ISO/TR 22455:2021

TECHNICAL ISO/TR
REPORT 22455
First edition
2021-11
Nanotechnologies — High throughput
screening method for nanoparticles
toxicity using 3D model cells
Nanotechnologies — Méthode de criblage à haut débit de la toxicité
des nanoparticules utilisant des systèmes cellulaires 3D
Reference number
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Background . 2
4.1 General . 2
4.2 Effects of optical properties of NPs on in vitro cell viability assays . 2
4.3 New assay platform for in vitro toxicity screening of NPs diminishing optical
interference . 4
4.4 Characteristics of 3D model cells . 7
4.5 Cell viability in response to NPs assessed using 3D model cells on a pillar insert . 9
4.6 Cellular uptake of NPs using 3D model cells on a pillar insert .13
4.7 Discussion of alternative strategies to evaluate in vitro toxicity testing of NPs . 16
5 Methods for cell viability screening of NPs using 3D-model cells .17
5.1 General . 17
5.2 Cell culture . 17
5.3 Preparation of the pillar insert for in vitro screening . 17
5.4 Encapsulation of cells on a micropillar chip to generate 3D-model cells . 18
5.5 NPs sample preparation . 18
5.6 Exposing 3D-model cells to NPs . 18
5.7 Cell viability analysis using a WST assay . 19
5.8 Cell viability analysis using live-cell imaging . 19
Bibliography .21
iii
Foreword
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This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
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iv
Introduction
With an increasing number of nano-products including nanoparticles (NPs), potential exposure of
consumers to NPs has increased. Therefore, the human and environmental impacts of NPs have recently
emerged as an issue. High-throughput screening (HTS) approaches are often used for NPs toxicity
screening. However, there are still limitations to provide the reproducible and reliable results based on
a HTS method. To assess the potential toxicity of manufactured or engineered NPs, traditional in vitro
toxicity studies have been performed using a surface attached two-dimensional (2D) culture system.
2D assays for cellular metabolic activity, cytotoxicity, or oxidative stress have been widely used in the
first stage of hazard evaluation. However, several problems were encountered during assay validation,
ranging from particle agglomeration in biological media to optical interference with the assay platform.
There are ISO documents on the cytotoxic effects of NPs using cell viability assays and detection
of reactive oxygen species (ROS) levels, but they can be applicable for a few classes of NPs that are
well-dispersed in the media. Additionally, reagents used in the assays can interact with tested NPs or
interfere with spectrophotometric reading.
This document describes a new assay platform, consisting of three-dimensional (3D) arrangement of
cells on pillar inserts to evaluate cell viability and diminish artefacts arising from optical interferences
and NP reactivity with assay components.
This document aims to overcome the optical interference of NPs and obtain reliable and reproducible
cell viability results. The 3D-model cells are exposed to fresh cell viability reagent by simply transferring
and immersing the pillar insert from one well to another well without optical interference from the
NPs. In addition, 3D-model cell culture approaches facilitate cell-cell interactions and enhance cell-to-
cell or cell-to-extracellular matrix (ECM) adhesion/signalling, ultimately leading to the expression of
phenotypic proteins/genes and the formation of in vivo tissue-like morphology. It generates uniform
cell-containing hydrogel droplets on the pillar insert and allows to easily change cell growth media
or expose 3D-model cells to analytical reagents by immersing the tip of the pillar insert in different
reaction plates.
v
TECHNICAL REPORT ISO/TR 22455:2021(E)
Nanotechnologies — High throughput screening method
for nanoparticles toxicity using 3D model cells
1 Scope
This document describes a method for high throughput evaluation of cytotoxic response of 3D model
cells exposed to NPs without optical interference.
The method in this document is intended to be used in biological testing laboratories that are competent
in the culture and growth of cells and the evaluation of cytotoxicity of NPs using 3D-model cells.
This method applies to materials that consist of nano-objects such as nanoparticles, nanopowders,
nanofibres, nanotubes, and nanowires, as well as aggregates and agglomerates of these materials.
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 terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
agglomerate
collection of weakly bound particles or aggregates or mixtures of the two where the resulting external
surface area is similar to the sum of the surface areas of the individual components
Note 1 to entry: The forces holding an agglomerate together are weak forces, for example, van der Waals forces,
or simple physical entanglement.
Note 2 to entry: Agglomerates are also termed secondary particles, and the original source particles are termed
primary particles.
[SOURCE: ISO/TS 80004-2:2015, 3.4, modified — "weakly or medium strongly bound particles" has
been replaced with " weakly bound particles or aggregates or mixtures of the two".]
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 super emulsion consists of both solid
and liquid phases dispersed in a continuous liquid phase.
[SOURCE: ISO 19007:2018, 3.2]
3.3
nano-object
material with one, two or three external dimensions in the nanoscale (3.5)
Note 1 to entry: This is a generic term for all discrete nanoscale objects.
[SOURCE: ISO/TS 80004-2:2015, 2.2, modified — "discrete piece of" has been added to the definition
and Note 1 to entry has been replaced.]
3.4
nanoparticle
NP
nano-object (3.3) with all three dimensions in the nanoscale (3.5)
Note 1 to entry: If the lengths of the longest to the shortest axes of the nano-object differ significantly (typically by
more than three times), the terms nanorod or nanoplate are intended to be used instead of the term nanoparticle.
3.5
nanoscale
size range from approximately 1 nm to 100 nm
Note 1 to entry: Properties that are not extrapolations from a larger size will typically, but not exclusively, be
exhibited in this size range. For such properties, the size limits are considered approximate.
Note 2 to entry: The lower limit in this definition (approximately 1 nm) is introduced to avoid single and small
groups of atoms from being designated as nano-objects (3.3) or elements of nanostructures, which can be implied
by the absence of a lower limit.
3.6
high-throughput screening
method that comprises the screening of a large number of chemicals via automation, miniaturized
assays and large-scale data analysis
Note 1 to entry: This protocol can be applied to screen the toxicity of NPs based on 96-well plate or 532 microchip.
4 Background
4.1 General
With the increase in the number of consumer products containing NPs, potential exposure to NPs has
increased, and potential human and environmental hazards of NPs have emerged. To assess the effects
of NPs, a high-throughput screening method to evaluate cell viability following exposure to NPs is
[1]
needed. High-throughput approaches have been used to screen for toxicity of manufactured NPs .
4.2 Effects of optical properties o
...