2. Impedance measurements in 3D cell culture
The implementation of EIS in 2D cultures relies on cells attaching to
the electrodes, this does not occur in 3D cultures as they contain
matrices such as biopolymers (collagen, fibrin), polymer scaffolds or
discrete suspended cell clusters. As a result, there are a number of
areas that the EIS system needs to be adapted for 3D applications:
1) Ensuring sensitivity of the electrode array to cells suspended in or
attached to the matrix/scaffold by ensuring there is an even electric
field through the whole system.
2) Adaptable to a range of different 3D cell culture formats – systems
are often designed to address a specific biological research question or
tissue function and so their architecture varies.
3) Ensuring that electrodes do not influence cell responses or change
the mechanical environment in 3D cell culture systems.
The design and integration of electrodes within culture systems appears
to be central to the effective monitoring of cells within scaffolds and
hydrogels. It has been demonstrated that the electrical properties of
cells can be monitored when they are immersed within a hydrogel rather
than directly attached to a planar electrode.(S.-M. Lee et al., 2016)
Lee et al. incorporated a matrix of vertical electrodes (Figure
1a) that were able to monitor the migration, proliferation and
apoptosis of cells through a gel matrix in 3D. In this system, cells
were grown in an alginate hydrogel scaffold and vertical parallel
electrodes were used to measure the capacitance of the system. An
increase in capacitance was shown to be proportional to an increase in
seeded cell density within the hydrogel (Figure 1b and Figure
1c) , showing that by measuring capacitance of the system it is possible
to monitor cell number. To monitor cell proliferation and migration, one
frequency (1 kHz) was used to obtain the capacitance of the system
rather than the whole impedance spectrum. The chosen frequency coincides
with the frequency typically used to monitor cells in 2D,(Benson et al.,
2013; Srinivasan et al., 2015) but the study did not explore how cell
death or migration affects the whole spectrum or whether other changes
in the spectrum could be correlated to these cellular processes.
In many instances, cells are grown in a 3D spheroidal structure in which
the cells clump together and proliferate. These models are used
extensively in cancer research to understand drug treatments for tumors.
Lei et al. used in-plane electrodes to monitor the viability of
spheroids developed in an agarose hydrogel using the hanging drop
technique (Figure 2a) .(Lei et al., 2018) Changes in the
impedance magnitude was successfully correlated with different cell
numbers seeded in the hydrogel (Figure 2b) . It is interesting
to note here that while the authors chose the standard 1 kHz as the
optimal sensing frequency, as was seen in the earlier study, there were
larger variations in the impedance magnitude at lower frequencies. These
variations were accounted for by the authors as being related to the
increases in the noise as the frequency is reduced, due to noise being
proportional to 1/f.
The position of the electrodes within 3D culture systems (parallel vs
in-plane) has also been shown to affect detection outcomes.(Y. Xu et
al., 2015) Pan et al. found that in-plane electrodes were not able to
significantly detect the proliferation of spheroids in a matrigel,
whereas parallel electrodes successfully monitored their proliferation
over time (Figure 3a and Figure 3c) . This data corresponds with
the theory that parallel electrodes can achieve more sensitive detection
due to a more uniform electric field.(Y. Xu et al., 2015) Nevertheless,
this challenges the results from the earlier studies from Lei et al.
using in-plane electrodes. Factors influencing these differences may be
gel thickness and electrode design. When in-plane electrodes are used in
a 3D system, the applied current can only penetrate a small volume and
distance, whereas parallel electrodes force the applied current to go
across the entire sample. In this instance it is to be expected that
more signal can be detected and signal to noise increases as the current
penetrates a larger volume of the 3D culture.
While the integration of standard metal electrodes into 3D culture is
challenging due to the stiffness of the electrode materials and the need
to connect them back to the outside world, one group has been exploring
the application of conductive polymers as both the cell scaffold and the
electrodes.(Khan et al., 2019) As the cells adhere to this conductive
scaffold, its impedance changes enabling cell adhesion to be detected(Figure 4a and Figure 4c) .(Del Agua et al., 2018)
Interestingly, the optimal sensing frequency found using a
poly(3,4-ethyenedioythiophene): xanthan scaffold was between 0.1 and 100
Hz (Figure 4b) . As mentioned in the previous example, noise
varies with 1/f, so at lower frequencies we would expect that noise
would increase significantly. The potentiostat used for the impedance
measurements may be able to compensate, however, little information is
given by the authors of this study to determine if this approach has
been implemented. Acquisition rate may also be problematic at low
frequencies, though in the case of cell monitoring this may not be an
issue since these are long-term cultures of days and weeks rather than
minutes and hours.
In 2D, it is well established that low frequency signals are dominated
by the double layer capacitance of the electrodes.(Benson et al., 2013)
Nevertheless, in this study the electrode properties change as the cells
are growing within the conductive hydrogel and is consistent with other
studies using conductive scaffolds.(Inal et al., 2017) Further,
modifying the properties of the 3D scaffolds (physical and chemical
properties) should be done with caution as scaffold properties can have
a big influence on cell behavior and function (Bahcecioglu, Hasirci, &
Hasirci, 2019; Caliari & Burdick, 2016; Sridharan, Ryan, Kearney,
Kelly, & O’Brien, 2019) and these influences need to be understood and
cross-validated with other characterization tools prior to
implementation. This suggests that the nature of the scaffold, its
potential use as a working electrode and the nature of the 3D model
(spheroids or tissue) plays a significant role in the final outcome of
the measurements and as such models are needed to compare results with
theoretical systems.
Several equivalent circuits have been designed to model and quantify the
electrical properties of cells grown in 2D systems. Figure 5shows the equivalent circuit generally used to describe live cells in a
monolayer.(Benson et al., 2013) This circuit takes into account the
resistance of the solution or electrolyte (phosphate buffered saline
(PBS) or cell media) Rmedium, the resistance and
capacitance of the cell monolayer that contributes to the total spectrum
RTEER and Ccl, respectively, and the
capacitance of the electrodes and electrode-medium interphase
CEl.(Benson et al., 2013) The electrical circuit and
electrode setup for 2D adherent cells are well developed in EIS and
comprehensive reviews of impedance measurements of cells are available
and demonstrated.(Benson et al., 2013; Canali et al., 2016; Elbrecht et
al., 2018; Srinivasan et al., 2015) In undertaking this review we could
find only one study that has directly compared equivalent circuits for
2D and 3D systems.(Pan et al., 2019) They demonstrated that a matrigel
matrix resistance component was in parallel with the electrical
components representing cell impedance (Figure 3b). This in contrast to
a typical 2D equivalent circuit which shows that the media component is
in series with the components representing cell impedance (Figure 5).
This is explained by looking at the fundamental differences in cell
interactions occurring in the two different models. In 2D, the cells
reside below the media (series circuit) while in 3D, the cells reside
within the scaffold, which resembles more of a parallel circuit.
Furthermore, Pan and colleagues paired simulations derived from their 3D
equivalent circuit with their experimental data using epithelial cells
and found that the presence of cells in 3D decreased the overall
impedance of the system. This was due to an increase in gel conductivity
after the addition of cells, which was attributed to the presence of gap
junctions between cells that allowed electrical connection between
neighboring cell membranes.(Pan et al., 2019) However, other studies
with similar cell types have shown the opposite effect, with the
introduction of cells increasing the overall impedance of 3D
systems.(Inal et al., 2017; S.-M. Lee et al., 2016; Lei et al., 2018)
Groeber et al. are amongst the only published groups using the whole EIS
spectrum and information provided by an equivalent circuit to monitor
tissue growth in a co-culture system. They used parallel electrodes to
measure the impedance of an artificially grown epidermis at different
levels of maturity as shown in Figure 6b .(Groeber et al., 2015)
They developed an equivalent circuit in which there are a series of
connections of n parallel resistor-capacitor circuits which correlated
with a different maturity phase of the artificially grown epidermis (seeFigure 6a ). The data obtained was validated against an isolated
human epidermis showing that the artificially grown epidermis at days
9-12 had the same electrical characteristics as the human epidermis. In
the study, 1kHz was found to be an optimal sensing frequency, suggesting
that it may well be the nature of the conductive scaffold that reduced
the frequency in the study by Agua et al. To measure the impedance of
the tissue, Groeber et al. grew the tissue in a transwell and
transferred the transwell to an impedance platform every time a
measurement was required. It is possible to use ideas from the
literature to envisage how electrodes could be inserted into the
transwell to monitor the system in situ or within a microfluidic device
as undertaken by Sriram et al.(Sriram et al., 2018) A natural extension
of this approach would be to design electrodes, so they are small enough
to contact only one layer of tissue construct. This could enable the
extraction of information of cells within a select region and monitoring
of the individual features as the tissue grows. While Groeber and
colleagues monitored growth and differentiation of epidermal cells,
electrodes placed at different layers of a full thickness skin
equivalent may allow for multicellular monitoring as well as the
potential monitoring of skin processes such as wound healing and barrier
function. Currently, skin tissue maturity is assessed by histology and
immunostaining at different time points, so a layer-by-layer electrode
setup which monitors tissue impedance as it grows could allow
researchers to track growth and cellular processes quickly and without
destroying the model.