ISO 19007:2018

INTERNATIONAL ISO
STANDARD 19007
First edition
2018-04
Nanotechnologies — In vitro MTS
assay for measuring the cytotoxic
effect of nanoparticles
Nanotechnologies - Analyse du MTS in vitro pour la mesure de l'effet
cytotoxique des nanoparticules
Reference number
©
ISO 2018
© ISO 2018
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Published in Switzerland
ii © ISO 2018 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms . 2
5 Materials . 3
5.1 Cell line . 3
5.2 Assay . 3
5.3 Controls . 3
6 Apparatus . 4
7 Nanoparticle test sample preparation . 4
8 Preparations . 5
8.1 General . 5
8.2 Culture medium . 5
8.3 Preparation of cell stock culture . 5
8.4 Verify viable cell growth . 6
8.5 Verification of plate reader uniformity . 6
8.6 Control preparation . 6
8.6.1 Control description . 6
8.6.2 CdSO stock solution preparation (10mM) . 7
8.6.3 Nanoparticle control suspension preparation . 7
8.7 Precision pipetting . 7
9 Characterization of nanoparticle impact on cell viability . 7
9.1 General . 7
9.2 Preparation of the cell plate . 8
9.3 Prepare the nanoparticle dosing plate . 9
9.4 Expose cells to nanoparticles in culture medium .11
9.5 Expose cells to MTS Assay .11
9.6 Measurement of formazan absorbance .12
10 Cell viability analysis.12
11 Interpertation of Assay Results .12
Annex A (informative) Potential cell lines and assays .13
Annex B (informative) Example: the MTS assay using the A549 cell line (EMPA-NIST protocol) .14
Annex C (informative) Example: MTS assay using the RAW 264.7 cell line (IANH protocol) .23
Bibliography .31
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
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For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
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World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www .iso .org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
iv © ISO 2018 – All rights reserved

Introduction
The field of nanotechnologies continues to advance rapidly through the development of new materials,
products and applications. At the same time, many questions have been raised relating to the potential
impact on human health and on the environment of some of these materials. Internationally, a large
program of research is underway to better understand and quantify potential hazards. Also the
chemicals used to coat the surface of nanoparticles in processing or in products can affect the toxicity
of nanoparticles, even more so due to their large surface to volume ratio.
Cellular systems are a fundamental element of living biological systems. It is likely that monitoring toxic
response of cellular model systems to nanoparticle exposure will provide insight into the “modes-of-
action” of nanoparticles and which of them would need to be further investigated for risk assessment.
In 2008, a number of international researchers concluded that some published results of nanomaterial
toxicity could not be replicated across laboratories and that accurate and reproducible nanotoxicology
tests were needed. As a result, the International Alliance for NanoEHS Harmonization (IANH) was
formed with the goal of developing testing protocols that would accurately assess toxicity and
biological interactions of nanoparticles in cellular systems and that these results be reproducible in
any laboratory. The IANH performed round robin characterization of particle size distributions in
liquid suspensions, and in vitro interactions of nanomaterials with cells with the several common
cytotoxicity assays (Annex A). This group identified a number of factors that increased variability and
developed techniques to reduce it. Research funded by the US NIEHS NanoGo further assessed some
of these protocols, in particular, the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
[1]
sulfophenyl)-2H-tetrazolium (MTS) assay protocol . A third team extended the IANH protocol and
performed experiments that employed a systematic plate layout to achieve improved analysis and
[2]
consistency of results (Annex B) . Importantly, each of these protocols used interlaboratory testing
between multiple laboratories to identify sources of variability and improve the assay protocols.
[3]
This document is a method to assess in vitro cell viability with the MTS assay. This assay produces
a colourmetric change (absorption peak at 490 nm) in a culture well due to generation of a formazan
product in the presence of cytoplasmic reductase enzymes. In general, changes in absorption intensity
is directly proportional to cell number although assay conditions that alter reductase activity or
reagent availability can result in colourmetric changes that are not directly due to changes in cell
viability (i.e. cell number). The MTS reagents are directly added to cell culture well which allows rapid
evaluation of potential intrinsic toxicity of nanoparticles. Due to the potential interference effects
that can occur with nanoparticles and colourmetric assays, it is important control experiments with
the nanoparticles and the MTS reagents are performed before the assay results are accepted. Direct
microscopic observation of cells after treatment also provides an orthogonal method to validate an
MTS assay result.  The normalized protocol presented here is limited to adherent cell types, but it could
be modified to be used with suspension cells.
This measurement of toxicity in this assay is a first-tier measurement of nanoparticle effects on
individual cellular systems. The normalized method presented here is based on the three MTS assay
protocols described above. Differences between the experimental systems are described in Table 1.
Table 1 — Summary of the studies used to develop a normalized MTS assay protocol
a
Study ID Cell line Nanoparticle Positive and Centrifuge step
b
tested negative control
materials
IANH RAW-264.7 +PS-NP, CeO CdSO ,no-particle No
2 4
treatment
NanoGo BEAS-2B, RLE-6TN ZnO, TiO , MWCNT No-particle treatment Yes
and THP-1
c +
EMPA-NIST A549 PS-NP CdCl , no-particle No
treatment
a  ATCC Cell Bank Name
b  +PS-NP is a positively charged polystyrene nanoparticle, CeO is cerium oxide, ZnO is zinc oxide, TiO is titanium dioxide,
2 2
and MWCNT is a multiwall carbon nanotube.
c  EMPA is the Swiss Federal Laboratories for Material Science and Technology.
As a result of these differences, some parts in the normalized protocol contains optional steps that
were presented in three interlaboratory studies.
[3]
Several methods can be used for determining cell viability, including MTS, 3-(4,5-dimethylthiazol-
[4]
2-yl)-2,5-diphenyltetrazolium bromide (MTT ), (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-
[5] [6] [7]
tetrazolium-5-carboxanilide) (XTT ), lactate dehydrogenase (LDH ), trypan blue exclusion and
[8]
neutral red assay , The MTS assay was used in a multi-group round robin characterization. The MTS
assay is an improved version of the MTT assay and provides a simple high throughput characterization
[1][9]
for cell viability . The optical density of the MTS assay solution increases upon its reduction by the
functioning cell enzymes in live cells.
Control experiments are required to determine a baseline optical density of cell viability for untreated
cells, and to verify that cells have an expected response to known non-toxic nanoparticles, toxic
[10]
chemicals and toxic nanoparticles as measured with the assay . Furthermore, it is important to
determine whether nanoparticles interfere with the optical readout of the assay and potentially
[11]
invalidate assessment of the nanoparticle cytotoxicity response.
It is important to note that the MTS assay described here is one of many commercially assays available
to assess the cytotoxicity of nanomaterials. Although assays such as the LDH assay which assesses
plasma membrane integrity, the ATP assay which evaluates energy metabolism and the BrdU assay for
DNA synthesis are not discussed here, the results from these assays in addition to the MTS assay allow
for a more comprehensive evaluation of the overall impact of nanoparticles on cells.
vi © ISO 2018 – All rights reserved

INTERNATIONAL STANDARD ISO 19007:2018(E)
Nanotechnologies — In vitro MTS assay for measuring the
cytotoxic effect of nanoparticles
1 Scope
This document specifies a method for evaluating the effects of nano-objects and their aggregates and
agglomerates (NOAA) on cellular viability using the MTS assay. The assay design includes performance
requirements and control experiments to identify and manage variability in the assay results.
This document is applicable to the use of a 96-well plate.
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 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO/TS 80004-2 and the
following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:/ /www.e lectropedia. org/
— ISO Online browsing platform: available at https:/ /www. iso. org/obp
3.1
culture vessel
example assay vessel described in this document based a 96-well tissue culture-grade plate format
Note 1 to entry: Other tissue culture grade vessels (i.e. 384 well plates, 24 well plates, 6 well plates) can be used
interchangeably in these methods provided that they meet the requirements of tissue culture grade and are
suitable for use with mammalian cells.
Note 2 to entry: Adjustments to the protocol such as cell seeding volumes, cell rinsing volumes, and cell dosing
volumes may be required if other tissue culture grade vessels are used during this procedure.
[SOURCE: ISO 10993-5:2009, 3.1]
3.2
dispersion
microscopic multi-phase system in which discontinuities of any state (solid, liquid or gas: discontinuous
phase) are dispersed in a continuous phase of a different composition or state
Note 1 to entry: If solid particles are dispersed in a liquid, the dispersion is referred to as a suspension. If the
dispersion consists of two or more liquid phases, it is termed an emulsion. A superemulsion consists of both solid
and liquid phases dispersed in a continuous liquid phase.
3.3
endotoxin
part of the outer membrane of the cell envelope of Gram-negative bacteria
Note 1 to entry: The main active ingredient is lipopolysaccharides (LPS).
[SOURCE: ISO 29701:2010, 2.3]
3.4
negative control material
material or chemical which, when tested in accordance with this document, does not produce a
cytotoxic response
Note 1 to entry: The purpose of the negative control is to demonstrate the basal level response of the cells. This
control is often composed of the vehicle solvent used to store the nanomaterial in stock concentrations.
[SOURCE: ISO 10993-5:2009, 3.4]
3.5
positive control material
material or chemical which, when tested in accordance with ISO 10993-5, provides a reproducible
cytotoxic response
Note 1 to entry: The purpose of the positive control is to demonstrate an appropriate test system response. For
example, a nanomaterial positive control would be positively charged polystyrene.
[SOURCE: ISO 10993-5:2009, 3.2, modified]
3.6
sedimentation
settling (separation) of the dispersed phase due to the higher density of the dispersed particles
compared to the continuous phase
Note 1 to entry: The accumulation of the dispersed phase at the bottom of the container is evidence that
sedimentation has taken place.
[SOURCE: ISO/TR 13097:2013, 2.13]
3.7
test sample
material that is subjected to biological or chemical testing or evaluation
[SOURCE: ISO 10993-5:2009, 3.5]
4 Symbols and abbreviated terms
cells/mL cells/mL (cells per millilitre)
MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
NPS nanoparticle suspension
PMS phenazine methosulfate
PS polystyrene
2 © ISO 2018 – All rights reserved

5 Materials
5.1 Cell line
Established cell lines are preferred and where used shall be obtained from recognized repositories.
Follow the basic principles of cell culture techniques regarding expanding a frozen stock of cells so that
[12]
the MTS assay for nanocytotoxicity can be performed .
If a stock culture of a cell line is stored, storage shall be at −80 °C or below in the corresponding culture
medium but containing a cryoprotectant, e.g. dimethylsulfoxide or glycerol. Long-term storage (several
months up to many years) is only possible at −130 °C or below.
Only cells free from mycoplasma shall be used for the test. Before use, stock cultures should be tested
for the absence of mycoplasma.
NOTE 1 It is important to check cells regularly [e.g. morphology, doubling time, modal chromosome number,
short tandom repeat (STR) testing] because sensitivity in tests can vary with passage number.
NOTE 2 Nanoparticle can interact with cells through different mechanisms. It is useful to include both a
phagocytic cell line (i.e. macrophage) and a non-phagocytic cell line (i.e. epithelial or fibroblast) in these studies.
Assay results with the use of these two cell types can provide insight into the mode of action for nanoparticle
toxicity.
5.2 Assay
5.2.1 MTS[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium]\PMS- phenazine methosulfate [CAS#138169-43-4].
The reagent is reduced in the presence of cellular enzymes and forms a coloured product that is soluble
in the culture media. The optical density of the culture media is correlated with cell count in a culture
vessel in the absence of artefacts that can occur if the culture conditions affect reductase activity
within the cells and if the nanoparticle causes interference effects in the assay readout. The reagent is
described in Reference [2] and the reagent materials are available from different vendors.
5.3 Controls
5.3.1 Chemical positive control material, CdSO , shall be used as positive chemical control.
+2
NOTE 1 Cd ions are toxic to animals and cells through an oxidative stress mechanism, see Reference [13].
NOTE 2 Cadmium containing compounds, including water soluble compounds such as CdCl and CdSO are
2 4
assigned the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) signal word of Danger.
Cadmium (Cd) is a toxic heavy metal and its disposal and use are regulated in some countries. In the
case where Cd cannot be used as a positive chemical control, an alternative chemical control shall be
selected. The control compound should be soluble in aqueous media, sufficiently stable for the time
course of the experiment and readily available as a purified product from commercial vendors. Non-
metallic chemicals such as phenol, DMSO and detergents such as Tween 80 can be used as positive
chemical controls, with the protocol undergoing additional validation for the use of these chemicals.
5.3.2 Positively charged polystyrene nanoparticles, (diameter 60 nm, dispersed in water) shall be used
as a nanoparticle positive control material. The use of these nanoparticles as positive controls in A549
and Raw 264.7 cells has been validated in interlaboratory studies (see Table 1).
NOTE 1 For dispersion protocol and biological activity of the cationic polystyrene nanoparticles, see
Reference [10].
NOTE 2 Positively charged polystyrene (amine terminated) induce toxic oxidative stress in many cells, see
Reference [14].
NOTE 3 Nanoparticles of quartz, silica and silver are also cytotoxic to many cell types and could be used as
positive controls, see Reference [15].
6 Apparatus
6.1 Incubator, 37 °C ± 1 °C, humidified, 5 % CO /air.
6.2 Flat bottom 96 multi-well plates.
6.3 96 multi-well plates with U bottom, for dosing plate use.
6.4 24 multi-well plates with flat bottom, for cell health and growth rate only.
6.5 96 well plate photometer microtitre plate reader.
6.6 Centrifuge, capable of at least 2 000 g acceleration.
6.7 Multichannel pipette (at least 8 position), with 200 μL volume/pipette.
6.8 Laminar flow cabinet, standard biological hazard.
2 2
6.9 Tissue culture flasks, 25 cm and 75 cm .
6.10 Inverted phase contrast microscope.
6.11 Stereomicroscope.
6.12 Laboratory balance.
6.13 Electronic cell counter or hemocytometer
6.14 Micropipette.
6.15 Vortex mixer.
7 Nanoparticle test sample preparation
Following the basic principle of sample preparation, nanoparticles shall be dispersed in a biologically
compatible fluid with a reproducible procedure. These can include sonication and mixing by vortexing.
Alternatively, nanoparticles can be dispersed with biologically compatible chemical stabilizers,
coatings, such as albumin, or directly in culture medium using the appropriate serum. Specific
dispersion techniques are not discussed in this document. Details for dispersion can be found in the
references cited in the NOTEs and in ISO/TS 19337.
NOTE 1 Several procedures have been published that identify methods to reproducibly disperse
[15][16][17]
nanoparticles and characterize nanosuspensions and their stability. Dispersion protocols from the
NANOGENOTOX Joint Action are publicly available on the internet.
NOTE 2 For biologically compatible chemical stabilizers see Reference [19]. For coatings such as albumin see
Reference [20]. For compatible culture medium, see Reference [21].
NOTE 3 Chemical stabilizers such as albumin can introduce high background levels in cell viability assays. It
is important to use control experiments (i.e. stabilizer only) to determine the effect of the stabilizer on the assay
readout.
4 © ISO 2018 – All rights reserved

With nanoparticles dispersed in a liquid media such as H O, the volume fraction of the nanoparticle
media in the cell culture media shall be below the fraction that is toxic to the cell culture.
The liquid media supporting the nanoparticle suspension can be toxic to cells and cause a false positive
toxicity measurement. Control experiments with liquid media should be performed to determine at
what volume fraction is the liquid media toxic to the cells.
NOTE 1 A 1 mg/1 ml suspension would produce a water content of ~10 % in cell culture media for a 100 μg/ml
exposure. When using water as a dispersion vehicle, a guideline is to keep the final concentration of water below
10 % of the total volume to reduce significant vehicle effects. If higher concentrations of vehicle are required for
nanoparticle dose preparation, it is important to validate the higher concentration of vehicle does not interfere
with the assay results.
The type of suspension process used shall be carefully considered in order to rule out false positive
cytotoxic effects that are not due to the nanoparticles
For nanosuspension stability evaluation, two factors shall be evaluated:
a) stability against agglomeration (reflected in the mean particle size); and
b) stability of the colloidal suspension (reflected by precipitation and sedimentation).
Nanosuspensions should be tested for the presence of endotoxins in accordance with ISO 29701.
8 Preparations
8.1 General
All solutions (except culture medium), glassware, etc., shall be sterile and all procedures should be
performed under aseptic conditions and in the sterile environment of a laminar flow cabinet (biological
hazard standard).
8.2 Culture medium
The culture medium shall be sterile.
The culture medium with or without serum shall meet the growth requirements of the selected cell
line. Antibiotics may be included in the medium provided that they do not adversely affect the assays.
Storage conditions such as refrigerator temperature shall be validated.
NOTE The stability of the culture medium varies with the composition and storage conditions.
8.3 Preparation of cell stock culture
Using the chosen cell line and culture medium, prepare sufficient cells to complete the test. If the cells
are to be grown from cultures taken from storage, remove the cryoprotectant, if present. Subculture
the cells at least once before use.
When subculturing cells, remove and resuspend the cells by enzymatic and/or mechanical
disaggregation using a method appropriate for the cell line. Additional cell line information is in
Annex A.
Good cell culture practices should be used. See Reference [12] additional instructions if required.
8.4 Verify viable cell growth
Prior to performing experiments on nanoparticles, characterize viability and doubling rates of the
cells. Cell growth rates: viability and doubling rates shall be characterized and monitored. Cell viability
should remain > 95 % by using a trypan blue exclusion assay:
a) Grow the cells in 24 well plates for 24 h and 48 h:
1) transfer 200 000 cells/ml in 500 μL of culture medium per well with eight replicates per
time period;
2) use one plate for each time period (24 h and 48 h);
3) gently move the plates into the incubator without agitation to avoid disturbing cell attachment
resulting in non-uniform deposition;
4) verify that incubators have been recently calibrated for: temperature, humidity, CO
concentration. Record metrics in a laboratory notebook to establish charting metrics;
5) at each time point (24 h and 48 h), remove one plate from the incubator;
6) make note of the apparent health and morphology of the cells with a stereomicroscope.
b) Assess cell number and viability with trypan blue:
1) remove culture medium from the wells with gentle pipetting;
2) harvest the cells using the manufacturer’s instructions;
3) collect the culture medium containing cells in a centrifuge tube;
4) spin the supernatant with the added cells in a centrifuge at 400 g for 5 min to form a pellet;
5) discard the supernatant;
6) add 25 μL (0,4 % trypan blue in PBS) to 100 μL culture medium;
7) resuspend the pellet in trypan blue/culture medium with a pipette;
8) deposit the cells on a hemacytometer;
9) record the total number of live and dead (blue) cells and the percent viability (live/total)
by counting the cells in the hemacytometer with a stereomicroscope. See Reference[22] for
details;
10) cell doubling times should be consistent with those expected for the cell line and the percentage
of viable cell through 48 h should be > 95 % prior to continuing with nanoparticle exposure
experiments.
8.5 Verification of plate reader uniformity
Ensure that the instrument is operating properly prior to performing the measurement.
8.6 Control preparation
8.6.1 Control description
Positive control materials shall be CdSO and positively charged polystyrene. See Clause 5 for more
detail on the selection of these positive controls.
Separate experiments shall be performed to determine whether the nanoparticles and antibiotics can
potentially interfere with the assay.
6 © ISO 2018 – All rights reserved

Prepare stock solutions in sterile and endotoxin free ultra pure water (<1,1 μS/cm at 20 °C).
8.6.2 CdSO stock solution preparation (10mM)
Cells shall be exposed to CdSO at final concentrations of 1 μM, 10 μM, 25 μM, 50 μM, and 100 μM.
a) Dissolve and dilute CdSO in ultra pure water to 10mM concentration;
b) Store the 10mM CdSO at 4 °C. Sterile filtration is not required.
8.6.3 Nanoparticle control suspension preparation
Adjust positive polystyrene concentration to 10 mg/ml with ultra pure water.
NOTE At this concentration, the maximum dosing concentration in the test plate (100 µg/ml) will result in
1.0 % (w/v) water vehicle in the cell culture media. This stock preparation, which was used in study described in
Annex B, is used as an example for all the following procedures. If a more dilute nanomaterial is used as a stock
solution, the fraction of vehicle in the cell culture media will be increased. See Clause 7 for preparation of other
nanoparticle suspensions.
8.7 Precision pipetting
A calibrated pipette shall be used to dispense reagents into the 96-well plate used for this assay. If
possible, a calibrated multichannel pipette that is capable of simultaneously dispensing from at least
6 pipette tips during a single ejection in a 96-well plate should be used to perform this procedure. A
previous study indicated that variability in cell seeding between wells using a multichannel pipette is
[21]
lower than the variability from separate pipetting steps.
NOTE With the small volumes of fluids, cells and nanoparticles used in the 96 well experiments, it is
important that the pipette system is carefully calibrated and procedures used to dispense fluids precisely. A
more detailed description of procedures vendor. As an example, Cerionix application note AN1-12 12/06 describes
“Precision- and Accuracy-Based Validation of Pipette Tips Used on Automated Liquid Handling Platforms
Following Multiple Cleanings with ‘Cold’ Atmospheric Plasma”.
9 Characterization of nanoparticle impact on cell viability
9.1 General
Due to potential variability in nanosuspensions, three independent replicate assays should be conducted
on different days with new suspensions. The protocol steps for each assay are summarized in the flow
chart shown in Figure 1.
Figure 1 — A simplified process flow for characterizing the potential cytotoxicity of
nanoparticle dispersions
9.2 Preparation of the cell plate
9.2.1 The cells should be collected, counted and then re-suspended in culture medium at
~7,5 × 10 cells/mL, as verified with a cell counter or hemacytometer and stereomicroscope.
— Cells shall be seeded in culture medium at the appropriate density so that cultures will not reach
confluence by the end of the test;
— Approximately 1 000 000 cells are required for a single plate (~30 % excess).
NOTE The cell concentration described here is based on a cell seeding density of 1.5x10 cells/well in a 96
well plate. This is appropriate for the A549 cells described in Annex B. Depending on the cell type, doubling
time, and MTS reduction activity, this cell seeding density can be validated in preliminary experiments. See
NOTE 1 in 9.5.2.
9.2.2 Transfer culture with cells (200 μL) into each well in columns 3-6 and 8-10, as shown in Figure 2.
9.2.3 Transfer complete cell culture media with no cells (200 μL) into each well in columns 2, 7 and 11,
as shown in Figure 2, hashed wells. Cells shall be seeded at 1,5 × 10 cells/well in each of the clear wells.
Columns 2, 7 and 11 shall have medium alone and column 6 shall have cells alone in the culture medium.
NOTE Wells in a single column are seeded with a single multichannel pipetting step. See 8.7.
8 © ISO 2018 – All rights reserved

Figure 2 — Cell culture plate layout
9.2.4 Gently transfer the plate to a humidified incubator at 37 °C ± 1°C with 5 % CO so cells are evenly
distributed.
a) Incubate the cells for 24 h ± 2 h.
b) After the cell plate has incubated for 24 h, inspect the cells with a microscope to determine whether
the cells are uniformly distributed and also make note of the apparent health and morphology of
the cells.
9.3 Prepare the nanoparticle dosing plate
9.3.1 Figure 3 shows the basic layout of a dosing plate.  Columns 6 and 7 wells shall contain cell culture
media + 1 % H O. Columns 2–5 and columns 8–11 contain positive control material and test sample
nanoparticle doses, respectively. Column 2 and column 11 serve as assay interference wells for both the
positive chemical control and the NP test sample, respectively. The volumes shall be in accordance with
Table 2. The content of these wells shall be transferred to the cell plate with an 8-channel multichannel
pipette steps for each row.
Figure 3 — Dosing plate layout
NOTE The NPS weight percentage shown in Figure 3 is for the +PS-NP suspension described in Clause 7. For
other nanoparticle preparations that require higher weight percentages in the dose preparations, see guidance
in Clause 7 for determining the amount of NP suspension to add to each dose and maximum vehicle percentages.
9.3.2 Dosing plate preparation.
a) Columns 2, 3, 4 and 5 shall receive doses of CdSO in culture medium specified in Table 2.
b) Column 8, 9, 10 and 11 shall receive positively charged polystyrene nanoparticles or test sample
nanoparticles in culture medium with doses specified in Table 2.
c) Column 6 and 7 receive 200 μL of culture medium with 1 % H O.
NOTE 1 The 1 % H O concentration in the cell culture media will depend on the stock nanoparticle
concentration. See Clause 7 for guidance.
NOTE 2 Prepare the nanoparticle dosing plate approximately 3 h before the cell plate is removed from the
incubator. This allows immediate transfer of the contents in the dosing plate to the cells after removal of media
from the cells. This also reduces long term precipitation effects that can occur when the NP are suspended in the
cell culture media.
10 © ISO 2018 – All rights reserved

Table 2 — Preparation of culture media, chemicals and nanoparticles (NP) in dosing plate
Row Letter Complete Cell CdSO positive Positive charged PS Test sample NP (1 %
Culture Media (µL) control material (100 µg/ml) in suspension concen-
in Columns 2–5 and (100 mmol/L) in complete cell culture tration) in complete
8–11 complete cell culture media in column 4 cell culture media in
media in columns 2 (µL) columns 8–11
and 3 (µL)
(µL)
B 200 0 0 0
C 198 2 2 2
D 180 20 20 20
E 150 50 50 50
F 100 100 100 100
G 0 200 200 200
9.4 Expose cells to nanoparticles in culture medium
9.4.1 After 24 h incubation of the cell plate, remove cell culture media from each well, with a pipette
positioned at the bottom edge of the well.
9.4.2 The contents of each column in the dosing plate shall be carefully transferred to the cell plate
with a pipette set to 200 μL (e.g. a multichannel pipette).
9.4.3 The plates shall then be transferred to the incubator for 24 h.
NOTE If extended incubation times including 48 h or 72 h are used for the assay, then the protocol steps in
this document are validated for these incubation times.
9.4.4 After the indicated exposure time, remove the plates from the incubator.
9.4.5 Remove the dosing treatments and non-adhered unhealthy cells using a pipette and gentle
aspiration.
9.5 Expose cells to MTS Assay
9.5.1 Transfer 200 μL of the MTS (317 μg/ml) reagent in culture medium into each column of the
cell plate.
Do not use the expel step in the pipetting procedure to prevent the formation of air bubbles.
9.5.2 Incubate the cells with the MTS reagent in the dark for 60 min at 37 °C in a humidified, 5 % CO
atmosphere.
NOTE 1 The incubation time might vary depending on the cell type, cell concentration, and MTS reagent
concentration. This is especially true if primary cells are used. For a new cell type, preliminary experiments are
performed to determine the appropriate incubation time. For example, the time required for a culture well of
non-treated cells to have an optical density between 1.0 and 2.0 (490 nm) after the addition of the MTS reagent
are used as an appropriate incubation time.
NOTE 2 The MTS reagent is relatively non-toxic to cells and can be directly added to the NP exposure media
without the use of the separate media removal step described in this document. Although this is a simplification
of the procedure, this modification is only be useful for NP suspensions that do not introduce optical absorption
artefacts. This modification is only valid for the use with a particular nanoparticle.
9.6 Measurement of formazan absorbance
9.6.1 Set the plate reader so that raw absorbance measurements are recorded for every well in the plate.
9.6.2 Absorbance shall be measured at 490 nm using a plate reader.
9.6.3 Measure the absorbance in each well. Take note of any wells that have air bubbles.
NOTE 1 An additional procedure that can be used to detect optical path issues in the culture wells (e.g. air
bubbles, fingerprints, etc) is to take absorbance measurements at a reference wavelengths that does not detect
the MTS reagent (e.g. 650 nm). Significant variations between the wells for this reference measurement could
indicate the presence of an optical path obstruction in a well. Such a procedure is validated before it is used as
part of the protocol.
NOTE 2 The protocol from the NanoGo interlaboratory comparison of nanoparticle cytotoxicity used a
separate centrifugation step to remove any residual NP from the MTS cell culture media mixture before it is read
on a absorbance plate reader. After incubation with the MTS reagent, the plate was centrifuged at 2000g for 10
[1]
min and the supernatants were transferred to a new plate and read on the plate reader . The protocol in this
document can be modified with this procedure, but the procedure undergoes validation before general use.
10 Cell viability analysis
Perform a simple spreadsheet-based analysis of the toxicity data.
a) The average background level for nanoparticles in culture medium (from column 11) shall be
subtracted from each well.
b) The resulting absorbance from each dosing well in a technical replicate shall be normalized to the
absorbance value in the no-treatment well in the technical replicate (row B). These background-
subtracted and normalized values represent the fraction of cells that remain after dose treatment.
c) After normalization, the values from a single row in the technical replicates (i.e. columns 3-5 or
8-10) shall be averaged and standard deviations for each dosing condition shall be determined.
d) If toxicity was observed, estimate an EC value and and a 95 % confidence interval from the data
set using GraphPad Prism or other statistical software applications.
If a treatment causes enhanced cell proliferation, viability will appear to increase. Additional
proliferation assays such as BrdU or cell counting should be used to confirm this result.
Assement of potential optical path interference artefacts due to NP dosing can occur by evaluating the
mean and variance of the absorbance of wells in column 11. If the mean absorbance and absorbance
variance is significantly different than the MTS reagent wells (column 7), it may suggest that the NP are
[23]
introducing interference artefacts.
11 Interpertation of Assay Results
The MTS assay is a screening assay and provides a rapid method to assess potential toxic interactions
with biological cells. In general, a single screening assay is not adequate to fully interpertate the
toxicity of a nanoparticle. The results of this assay should be used with the results of other assays
to potentially understand the mechanism of action of toxicity and how the data can be used in a risk
assessment model.
12 © ISO 2018 – All rights reserved

Annex A
(informative)
Potential cell lines and assays
Cell lines that are applicable to use with the MTS assay include: mouse macrophage; Abelson murine
leukaemia virus transformed (RAW 264.7), human lung epithelial cell (A549), human bronchial
epithelial (BEAS-2B), rat alveolar type II epithelial (RLE-6TN), mouse liver epit
...