ASTM F813-83(1996)e1

NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
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e1
Designation: F 813 – 83 (Reapproved 1996)
Standard Practice for
Direct Contact Cell Culture Evaluation of Materials for
Medical Devices
This standard is issued under the fixed designation F 813; 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.
e NOTE—Footnote 4 was corrected editorially December 1996.
1. Scope USP Negative Control Plastic Reference Standard
1.1 This practice covers a reference method of direct contact
3. Summary of Practice
cell culture testing which may be used in evaluating the
3.1 Cell cultures are grown to a confluent monolayer in Petri
cytotoxic potential of materials for use in the construction of
dishes. The growth medium is aspirated and replenished to
medical materials and devices.
provide a resting, confluent cell layer. Test and control speci-
1.2 This method may be used either directly to evaluate
mens are placed in direct contact with the cell layer to provide
materials or as a reference against which other cytotoxicity test
an accelerated assessment of the presence or absence of a
methods may be compared.
cytotoxic effect from a given material or device.
1.3 This is one of a series of reference test methods for the
assessment of cytotoxic potential, employing different tech-
4. Significance and Use
niques.
4.1 This practice is useful for assessing cytotoxic potential
1.4 Assessment of cytotoxicity is one of several tests
both when evaluating new materials or formulations for
employed in determining the biological response to a material,
possible use in medical applications, and as part of a quality
as recommended in Practice F 748.
control program for established medical materials and medical
1.5 The L-929 cell line was chosen because it has a
devices.
significant history of use in assays of this type. This is not
4.2 This practice assumes that assessment of cytotoxicity
intended to imply that its use is preferred; only that the L-929
potential provides one method for predicting the potential for
is an established cell line, well-characterized and readily
cytotoxic or necrotic reactions to medical materials and devices
available, that has demonstrated reproducible results in several
during clinical applications to humans. In general, cell culture
laboratories.
testing methods have shown good correlation with animal
1.6 This standard does not purport to address all of the
assays and are frequently more sensitive to toxic moieties.
safety concerns, if any, associated with its use. It is the
4.3 This cell culture test method is suitable for adoption in
responsibility of the user of this standard to establish appro-
specifications and standards for materials for use in the
priate safety and health practices and determine the applica-
construction of medical devices that are intended to be im-
bility of regulatory limitations prior to use.
planted in the human body or placed in contact with tissue,
tissue fluids, or blood on a long-term basis.
2. Referenced Documents
2.1 ASTM Standards:
5. Apparatus
F 748 Practice for Selecting Generic Biological Test Meth-
5.1 The following apparatus shall be used:
ods for Materials and Devices
5.2 Incubator, to maintain 37 6 2°C and4to6%CO with
2.2 Other Documents:
greater than 90 % relative humidity.
The American Type Culture Collection (ATCC), Catalogue
3 5.3 Tissue Culture Grade Petri Dishes, that are sterile and
of Strains II
35 mm in diameter by 10 mm deep.
NOTE 1—Plastic dishes are recommended because they provide a flat
surface that contributes to the formation of a uniform cell monolayer.
1 5.4 Disposable, Sterile, Centrifuge Tubes.
This practice is under the jurisdiction of ASTM Committee F-4 on Medical and
5.5 Inverted Optical Microscope, with magnifications of
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.16 on Biocompatibility Test Methods.
403, 1003, and 2003.
Current edition approved April 29, 1983. Published June 1983.
Annual Book of ASTM Standards, Vol 13.01.
3 4
American Type Culture Collection, 12031 Parklawn Drive, Rockville, MD U.S. Pharmacopeia, Vol 23, Rand McNally, Taunton, MA, 1994, pp.
10852. 1652–1653. Use latest supplement to ensure current cumulative revisions are used.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
F 813
5.6 Clinical Centrifuge. 9. General Technique
5.7 Filter Disks—10 mm in diameter (for evaluation of
9.1 Use the aseptic technique throughout this assay to
liquids).
minimize microbial contamination.
NOTE 2—Millipore AP2501000 filter disks have been found satisfac-
NOTE 7—Mouth pipetting should not be employed to transfer cells,
tory for use in cytotoxicity evaluations because they elicit no cytopathic
medium, or reagents.
effect. Other filter disks that do not elicit a cytopathic effect may also be
9.2 Warm all solutions and materials to a temperature of 37
used.
6 2°C before placing in contact with cells.
5.8 Water Bath, capable of maintaining a temperature of 37
6 2°C.
10. Preparation of Specimens
NOTE 3—A laminar flow work area capable of filtering out 99.99 % of
10.1 Sterilize all specimens by a method appropriate to the
all particles greater than 0.5 μm in diameter, or a class 100 clean room may
end use of the device.
be necessary to prevent contamination of cultures.
10.2 Where a device is sufficiently small (see 10.3 and 10.4)
6. Reagents
to fit into the Petri dish leaving an adequate margin of cells for
6.1 The following reagents shall be used: evaluation, use the entire device as a specimen.
6.1.1 Minimum Essential Medium (MEM), prepared with- 10.3 Cut large solid materials and devices in cross-section
out L-glutamine and augmented by the addition of Earle’s salts to obtain a flat surface having an area of 100 to 250 mm to be
and 10 % fetal bovine serum. placed in direct contact with the cell monolayer.
10.4 Prepare specimens of rod or tubing or of rod- or
NOTE 4—Glutamine is omitted from this formulation in order to
tube-shaped devices as follows:
maximize the shelf life of the medium. Immediately before use, 5 mL of
10.4.1 Where the diameter is less than 6.4 mm, cut 5 to 15
L-glutamine solution (see 6.1.2) are added to each 500 mL of MEM.
mm in length.
NOTE 5—Opened containers of MEM may be stored at a temperature of
2 to 8°C for periods of not more than one week.
10.4.2 Where the diameter is 6.4 to 15 mm, cut 2 to 8 mm
NOTE 6—Antibiotics, such as penicillin G and streptomycin, may be
in length.
added to the medium to reduce the incidence of bacterial contamination.
10.4.3 Where the diameter exceeds 15 mm, prepare cross-
This may, however, have an adverse effect on the viability of the cell
sections as described in 10.3.
cultures.
10.5 Obtain specimens from larger medical items from
6.1.2 L-glutamine Solution, 29.2 mg/mL of sterile water.
locations with relatively large cross-sections in order to expose
6.1.3 Hanks’ Solution, calcium-and magnesium-free (store
interior material.
at room temperature).
10.6 If a device is constructed of two or more materials, cut
6.1.4 Trypsin, 0.1 % solution in Hanks’ solution or calcium-
either the test specimen from the materials’ interface or test
and magnesium-free, phosphate-buffered saline (store frozen).
separate specimens from each material.
6.1.5 Water, distilled, deionized, and sterile, with a mini-
10.7 Prepare specimens for evaluating the cytotoxicity of
mum resistivity of 1 MV·cm.
liquids or extracts by saturating a sterile filter disk and allowing
6.2 All reagents shall be tissue-culture grade or equivalent.
the excess liquid to drain off while maintaining asepsis. Use the
6.3 Reagents shall be reconstituted in accordance with the
saturated filter disk as a test specimen.
manufacturer’s directions, using aseptic technique.
NOTE 8—When ethylene oxide or other chemical sterilants are used,
6.4 Reagents shall be stored in accordance with the manu-
adequate aeration time should be allowed, to permit dissipation of residues
facturer’s directions unless otherwise indicated in 6.1.
which may adversely affect the results recorded in this assay.
7. Cell Cultures
NOTE 9—In general, specimens should be cleaned to remove any
residues from specimen preparation, and sterilized after they have been
7.1 Cell cultures used in this assay shall be the ATCC, CCL
cut to size. If the large solid materials are very hard, like ceramics, which
1 NCTC clone 929 strain (clone of Strain L, mouse connective
require cutting with metal or diamond saws, care should be taken to
tissue) designated L-929.
remove any contamination from the metal blade or from the metal bonding
the diamonds to the blade. When evaluating the cytotoxicity potential of
8. Control Materials
medical materials or devices that are contained in the final sterile package,
8.1 Prepare negative control specimens in accordance with
resterilization, further processing, or delay between the time of opening
Section 10 from a mat
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