ASTM F895-84(2001)
(Test Method)Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
SCOPE
1.1 This test method is appropriate for materials in a variety of shapes and for materials which are not necessarily sterile. This test method would be appropriate in situations where the amount of material is limited. For example, small devices or powders could be placed on the agar and the presence of a zone of inhibition of cell growth could be examined.
1.1.1 This test method is not appropriate for leachables which do not diffuse through agar or agarose.
1.1.2 While the agar layer can act as a cushion to protect the cells from the specimen, there may be materials which are sufficiently heavy to compress the agar and prevent diffusion or to cause mechanical damage to the cells. This test method would not be appropriate for these materials.
1.2 The L-929 cell line was chosen because it has a significant history of use in assays of this type. This is not intended to imply that its use is preferred, only that the L-929 is an established cell line, well-characterized and readily available, that has demonstrated reproducible results in several laboratories.
1.3 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 and health practices and determine the applicability of regulatory limitations prior to use.
General Information
Relations
Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or discontinued.
Contact ASTM International (www.astm.org) for the latest information.
Designation: F 895 – 84 (Reapproved 2001)
Standard Test Method for
Agar Diffusion Cell Culture Screening for Cytotoxicity
This standard is issued under the fixed designation F 895; 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.
This standard has been approved for use by agencies of the Department of Defense.
TABLE 1 Zone Description
1. Scope
Zone
1.1 This test method is appropriate for materials in a variety
Description of Zone
Index
of shapes and for materials which are not necessarily sterile.
0 No detectable zone around or under specimen
This test method would be appropriate in situations in which
1 Zone limited to area under specimen
the amount of material is limited. For example, small devices
2 Zone extends less than 0.5 cm beyond specimen
3 Zone extends 0.5 to 1.0 cm beyond specimen
or powders could be placed on the agar and the presence of a
4 Zone extends greater than 1.0 cm beyond specimen but
zone of inhibition of cell growth could be examined.
does not involve entire dish
1.1.1 This test method is not appropriate for leachables that
5 Zone involves entire dish
do not diffuse through agar or agarose.
1.1.2 While the agar layer can act as a cushion to protect the
3. Summary of Test Method
cells from the specimen, there may be materials that are
sufficiently heavy to compress the agar and prevent diffusion or
3.1 Cell cultures are grown to a confluent monolayer in
to cause mechanical damage to the cells. This test method
culture dishes. The medium is aspirated and replaced with an
would not be appropriate for these materials.
agar-containing medium that is allowed to solidify. Test control
1.2 The L-929 cell line was chosen because it has a
articles are placed on the agar surface to evaluate the cytotoxic
significant history of use in assays of this type. This is not properties of a given material or device. Toxic components in
intended to imply that its use is preferred, only that the L-929
the test article can diffuse into the culture medium forming a
is an established cell line, well characterized and readily concentration gradient and adversely affecting cells at varying
available, that has demonstrated reproducible results in several
distances from the test article. This method is well suited for
laboratories. low-density materials (film, paper, and so forth), powders,
1.3 This standard does not purport to address all of the
liquids, and high-density materials that could physically dam-
safety concerns, if any, associated with its use. It is the age the cells if placed in direct contact with the cell monolayer.
responsibility of the user of this standard to establish appro-
4. Significance and Use
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. 4.1 This test method is useful for assessing the cytotoxic
potential of new materials and formulations and as part of a
2. Referenced Documents
quality control program for established medical devices and
2.1 ASTM Standards:
components.
F 748 Practice for Selecting Generic Biological Test Meth-
4.2 This test method assumes that assessment of cytotoxic-
ods for Materials and Devices
ity provides useful information to aid in predicting the potential
2.2 ATCC Document:
clinical applications in humans. Cell culture methods have
American Type Culture Collection, (ATCC) Catalogue of
shown good correlation with animal assays and are frequently
Strains II
more sensitive to cytotoxic agents.
USP Negative Control Plastic Reference Standard
4.3 This cell culture test method is suitable for incorporation
into specifications and standards for materials to be used in the
TABLE 2 Lysis Description
This test method is under the jurisdiction of ASTM Committee F04 on Medical
Lysis
Description of Zone
and Surgical Materials and Devices and is the direct responsibility of Subcommittee
Index
F04.16 on Biocompatibility Test Methods.
0 No observable cytotoxicity
Current edition approved Sept. 28, 1984. Published February 1985.
2 1 Less than 20 % of zone affected
Annual Book of ASTM Standards, Vol 13.01.
2 20 to 39 % of zone affected
Fourth edition, 1983, is available from American Type Culture Collection,
3 40 to 59 % of zone affected
12031 Parklawn Dr. Rockville, MD 10892. Library of Congress No. 76-640122.
4 60 to 80 % of zone affected
U.S. Pharmacopeia, Vol XXIII, Mack Publishing Co., Easton, PA, 1995, p.
5 Greater than 80 % of zone affected
1652–1653. Use lastest supplement to ensure current cumulative revisions are used.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
F 895
construction of medical devices that are to be implanted into 2-mL 100X nonessential amino acid (L-glutamine). Bring to
the human body or placed in contact with tissue fluids or blood final volume (100 mL) with sterile distilled water. Filter
on a long-term basis. sterilize the 2X media. Mix with equal amounts of sterilized
4.4 Some biomaterials with a history of safe clinical use in 3 % agar nobel to give the final concentration of the media as
medical devices are cytotoxic. This test method does not imply 1X.
that all biomaterials must pass this assay to be considered safe 6.1.3 L-Glutamine Solution (Lyophilized), 29.2 mg/mL.
for clinical use (Practice F 748). Rehydrate with sterile distilled water. (Store frozen.)
6.1.4 Hanks’ Balanced Salt Solution, calcium- and
5. Apparatus
magnesium-free (store at room temperature).
5.1 The following apparatus shall be used:
6.1.5 Trypsin, 0.1 % solution in Hanks’ balanced salt solu-
5.2 Incubator, which maintains the cultures at 37 6 2°C, 5
tion or calcium- and magnesium-free, phosphate-buffered sa-
6 1% CO , and greater than 90 % relative humidity.
line (store frozen).
5.3 Water Bath, capable of maintaining a temperature of 37
6.1.6 Water, sterile, deionized, or distilled water should be
6 2°C and 45 6 2°C.
used.
5.4 Microscope, with inverted phase contrast optics and
6.1.7 Noble Agar,3%.
magnifications of 40, 100, and 200X.
6.1.8 Neutral Red Stain, 0.01 % by weight in phosphate-
5.5 Clinical Centrifuge, capable of attaining 1000 gravities.
buffered saline.
5.6 Sterile, Disposable 150-cm Tissue Culture Flasks.
6.2 All reagents shall be tissue-culture grade or equivalent.
5.7 Sterile, Tissue Culture Dishes, 35 mm in diameter and
6.3 Reagents shall be reconstituted in accordance with the
10 mm deep.
manufacturer’s directions, using aseptic technique.
NOTE 1—Plastic dishes are recommended because they provide a flat
7. Cell Culture
surface that promotes the formation of a uniform monolayer of cells.
7.1 Cell cultures used in this assay shall be the ATCC, CCL
5.8 Sterile, Disposable, Centrifuge Tubes.
I NCTC clone 929 strain (clone of Strain L, mouse connective
5.9 Sterile Pipettes, 1, 5, and 10 mL.
tissue) designated L-929.
5.10 Filter Disks, 10 mm in diameter for evaluation of
liquids.
8. Control Materials
NOTE 2—Millipore AP2501000 filter disks have been found satisfac-
8.1 Prepare negative control specimens in accordance with
tory for use in cytotoxicity evaluations because they elicit no cytopathic
Section 10 from a material that consistently elicits negligible
effect. Other filter disks that do not elicit a cytopathic effect may also be
cellular response in this assay (for example, USP Negative
used.
Control Plastic Reference Standard).
NOTE 3—A laminar flow work area capable of filtering out 99.99 % of
all particles greater than 0.3 μm in diameter, or a Class 100 clean room
8.2 Prepare positive control specimens in accordance with
may be necessary to prevent contamination of cultures.
Section 10 from a material that consistently elicits a moderate
and reproducible degree of cytotoxicity (for example, an
6. Reagents
aqueous solution of phenol (0.45 6 0.05 % by volume), or
6.1 The following reagents shall be used:
other material producing a known cytotoxic response, for
6.1.1 For Cell Culture Maintenance, 1X Media. Minimum
example, latex rubber).
Essential Medium (MEM) is prepared by mixing 90-mL
8.2.1 Use an aqueous solution of phenol to give a diffuse
Eagle’s MEM (with Earle’s salts, without L-glutamine), adjust
reaction of cellular degeneration and sloughing; a latex rubber
solution to pH 7.15, add 10-mL fetal bovine serum, and 1-mL
will give a zone of toxicity.
100X nonessential amino acids (L-glutamine).
8.2.2 Take care when preparing aqueous solutions of phenol
6.1.1.1 Opened containers of prepared MEM may be stored
to ensure the homogeneity of the solution since phase separa-
at a temperature of 2 to 8°C for periods of not more than two
tions may occur.
weeks. Glutamine is omitted from this formulation to maxi-
8.2.3 Latex rubber is a widely used control material that has
mize the shelf life. Immediately before use, 1 mL of
demonstrated reproducible results in several laboratories.
L-glutamine solution (see 6.1.3) is added to each 100 mL of
MEM.
9. General Technique
6.1.1.2 Antibiotics, such as penicillin G10 000 I.V./mL, and
9.1 Use aseptic technique throughout this assay to minimize
streptomycin 10 000 I.V./mL, may be added to the medium to
microbial contamination.
reduce the incidence of bacterial contamination. Use 1 mL of
NOTE 4—Mouth pipetting should not be used to transfer cells, medium,
antibiotic per 100-mL media. Care shall be taken to ensure that
or reagents.
the antibiotics do not have an adverse effect on the viability of
the cell cultures. 9.2 Warm all solutions and material to a temperature of 37
6.1.2 For Agar Media Overlay, to prepare 2X Media 6 2°C before being placed in contact with cells.
(100-mL final volume). Twice concentrated (2X) MEM is 9.3 Wash all glass vessels thoroughly with a cleaning
prepared by mixing 20 mL of 10X Eagle’s MEM (with Earle’s solution and rinse thoroughly with copious amounts of deion-
Salts, without L-glutamine), 0.22-g sodium bicarbonate ized water.
(buffer) and sterile distilled water to bring to 70 % volume (70 9.4 Clean all work surfaces with a disinfectant solution
mL). Adjust pH to 7.15. Add 20-mL fetal bovine serum and before use.
F 895
9.5 Record the culture history of the cells. 11.4 Aspirate the rinse solution.
9.6 Stock cultures should be periodically screened for my- 11.5 Add a sufficient volume of trypsin solution (0.1 %) to
coplasma contamination. the flask to cover the cell monolayer (approximately 5 mL).
11.6 Incubate for 5 to 10 min to suspend the cells.
10. Specimen Preparation
11.7 Transfer the cell suspension to a centrifuge tube.
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.