ISO 19007:2018
(Main)Nanotechnologies — In vitro MTS assay for measuring the cytotoxic effect of nanoparticles
Nanotechnologies — In vitro MTS assay for measuring the cytotoxic effect of nanoparticles
ISO 19007:2018 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. ISO 19007:2018 is applicable to the use of a 96-well plate.
Nanotechnologies - Analyse du MTS in vitro pour la mesure de l'effet cytotoxique des nanoparticules
General Information
Standards Content (Sample)
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 19007:2018(E)
ISO 2018
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ISO 19007:2018(E)
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Published in Switzerland
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ISO 19007:2018(E)
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
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ISO 19007:2018(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 on 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 the following
URL: www .iso .org/iso/foreword .html.This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
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ISO 19007:2018(E)
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.
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ISO 19007:2018(E)
Table 1 — Summary of the studies used to develop a normalized MTS assay protocol
Study ID Cell line Nanoparticle Positive and Centrifuge step
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 2and 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.
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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-objects3 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 stateNote 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.© ISO 2018 – All rights reserved 1
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ISO 19007:2018(E)
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 responseNote 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 responseNote 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 phaseNote 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 suspensionPMS phenazine methosulfate
PS polystyrene
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ISO 19007:2018(E)
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 Controls5.3.1 Chemical positive control material, CdSO , shall be used as positive chemical control.
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 4assigned 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].© ISO 2018 – All rights reserved 3
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ISO 19007:2018(E)
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
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ISO 19007:2018(E)
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 nanoparticlesFor 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 Preparations8.1 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 cultureUsing 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.
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ISO 19007:2018(E)
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...
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