ASTM F895-84(2006)
(Test Method)Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
Standard Test Method for Agar Diffusion Cell Culture Screening for Cytotoxicity
SIGNIFICANCE AND USE
This test method is useful for assessing the cytotoxic potential of new materials and formulations and as part of a quality control program for established medical devices and components.
This test method assumes that assessment of cytotoxicity provides useful information to aid in predicting the potential clinical applications in humans. Cell culture methods have shown good correlation with animal assays and are frequently more sensitive to cytotoxic agents.
This cell culture test method is suitable for incorporation into specifications and standards for materials to be used in the construction of medical devices that are to be implanted into the human body or placed in contact with tissue fluids or blood on a long-term basis.
Some biomaterials with a history of safe clinical use in medical devices are cytotoxic. This test method does not imply that all biomaterials must pass this assay to be considered safe for clinical use (Practice F 748).
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
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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation:F895–84 (Reapproved 2006)
Standard Test Method for
Agar Diffusion Cell Culture Screening for Cytotoxicity
ThisstandardisissuedunderthefixeddesignationF895;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginal
adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.Asuperscript
epsilon (´) indicates an editorial change since the last revision or reapproval.
This standard has been approved for use by agencies of the Department of Defense.
1. Scope American Type Culture Collection, (ATCC) Catalogue of
Strains II
1.1 This test method is appropriate for materials in a variety
USP Negative Control Plastic Reference Standard
of shapes and for materials that are not necessarily sterile.This
test method would be appropriate in situations in which the
3. Summary of Test Method
amount of material is limited. For example, small devices or
3.1 Cellculturesaregrowntoamonolayerinculturedishes.
powderscouldbeplacedontheagarandthepresenceofazone
The medium is aspirated and replaced with an agar-containing
of inhibition of cell growth could be examined.
medium that is allowed to solidify. Test control articles are
1.1.1 This test method is not appropriate for leachables that
placed on the agar surface to evaluate the cytotoxic properties
do not diffuse through agar or agarose.
of a given material or device. Toxic components in the test
1.1.2 While the agar layer can act as a cushion to protect the
article can diffuse into the culture medium forming a concen-
cells from the specimen, there may be materials that are
tration gradient and adversely affecting cells at varying dis-
sufficientlyheavytocompresstheagarandpreventdiffusionor
tances from the test article. This method is well suited for
to cause mechanical damage to the cells. This test method
low-density materials (film, paper, and so forth), powders,
would not be appropriate for these materials.
liquids, and high-density materials that could physically dam-
1.2 The L-929 cell line was chosen because it has a
age the cells if placed in direct contact with the cell monolayer.
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
4. Significance and Use
is an established cell line, well characterized and readily
4.1 This test method is useful for assessing the cytotoxic
available, that has demonstrated reproducible results in several
potential of new materials and formulations and as part of a
laboratories.
quality control program for established medical devices and
1.3 This standard does not purport to address all of the
components.
safety concerns, if any, associated with its use. It is the
4.2 This test method assumes that assessment of cytotoxic-
responsibility of the user of this standard to establish appro-
ityprovidesusefulinformationtoaidinpredictingthepotential
priate safety and health practices and determine the applica-
clinical applications in humans. Cell culture methods have
bility of regulatory limitations prior to use.
shown good correlation with animal assays and are frequently
more sensitive to cytotoxic agents.
2. Referenced Documents
2 4.3 Thiscellculturetestmethodissuitableforincorporation
2.1 ASTM Standards:
into specifications and standards for materials to be used in the
F748 Practice for Selecting Generic Biological Test Meth-
construction of medical devices that are to be implanted into
ods for Materials and Devices
the human body or placed in contact with tissue fluids or blood
2.2 ATCC Document:
on a long-term basis.
4.4 Some biomaterials with a history of safe clinical use in
This test method is under the jurisdiction ofASTM Committee F04 on Medical
medical devices are cytotoxic.This test method does not imply
andSurgicalMaterialsandDevices andisthedirectresponsibilityofSubcommittee
that all biomaterials must pass this assay to be considered safe
F04.16 on Biocompatibility Test Methods.
for clinical use (Practice F748).
Current edition approved March 1, 2006. Published April 2006. Originally
´1
approved in 1984. Last previous edition approved in 2001 as F895 – 84 (2001) .
DOI: 10.1520/F0895-84R06.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Fourth edition, 1983, is available from American Type Culture Collection,
Standards volume information, refer to the standard’s Document Summary page on 12031 Parklawn Dr., Rockville, MD 10892. Library of Congress No. 76-640122.
the ASTM website. U.S. Pharmacopeia, current edition, Rockville, MD.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F895–84 (2006)
5. Apparatus 6.1.4 Hanks’ Balanced Salt Solution, calcium- and
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-
61%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 2003.
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
7. Cell Culture
surface that promotes the formation of a uniform monolayer of cells.
7.1 Cell cultures used in this assay shall be theATCC, 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. Other suitable validated cell lines
5.10 Filter Disks, 10 mm in diameter for evaluation of
may be considered.
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
used. cellular response in this assay (for example, USP Negative
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,13 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 5- to 10-mL fetal bovine serum, and will give a zone of toxicity.
1-mL 1003 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-
tions may occur.
at a temperature of 2 to 8°C for periods of not more than two
weeks. Glutamine is omitted from this formulation to maxi- 8.2.3 Latex rubber is a widely used control material that has
demonstrated reproducible results in several laboratories.
mize the shelf life. Immediately before use, 1 mL of
L-glutamine solution (see 6.1.3) is added to each 100 mL of
9. General Technique
MEM.
9.1 Use aseptic technique throughout this assay to minimize
6.1.1.2 Antibiotics, such as penicillin G10 000 I.U./mL, and
microbial contamination.
streptomycin 10 000 I.U./mL, may be added to the medium to
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-mLmedia. Care shall be taken to ensure that or reagents.
the antibiotics do not have an adverse effect on the viability of
9.2 Warm all solutions and material to a temperature of 37
the cell cultures.
6 2°C before being placed in contact with cells.
6.1.2 For Agar Media Overlay, to prepare 23 Media
9.3 Wash all glass vessels thoroughly with a cleaning
(100-mL final volume). Twice concentrated (23) MEM is
solution and rinse thoroughly with copious amounts of deion-
prepared by mixing 20 mLof 103 Eagle’s MEM (with Earle’s
ized water.
SaltswithoutL-glutamine),0.22-gsodiumbicarbonate(buffer)
9.4 Clean all work surfaces with a disinfectant solution
and sterile distilled water to bring to 70 % volume (70 mL).
before use.
Adjust pH to 7.15. Add 20-mL fetal bovine serum and 2-mL
9.5 Record the culture history of the cells.
1003 nonessential amino acid (L-glutamine). Bring to final
9.6 Stock cultures should be periodically screened for my-
volume (100 mL) with sterile distilled water. Filter sterilize the
coplasma contamination.
23 media. Mix with equal amounts of sterilized 3 % agar
10. Specimen Preparation
nobel to give the final concentration of the media as 13.
6.1.3 L-Glutamine Solution (Lyophilized), 29.2 mg/mL. 10.1 Sterilize all specimens by a method appropriate to the
Rehydrate with sterile distilled water. (Store frozen.) end use of the device.
F895–84 (2006)
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 P
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