ASTM F1903-18

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it may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current version
of the standard as published by ASTM is to be considered the official document.
Designation: F1903 − 10 F1903 − 18
Standard Practice for
Testing For Biologicalfor Cellular Responses to Particles In
Vitroin vitro
This standard is issued under the fixed designation F1903; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope
1.1 This practice covers the production of wear debrisassessment of cellular responses to wear particles and degradation
products from implanted materials that may lead to a cascade of biological responses resulting in damage to adjacent and remote
tissues. In order to ascertain the role of particles in stimulating such responses, the nature of the responses, and the consequences
of the responses, established protocols are needed. This is an emerging, rapidly developing area, and the information gained from
standard protocols is necessary to interpret cellular responses to particles and to determine if there is correlation with the these
correlate with in vivo responses. Since there are many possible and established ways of determining responses, a single standard
protocol is not stated. However, well described protocols are needed to compare results from different investigators using the same
materials and to compare biological responses for evaluating (ranking) different materials. For laboratories without established
protocols, recommendations are given and indicated with an asterisk*.asterisk (*).
1.2 Since the purpose of these studies the following test procedures is to predict the response in humans, human tissues, the use
of human cells would provide much information. However, in this practice, (preferably macrophage lineage) cells is recommended.
However, the use of non-macrophage cell lineage or the use of cells from non-human and non-primate cells is described. If the
user should wish to employ cell lines from humans, cell lines are available from ATCC and most of the information and
recommendations will still apply.sources may be acceptable. The source of the cells or the cell line used should be justified based
on the cellular responses under test and/or tissue of interest. Non-human cells should not be used if there is evidence of possible
cross-species difference for specific test results as the results of this in vitro test may not correspond to actual human response.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.
1.5 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.
2. Referenced Documents
2.1 ASTM Standards:
F619 Practice for Extraction of Medical Plastics
F748 Practice for Selecting Generic Biological Test Methods for Materials and Devices
F1877 Practice for Characterization of Particles
3. Summary of Practice
3.1 BiologicalCellular responses to particles may be evaluated using specimens from animals being tested according to the
Practice F748 matrix for irritation and sensitivity, or for implantation. Blood, organs, or tissues from the animals may be used.
This practice is under the jurisdiction of ASTM Committee F04 on Medical and Surgical Materials and Devices and is the direct responsibility of Subcommittee F04.16
on Biocompatibility Test Methods.
Current edition approved June 1, 2010Oct. 1, 2018. Published June 2010October 2018. Originally approved in 1998. Last previous edition approved in 20032010 as
F1903 – 98 (2003).F1903 – 10. DOI: 10.1520/F1903-10.10.1520/F1903-18.
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 the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F1903 − 18
3.2 BiologicalCellular responses to particles may be evaluated using materials or extracts according to Practice F619. These
materials or extracts may be used for in vivo tests or for the in vitro tests. Particles generated by other methods methods (for
example, derived from in vitro mechanical testing or retrieved from ex vivo peri-implant tissues either from clinical retrievals or
animal models) may also be used.used as long as they have characteristics similar to those produced by the implant or device being
tested with appropriate justification.
3.3 The purpose of this practice is to assess the response of cells in direct contact with particles and,and therefore, this practice
is primarily intended to cover the testing of particles placed into culture with the cells. This practice should be equally appropriate
for the testing of the response to nanoparticles placed in culture, if particles of that size are the particles of interest. The size range
of particles (among other particle characteristics) should be clearly defined and stratification of the test results based on the particle
size and other characteristics is recommended.
4. Significance and Use
4.1 This practice is to be used to help assess the biocompatibility of materials used in medical devices. It is designed to test the
effect of particles from the materials on macrophages. The use of nonhuman, nonprimate cells is recommended in this practice.
For laboratories equipped and approved to work with human blood and tissue, the use of these same protocols would be
advantageous for development of understanding of the interaction of cells and particles.released from medical devices and
biomaterials on macrophages or other cells.
4.2 The appropriateness of the methods should be carefully considered by the user since not all materials or applications need
to be tested by this practice.
4.3 Abbreviations:
4.3.1 LPS—lipopolysaccharide (endotoxin).
4.3.2 LAL—Limulus Amebocyte Lysate.
4.3.3 ATCC—American Type Culture Collection.
4.3.1 FCS (FBS)—Fetal Calf Serum.Serum (Fetal Bovine Serum)
4.3.2 NCS—FGFs—Newborn Calf Serum.Fibroblast Growth Factors
4.3.3 HBSS—Hank’s Balanced Salt Solution
4.3.4 HEPES—A buffering salt (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
4.3.5 IL17—Interleukin 17
4.3.6 IL18—Interleukin 18
4.3.7 IL1β—Interleukin 1 beta
4.3.8 IL6—Interleukin 6
4.3.9 IL8—Interleukin 8
4.3.10 LAL—Limulus Amebocyte Lysate
4.3.11 LPS—lipopolysaccharide (endotoxin)
4.3.12 MCP1—Monocyte Chemotactic Protein-1
4.3.13 MMPs—Matrix Metalloproteinases
4.3.14 NO—Nitric Oxide
4.3.15 PBS—Phosphate Buffered Saline.Saline
4.3.16 HANKS—PGE2—A balanced salt solution.Prostaglandin E2
4.3.8 MMPS—Matrix Metallo Proteases.
4.3.17 RPMI 16401640——Specific Growth Medium (Roswell Park Memorial Institute).Institute)
4.3.18 HEPES—TGFβ—A buffering salt.Transforming growth factor beta
4.3.19 TNFα–—Tumor Necrosis Factor alpha
4.3.20 VEGF—Vascular Endothelial Growth Factor
5. Responses from Cells Grown In Vitroin vitro
5.1 Particles—Define the nature of the particles used:used (see Practice F1877 for detailed particle characterization
methodology):
5.1.1 Source,Source;
5.1.2 Chemistry,Chemical composition;
5.1.3 Size (mean and range),range, refer to Practice F1877 for additional information regarding how size should be determined);
5.1.4 Shape,Shape;
5.1.5 Method of sterilization,sterilization;
5.1.6 If the presence of bacterial lipopolysaccharide (LPS) was determined, specify how this was done and the sensitivity of the
method.method (LAL testing with a sensitivity of at least 0.06 EU is recommended),endotoxin units (EU) is recommended, see
X1.5);
5.1.7 Concentration of particles used as weight or number or surface area/10weight, number, or surface area/106 cells, andcells;
5.1.8 Surface charge (if known).known);
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5.1.9 Since some particles may have a density less than that of the culture medium being used, careful consideration should be
given to determining appropriate procedures for ensuring that the particles and cells are in contact. come into contact with each
other. Techniques in which cells are grown on a substrate and then the substrate is inverted in culture may be appropriate.
5.2 Cells—Define the nature of the cells used:
5.2.1 Established Cell LineLines (if not, go to 5.2.2)—(if not, go to The 5.2.2)—The use of established cell lines provides a
known cell type with a reproducible response. Although the correlation with the in vivo system may not be known at this time,
careful studies with established cell lines could eventually allow determination of correlation between in vivo and in vitro systems.
5.2.1.1 Source Specify source of cell and identifying number or code,code.
5.2.1.2 Nature of the cell (for example, macrophage), andSpecify type of cells.
5.2.1.3 Special Specify special attributes of the cell line (for example, nonphagocytic),line.
5.2.1.4 *ATCC *Human macrophages such as U-937 are recommended; however, non-human murine macrophages such as
RAW 264.7, J774A,J774A.1, P388D1, or IC-21 are recommended.may be used.
5.2.2 Primary IsolateIsolate: (if not, go to 1.2.1):
5.2.2.1 Source Specify source of cellcells including species and location (for example, murine, alveolar),tissue origin (e.g.,
human, alveolar).
5.2.2.2 Nature of the cell (for example, macrophage),Specify type of cells.
5.2.2.3 Mechanism Specify mechanism of isolation (for example, lavage), and(e.g., lavage, enzymatic digestion).
5.2.2.4 Specify if stimulant used and if so, which one (for example, (e.g., mineral oil).
5.2.2.5 *Mouse (specify strain, *Strain, age, and sex used) peritoneal exudate cells are recommended with a mild stimulant such
as nutrient broth.used should be specified; use of animals of both sexes should be considered for tests involving in vivo models.
5.3 Culture Conditions:
5.3.1 Specify source and type of medium. If not from a commercial source, provide the catalog and/or reference number. If not
from a commercial source, provide a list of ingredients and sources of ingredients.their sources.
5.3.2 Specify source and type of serum, and whether it was heat inactivated. heat-inactivated. If the presence of LPS was
determined, specify the amount present as well as the method and sensitivity of the method.
5.3.3 Specify culture conditions (for example, 37°C, humidified, 5 % CO incubator).
5.3.4 Specify when and how the particles were added to the system.
5.3.5 Specify time of termination of culture or sampling of culture duration, test exposure, and/or time course of sampling of
the culture medium.
5.3.6 If cell counts were determined(in units of cell number/mL) were determined, specify as to when and how. If estimates of
cell number/mL specify when and how.how (for example, hemocytometer, Coulter counter).
5.3.6.1 *Medium and serum specified by the supplier of the cells are recommended. Generally RPMI 1640 with heat inactivated
10 % newborn or fetal calf serum are recommended. *Cells should be cultured using the culture medium and serum
specified/recommended by the supplier. LPS levels are generally provided or available from the distributor. Recommended culture
conditions are 37°C, with 5 % CO , in a humidified incubator. Cell counts at the time of initial plating and at the termination of
the culture are recommended using a hemocytometer with monolayer cells resuspended by trypsin solution (not recommended for
macrophages), washing with Ca and Mg free PBS or Hanks, or scraping in 1 mL of Ca and Mg free PBS or Hanks. The addition
of trypan blue is helpful. The supernatant of the medium from recommended. For adherent cell populations, cells should be
detached from the culture surface and suspended either by gentle pipetting and/or scraping, or by using a cell dissociation solution,
depending on the cell supplier recommendations. After the cells are resuspended, they should be washed with Ca- and Mg-free
PBS, HBSS, or culture medium. During counting with a hemocytometer, it may be helpful to ascertain the percentage of dead cells
in the population using trypan blue viability staining. The responses of the macrophages exposed to particles for specified time
periods is assayed.should be assayed as described in 5.4.
5.3.7 Controls:
5.3.7.1 Cells not stimulated with particles particles, but otherwise cultured under the same experimental culture conditions,
should be maintained at the same time under the same conditions.in parallel with test condition(s), positive control, and/or
reference standard.
5.3.7.2 Polystyrene particles, spherical, size range 1 to 5 μm, should (PS) particles may be used as a reference control. The
choice of control may be affected by the size range, chemical composition, and other characteristics of particles of interest (e.g.,
special consideration should be given if nanoparticles are relevant to the device).
5.3.7.3 LPS is a known cell stimulant and is a good positive control. may be used as a positive control for certain immune
responses. A concentration between 0.25 and 1 ng/mL of culture medium is sufficient.
5.3.7.4 Culture medium is a recommended diluent for the assays.
5.3.8 Test Conduct:
5.3.8.1 Culture cells for a dose ranging study to determine the Lethal Concentration (LC50) and investigate dosing effects on
cellular uptake of particles and corresponding cellular response.
5.3.8.2 Record when and how particles are added to the test system, and explain how the doses for the study were chosen to
include how the dosing concentration compares to a proposed clinical dose, if applicable.
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5.3.8.3 Determine which subset of cellular responses identified in 5.4 will be chosen for inclusion in your study and document
the rationale for the selection.
5.3.8.4 Items to include in test reports:
(1) Description of the particle sample;
(2) Justification of the choice of cells and cell source(s);
(3) Assay method(s) and rationale;
(4) Description of controls (including positive, negative, reference particles);
(5) Assay response and other observations.
5.4 Products or Response Determined—Responses Determined: One or more of the following:
5.4.1 Particle Uptake Determination—Special attention should be paid to in vitro analysis of the uptake of particles by
macrophages (with the results stratified by the size and other particle characteristics), since the internalization and subcellular
localization of particles could pre-determine the subsequent cell survival and cytokine release.
5.4.1.1 For metallic particles, uptake is most easily ascertained, and may be assessed using optical emission spectroscopy
(ICP-MS/OES) or various microscopy techniques (e.g., transmission electron microscopy or confocal microscopy).
5.4.1.2 For polymeric particles, quantitative assessment of uptake is more difficult, and so qualitative assessment may be
performed, such as by visualizing uptake using various microscopy techniques (e.g., transmission electron microscopy or confocal
microscopy).
5.4.2 Cell death and viability can be determined by any of several methods:several methods including, but not limited to:
5.4.2.1 From the The total cell counts and viable cell countcounts with trypan blue dye exclusion, neutral red uptake, etc.;
5.4.2.2 Metabolic assays such as MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide);
5.4.2.3 From flow Low cytometry with vital stains,stains such as calcein AM fluorescence/propidium iodide exclusion, etc.;
5.4.2.4 FromThe uptake of DNA precursors, andprecursors;
5.4.2.5 FromThe total DNA or RNA in the culture.content of cell populations. Special consideration should be given to
determine the type(s) of cell death, including apoptosis (e.g., from Annexin V staining) and necrosis (
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