ASTM F1903-98(2003)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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:F1903–98 (Reapproved 2003)
Standard Practice for
Testing For Biological Responses to Particles in 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 F748 Practice for Selecting Generic Biological Test Meth-
ods for Materials and Devices
1.1 This practice covers the production of wear debris and
degradationproductsfromimplantedmaterialsthatmayleadto
3. Summary of Practice
a cascade of biological responses resulting in damage to
3.1 Biologicalresponsestoparticlesmaybeevaluatedusing
adjacent and remote tissues. In order to ascertain the role of
specimens from animals being tested according to the Practice
particles in stimulating such responses, the nature of the
F748 matrix for irritation and sensitivity, or for implantation.
responses, and the consequences of the responses, established
Blood, organs, or tissues from the animals may be used.
protocols are needed. This is an emerging, rapidly developing
3.2 Biologicalresponsestoparticlesmaybeevaluatedusing
area and the information gained from standard protocols is
materials or extracts according to Practice F619. These mate-
necessary to interpret responses and to determine if there is
rials or extracts may be used for in vivo tests or for the in vitro
correlation with the in vivo responses. Since there are many
tests. Particles generated by other methods may also be used.
possible and established ways of determining responses, a
single standard protocol is not stated. However, well described
4. Significance and Use
protocols are needed to compare results from different inves-
4.1 This practice is to be used to help assess the biocom-
tigators using the same materials and to compare biological
patibility of materials used in medical devices. It is designed to
responses for evaluating (ranking) different materials. For
test the effect of particles from the materials on macrophages.
laboratories without established protocols, recommendations
For safety reasons, the use of non human, non primate cells is
are given and indicated with an *.
recommended in this practice. For laboratories equipped and
1.2 Since the purpose of these studies is to predict the
approvedtoworkwithhumanbloodandtissue,theuseofthese
response in humans, the use of human cells would provide
same protocols would be advantageous for development of
much information. However in the interest of safety in labo-
understanding of the interaction of cells and particles.
ratoryprotocols,theuseofnon-humanandnon-primatecellsis
4.2 The appropriateness of the methods should be carefully
described.
considered by the user since not all materials or applications
1.3 The values stated in SI units are to be regarded as the
need be tested by this practice.
standard.
4.3 Abbreviations:
1.4 This standard does not purport to address all of the
4.3.1 LPS—lipopolysaccharide (endotoxin).
safety concerns, if any, associated with its use. It is the
4.3.2 LAL—Limulus Amebocyte Lysate.
responsibility of the user of this standard to establish appro-
4.3.3 ATCC—American Type Culture Collection.
priate safety and health practices and determine the applica-
4.3.4 FCS (FBS)—Fetal Calf Serum.
bility of regulatory limitations prior to use.
4.3.5 NCS—Newborn Calf Serum.
2. Referenced Documents 4.3.6 PBS—Phosphate Buffered Saline.
2 4.3.7 HANKS—A balanced salt solution.
2.1 ASTM Standards:
4.3.8 MMPS—Matrix Metallo Proteases.
F619 Practice for Extraction of Medical Plastics
4.3.9 RPMI1640—Specific Growth Medium (Roswell Park
Memorial Institute).
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
4.3.10 HEPES—A buffering salt.
Surgical Devices and is the direct responsibility of Subcommittee F04.16 on
Biocompatibility Test Methods.
5. Responses from Cells Grown In Vitro
Current edition approved Nov. 1, 2003. Published December 2003. Originally
5.1 Particles—Define the nature of the particles used:
approved in 1998. Last previous edition approved in 1998 as F1903 – 98. DOI:
10.1520/F1903-98R03.
5.1.1 Source,
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
5.1.2 Chemistry,
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
5.1.3 Size (mean and range),
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. 5.1.4 Shape,
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F1903–98 (2003)
5.1.5 Method of sterilization, 5.3.6.1 Cells not stimulated with particles should be main-
5.1.6 If the presence of bacterial lipopolysaccharide (LPS) tained at the same time under the same conditions.
was determined, specify how this was done and the sensitivity
5.3.6.2 Polystyrene particles, spherical, size range 1 to 5
of the method. (LAL testing with a sensitivity of at least 0.06
µm, should be used as a reference control.
EU is recommended),
5.3.6.3 LPS is a known stimulant and is a good positive
5.1.7 Concentrationofparticlesusedasweightornumberor
control. A concentration between 0.25 and 1 ng/mL of culture
surface area/10 cells, and
medium is sufficient.
5.1.8 Surface charge (if known).
5.3.6.4 Culture medium is a recommended diluent for the
5.2 Cells—Define the nature of the cells used:
assays.
5.2.1 EstablishedCellLine (ifnot,goto5.2.2)—Theuseof
5.4 Products or Response Determined—One or more of the
established cell lines provides a known cell type with a
following:
reproducible response. Although the correlation with the in
vivosystemmaynotbeknownatthistime,carefulstudieswith 5.4.1 Cell viability can be determined by any of several
established cell lines could eventually allow determination of
methods:
correlation between in vivo and in vitro systems.
5.4.1.1 From the total cell counts and viable cell count with
5.2.1.1 Source of cell and identifying number or code,
trypan blue dye exclusion,
5.2.1.2 Nature of the cell (for example, macrophage), and
5.4.1.2 From flow cytometry with vital stains,
5.2.1.3 Special attributes of the cell line (for example,
5.4.1.3 From uptake of DNA precursors, and
nonphagocytic),
5.4.1.4 From total DNA or RNA in the culture.
5.2.1.4 *ATCC murine macrophages such as RAW 264.7,
5.4.2 Soluble Cell Products Elaborated:
J774A, P388D1, or IC-21 are recommended.
5.2.2 Primary Isolate (if not, go to 1.2.1):
5.4.2.1 Reliable reagents or kits are available to detect
5.2.2.1 Source of cell including species and location (for products. The products are classified into three classes: growth
example, murine, alveolar), factors such as TNFa, TGF beta, and PGE2, interleukins such
5.2.2.2 Nature of the cell (for example, macrophage),
as IL-1, IL-1 receptor antagonist, IL-1 beta, and IL-6, and
5.2.2.3 Mechanism of isolation (for example, lavage), and reactive oxygen species such as superoxide or nitric oxide
5.2.2.4 Specify if stimulant used and if so which one (for (NO) (usually measured as nitrite produced using the Griess
example, mineral oil). reagent). The release of at least two products, which are from
different classes, should be determined.
5.2.2.5 *Mouse (specify strain, age, and sex used) perito-
nealexudatecellsarerecommendedwithamildstimulantsuch
5.4.2.2 The time in culture should be specified since these
as nutrient broth.
products peak at different times (for example, TNFa peaks by
5.3 Culture Conditions:
24 h, NO peaks around 72 h, the others generally peak at 48 to
5.3.1 Specify source and type of medium. If not a commer-
72 h). The detection of NO and TNFa are strongly recom-
cial source, list ingredients and sources of ingredients.
mended and the assay procedures are described in some detail
5.3.2 Specify source and type of serum, and whether it was
in this recommended practice.
heat inactivated. If the presence of LPS was determined,
5.4.3 Assay for Nitric Oxide:
specify method and sensitivity of the method.
5.4.3.1 The production of NO is most conveniently mea-
5.3.3 Specify culture conditions (for example, 37°C, hu-
sured by detection of the stable endproduct nitrite.This reflects
midified, 5 % CO incubator).
the action of NO on arginine. The technique is simple,
5.3.4 Specify time of termination of culture or sampling of
inexpensive and quantitative. The reagent used to quantitate
culture medium.
nitrite is the Griess reagent. This is prepared by dissolving 100
5.3.5 If cell counts were determined specify as to when and
mg of Sulfanilamide and 10 mg of N-(1-
how. If estimates of cell number/mL specify when and how.
naphthyl)ethylenediamine in 2.5 % phosphoric acid. Assay of
5.3.5.1 *Medium and serum specified by the supplier of the
culture medium: 100 uL of the culture medium is placed in a
cells are recommended. Generally RPMI 1640 with heat
96-well microtiter plate and then 100 uL of the Griess reagent
inactivated 10 % newborn or fetal calf serum are recom-
is added.This is incubated at room temperature for 10 min and
mended. LPS levels are generally provided or available from
the optical density determined at 540 nm wavelength. A
the distributor. Recommended culture conditions are 37°C,
standard curve is prepare
...