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

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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/TS 21633:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 21633:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 21633:2021(E)
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

© ISO 2021 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO/TS 21633:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

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

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/

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

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
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

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
© ISO 2021 – All rights reserved 1
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ISO/TS 21633:2021(E)
3.5
nanoparticle

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
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
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ISO/TS 21633:2021(E)
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
© ISO 2021 – All rights reserved 3
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ISO/TS 21633:2021(E)
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
---------------------- Page: 8 ----------------------
ISO/TS 21633:2021(E)

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
© ISO 2021 – All rights reserved 5
---------------------- Page: 9 ----------------------
ISO/TS 21633:2021(E)
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

(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

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
Z = (2)
()2πfC
[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

−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

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
---------------------- Page: 10 ----------------------
ISO/TS 21633:2021(E)
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.

© ISO 2021 – All rights reserved 7
---------------------- Page: 11 ----------------------
ISO/TS 21633:2021(E)
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

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
---------------------- Page: 12 ----------------------
ISO/TS 21633:2021(E)

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.
© ISO 2021 – All rights reserved 9
---------------------- Page: 13 ----------------------
ISO/TS 21633:2021(E)
[88]
The CI values are then calculated as follows :
Rf() 
cell i
I =max −1 (3)
Cii=…1, N  
 
...

TECHNICAL ISO/TS
SPECIFICATION 21633
First edition
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
PROOF/ÉPREUVE
Reference number
ISO/TS 21633:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO/TS 21633:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii PROOF/ÉPREUVE © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/TS 21633:2021(E)
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) Basics 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

© ISO 2021 – All rights reserved PROOF/ÉPREUVE iii
---------------------- Page: 3 ----------------------
ISO/TS 21633:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

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

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

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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, in whole or in part, are normatively referenced in this document and are

indispensable for its application. 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 terminological 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

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
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ISO/TS 21633:2021(E)
3.5
nanoparticle

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
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
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ISO/TS 21633:2021(E)
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
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ISO/TS 21633:2021(E)
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 contains 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.
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ISO/TS 21633:2021(E)

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 can be assessed with different cell lines using this technique, 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
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ISO/TS 21633:2021(E)
6 Basic principles, instruments
6.1 Basics of electrochemical impedance technique

In the EIS system, an electrical equivalent circuit is used to curve 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

(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

C + R + C where the impedence 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
Z = (2)
()2πfC
[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

−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

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.
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ISO/TS 21633:2021(E)
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.

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ISO/TS 21633:2021(E)
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

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.

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ISO/TS 21633:2021(E)

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 indeces. 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.
© ISO 2021 – All rights reserved PROOF/ÉPREUVE
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