ASTM E2526-08

Standard Test Method for Evaluation of Cytotoxicity of Nanoparticulate Materials in Porcine Kidney Cells and Human Hepatocarcinoma Cells

SIGNIFICANCE AND USE
Assessing the propensity of a nanomaterial to cause cytotoxicity to the cells of a target organ can assist in preclinical development.
The standard historical cytotoxicity testing of materials and extracts of materials has used fibroblasts and is well documented in Practice F 813, Test Method F 895, and ISO 10993-5. The use of macrophages and micron size particles has also provided information on cytotoxicity and stimulation using Practice F 1903.
This test method adds to the cytotoxicity test protocols by using target organ cells. Two quantitative assays measuring LDH leakage and MTT reduction are used to estimate cytotoxicity.
This test method may not be predictive of events occurring in all types of nanomaterial applications and the user is cautioned to consider the appropriateness of the test for various types of nanomaterial applications. This procedure should only be used to compare the cytoxicity of a series of related nanomaterials. Meaningful comparison of unrelated nanomaterials is not possible without additional characterization of physicochemical properties of each individual nanomaterial in the assay matrix.
SCOPE
1.1 This test method provides a methodology to assess the cytotoxicity of suspensions of nanoparticulate materials in porcine proximal tubule cells (LLC-PK1) and human hepatocarcinoma cells (Hep G2) which represents potential target organs following systemic administration
1.2 This test method is part of the in vitro preclinical characterization cascade.
1.3 This test method consists of a protocol utilizing two methods for estimation of cytotoxicity, 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

General Information

• Status: Historical
• Publication Date: 31-Jan-2008
• ICS: 07.100.10 - Medical microbiology
• Technical Committee: E56 - Nanotechnology
• Drafting Committee: E56.03 - Environment, Health, and Safety

Standards Content (Sample)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: E2526 − 08
StandardTest Method for
Evaluation of Cytotoxicity of Nanoparticulate Materials in
Porcine Kidney Cells and Human Hepatocarcinoma Cells
This standard is issued under the fixed designation E2526; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 3. Terminology
1.1 This test method provides a methodology to assess the 3.1 Abbreviations:
cytotoxicity of suspensions of nanoparticulate materials in 3.1.1 APAP—acetaminophen- positive control
porcine proximal tubule cells (LLC-PK1) and human hepato-
3.1.2 DMSO—dimethyl sulfoxide
carcinoma cells (Hep G2) which represents potential target
3.1.3 DMEM—Dulbelcco’s modified eagles media
organs following systemic administration
3.1.4 Hep G2—human hepatocarcinoma cells
1.2 This test method is part of the in vitro preclinical
3.1.5 LDH—lactic dehydrogenase
characterization cascade.
3.1.6 LLC-PK1—porcine proximal tubule cells
1.3 This test method consists of a protocol utilizing two
3.1.7 LPS—lipopolysacchride, bacterial endotoxin
methods for estimation of cytotoxicity, 3-(4,5-
Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT)
3.1.8 MTT—3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetra-
reduction and lactate dehydrogenase (LDH) release.
zolium bromide
1.4 This standard does not purport to address all of the
3.1.9 Physiologic solution—isotonic with a pH 7.2 6 0.2
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro- 4. Summary of Test Method
priate safety and health practices and determine the applica-
4.1 Nanoparticulate test materials in suspension in cell
bility of regulatory limitations prior to use.
culture media and appropriate controls are added to cell
cultures.The release of LDH indicates membrane damage and
2. Referenced Documents
thediminutionofMTTreductionindicateslossofcellviability.
2.1 ASTM Standards: These are quantitative indicators of cytotoxicity. Aseptic pro-
F813Practice for Direct Contact Cell Culture Evaluation of
cedures are required.
Materials for Medical Devices
5. Significance and Use
F895TestMethodforAgarDiffusionCellCultureScreening
for Cytotoxicity
5.1 Assessing the propensity of a nanomaterial to cause
F1877Practice for Characterization of Particles
cytotoxicity to the cells of a target organ can assist in
F1903Practice for Testing For Biological Responses to
preclinical development.
Particles In Vitro
5.2 The standard historical cytotoxicity testing of materials
2.2 ISO Standard:
and extracts of materials has used fibroblasts and is well
ISO10993-5BiologicalEvaluationofMedicalDevices:Part
documented in Practice F813, Test Method F895, and ISO
5 Tests for in vitro Cytotoxicity
10993-5.Theuseofmacrophagesandmicronsizeparticleshas
also provided information on cytotoxicity and stimulation
using Practice F1903.
This test method is under the jurisdiction of ASTM Committee E56 on
5.3 This test method adds to the cytotoxicity test protocols
Nanotechnology and is the direct responsibility of Subcommittee E56.03 on
by using target organ cells.Two quantitative assays measuring
Environment, Health, and Safety.
CurrenteditionapprovedFeb.1,2008.PublishedFebruary2008.DOI:10.1520/
LDH leakage and MTT reduction are used to estimate cyto-
E2526-08.
toxicity.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
5.4 This test method may not be predictive of events
Standards volume information, refer to the standard’s Document Summary page on
occurringinalltypesofnanomaterialapplicationsandtheuser
the ASTM website.
is cautioned to consider the appropriateness of the test for
Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
4th Floor, New York, NY 10036, http://www.ansi.org. various types of nanomaterial applications. This procedure
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2526 − 08
should only be used to compare the cytoxicity of a series of 7.2.2 Hepatocyte Acetaminophen (APAP) positive control:
related nanomaterials. Meaningful comparison of unrelated 20 mM APAP in RPMI 1640 cell culture media.
nanomaterials is not possible without additional characteriza-
7.2.3 Triton X100 is diluted to 1% in cell culture medium.
tion of physicochemical properties of each individual nanoma- This is the positive control for the LDH assay.
terial in the assay matrix.
7.3 MTT Assay Reagents:
7.3.1 MTT solution-5mg/mL MTT in PBS, store for up to
6. Reagents and Materials
one month at 4°C in the dark.
6.1 Purity of Reagents—Reagent grade chemicals shall be
7.3.2 Glycine Buffer-0.1M glycine (MW 75.07), 0.1 M
used in all tests. Unless otherwise indicated, it is intended that
NaCl (MW 58.44), pH 10.5, store at room temperature.
all reagents conform to the specifications of the Committee on
7.4 Biovision LDH-Cytotoxicity Assay Kit Reagents:
Analytical Reagents of theAmerican Chemical Society where
7.4.1 Reconstitute catalyst in 1 mL dH 0 for 10 min and
such specifications are available. Other grades may be used, 2
vortex (stable for 2 weeks at 4°C).
provided it is first ascertained that the reagent is of sufficiently
7.4.2 Reactionmixture(forone96-wellplate):Add250mL
high purity to permit its use without lessening the accuracy of
of reconstituted, catalyst solution to 11.25 mL of dye solution
the determination.
(stable for 2 weeks at 4°C).
6.2 Reagents and supplies (aseptic procedures are needed
7.4.3 For other LDH Cytotoxicity assay kits, follow their
andcareshouldbetakentousesterilereagentsandsuppliesas
instructions.
necessary).
7.5 Cell Culture:
6.2.1 MTT (3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetra-
zolium bromide). 7.5.1 LLC-PK1 Cell Preparation:
6.2.2 Acetaminophen. 7.5.1.1 Harvest cells from flasks prepared from cryopre-
6.2.3 Dimethyl sulfoxide. served cells according to the instructions from the supplier
6.2.4 Glycine. (limit passages to 20). An example of the appearance of the
6.2.5 Sodium Chloride. cells is in Fig. 1.
6.2.6 Medium 199 Cell Culture Media. 7.5.1.2 Count cell concentration using a Coulter type coun-
6.2.7 Triton X 100. ter or hemocytometer.
6.2.8 LDH-CytotoxicityAssay Kit (Biovision Cat. # K311- 7.5.1.3 Dilute cells to a density of 2.5 × 10 cells/mol in
400 was used in developing this test method)*
M199 (3% FBS) cell culture media.
6.2.9 96 well flat bottom cell culture plates.
7.5.1.4 4 Plate 100 µL cells/well as per plate format de-
6.2.10 RPMI 1640.
scribed in Fig. 3 for 4 plates (time zero, 6 hour sample
6.2.11 L-glutamine.
exposure,24hoursampleexposure,48hoursampleexposure).
6.2.12 Fetal bovine serum (FBS).
The format indicates no cells in rows D&E and they serve as
particle controls. Each plate accommodates two samples
6.3 Cell Lines:
(RowsA-CandF-H).Eachnanoparticulatematerialistestedat
6.3.1 LLC-PK1(porcineproximaltubulecell)(ATCC#CL-
9 dilutions. Column 11 receives the positive control and
101)*
column 12 receives Triton X 100.
6.3.2 Hep G2 (human hepatocarcinoma)(ATCC # HB-
7.5.1.5 Incubate plates for 24 hours at 5% CO , 37°C and
8065)*
95%humidity(cellsshouldbeapproximately80%confluent).
6.4 Equipment:
7.5.2 Hep G2 Cell Preparation:
6.4.1 Plate reader.
7.5.2.1 Harvest cells from flasks prepared from cryopre-
6.4.2 Plate Centrifuge set at 700-800 g.
served cells according to the instructions from the supplier.
6.4.3 Cell Culture Microscope.
(limit passages to 20). An example of the appearance of the
NOTE 1—Commercial sources are indicated for informational purposes
cells is in Fig. 2.
only to aid laboratories initiating these test procedures. This does not
7.5.2.2 Count cell concentration using a Coulter type coun-
indicate endorsement by ASTM. Other equivalent sources may be
ter or hemocytometer.
available.
7.5.2.3 Dilute cells to a density of 5.0 × 10 cells/mol in
7. Experimental Procedure
RPMI 1640 (2 mM L-glutamine, 10% FBS) cell culture
media.
7.1 Aseptic precautions are required.
7.5.2.4 Plate100µLcells/wellasperplateformatdescribed
7.2 Positive Control Preparation:
in Fig. 3 for 4 plates (time zero, 6 hour sample exposure, 24
7.2.1 LLC-PK1 Acetaminophen (APAP) positive control:
hour sample exposure, 48 hour sample exposure). The format
25 mM APAP in M199 cell culture media.
ind
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