ASTM E3238-20

Standard Test Method for Quantitative Measurement of the Chemoattractant Capacity of a Nanoparticulate Material <emph type="bdit">in vitro</emph>

SIGNIFICANCE AND USE
5.1 This test method will assess whether the test nanoparticulate material has chemoattractant activity.  
5.2 This test method will provide a rapid and quantitative measure of the ability of nanoparticulate material to recruit immune cells.  
5.3 Recruitment of immune cells by chemotaxis plays an important part in all phases of both humoral and cell-mediated immune responses.  
5.4 Testing the capacity of a nanoparticulate material to recruit immune cells in vitro helps in predicting the influence of such material on the immune cell response.
SCOPE
1.1 This test method provides a protocol for rapid and quantitative measurement of the chemoattractant capacity of a nanoparticulate material (nanoparticles and their aggregates and agglomerates).  
1.2 Immune cells recruitment (by chemotaxis) plays a central role in the immune system function especially in the inflammatory process.  
1.3 This test method uses an in vitro model. In this model, peripheral blood human acute promyelocytic leukemia cells HL-60 are separated from control chemoattractant or test nanoparticulate material by a 3-µm pore size filter; the cell migration through the filter is monitored and quantified using the fluorescent dye calcein AM (Figs. 1 and 2).
FIG. 1 Chemotaxis Chamber (Boyden Chamber)
FIG. 2 Chemotaxis Assay
a (left)—Parts of the chemotaxis assay assembly.
b (right)—Procedure for testing the chemoattractant capacity of a nanoparticulate material.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 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.

General Information

• Status: Published
• Publication Date: 31-Dec-2019
• ICS: 07.120 - Nanotechnologies
• ICS: 11.020.20 - Medical science
• Technical Committee: E56 - Nanotechnology
• Drafting Committee: E56.08 - Nano-Enabled Medical Products

Standards Content (Sample)

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
1
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
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
1
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.
2
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 [e-mail]. 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
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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.
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E3238 − 20
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