was 31 mW (15 dB) and
transmission losses were found to be at most by
370 mW (2 dB) for an input power of 1 W. For
relevant measurement frequency of 900 MHz the
situation is especially better as the S11 was 18 dB
and S21 was 1.6 dB. These levels were quite equal
to those measured in the empty Stripline, conclud-
ing that the system’s performance was not affected
by presence of cell culture dishes. Due to experi-
mental constraints, electric field distribution was
not measured within a medium-filled Petri dish or
MEA chip.
Experimentally, SAR was assessed by measure-
ment of the local temperature increase induced
by 1 W input power at 900 MHz in the medium-
filled (“replaced”) MEA chip. The SAR was calcu-
lated with
dT
SAR ¼ c jt¼0 ð4Þ
dt
where c is the specific heat of the medium
(c ¼ 4186 J/kg K) and dT/dt the initial temperature
gradient [K€uhn and Kuster, 2007]. In particular, we
employed a non-interfering fiber-optic sensor (OTG-F,
OPSENS, Quebec, Canada) to acquire temperature in
the “recording domain.” Measurement noise (
sd) of

Fig. 9. Simulation results of SAR distribution in a MEA chip with its delicate electrode array
structure. Vertical cross-sections at varying medium conditions (ac) through center of the
MEA chip illustrate effect of the medium volume/height (for filling levels compare Table 3) on
SAR values. (d) Horizontal cross-section 1 m above the medium container bottom, medium
level: replaced, shows SAR distribution in the center of the MEA chip at an input power of 1W,
and a frequency of 900 MHz. (e) Alterations of SAR value caused by the electrodes presence
are shown 1 m above electrode array. Influence of the metallic microelectrodes is minor and
limited to the electrode’s area (f).
Bioelectromagnetics
274 Oster et al.

TABLE 4. Detailed Dosimetry for Different Medium Conditions and for Flexible Sample Positioning in the Stripline

SAR þ standard deviation (sd)/mW/kg

Exact exposure domain Coverslip in Petri dish one sample MEA chip (recording domain) one sample
“Replaced” 135.7
30.9 361.7
6.9
“Evaporation loss” 83.3
19.2 202.3
6.9
“Replacement needed” 58.8
11.4 100.9
5.6
Utilizable exposure domain Coverslip in Petri dish eight samples MEA chip (recording domain) eight samples
“Replaced” 121.7
28.4 362.5
11.4

Results representing one sample were determined in the “exact exposure domain” and results representing eight/six samples were
obtained in the “utilizable exposure domain.” For both, values were obtained in a horizontal slice 1 mm above the coverslip or MEA
chip bottom.

the probe, determined in the incubator at 37 8C, was
calculated to be
2 mK. By linear regression method
in the first 200 s, a temperature increase dT/dt of
0.05 mK/s was calculated (n ¼ 31), which corresponded
to an SAR value of 210
63 mW/kg. Total thermal
rise was limited to 0.05
0.013 K after 15 min.
Cell Exposure Experiments
Long-term exposure studies were executed with
the carefully characterized Stripline to address possi-
ble RF-EMF induced effects on developing neuronal
networks. Cortical neurons from embryonic rats were
cultivated on MEA chips and reorganized themselves
into functional two-dimensional (2-D) networks. Fol-
lowing an empiric exposure protocol, cell monolayers
were subjected to RF-EMFs, starting from first day in
vitro. Analysis of electric activity and network
communication was done off-line following spike and
burst events.
Cell culture exposure started from first day in
vitro (1 div). We applied 900 MHz continuous wave
signal at 1 W Stripline input power, resulting in
0.36 W/kg SAR within the cell monolayer. Every
“exposure cycle” lasted 2 h, starting with 15 min RF-
exposure period followed by 105 min non-exposure
period (see Fig. 10b). There were 12 cycles per day,
repeated 7 days per week. Exposure cycles were
employed for up to several weeks. In parallel experi-
ments, we employed a fiber-optic sensor for non-
interfering temperature evaluation within cell culture
medium during RF exposure. Following exposure
protocol, we found marginal temperature alterations
that did not exceed 0.1 K (Fig. 10a). Consequently, it
can be assumed that temperature rise was not signifi-
cant in terms of network signaling and allowed for
investigation of possible athermal effects.
Fully dissociated cells from rat embryonic cortex
reestablished functional networks (Fig. 11a) under
exposure (0.36 W/kg) and sham-exposure (0 W/kg).
Bioelectromagnetics
First, intrinsic activity of neurons was detected around
14 div in terms of single, spontaneous spikes. Cellular
signals typically had amplitudes in the range 20 to
130 mV and were isolated from noise using a
threshold-based algorithm. In the following days,
development of network synchronous burst events
was observed and after approximately 3 weeks, burst
occurrence stabilized to a robust pattern. Measure-
ments were performed for several weeks and after
60 div, both exposed and sham-exposed networks still
exhibited intrinsic network communication, which
proved long-term feasibility of the setup (Fig. 11b).
Exposed and sham-exposed neurons had similar
electrophysiological characteristics in terms of spike
and burst formation occurring.
DISCUSSION
We developed a flexible experimental setup for
long-term electromagnetic exposure of developing
neuronal networks in vitro. We state that the homoge-
neous field, which is prevalent between parallel plates
of the so-called Stripline in combination with MEA
chips enabling non-invasive and label-free assessment
of neuronal network communication, is the most
suitable approach to investigate possible athermal
EMF-induced effects on electrogenic cells or tissue.
Modified recording setups [Koester et al., 2007;
Moretti et al., 2013] or resonant systems [Laval et al.,
2000; Paffi et al., 2010b] are limited in sample
positioning due to setup adaptions or local field
interferences. We satisfy statistical requirements as a
large uniform exposure area can be used for flexible
sample positioning up to eight Petri dishes or six
MEA chips. However, its main advantage is variety of
frequencies including common mobile phone carrier
bands, allowing for cell monolayer irradiation without
repositioning samples or any resizing setup modifica-
tion. Simulations of the empty Stripline revealed

Fig. 10. Temperature drift was measured with a fiber-optic sensor within cell culture medium.
During 15 min-RF exposure period, temperature increase did not exceed 0.1K. (a) Eight-hour
course of a 24-h measurement. (b) Exposure cycles lasted each 120 min (2 h) with alternating
exposure (15 min) and non-exposure (105 min) periods.
high constancy (
0.2 dB) of electric field in lower
frequency domain (DC to 1.0 GHz), whereas higher
frequency domain (1.0–1.7 GHz) showed decreasing
stability (
1.1 dB). This effect can be explained
by parasitic effects, like standing waves caused by
incubator walls and the Stripline. Still, high frequency
domain is also applicable if one keeps some limita-
tions in mind.
In agreement with simulations, measurements of
S-parameters indicated good system matching up to
the cutoff frequency at 1.7 GHz. For frequencies up to
1.0 GHz, input reflection coefficient S11 was found
smaller than 10 dB and transmission losses S21 were
larger than 2.5 dB, where an open front door
resulted in less scattered fields and parasitic effects
diminished. Particularly at 900 MHz, an open front
door had negligible impact on S11 and S21 coefficient.
This was important for interpretation of comprehen-
sive experiments with DASY 3 near-field scanning
technology, performed to exactly determine field
characteristics. At 900 MHz, deviation of averaged
electric field strength between experimental and
simulated data was 1.1 dB and slightly lower for
adjacent frequencies, 880 MHz (0.81 dB) and
920 MHz (0.36 dB). This deviation cannot fully be
explained by deviation from isotropy of the field
probe (
0.3 dB). We assume that measurement uncer-
tainty and minor environmental disturbances, revealed
by measured Standing Wave Ratio (SWR) of 1.15,
may be the origin for these undesired deviations.
S-parameters were not considerably affected by pres-
ence of liquid-filled MEA chips in the Stripline.
At 900 MHz and 1 W incident power, a homoge-
neous electric field of 267
5 Vm1 was generated in
Exposure of Developing Neuronal Networks 275
a large so called “utilizable exposure domain” of the
Stripline. Excellent field homogeneity of 270
1 Vm1
was calculated in the smaller “exact exposure domain.”
For the latter, corresponding SAR values were
135.7
30.9 mW/kg on a coverslip (in a Petri dish) and
361.7
6.9 mW/kg in the “recording domain” of a
MEA chip. It is notable that verified SAR values did
not depend on position of the sample container due to
predominant uniform field distribution. Overall homo-
geneity was likewise very good, as for the Stripline
loaded with eight Petri dishes (with coverslips) deter-
mined SAR across all coverslips deviated by 1 dB
(121.7
28.4 mW/kg). For MEA chips, uniformity was
even better as throughout all chips maximum deviation
was assessed to be 0.15 dB (362.5
11.4 mW/kg).
Schuderer et al. [2004b] determined nonuniformity of
SAR in cell-monolayers seeded in Petri dishes for
several exposure setups between 0.8 and 1.4 dB.
It appeared that the level of medium had
considerable effect on SAR value as also found by
other groups like Laval et al. [2000], Sch€onborn et al.
[2001], and Schuderer et al. [2004b]. Generally, SAR
values decreased with reducing medium level as the
inductively coupled part of the electric field in the
solution contributed to SAR value as a quadratic
function of medium height [Burkhardt et al., 1996].
Simulations revealed that SAR on coverslips (in Petri
dishes) was reduced by 2.1 dB if the fluid level was
three quarters (“evaporation loss”) and by 3.6 dB if
the level was half (“replacement needed”) of maxi-
mum (“replaced”). For the “recording domain” of
MEA chips, influence was likewise, as SAR
decreased 2.5 dB at three quarters and 5.6 dB at
half of medium level. These values are reasonable, as
Bioelectromagnetics
276 Oster et al.
Fig. 11. Rat cortical neurons on a MEA chip. Electrode diame-
ter was 30 mm and electrodes interspace was 200 mm. Elec-
trogenic cells (a) reestablished functional networks under
exposure and sham-exposure. (b) Intrinsic neuronal activity in
terms of burst or spikes on a single electrode at 62 div.
above mentioned groups found SAR deviations up
to 3 dB. Strong sensitivity of exposure dosimetry
toward unavoidable evaporation of cell culture me-
dium should be kept in mind if performing long-term
experiments. Lids or gas permeable membranes could
be employed to reduce evaporation to a minimum. On
the other hand, SAR inhomogeneity inside the me-
dium container was relatively low. We calculated a
value of 1 dB for coverslips (in Petri dishes) and
0.1 dB for MEA chips (in the “recording domain”);
hence both are therefore applicable for in vitro
studies. It should be noted that we did not consider the
meniscus profile of the medium in the container as
recommended for cells in suspension by Schuderer
et al. [2004a]. This is reasonable since we assessed
only cell monolayers at the central bottom of sample
container.
We found that MEAs were suitable devices to
investigate neuronal network communication during
or after EM exposure. As their recording electrodes
are metallic structures, they are also supposed to
affect electric field and SAR value. A recent study
[Merla et al., 2011]; addressed this problem by
implementing different simulation strategies to char-
acterize electrodes’ impact on the field and found
some considerable inhomogeneity in SAR uniformity
Bioelectromagnetics
near recording electrodes. However, electrodes used
by Merla had a high aspect ratio, which likely
accounted for field intensification. For dimensions
of our metal microelectrodes (30 mm in diameter
and 1 mm thick), we found SAR values of
429.9
79.0 mW/kg on top of the electrode and
358.5
0.02 mW/kg for interspaces. Taking into
account that electrodes represent approximately 1% of
“recording domain,” we assume that inhomogeneities
can be neglected, as we study complex functional
networks that grow within the entire “recording
domain.” Our models showed that electrodes’ pres-
ence altered mean SAR value in this area insignif-
icantly by 0.01 dB.
More incident power implicated higher energy
deposition within culture medium resulting in a
reinforced temperature rise. As the Stripline was
placed in a temperature-stable incubator, field induced
temperature elevation could not be regulated by
ambient air exchange. To overcome this problem,
another study employed active air [Schuderer et al.,
2004b] cooling mechanisms. Our system was limited
to low SAR values reflecting typical exposure sit-
uations arising from mobile phone use. We employed
an exposure protocol based on empirical studies
and found that temperature alterations did not exceed
0.1 K, which satisfied elementary requirements to
analyze potential athermal (DT < 0.1 K) EMF-induced
effects [Schuderer et al., 2004b]. A fiber-optic sensor
facilitated non-interfering temperature monitoring in
the EMF environment with high repeatability and
reliability. We used linear regression method to
calculate SAR values based on temperature rise as
others did [Merla et al., 2011] and satisfied statistical
power as n ¼ 31 heating curves were assessed. Our
measurements revealed an SAR of 210
63 mW/kg,
matching well with simulation results as values
differed by only 0.2 dB for numeric condition “evapo-
ration loss.”
We performed exposure experiments with pri-
mary cortical neurons obtained from embryonic rats
for up to two months. These initially dissociated cells
developed functional networks and could be consid-
ered an appropriate paradigm to mimic mammalian in
vivo-like conditions [Dichter, 1978]. In contrast to
other protocols in literature, which carried out expo-
sure from 15 to 21 div in daily 3 min intervals [Moretti
et al., 2013], or one-time exposure of mature networks
with different EMF power patterns [Koester et al.,
2007], we addressed early exposure of developing
neuronal networks to see potential impact of pre- or
post-natal exposure on functionality. After 12 days in
vitro (div), first spikes of the neural network were
detected. Ongoing development of intercellular and

synaptic connections resulted in an enhanced signal
density alluding to a distinct functional network. In
good agreement with literature [Otto et al., 2003;
Jungblut et al., 2009], we observed at day 16 in vitro
the onset of volleys of spikes, so-called bursts. In the
following weeks, network activity stabilized in regular
bursting patterns. After around 55 div, signal ampli-
tude and burst rate began to decrease. A significant
difference between sham-exposed and exposed sam-
ples was not observed during these preliminary
experiments.
CONCLUSION
Neuronal networks and their functionality are
considered one of the most promising targets of
research to assess potential EMF-induced effects on
neuronal activity and communication patterns. A
number of exposure studies revealed there is currently
no evidence of any adverse health effects caused by
weak EMFs. However, there is still a lack of expertise
and some inconsistent findings regarding whether
adolescents are particularly sensitive to EMFs or if
pre- or neonatal exposure is detrimental regarding
development of neuronal networks. To address possi-
ble long-term effects on developing or mature neuro-
nal tissue at a cellular level, in vitro studies are
favored approaches. This implicates a demand on
precisely characterized exposure setups providing
well-defined environmental conditions to research on
proper in vitro systems.
We present a flexible experimental setup for
long-term electromagnetic exposure of developing
neuronal networks in vitro, and employed comprehen-
sive characterizations in terms of numerical dosime-
try, electrical field, and temperature measurements.
Our setup provides well-defined environmental con-
ditions confirmed by experimental findings, which
were in good agreement with numerical results.
Prenatal neuronal cells were plated onto MEA chips
and exposed to EMFs at GSM 900. Daily recordings
in the course of their network formation and matura-
tion (for 60 days) revealed that exposed groups
developed intrinsic electric activity indicating func-
tional network communication. No significant devi-
ations from the results of sham group have been
observed.
We expect our experimental setup will help to
promote experiments that would answer the question
of whether and how the developing brain of unborn
children or infants up to adolescents might be affected
by electromagnetic radiation. In further studies, we
will use the system presented to expose neurons
derived from human iPS cells. It can be assumed that
Exposure of Developing Neuronal Networks 277
these cells can enhance the physiological relevance of
in vitro studies and can bring new findings concerning
potential health risks of EMF to humans.
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