ISO/TS 21633:2021

Label-free impedance technology to assess the toxicity of nanomaterials in vitro

This document describes a methodology of a label free and real-time detection for non-invasive monitoring of cell-based assays to assess toxicity of nanomaterials to eukaryotic and prokaryotic cells.

Technologie de l'impédance électrique sans marqueur pour évaluer la toxicité des nanomatériaux in vitro

General Information

• Status: Published
• Publication Date: 02-Aug-2021
• ICS: 07.120 - Nanotechnologies
• Technical Committee: ISO/TC 229 - Nanotechnologies
• Drafting Committee: ISO/TC 229/WG 3 - Health, Safety and Environmental Aspects of Nanotechnologies
• Current Stage: 9093 - International Standard confirmed
• Start Date: 07-Jan-2025
• Completion Date: 13-Dec-2025

Standards Content (Sample)

TECHNICAL ISO/TS
SPECIFICATION 21633
First edition
2021-08
Label-free impedance technology to
assess the toxicity of nanomaterials in
vitro
Technologie de l'impédance électrique sans marqueur pour évaluer la
toxicité des nanomatériaux in vitro
Reference number
©
ISO 2021
© ISO 2021
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ii © ISO 2021 – All rights reserved

Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviations. 2
5 Background . 4
5.1 General . 4
5.2 Electrochemical impedance technique . 4
6 Basic principles, instruments . 6
6.1 Basics of electrochemical impedance technique . 6
6.2 Types of instrument . 6
6.2.1 Electrochemical impedance-based instruments for in vitro analysis of
toxicity on cell monolayers . 6
6.2.2 Impedance-based flow cytometry . 6
6.2.3 Electrochemical impedance-based spectroscopy . 7
6.2.4 Electrical impedance tomography . 7
7 Application for in vitro toxicity assessment . 7
7.1 General . 7
7.2 Normalized cell index.10
8 Technical limitations .11
Annex A (informative) Basic procedures using the xCELLigence system .12
Annex B (informative) Case studies using standard operating procedure for setting up an
xCELLigence experiment with various cellular models .17
Bibliography .21
Foreword
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electrotechnical standardization.
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described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
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iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
Any feedback or questions on this document should be directed to the user’s national standards body. A
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iv © ISO 2021 – All rights reserved

TECHNICAL SPECIFICATION ISO/TS 21633:2021(E)
Label-free impedance technology to assess the toxicity of
nanomaterials in vitro
1 Scope
This document describes a methodology of a label free and real-time detection for non-invasive
monitoring of cell-based assays to assess toxicity of nanomaterials to eukaryotic and prokaryotic cells.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO/TS 10993-1, Biological evaluation of medical devices — Part 1: Evaluation and testing within a risk
management process
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
nanoscale
length range approximately from 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from larger sizes are predominantly exhibited in this
length range.
3.2
nanomaterial
NM
material with any external dimension in the nanoscale (3.1), or having internal structure or surface
structure in the nanoscale
Note 1 to entry: This generic term is inclusive of nano-object (3.3) [and nanostructured material (3.4)].
3.3
nano-object
discrete piece of material with one, two or three external dimensions in the nanoscale (3.1)
3.4
nanostructured material
material having internal nanostructure or surface nanostructure
3.5
nanoparticle
NP
nano-object (3.3) with all external dimensions in the nanoscale (3.1) where the lengths of the longest
and the shortest axes of the nano-object do not differ significantly
[SOURCE: ISO/TS 80004-2:2015, 4.4, modified — Note 1 to entry has been deleted.]
3.6
test sample
material, device, device portion, component, extract or portion thereof that is subjected to biological or
chemical testing or evaluation
3.7
cell index
CI
dimensionless parameter obtained from the electrochemical impedance measurement
3.8
electrochemical impedance
effective resistance of an electric circuit or component to alternating current, arising from the combined
effects of ohmic resistance and reactance.
3.9
impedance-based flow cytometry
IFC
technique used to detect and measure physical and chemical characteristics of a population of cells or
particles
Note 1 to entry: A sample containing cells or particles is suspended in a fluid and injected into the flow cytometer
instrument.
3.10
electrochemical impedance spectroscopy
EIS
method that measures the impedance of a system in dependence of the AC potentials frequency and
therefore that determines both the resistive and capacitive (dielectric) properties of materials
3.11
electrical impedance tomography
EIT
technique in which electrical measurements between many pairs of appropriately positioned surface
electrodes are used to produce images of underlying body structures
4 Abbreviations
AC Alternating current
AgNPs Silver nanoparticles
AuNPs Gold nanoparticles
BSA Bovine serum albumin
CB Carbon black
CI Cell index
CeO Cerium oxide
2 © ISO 2021 – All rights reserved

CuO Copper oxide
DIC Differential interference contrast
DMEM Dulbecco’s modified Eagle’s medium
DMSO Dimethylsulfoxide
ECIS Electric cell-substrate impedance sensing
ECM Extracellular matrix
EDTA Ethylenediaminetetraacetic acid
EIS Electrochemical impedance spectroscopy
EIT Electrical impedance tomography
EMEM Eagle's minimum essential medium
Fe O Ferric oxide
2 3
FBS Fetal bovine serum
HTS High throughput system
IC half-maximal inhibition concentration
IFC Impedance-based flow cytometry
IMEs Interdigitated microelectrodes
Mn O Manganese oxide
2 3
NCI Normalized CI
Ni Nickel
PBS Phosphate buffered saline
QD Quantum dot
RTCA Real-time cell analyzer
RPMI Roswell park memorial institute medium
SiO Silicon dioxide
SPR Surface plasmon resonance
TiO Titanium dioxide
ZrO Zirconium oxide
ZnO Zinc oxide
5 Background
5.1 General
Several in vitro assay systems that have been developed for the assessment of the toxicity of
different chemical compounds have also been implemented to assess the toxicity of NMs. Due to their
physicochemical properties, NMs may behave differently than the chemical compounds for which
these assay systems were developed and therefore, when they were used with NMs, discrepancies in
[1]
results among assays were often observed . As a result, investigators were prompted to consider the
interaction of NMs with the assay systems as a possible source for the observed discrepancies.
The detection systems of these toxicity assays are mostly optical in nature and rely on absorbance,
luminescence or fluorescence to quantify the products of the assay systems (e.g. tetrazolium salts).
NMs may therefore interfere directly with the assay readout by altering the absorbance, luminescence
[2]
or fluorescence of the products of these assay systems . Depending on their material, shape and size,
certain NMs may absorb, scatter and emit light at the assay detection wavelength. Carbon-based NPs, for
example, CB are known to absorb light in the visible spectrum whereas metal oxides, metal hydroxides,
[3]
and metal carbonate NPs are known to scatter light . AuNPs with a strong SPR absorb more light
[1]
than iron oxide NPs and larger NPs absorb more light than smaller NPs . Similar to AuNPs, AgNPs
[4]
also have strong plasmon resonances . Such absorptive abilities of these NPs may therefore interfere
with the absorptive properties of products obtained from different assay systems. NMs may also
interfere directly with the assay by interacting with the chemical reaction product. Due to their large
surface area per unit mass and surface reactivity, compared to large particles NMs may also display
an increased adsorption capacity thereby increasing the possible interaction between nanoparticles
[3][5]
and assay components . Finally, NMs may also catalyse the conversion of substrate to product. The
large surface area per unit mass and surface reactivity may lead to an excess in surface energy with
[6]
subsequent enhancement in the catalytic activity of NMs .
This document is therefore based on current information about electrochemical impedance technique
that does not rely on optical measurements to determine the degree of cell viability or cytotoxicity and
also provide kinetic information non-invasively and with high temporal resolution through recorded
growth curves. The electrochemical impedance technique can therefore be used as an alternative assay
system for the study of the viability and toxicity of NMs in vitro with no interference. It also directs
further studies into the mode of action (toxicity) of a NM.
5.2 Electrochemical impedance technique
[7][8][9]
ECIS was developed in the 1980s for studying cellular processes in real time . In this assay,
cells are cultured in wells, which contain a large reference electrode as well as number of detection
electrodes that cover 80 % of surface area of each well bottom. Upon application of the low amplitude
sinusoidal potential, the electrochemical impedance between the electrodes is measured. As the
cells attach and spread on the electrode surface, they alter the effective area available for current
[10]
flow causing an increase in the impedance of the system . Increase in impedance is possible due
to insulating properties of cellular bilipid membranes which act as dielectric objects and therefore it
should correlate to the number of cells on the electrodes. Subsequently, the technique was applied to
[11][12][13][14] [7][10][15]
assess cellular toxicity as well as motility . Impedance spectroscopy of cellular
[12][16][17][18][19][20]
activity was also developed based on this same impedance technique .
Based on this ECIS technique, a new electronic sensing RTCA was developed with improvements on
[21][22][23][24][25]
the electrode structure to allow detection of almost all cell types in a culture well
[26][27][28]
. Here, cells are allowed to grow and attach to the electrodes and with change in the flow of
current around and through cells, concomitant increase in impedance may ensue and thus providing
information on their count, morphology and viability. Upon addition of a test material, cells may
[11][12]
detach causing a drop of impedance indicating toxicity through reduction in cellular viability
[29]
. To ensure that cells do not detach due to overcrowding, a cell proliferation assessment should be
performed prior to experimentation to determine an ideal seeding concentration for the cell type in
question. In addition, untreated control cells should be assayed alongside the treated cells to monitor
confluency.
4 © ISO 2021 – All rights reserved

In the past, an impedance measurement technique was applied to quantitatively monitor cell number
and cell viability in monolayer cultures through various impedance measurements of cellular responses,
[30][31][32][33][34][35][36][37][38]
such as proliferation and toxicity, in a real-time and label-free manner
[39]
. With this technique, it is also possible to assess cell differentiation, cell invasion and migration,
cell-cell interaction using co-cultures, and cellular mechanistic investigation such as intracellular
[40][41][42][43]
levels of calcium and DNA damage . The electrochemical impedance technique was also
used successfully for in situ monitoring of NM-induced cellular toxicity and other aspects of cell
[44]
physiology such as proliferation, morphology, attachment, and intercellular adhesion . For example,
electrochemical impedance-based monitoring was used to investigate the cytotoxic effects of various
carbon-based NMs with different cell lines, see Table 1.
Table 1 — Cell types used for the toxicity assessment of nanomaterials using the impedance
technique
Cell type Nanomaterials Reference
[45][46][47]
endothelial EA.hy926 (ATCC ® CRL- Carbon nanotubes
2922™) cells
L929 (ATCC® CCL-1™) and V79-4
(ATCC® CCL-93™) fibroblasts
GC-2spd(ts) (ATCC® CRL-2196™)
cell line, derived from immortalized
mouse spermatocyte
THP-1 (ATCC® TIB-202™) human
monocytic cells
[48]
Human glioblastoma U87-MG Graphene
(ATCC® HTB-14™)
Rat astrocytes (CRL-2006) and
mouse endothelial (ATCC® CRL-
2583™) cells
[49]
Hepatoma C3A (ATCC® CRL-
10741™) cells
[50]
Epithelial lung cell line, A549 CuO and TiO
(ATCC® CCL-185™)
[51]
16HBE14o, an adherent, immortal- CeO , SiO , Fe O , Mn O , ZnO and ZrO
2 2 2 3 2 3 2
ized human bronchial epithelial cell
line
[52]
Human epithelial intestinal HT-29 SiO
® ™
(ATCC HTB-38 ) cell line
® [53]
Human intestinal Caco-2 (ATCC AgNPs
™
HTB-37 ) cell line
[54]
Bronchial epithelial BEAS-2B AuNPs
(ATCC® CRL-9609™) cell line
Chinese hamster ovary cell line CHO
(CRL-12023)
Human embryonic kidney cell line
HEK 293 (ATCC® CRL-1573™)
[55]
Hepatocellular carcinoma cells Ni NPs
(SMMC-7721)
[56]
HeLa (ATCC® CCL-2™) human cer- Polymeric nanoparticles
vix epithelia cell line
6 Basic principles, instruments
6.1 Basics of electrochemical impedance technique
In the EIS system, an electrical equivalent circuit is used to curve fit the experimental data. For cellular
detection, number of electrical equivalent circuits were proposed (Reference [57] and its impedance
[58]
spectrum for IMEs were reported ). In a simplified electrical equivalent circuit, it is suggested that
two identical double layer capacitances at each electrode (C ) are connected to the medium resistance
di
(R ) in series, where the dielectric capacitance of the medium (C ) is introduced in parallel with
sol di
[30]
these series elements . In this equivalent circuit, there are two parallel branches namely C and
di
C + R + C where the impedance Z and Z in each branch can be expressed with Formula (1) for
di sol di 1 2
branch C + R + C : and with Formula (2) for branch C :
di sol di di
ZR=+ (1)
1 sol
()πfC
di
Z = (2)
()2πfC
di
[32]
As biological cells are very poor conductors at frequencies below 10 kHz the presence of the
electrically insulated cell membranes alters the C . The conductivity of the cell membrane is around
di
−7 [59]
10 S/m, whereas the conductivity of the interior of a cell can be as high as 1 S/m ( ). Therefore, cell
[30]
proliferation can be estimated by the total impedance at low frequencies .
6.2 Types of instrument
6.2.1 Electrochemical impedance-based instruments for in vitro analysis of toxicity on cell
monolayers
1)
The xCELLigence®, CellSine, and ECIS (ECIS Zθ) systems are the examples of current commercially
available electrochemical impedance-based instruments for in vitro analysis of toxicity where they use
cell monolayers to monitor the changes in impedance properties of cells after exposure to bioactive
agents including NMs.
The design of the cell culture plates with gold-plated electrodes attached to the bottom of the wells
implemented in HTS format making it possible for real-time observations to be made of cell changes
[14][15][29][60]
throughout an experiment without the need for destructive cell sampling . Information
may thus be provided on cell proliferation and their reaction to the bioactive agent including NMs in
[61]
question .
A portable automated bench-top mammalian cell-based toxicity sensor that incorporates enclosed
fluidic biochips containing endothelial cells monitored by ECIS technique was also developed to assess
[62]
the toxicity of drinking water .
6.2.2 Impedance-based flow cytometry
In addition to surface-attached cell-based electrochemical impedance technique, an IFC system, a
microfluidic chip-based IFC, can analyze single cell impedance without specific sample preparation
[63]
This technique is also able to provide information on size and number of cells as well as on their
membrane capacitance and cytoplasmic conductivity.
1) xCELLigence®, CellSine, and ECIS (ECIS Zθ) are examples of suitable products available commercially. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO

of these products.
6 © ISO 2021 – All rights reserved

6.2.3 Electrochemical impedance-based spectroscopy
EIS is a non-invasive, label-free method to measure the dielectric properties of samples while applying
a varying frequency AC electrical field by means of electrodes. EIS was initially developed to elucidate
double-layer capacitance but it is now used to characterize electrochemical processes under complex
[64][65]
potential modulation . With EIS, in addition to monitoring cell growth rate in a non-invasive
[66][67]
manner, it is also possible to obtain high-resolution imaging of non-adherent or suspended cells .
EIS is a label-free, non-invasive analysis method for a wide variety of biological samples, ranging
from single cells to multicellular aggregates and organisms. For example, real time cell viability
[16][20]
and toxicity of test materials can be assessed . Moreover, it was also shown that with EIS it is
possible to measure the alterations in morphology of cell aggregates due to necrosis and apoptosis by
hydrodynamically positioning cell spheroids in a capillary featuring a four-electrode measurement
[68][69]
setup . EIS measurements was used in environmental toxicology to characterize and manipulate
[70][71][72][73]
multicellular organisms, such as trapping, sorting, and counting of Caenorhabditis elegans
or measuring the responses of fish embryos to cryoprotective chemicals, such as methanol and DMSO
[74][75]
. Moreover, EIS measurements can also be used for the detection of parasites in drinking water
[76] [77][78]
and for testing anthelmintic drugs by monitoring parasite motilities .
[79]
Using EIS, the toxicity of silica nanowires on breast epithelial cancer cells and of QD and AuNPs to
2) [80]
fibroblastic V79 V794 (e.g. ATCC® CCL93™ ) cells can be assessed.
6.2.4 Electrical impedance tomography
[81]
EIT, in addition for its possible clinical applications , is also used to assess cell growth and cell
activity in live 3D structures as well as cell viability that can spatially resolve cell viability for single 3D
[82]
spheroids .
7 Application for in vitro toxicity assessment
7.1 General
It is possible to apply the electrochemical impedance-based technique in the assessment of the toxicity
of NMs. Cells are cultured and when adherent cells attach and spread across the sensor surface of an
electrode, increases in impedance are recorded. Conversely, cells that round up or detach even for a
short time will cause impedance values to drop. The presence of cells on top of the E-Plates electrodes, a
multiple-well electronic microtiter plates, affects the local ionic environment at the electrode/solution
interface, leading to a change in circuit impedance (see Figure 1).
2) ATCC® CCL93™ is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of this product.
Key
1 electrode 7 electrode with two strongly-attached cells
2 cell 8 z = z    baseline
3 cells 9 z = z  impedance
cell 1
4 electrode without cell 10 z = z  impedance doubled
cell 2
5 electrodes with one cell attached 11 z = z  impedance further increased
cell 3
6 electrode with two cells attached
Figure 1 — Schematic illustration of the biological cell electrochemical impedance
[83]
measurement and integrated microelectrodes on the well bottom
Cell death leads to the release of cells from the surface of the measurement electrode that induces
the decrease of the impedance measured across the electrodes. Recording of the impedance across
electrodes at the bottom of the plate (a complex resistance in Ohm's law) is expressed as a parameter
in different systems described in 6.1. For example, it is presented as CI with xCELLigence, Ziec (Ω) with
3)
CellKey™ , Δ|Z| (Ω) with CellSine, and Impedance (Ω) with ECIS Zθ system.
The following discussion provides an example of how the xCELLigence system calculates and utilizes
the cell index. As the cells interact with the biocompatible microelectrode surface in the E-Plate well
with the aid of specialized software, the electrochemical impedance signal is converted to a specific
parameter called the CI.
3) CellKey™ is an example of a suitable product available commercially. This information is given for the convenience
of users of this document and does not constitute an endorsement by ISO of this product.
8 © ISO 2021 – All rights reserved

Monitoring of cell viability is critical and the cell impedance system enables continuous measurement
[84][85]
and quantification of cells . The CI reflects the cell viability, which in turn measures cell number,
[25][26]
attachment quality and cell type . The CI, a unit-less parameter, is derived to represent cell status
based on the measured relative change in impedance that occurs in the presence and absence of cells
[13][86]
in the wells . Figure 2 represents four different cell lines displaying different proliferation curves
with different cell indices. This in turn, indicates that cell index relates to cell type where each has a
relatively unique lag phase, exponential phase and stationary phase.
Key
X time (h)
Y cell index
MCF 7 (30 000 cells)
COS 7 (6 250 cells)
HT 29 (50 000 cells)
PC3 (6 250 cells)
NOTE Cell lines were seeded in triplicate to calculate the average and ± SD of CI values of corresponding cell
[83]
lines .
Figure 2 — Cell proliferation curves of four different cell lines displayed as cell index on the cell
impedance system
The instrument software converts impedance in ohms (Ω) into a CI value, where CI = Ω/15. Cell
impedance is displayed as CI values and can be used to monitor cell viability, proliferation, degree
of cell adhesion, cell number and morphology. Low CI values, compared to untreated cells, indicate
cytotoxicity while high CI values indicate cell adhesion and proliferation. It is indicated that impedance
change may occur depending on mainly two factors:
a) the number of cells attached to the electrodes where, if there are no cells on an electrode surface,
the sensor’s electronic feature is not be affected and the impedance change is 0;
b) the dimensional change of the attached cells on the electrodes where despite the same cell numbers,
dimensional changes such as an increase in cell adhesion or cell spread of the attached cells on the
[87]
electrodes leads to a higher CI value.
On the other hand, a decrease in CI value indicates cell detachment due to a decrease
in resistance measured by the electrodes.
[88]
The CI values are then calculated as follows :
Rf() 
cell i
I =max −1 (3)
Cii=…1, N  
Rf
()
 
bi
where
R ( f ) is the frequency-dependent electrode impedance;
cell i
R ( f ) is the background impedance measured at the initial time without cells;
b i
N is the number of frequency points at which the impedance is measured.
Key
X time (h)
Y cell index
BEAS-2B (10 000 cells)
particle control (media and particles only)
media control (media only wells)
NOTE Cell viability curve of human bronchial epithelial (BEAS-2B) cells, media control and particle control displayed
[89]
as cell index after exposure to 14 nm AuNPs .
Figure 3 — Real-time monitoring of cell viability
As can be seen in Figure 3, where there are no cells or only nanoparticles added, the CI value is zero
because R ( f ) = Rb( f ) in Formula (3) indicating no interference by the nanoparticles tested. On the
cell i i
other hand, when there is an increase in cell attachment, the CI values are larger. Thus, CI is related to
the cell number present in a well. Moreover, changes in CI values will give an indication in changes in
[36]
cell morphology, cell adhesion, or cell viability .
7.2 Normalized cell index
In order to allow comparisons amongst various treatments, it is required to normalize the CI of the
wells to minimize inter-well variability at the time point just before NP addition.
The NCI value is calculated as given in Formula (4):
It
()
Ci
I = (4)
CNi ,
It()
Coi f dose
where
10 © ISO 2021 – All rights reserved

C (t) is CI at any time;
Ci
I (t ) is CI at the time of NP dosing.
Ci of dose
The NCI for all wells is therefore set to one at the normalization time point, as illustrated in Figure 4.
Key
X time (h)
Y normalized cell index
cell line 1
cell line 2
cell line 3
cell line 4
cell line 5
NOTE The normalization time point was chosen at the time just before the addition of NPs, as indicated by
[54]
the black vertical line .
Figure 4 — NCI of BEAS-2B cells treated with AuNPs
A procedure for use of the xCELLigence system is given in Annex A and case studies using standard
operating procedure for setting up an xCELLigence experiment with various cellular models are given
in Annex B.
8 Technical limitations
Cell adhesion is required for the application of the impedance technique. Adhesion of cells in suspension
[90]
is achieved by using a suitable ECM to facilitate this attachment. These include collagen ( ), fibronectin
[91] [92]
or poly-L-lysine .
However, a limitation of the ECIS technique was said to include the provision of an average measure of
adhesion changes over all cells present on the microelectrode surface with no information about the
precise nature of adhesion or morphological changes at the level of individual cells. It was therefore
proposed that a live cell DIC microscopy and image analysis will provide a complementary measure of
[91]
adhesion changes and will reveal the morphological changes of individual cells .
It is also important not to overload the electrodes and therefore assess interference of NMs tested by
conducting experiments without cells at the concentrations of NMs used for the toxicity assessment to
different cells, as indicated in Figure 3.
Annex A
(informative)
Basic procedures using the xCELLigence system
A.1 General
According to the literature, currently the most widely used electrochemical impedance system to
assess the toxicity of NMs is the xCelligence instrument. It is therefore justifiable to describe in more
detail the methodologies involved in the assessment of the toxicity of NMs.
A.2 System validation
The RTCA system cannot be validated; however, system verification with a dummy electrochemical cell
can be performed.
A.3 System verification
The xCELLigence system has a resistor plate that is used to verify the instrument. The resistor plate
has the same size as the corresponding e-plate, i.e. the same number of wells. Where, instead of a micro
sensor array, a resistor array is placed at each well position in such a way that it shows resistance values
close to that of the corresponding e-plate when only cell media are put into each well. Therefore, system
functionality can be verified. Resistor plates are not instrument-specific. Each resistor plate can be
used to perform resistor plate verification on another station (the dedicated place that accommodates
the electronic microtiter plate (e-plate) of the same type).
A.4 Cells and cell culture — Testing for seeding concentration
In both 16-well and a 96-well electronic microtiter plate, the volume per well is 243 µl. It is however
advisable not to exceed 200 µl per well. To determine the optimal cell seeding concentration and
cell growth within this specified volume, preliminary “cell titration” experiments should be done to
determine the optimized cell seeding density and the preferable treatment point in the exponential
growth phase. Once the seeding density has been established, this information is used for toxicity
assessments of the NMs. One would however, include ‘control untreated cells’ that would confirm
expected cell growth and confluency during the toxicity testing experiments.
It is important to note that different seeding densities will produce different proliferation curves (see
[90][93]
Figure A.1) and this would influence at which time point the cells will be treated . As shown in
Figure A.1, the growth profile and optimal seeding density for the two cell lines varies. In Figure A.1a, a
density of 20 000 cells can be used for experimentation where the cells per well are allowed to recover
for 24 h followed by treatment for up to 72 h. In Fig. A.1b, a density of 6 250 cells can be used where the
cells are seeded and allowed to recover for 24 h followed by treatment for up to 5 d.
12 © ISO 2021 – All rights reserved

a)  Adhesion of HMEC-1 endothelial cells
b)  NTera2/D1 (NT2)-astrocytes
Key
X time (h)
Y cell index
1 40 000 cells
2 20 000 cells
3 10 000 cells
4 5 000 cells
5 0 cells
6 12 500 cells
7 6 250 cells
8 3 125 cells
9 1 500 cells
10 750 cells
[90]
NOTE Curves represent the mean cell index value from > 4 wells ±SD .
Figure A.1 — Optimization of ideal seeding quantities
A.5 Reagents
Each instrument requires a particular disposable plate for cell measurements. Test cells are maintained
using growth conditions and supplements required for the cell line and using optimal culture
conditions. Test substances for growth assays are purchased as required and used with appropriate
solvent controls.
As an example, to perform a basic cell titration or toxicity experiment the following reagents are
required:
— cell line of interest, e.g., BEAS-2B cells, CHO cells, HEK 293 cells, HepG2 cells, U937 monocyte-derived
macrophages;
— ECM molecule of choice, e.g. fibronectin, collagen, poly-L-lysine (when necessary);
— appropriate growth medium for culturing cell line of interest, for example, DMEM, EMEM and RPMI;
— appropriate base medium to be used in experiment;
— L-Glutamine (when needed);
— PBS;
— FBS (when needed);
— trypsin-EDTA for cell culture;
— ≥96 % pure BSA (when needed).
A.6 Growth measurements
Growth assay measurements are carried out as per manufacturer’s instructions. As an example, this is
determined by a change in the impedance values, where proliferation and attachment of cells will be
displayed as an increase in CI. The measurements can be set on the “Schedule” page. Experiments can
be divided into multiple steps, and each step consists of a minimum of one sweep. Each sweep is one
measurement across all selected wells and can be set at a desired interval.
A.7 IC estimation
[26]
The system allows for the calculation of time dependent IC values in real-time (see Figure A.2) . For
example, where there are no cells or there is no cell adherence, the CI value is zero. On the other hand,
when there is an increase in cell attachment, the CI values are larger. Thus, CI is related to cell number
present in a well. Moreover, changes in CI values give an indication of the changes in cell morphology,
cell adhesion, or cell viability (see Reference [89]). Using the instrument software, it is possible to
display the standard deviation of the CIs of replicate samples as well as plotting of a dose response
curve and IC value.
14 © ISO 2021 – All rights reserved

Key
X log of concentration (µg/cm )
Y normalized cell index
[26]
Figure A.2 — Dose-response curve and IC value of BEAS-2B cells treated with AgNPs
The software is able to calculate and display IC values of data obtained, where cells were treated with
a compound at different concentrations and can be derived using various methods.
— From the dose response curve, this can be achieved by using the “sigmoidal dose-response equation”
to apply curve-fitting to the data points, from which the IC values are calculated.
— The time dependent change in IC values over a time interval following a compound treatment,
providing kinetic data on the effectiveness and potency of a compound (e.g. compound-induced
cytotoxicity).
A.8 Testing
When adding a test compound (e.g. NM), prepare the dispersion, at a desired concentration, followed by
carefully removing and replacing the old media with fresh media containing the test compound.
The test sample should be pre-equilibrated to room temperature to minimize handling time (to
preferably less than 5 min) as cooling the cells to room temperature will have an impact on cellular
behavior and the CI values. Solution volume in wells should not exceed 200 µl/well.
A.9 Basic experimental design
— Prior to every experiment, control wells should be included to assess whether nanoparticles and
nanofibres cause direct changes of the readings, i.e. in the absence of cells.
— Wells with media only.
— Wells with media and dispersion medium (if not growth media).
— Well with media and the dispersed NM (at the highest concentration).
— Establish a baseline reading for the instrument, by adding 50 µl of supplemented media into each
well of the e-plate, prior to cell seeding.
— Seeding of different cellular models to a final volume of 150 µl supplemented media.
— Allow cells to equilibrate for approximately 30 min prior to being placed into the RTCA station in an
incubator.
— The cells are then allowed to proliferate/mature prior to treatment for appropriate time (the optima
cell number is determined in cell titration experiment), during which time CI scans are acquired
every 15 min.
— Treatment of cells with the NPs, over the selected concentration range, must be done in fresh
supplemented media (maximum volume 200 µl).
o
— Return the E-plate to the RTCA station at 37 C with 5 % CO in a humidified atmosphere.
— Readings are acquired every 5 min for the first 24 h and every 15 min for the remainder of the
experiment.
— Untreated cells receive fresh supplemented media only.
— Control wells containing supplemented media must be included.
— The CI values should be normalized, at the treatment point, to minimize inter-well variability
and allow comparison between wells, for data analysis.
A.10 Data output
Some of the parameters that can be obtained for cell-based assays include:
— average CI (line graph – CI versus time);
— standard deviation of the CI;
— IC values for experimental data, including various methods for obtaining the IC values;
50 50
— time dependent IC value;
— dose-response curves;
— graphical representation of all data indicated.
A.11 Maintenance and cleaning
The manufacturer's instructions for the particular instrument should be followed for maintenance and
cleaning.
16 © ISO 2021 – All rights reserved

Annex B
(informative)
Case studies using standard operating procedure for setting up an
xCELLigence experiment with various cellular models
B.1 Human bronchial epithelial (BEAS-2B) cells — Basic experimental design
— Establish a baseline reading for the instrument, by adding 50 µl of supplemented media into each
well of the e-plate, prior to cell seeding.
— Seeding of different cellular models in a final volume of 150 µl supplemented media: BEAS-2B cells
4 2
are seeded at 10 000 cells/well (5 × 10 cells/cm ).
— Allow cells to equilibrate for approximately 30 min prior to being placed into the RTCA station in an
incubator.
— The cells are then allowed to proliferate prior to treatment for 24 h, during which time CI readings
are acquired every 15 min.
— Treatment of cells with the NPs, over the selected concentration range, must be done in fresh
supplemented media (final volume of 150 µl).
— Return the e-plate to the RTCA station at 37 °C with 5 % CO in a humidified atmosphere.
— Readings are acquired every 5 min for the first 24 h and every 15 min for the remainder of the
experiment.
— Untreated cells receive fresh supplemented media only.
— Control wells containing supplemented media and wells containing highest NP concentration in
supplemented media must be included.
— The CI values should be normalized, at the treatment point, to minimize inter-well variability and
allow comparison between wells, for data analysis.
B.2 Chinese hamster ovary (CHO) cells — Basic experimental design
— Establish a baseline reading for the instrument, by adding 50 µl of supplemented media into each
well of the e-plate, prior to cell seeding.
— Seeding of different cellular models in a final volume of 150 µl supplemented media: CHO cells are
4 2
seeded at 3 000 cells/well (1,5 × 10 cells/cm ).
— Allow cells to equilibrate for approximately 30 min prior to being placed into the RTCA station in an
incubator.
— The cells are then allowed to proliferate prior to treatment for 24 h, during which time CI readings
are acquired every 15 min.
— Treatment of cells with the NPs, over the selected concentration range, must be done in fresh
supplemented media (final volume of 150 µl).
— Return the E-plate to the RTCA station at 37 °C with 5 % CO in a humidified atmosphere.
— Readings are acquired every 5 min for the first 24 h and every 15 min for the remainder of the
experiment.
— Untreated cells receive fresh supplemented media only.
— Control wells containing supplemented media and wells containing highest NP concentration in
supplemented media must be included.
— The CI valu
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