ASTM E3238-20

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3238 − 20
Standard Test Method for
Quantitative Measurement of the Chemoattractant Capacity
of a Nanoparticulate Material in vitro
This standard is issued under the fixed designation E3238; 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 of Nanomaterials in Suspension by Photon Correlation
Spectroscopy (PCS)
1.1 This test method provides a protocol for rapid and
E2834Guide for Measurement of Particle Size Distribution
quantitative measurement of the chemoattractant capacity of a
of Nanomaterials in Suspension by NanoparticleTracking
nanoparticulate material (nanoparticles and their aggregates
Analysis (NTA)
and agglomerates).
F1877Practice for Characterization of Particles
1.2 Immune cells recruitment (by chemotaxis) plays a
F1903Practice for Testing for Cellular Responses to Par-
central role in the immune system function especially in the
ticles in vitro
inflammatory process.
1.3 This test method uses an in vitro model. In this model, 3. Terminology
peripheral blood human acute promyelocytic leukemia cells
3.1 Acronyms:
HL-60 are separated from control chemoattractant or test
3.1.1 BSA—bovine serum albumin
nanoparticulate material by a 3-µm pore size filter; the cell
3.1.2 calcein AM—calcein acetoxymethyl ester
migration through the filter is monitored and quantified using
the fluorescent dye calcein AM (Figs. 1 and 2). 3.1.3 C —maximum serum concentration
max
3.1.4 CV—coefficient of variation
1.4 The values stated in SI units are to be regarded as
standard. No other units of measurement are included in this
3.1.5 FBS—fetal bovine serum
standard.
3.1.6 FU—fluorescence units
1.5 This standard does not purport to address all of the
3.1.7 g—relative centrifugal force
safety concerns, if any, associated with its use. It is the
3.1.8 NC—negative control
responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter-
3.1.9 PBS—phosphate buffered saline
mine the applicability of regulatory limitations prior to use.
3.1.10 PC—positive control
1.6 This international standard was developed in accor-
3.1.11 PK—pharmacokinetic
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the 3.1.12 RBC—reagent background control
Development of International Standards, Guides and Recom-
3.1.13 RPMI—Roswell Park Memorial Institute
mendations issued by the World Trade Organization Technical
3.1.14 SD—standard deviation
Barriers to Trade (TBT) Committee.
3.1.15 SM—starvation medium
2. Referenced Documents
3.1.16 TS—test sample (of nanoparticulate material dis-
2.1 ASTM Standards: solved in starvation medium)
E2490Guide for Measurement of Particle Size Distribution
3.1.17 U—units
4. Summary of Test Method
This test method is under the jurisdiction of ASTM Committee E56 on
Nanotechnology and is the direct responsibility of Subcommittee E56.08 on
4.1 This test method describes a protocol for assessing and
Nano-Enabled Medical Products.
measuring the chemoattractant capacity of nanoparticulate
Current edition approved Jan. 1, 2020. Published January 2020. DOI: 10.1520/
material.
E3238-20.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
4.2 The migration of cells through a filter towards the
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
nanoparticulate material is monitored and quantified after
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. staining with calcein AM dye.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3238 − 20
FIG. 1 Chemotaxis Chamber (Boyden Chamber)
a (left)—Parts of the chemotaxis assay assembly.
b (right)—Procedure for testing the chemoattractant capacity of a nanoparticulate material.
FIG. 2 Chemotaxis Assay
4.3 CalceinAM, a non-fluorescent hydrophobic compound, 5. Significance and Use
is transported through the cell membrane into live cells.
5.1 This test method will assess whether the test nanopar-
Intracellular esterases remove the acetomethoxy group of
ticulate material has chemoattractant activity.
calceinAM producing calcein, a hydrophilic green fluorescent
compound. Calcein is retained by the live cells.
E3238 − 20
5.2 This test method will provide a rapid and quantitative 9.6 Biohazard safety cabinetapprovedforLevelIIhandling
measure of the ability of nanoparticulate material to recruit of biological material.
immune cells.
9.7 Inverted microscope.
5.3 Recruitment of immune cells by chemotaxis plays an
9.8 Vortex mixer.
important part in all phases of both humoral and cell-mediated
9.9 Hemocytometer.
immune responses.
9.10 Fluorescence plate reader.
5.4 Testing the capacity of a nanoparticulate material to
recruit immune cells in vitro helps in predicting the influence
10. Preparation of Media, Calcein AM, Test Samples
of such material on the immune cell response.
and Controls
6. Materials
10.1 Three solutions of each nanoparticulate material test
concentration (referred to as test samples) and three solutions
6.1 Pipettes covering the range of 0.05 to 10 mL.
of each control (SM, PC, and NC) shall be independently
6.2 96-well filter plate, 3.0-µm pore size polycarbonate
prepared.
membrane, clear, sterile.
10.2 Preparation of Complete RPMI-1640 Medium—The
6.3 96-well feeding tray, clear, sterile (culture tray).
complete RPMI-1640 medium is a solution of RPMI-1640
6.4 Holding tray.
medium (basal medium) supplemented with 20 % FBS (heat-
inactivated), 4 mM L-glutamine, 1.5 g/L sodium bicarbonate,
6.5 Cover for holding tray.
50 µM β-mercaptoethanol, 100 U/mL penicillin, and 100
6.6 96-microwell clear bottom plates suitable for
µg/mL streptomycin sulfate. The medium should be stored at
fluorescent-based assays.
2–8°C protected from light for no longer than 1 month. Before
6.7 Polypropylene tubes, 5 and 15 mL.
use, warm in a water bath.
6.8 Cell culture flasks.
10.3 Preparation of Starvation Medium (SM)—The SM is a
solution of RPMI-1640 medium (basal medium) supplemented
7. Cell Line
with 0.2 % BSA, 4 mM L-glutamine, 1.5 g/L sodium
bicarbonate, 50 µM β-mercaptoethanol, 100 U/mL penicillin,
7.1 Peripheral blood human acute promyelocytic leukemia
and 100 µg/mL streptomycin sulfate. The medium is stored at
cells HL-60 (ATCC CCL-240 ).
2–8°C protected from light for no longer than 1 month. Before
use, warm in a water bath. Prepare three solutions of SM (see
8. Reagents
10.1).
8.1 Phosphate buffered saline (PBS), pH 7.4.
10.4 Postive Control (PC)—On the day of the experiment,
8.2 Bovine serum albumin (BSA), endotoxin-free and suit-
prepare a solution of RPMI-1640 medium supplemented with
able for cell culture.
20 % heat-inactivated FBS and 0.2 % BSA. Repeat twice to
8.3 Fetal bovine serum (FBS).
obtain three independently prepared solutions (see 10.1).
8.4 Commercial RPMI-1640 medium (basal medium).
10.5 Negative Control (NC)—Use PBS as a NC.
8.5 L-glutamine, 4 mM.
10.6 Calcein AM Working Solution—Dilute the calceinAM
1-mM stock solution in prewarmed (37°C) PBS to a final
8.6 Sodium bicarbonate.
concentration of 4 µM. The working dilution shall be prepared
8.7 β-mercaptoethanol, 50 µM.
immediately prior to use.
8.8 Calcein AM, 1 mM.
10.7 Specification of Nanoparticulate Material Test
8.9 Trypan blue solution.
Samples—The selected nanoparticulate material must be fully
dispersed in SM. This assay requires a minimum of 2.3 mLof
8.10 Penicillin.
nanoparticulate material dissolved/resuspended in SM. (This
8.11 Streptomycin sulfate.
volumeiscalculatedfromthenumberandconcentrationoftest
samplespreparedasspecifiedbelow,includingreplicates.)The
9. Apparatus
following factors shall be considered in choosing the nanopar-
9.1 Centrifuge.
ticulate material concentration:
(1)Dispersibility, stability, and homogeneity of the nano-
9.2 Water bath set at 37°C.
particulate material in a biocompatible buffer (that is, SM),
9.3 Refrigerator, 2–8°C.
(2)Maintaining the pH in the physiological range, and
9.4 Freezer, –20°C.
(3)Stability of nanoparticulate material during testing.
Before testing, the nanoparticulate material shall be charac-
9.5 Cell culture incubator with 5 % carbon dioxide (CO )
terized (for example, size, size distribution, and charge) under
and 95 % humidity.
physiological conditions in accordance with with standard
methods such as those recommended in Guides E2490 and
A trademark of ATCC in Manassas, VA. E2834 for nanomaterials and Practices F1877 and F1903 for
E3238 − 20
powders. In the absence of data from PK studies (see the 11.5 Place the feeding tray into the holding tray. Gently
following sentence), the highest concentration of nanoparticu- placethefilterplate(11.4)ontopofthefeedingtraycontaining
late material was selected to be 1.0 mg/mL. (When the the controls and test samples (11.3) and add the cover. Avoid
nanoparticulatematerial’splasmaconcentration C isknown shaking or tilting the chemotaxis assay assembly as it will
max
from in vivo PK studies, the highest concentration tested in disturb the concentration gradient.
vitro is chosen as 10×, 30×, or 100× of that C .)
max
11.6 Place the assembly in a cell culture incubator and
10.8 Preparation of Nanoparticulate Material Test incubate for4hwith5%CO and 95 % humidity. During
Samples—Prepare a stock solution containing nanoparticulate incubation, pre-warm PBS to 37°C and equilibrate calceinAM
material dissolved in SM at a concentration of 1.0 mg/mL. stock solution to room temperature.
Prepa
...