ASTM E2525-08(2013)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: E2525 − 08 (Reapproved 2013)
Standard Test Method for
Evaluation of the Effect of Nanoparticulate Materials on the
Formation of Mouse Granulocyte-Macrophage Colonies
This standard is issued under the fixed designation E2525; 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 2.2 ANSI Standard:
ANSI/ AAMI ST72 Bacterial Endotoxins—Test
1.1 This test method provides a protocol for quantitative
Methodologies, Routine Monitoring, and Alternatives to
analysis of the effect of nanoparticulate materials in physi-
Batch Testing
ologic solution on granulocyte-macrophage colony-forming
units.
3. Terminology
1.2 This test method employs murine bone marrow he-
3.1 Abbreviations:
matopoietic stem cells which proliferate and differentiate to
3.1.1 BM—bone marrow
form discrete cell clusters or colonies which are counted.
3.1.2 CFU-GM—colony forming unit of granulocyte and
1.3 This test method is part of the in vitro preclinical
macrophage
characterization cascade for nanoparticulate materials for sys-
3.1.3 Cisplatin—positive control
temic administration in medical applications.
3.1.4 DMSO—dimethyl sulfoxide
1.4 The values stated in SI units are to be regarded as
3.1.5 DPBS—Dulbecco’s phosphate buffered saline
standard. No other units of measurement are included in this
3.1.6 FBS—fetal bovine serum
standard.
3.1.7 IMDM—Iscove’s media
1.5 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
3.1.8 LPS—ipopolysaccharide
responsibility of the user of this standard to establish appro-
3.1.9 Physiologic Solution—isotonic, pH 7.2 6 0.2
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
4. Summary of Test Method
1.6 This international standard was developed in accor-
4.1 The effect of nanoparticulate materials on the formation
dance with internationally recognized principles on standard-
of granulocyte and macrophage colonies is assessed. Bone
ization established in the Decision on Principles for the
marrow cells are obtained from mice and cultured in stimula-
Development of International Standards, Guides and Recom-
tory media. The number of colony forming units following
mendations issued by the World Trade Organization Technical
contactwithnanoparticlesiscountedandcomparedtobaseline
Barriers to Trade (TBT) Committee.
and positive control. This determines if the nanoparticulate
material in physiologic solution is stimulatory or inhibitory to
2. Referenced Documents
bone marrow stem cells. Aseptic procedures are necessary.
2.1 ASTM Standards:
F1903Practice for Testing For Biological Responses to 5. Significance and Use
Particles In Vitro
5.1 Stemcellsofhematopoieticoriginarepluripotentialand
may be particularly sensitive to the effects of stimulation by
nanoparticulate materials.
This test method is under the jurisdiction of ASTM Committee E56 on
5.2 The effect of particles on macrophage responses has an
Nanotechnology and is the direct responsibility of Subcommittee E56.03 on
extensive history and can be assessed by Practice F1903. The
Environment, Health, and Safety.
Current edition approved Sept. 1, 2013. Published September 2013. Originally test method described here will assess the effect on stem cells
approved in 2008. Last previous edition approved in 2008 as E2525 – 08. DOI:
which can be progenitor cells to the macrophage line.
10.1520/E2525-08R13.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on Available fromAmerican National Standards Institute (ANSI), 25 W. 43rd St.,
the ASTM website. 4th Floor, New York, NY 10036, http://www.ansi.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2525 − 08 (2013)
6. Reagents and Materials 7. Procedure
6.1 Purity of Reagents—Reagent grade chemicals shall be 7.1 Aseptic Precautions are required.
used in all tests. Unless otherwise indicated, it is intended that
7.2 Reagent and Control Preparation:
all reagents conform to the specifications of the Committee on
7.2.1 MethoCult Medium—The MethoCult medium is sup-
Analytical Reagents of theAmerican Chemical Society where
plied in 100-mL size batches. It is recommended by manufac-
such specifications are available. Other grades may be used,
turer that the medium be thawed at room temperature or in a
provided it is first ascertained that the reagent is of sufficiently
refrigeratorovernight,vortexedtomixtheingredients,thenleft
high purity to permit its use without lessening the accuracy of
at a room temperature for approximately 5 min to allow air
the determination.
bubbles to dissipate. If using another source of media, follow
6.2 Reagents and Supplies: the instructions indicated by the supplier. Use a 16-gauge
6.2.1 MethoCult medium, StemCell Technologies Inc cat.#
blunt-end needle to dispense 3-mL aliquots of the MethoCult
03534. medium into sterile 15-mL tubes. Store the aliquoted medium
6.2.2 Fetal Bovine Serum prescreened for hematopoietic
at a nominal temperature of –20°C. Before the test thaw the
stem cells, StemCell Technologies Inc cat.# 06200.
required number of tubes at room temperature for approxi-
6.2.3 IMDM with 2% FBS, StemCell Technologies Inc
mately 20 min and keep on ice prior to use. Repeated freezing
cat.# 07700.
and thawing should be avoided.
6.2.4 Sterile distilled water.
7.2.2 50 mM Cisplatin (Positive Control)—Cisplatin is sup-
6.2.5 Cisplatin, (positive control) Sigma cat# P4394.
plied in a lyophilized form. Reconstitute the lyophilized
2+ 2+
6.2.6 Sterile Ca /MG -free DPBS, (negative control)
powderbyaddingtheappropriateamountofDMSOtomakea
Sigma cat.# D8537.
stock solution with nominal concentration of 50 mM. Prepare
small aliquots and store at a nominal temperature of –80 °C.
NOTE 1—The source of the reagents is shown for information purposes
Prior to use in the assay, thaw an aliquot of the stock solution
onlytoaidlaboratoriesinitiatingthisprocedure.Equivalentreagentsfrom
other suppliers may be used. at room temperature and dilute in IMDM supplemented with
2% FBS to bring the Cisplatin concentration to 2 mM. One
6.3 Equipment—Aseptic procedures are necessary and care
hundred fifty (150) µL of this intermediate solution is then
should be used in acquiring sterile equipment as needed.
added to 3 mL of culture medium. Final concentration of
6.3.1 Pipettes covering the range of 0.05 to 10 mL.
Cisplatin in the positive control sample is 100 µM.
6.3.2 35-mmculturedishesprescreenedtosupportstemcell
growth and differentiation, StemCell Technologies Inc cat.#
7.3 Preparation of Study Samples:
27100.
7.3.1 This assay requires 1200 µL of nanoparticles, 150 µL
6.3.3 Blunt-end 16-gauge needles, StemCell Technologies
samples in duplicate for each of 4 concentrations. The nano-
Inc cat.# 03534.
particles subjected to the biological test environment should
6.3.4 100-mm Petri dishes.
have been characterized as appropriate to allow adequate data
6.3.5 Plastic beakers.
interpretation and to help provide information to predict
6.3.6 Polypropylene tubes 50 and 15-mL.
biological responses. For example, lot-to-lot variations in
6.3.7 Centrifuge.
particle size and surface characteristics of the particles could
6.3.8 Refrigerator, 2 to 8°C.
result in different assay results. For this assay, the particles
6.3.9 Freezer, –20°C.
shall be provided in physiologic solution (isotonic with pH
6.3.10 Cell culture incubator with 5% CO and 95%
2 7.2 60.2) and this solution shall be defined. The preparation
humidity.
shallbesterileandthelevelofLPSprovidedordeterminedby
6.3.11 CO euthanasia box, or appropriate equipment ap-
2 the testing laboratory.ANSI/AAMI ST72 may be helpful. The
proved by institution.
number of particles/mL and mg/mL shall be indicated.
6.3.12 Scissors for tissue dissection.
7.3.2 The test sample shall be used at the highest concen-
6.3.13 Forceps.
tration possible and at three serial one to five (1:5) dilutions.
6.3.14 Biohazard safety cabinet approved for level II han-
Thehighestpossibleconcentrationisthatatwhichtheparticles
dling of biological material.
appear evenly dispersed in the liquid. If the concentration for
6.3.15 Inverted microscope.
its intended use is known, this may serve as the highest
6.3.16 Vortex.
concentration to be tested and at least three dilutions made.
6.3.17 Hemocytometer.
7.4 Isolation and Counting of Bone Marrow Ce
...