ASTM F1903-98

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.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: F 1903 – 98
Standard Practice for
Testing For Biological Responses to Particles In Vitro
This standard is issued under the fixed designation F 1903; 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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope 3. Summary of Practice
1.1 This practice covers the production of wear debris and 3.1 Biologicalresponsestoparticlesmaybeevaluatedusing
degradationproductsfromimplantedmaterialsthatmayleadto specimens from animals being tested according to the Practice
a cascade of biological responses resulting in damage to F 748 matrix for irritation and sensitivity, or for implantation.
adjacent and remote tissues. In order to ascertain the role of Blood, organs, or tissues from the animals may be used.
particles in stimulating such responses, the nature of the 3.2 Biologicalresponsestoparticlesmaybeevaluatedusing
responses, and the consequences of the responses, established materials or extracts according to Practice F 619. These mate-
protocols are needed. This is an emerging, rapidly developing rials or extracts may be used for in vivo tests or for the in vitro
area and the information gained from standard protocols is tests. Particles generated by other methods may also be used.
necessary to interpret responses and to determine if there is
4. Significance and Use
correlation with the in vivo responses. Since there are many
possible and established ways of determining responses, a 4.1 This practice is to be used to help assess the biocom-
patibility of materials used in medical devices. It is designed to
single standard protocol is not stated. However, well described
protocols are needed to compare results from different inves- test the effect of particles from the materials on macrophages.
For safety reasons, the use of non human, non primate cells is
tigators using the same materials and to compare biological
responses for evaluating (ranking) different materials. For recommended in this practice. For laboratories equipped and
approvedtoworkwithhumanbloodandtissue,theuseofthese
laboratories without established protocols, recommendations
are given and indicated with an *. same protocols would be advantageous for development of
understanding of the interaction of cells and particles.
1.2 Since the purpose of these studies is to predict the
response in humans, the use of human cells would provide 4.2 The appropriateness of the methods should be carefully
considered by the user since not all materials or applications
much information. However in the interest of safety in labo-
need be tested by this practice.
ratoryprotocols,theuseofnon-humanandnon-primatecellsis
described. 4.3 Abbreviations:
4.3.1 LPS—lipopolysaccharide (endotoxin).
1.3 The values stated in SI units are to be regarded as the
standard. 4.3.2 LAL—Limulus Amebocyte Lysate.
4.3.3 ATCC—American Type Culture Collection.
1.4 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the 4.3.4 FCS (FBS)—Fetal Calf Serum.
4.3.5 NCS—Newborn Calf Serum.
responsibility of the user of this standard to establish appro-
priate safety and health practices and determine the applica- 4.3.6 PBS—Phosphate Buffered Saline.
4.3.7 HANKS—A balanced salt solution.
bility of regulatory limitations prior to use.
4.3.8 MMPS—Matrix Metallo Proteases.
2. Referenced Documents
4.3.9 RPMI 1640—Specific Growth Medium (Roswell Park
2.1 ASTM Standards: Memorial Institute).
F 619 Practice for Extraction of Medical Plastics 4.3.10 PBS—Phosphate Buffered Saline.
F 748 Practice for Selecting Generic Biological Test Meth- 4.3.11 HEPES—A buffering salt.
ods for Materials and Devices
5. Responses from Cells Grown In Vitro
5.1 Particles—Define the nature of the particles used:
This practice is under the jurisdiction ofASTM Committee F-4 on Medical and
5.1.1 Source,
Surgical Devicesand is the direct responsibility of Subcommittee F04.16on Bio-
5.1.2 Chemistry,
compatibility Test Methods.
5.1.3 Size (mean and range),
Current edition approved June 10, 1998. Published August 1998.
Annual Book of ASTM Standards, Vol 13.01. 5.1.4 Shape,
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
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.
F 1903
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 prepared with sodium nitrite in water. It is
with
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