ISO 19007:2018

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

• Status: Published
• Publication Date: 09-Apr-2018
• ICS: 07.030 - Physics. Chemistry
• ICS: 07.120 - Nanotechnologies
• Technical Committee: ISO/TC 229 - Nanotechnologies
• Drafting Committee: ISO/TC 229 - Nanotechnologies
• Current Stage: 9020 - International Standard under periodical review
• Start Date: 15-Apr-2023
• Completion Date: 15-Apr-2023

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
---------------------- Page: 1 ----------------------
ISO 19007:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

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: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
---------------------- Page: 2 ----------------------
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

© ISO 2018 – All rights reserved iii
---------------------- Page: 3 ----------------------
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.
iv © ISO 2018 – All rights reserved
---------------------- Page: 4 ----------------------
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.

© ISO 2018 – All rights reserved v
---------------------- Page: 5 ----------------------
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 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
---------------------- Page: 6 ----------------------
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.
© ISO 2018 – All rights reserved 1
---------------------- Page: 7 ----------------------
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 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
---------------------- Page: 8 ----------------------
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 Controls

5.3.1 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 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].
© ISO 2018 – All rights reserved 3
---------------------- Page: 9 ----------------------
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
---------------------- Page: 10 ----------------------
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 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.

© ISO 2018 – All rights reserved 5
---------------------- Page: 11 ----------------------
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
...