ASTM F895-84(2001)e1
(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.
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e1
Designation:F895–84 (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.
e NOTE—Editorial corrections were made throughout and keywords were added in June 2001.
1. Scope USP Negative Control Plastic Reference Standard
1.1 This test method is appropriate for materials in a variety
3. Summary of Test Method
of shapes and for materials that are not necessarily sterile.This
3.1 Cellculturesaregrowntoamonolayerinculturedishes.
test method would be appropriate in situations in which the
The medium is aspirated and replaced with an agar-containing
amount of material is limited. For example, small devices or
medium that is allowed to solidify. Test control articles are
powderscouldbeplacedontheagarandthepresenceofazone
placed on the agar surface to evaluate the cytotoxic properties
of inhibition of cell growth could be examined.
of a given material or device. Toxic components in the test
1.1.1 This test method is not appropriate for leachables that
article can diffuse into the culture medium forming a concen-
do not diffuse through agar or agarose.
tration gradient and adversely affecting cells at varying dis-
1.1.2 While the agar layer can act as a cushion to protect the
tances from the test article. This method is well suited for
cells from the specimen, there may be materials that are
low-density materials (film, paper, and so forth), powders,
sufficientlyheavytocompresstheagarandpreventdiffusionor
liquids, and high-density materials that could physically dam-
to cause mechanical damage to the cells. This test method
age the cells if placed in direct contact with the cell monolayer.
would not be appropriate for these materials.
1.2 The L-929 cell line was chosen because it has a
4. Significance and Use
significant history of use in assays of this type. This is not
4.1 This test method is useful for assessing the cytotoxic
intended to imply that its use is preferred, only that the L-929
potential of new materials and formulations and as part of a
is an established cell line, well characterized and readily
quality control program for established medical devices and
available, that has demonstrated reproducible results in several
components.
laboratories.
4.2 This test method assumes that assessment of cytotoxic-
1.3 This standard does not purport to address all of the
ityprovidesusefulinformationtoaidinpredictingthepotential
safety concerns, if any, associated with its use. It is the
clinical applications in humans. Cell culture methods have
responsibility of the user of this standard to establish appro-
shown good correlation with animal assays and are frequently
priate safety and health practices and determine the applica-
more sensitive to cytotoxic agents.
bility of regulatory limitations prior to use.
4.3 Thiscellculturetestmethodissuitableforincorporation
into specifications and standards for materials to be used in the
2. Referenced Documents
construction of medical devices that are to be implanted into
2.1 ASTM Standards:
the human body or placed in contact with tissue fluids or blood
F 748 Practice for Selecting Generic Biological Test Meth-
on a long-term basis.
ods for Materials and Devices
4.4 Some biomaterials with a history of safe clinical use in
2.2 ATCC Document:
medical devices are cytotoxic.This test method does not imply
American Type Culture Collection, (ATCC) Catalogue of
3 that all biomaterials must pass this assay to be considered safe
Strains II
for clinical use (Practice F 748).
5. Apparatus
This test method is under the jurisdiction ofASTM Committee F04 on Medical
and Surgical Materials and Devices and is the direct responsibility of Subcommittee
5.1 The following apparatus shall be used:
F04.16 on Biocompatibility Test Methods.
Current edition approved Sept. 28, 1984. Published February 1985.
Annual Book of ASTM Standards, Vol 13.01.
Fourth edition, 1983, is available from American Type Culture Collection,
12031 Parklawn Dr., Rockville, MD 10892. Library of Congress No. 76-640122. U.S. Pharmacopeia, current edition, Rockville, MD.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F895
5.2 Incubator, which maintains the cultures at 37 6 2°C, 5 6.1.5 Trypsin, 0.1 % solution in Hanks’ balanced salt solu-
61%CO , and greater than 90 % relative humidity. tion or calcium- and magnesium-free, phosphate-buffered sa-
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 xg.
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
surface that promotes the formation of a uniform monolayer of cells.
7. Cell Culture
5.8 Sterile, Disposable, Centrifuge Tubes.
7.1 Cell cultures used in this assay shall be theATCC, CCL
5.9 Sterile Pipettes, 1, 5, and 10 mL. I NCTC clone 929 strain (clone of Strain L, mouse connective
5.10 Filter Disks, 10 mm in diameter for evaluation of tissue) designated L-929. Other suitable validated cell lines
may be considered.
liquids.
NOTE 2—Millipore AP2501000 filter disks have been found satisfac-
8. Control Materials
tory for use in cytotoxicity evaluations because they elicit no cytopathic
effect. Other filter disks that do not elicit a cytopathic effect may also be 8.1 Prepare negative control specimens in accordance with
used.
Section 10 from a material that consistently elicits negligible
NOTE 3—A laminar flow work area capable of filtering out 99.99 % of
cellular response in this assay (for example, USP Negative
all particles greater than 0.3 µm in diameter, or a Class 100 clean room
Control Plastic Reference Standard).
may be necessary to prevent contamination of cultures.
8.2 Prepare positive control specimens in accordance with
Section 10 from a material that consistently elicits a moderate
6. Reagents
and reproducible degree of cytotoxicity (for example, an
6.1 The following reagents shall be used:
aqueous solution of phenol (0.45 6 0.05 % by volume), or
6.1.1 For Cell Culture Maintenance, 1X Media. Minimum
other material producing a known cytotoxic response, for
Essential Medium (MEM) is prepared by mixing 90-mL
example, latex rubber).
Eagle’s MEM (with Earle’s salts, without L-glutamine), adjust
8.2.1 Use an aqueous solution of phenol to give a diffuse
solution to pH 7.15, add 5- to 10-mL fetal bovine serum, and
reaction of cellular degeneration and sloughing; a latex rubber
1-mL 100X nonessential amino acids (L-glutamine).
will give a zone of toxicity.
6.1.1.1 Opened containers of prepared MEM may be stored 8.2.2 Take care when preparing aqueous solutions of phenol
at a temperature of 2 to 8°C for periods of not more than two
to ensure the homogeneity of the solution since phase separa-
weeks. Glutamine is omitted from this formulation to maxi- tions may occur.
mize the shelf life. Immediately before use, 1 mL of
8.2.3 Latex rubber is a widely used control material that has
L-glutamine solution (see 6.1.3) is added to each 100 mL of demonstrated reproducible results in several laboratories.
MEM.
6.1.1.2 Antibiotics, such as penicillin G10 000 I.U./mL, and
9. General Technique
streptomycin 10 000 I.U./mL, may be added to the medium to
9.1 Use aseptic technique throughout this assay to minimize
reduce the incidence of bacterial contamination. Use 1 mL of
microbial contamination.
antibiotic per 100-mLmedia. Care shall be taken to ensure that
NOTE 4—Mouth pipetting should not be used to transfer cells, medium,
the antibiotics do not have an adverse effect on the viability of
or reagents.
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 mLof 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 (buffer)
ized water.
and sterile distilled water to bring to 70 % volume (70 mL).
9.4 Clean all work surfaces with a disinfectant solution
Adjust pH to 7.15. Add 20-mL fetal bovine serum and 2-mL
before use.
100X nonessential amino acid (L-glutamine). Bring to final
9.5 Record the culture history of the cells.
volume (100 mL) with sterile distilled water. Filter sterilize the
9.6 Stock cultures should be periodically screened for my-
2Xmedia.Mixwithequalamountsofsterilized3 %agarnobel
coplasma contamination.
to give the final concentration of the media as 1X.
6.1.3 L-Glutamine Solution (Lyophilized), 29.2 mg/mL.
10. Specimen Preparation
Rehydrate with sterile distilled water. (Store frozen.)
6.1.4 Hanks’ Balanced Salt Solution, calcium- and 10.1 Sterilize all specimens by a method appropriate to the
magnesium-free (store at room temperature). end use of the device.
F895
10.2 Whereadeviceissufficientlysmall(see10.3and10.4) 11.10 Resuspend the cells in 10 6 0.1 mL of fresh medium
to fit into the culture dish leaving an adequate margin of cells and mix the suspension thoroughly.
for evaluation, use the entire device as a specimen. 11.11 Distribute the cell suspension equally among each of
10.3 Cutlargesolidmaterialsanddevicesincrosssectionto two to eight 150-cm tissue culture flasks.
obtain a flat surface having an area of 100 to 250 mm to be
11.12 Add a sufficient volume of fresh medium so that each
placed in direct contact with the agar surface. flask will contain approximately 50 mL.
10.4 Prepare specimens of rod or tubing or of rod- or
11.13 Change the medium every two to three d
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