ASTM F895-11(2016)
(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
4.1 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.
4.2 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.
4.3 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.
4.4 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 F748).
SCOPE
1.1 This test method is appropriate for materials in a variety of shapes and for materials that are not necessarily sterile. This test method would be appropriate in situations in which 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 that 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 that 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 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 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)
This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: F895 − 11 (Reapproved 2016)
Standard Test Method for
Agar Diffusion Cell Culture Screening for Cytotoxicity
ThisstandardisissuedunderthefixeddesignationF895;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginal
adoptionor,inthecaseofrevision,theyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperscript
epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2.2 ATCC Document:
American Type Culture Collection, (ATCC) Catalogue of
1.1 Thistestmethodisappropriateformaterialsinavariety
Strains II
ofshapesandformaterialsthatarenotnecessarilysterile.This
USP Negative Control Plastic Reference Standard
test method would be appropriate in situations in which the
amount of material is limited. For example, small devices or
3. Summary of Test Method
powderscouldbeplacedontheagarandthepresenceofazone
3.1 Cellculturesaregrowntoamonolayerinculturedishes.
of inhibition of cell growth could be examined.
The medium is aspirated and replaced with an agar-containing
1.1.1 This test method is not appropriate for leachables that
medium that is allowed to solidify. Test control articles are
do not diffuse through agar or agarose.
placed on the agar surface to evaluate the cytotoxic properties
1.1.2 Whiletheagarlayercanactasacushiontoprotectthe
of a given material or device. Toxic components in the test
cells from the specimen, there may be materials that are
article can diffuse into the culture medium, forming a concen-
sufficientlyheavytocompresstheagarandpreventdiffusionor
tration gradient and adversely affecting cells at varying dis-
to cause mechanical damage to the cells. This test method
tances from the test article. This method is well suited for
would not be appropriate for these materials.
low-density materials (film, paper, and so forth), powders,
1.2 The L-929 cell line was chosen because it has a
liquids, and high-density materials that could physically dam-
significant history of use in assays of this type. This is not
agethecellsifplacedindirectcontactwiththecellmonolayer.
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
available, that has demonstrated reproducible results in several
4.1 This test method is useful for assessing the cytotoxic
laboratories.
potential of new materials and formulations and as part of a
quality control program for established medical devices and
1.3 The values stated in SI units are to be regarded as
components.
standard. No other units of measurement are included in this
standard.
4.2 This test method assumes that assessment of cytotoxic-
ityprovidesusefulinformationtoaidinpredictingthepotential
1.4 This standard does not purport to address all of the
clinical applications in humans. Cell culture methods have
safety concerns, if any, associated with its use. It is the
shown good correlation with animal assays and are frequently
responsibility of the user of this standard to establish appro-
more sensitive to cytotoxic agents.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
4.3 Thiscellculturetestmethodissuitableforincorporation
intospecificationsandstandardsformaterialstobeusedinthe
2. Referenced Documents
construction of medical devices that are to be implanted into
2.1 ASTM Standards: thehumanbodyorplacedincontactwithtissuefluidsorblood
F748PracticeforSelectingGenericBiologicalTestMethods on a long-term basis.
for Materials and Devices
4.4 Some biomaterials with a history of safe clinical use in
medicaldevicesarecytotoxic.Thistestmethoddoesnotimply
that all biomaterials must pass this assay to be considered safe
ThistestmethodisunderthejurisdictionofASTMCommitteeF04onMedical
for clinical use (Practice F748).
andSurgicalMaterialsandDevicesandisthedirectresponsibilityofSubcommittee
F04.16 on Biocompatibility Test Methods.
5. Apparatus
Current edition approved April 1, 2016. Published June 2016. Originally
approved in 1984. Last previous edition approved in 2011 as F895–11. DOI:
5.1 The following apparatus shall be used:
10.1520/F0895-11R16.
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 − 11 (2016)
5.2 Incubator, which maintains the cultures at 37 6 2°C, 5 6.1.4 Hanks’ Balanced Salt Solution, calcium- and
61%CO , and greater than 90% relative humidity. magnesium-free (store at room temperature).
6.1.5 Trypsin, 0.1% solution in Hanks’ balanced salt solu-
5.3 Water Bath, capable of maintaining a temperature of 37
tion or calcium- and magnesium-free, phosphate-buffered sa-
6 2°C and 45 6 2°C.
line (store frozen).
5.4 Microscope, with inverted phase contrast optics and
6.1.6 Water, sterile, deionized, or distilled water should be
magnifications of 40, 100, and 200×.
used.
6.1.7 Noble Agar,3%.
5.5 Clinical Centrifuge, capable of attaining 1000 xg.
6.1.8 Neutral Red Stain, 0.01% by weight in phosphate-
5.6 Sterile, Disposable 150-cm Tissue Culture Flasks.
buffered saline.
5.7 Sterile, Tissue Culture Dishes, 35 mm in diameter and
6.2 All reagents shall be tissue-culture grade or equivalent.
10 mm deep.
6.3 Reagents shall be reconstituted in accordance with the
NOTE 1—Plastic dishes are recommended because they provide a flat
manufacturer’s directions, using aseptic technique.
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
liquids.
may be considered.
NOTE2—MilliporeAP2501000filterdiskshavebeenfoundsatisfactory
foruseincytotoxicityevaluationsbecausetheyelicitnocytopathiceffect.
8. Control Materials
Other filter disks that do not elicit a cytopathic effect may also be used.
8.1 Prepare negative control specimens in accordance with
NOTE 3—Alaminar 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 Section 10 from a material that consistently elicits negligible
may be necessary to prevent contamination of cultures.
cellular response in this assay (for example, USP Negative
Control Plastic Reference Standard).
6. Reagents
8.2 Prepare positive control specimens in accordance with
6.1 The following reagents shall be used:
Section 10 from a material that consistently elicits a moderate
6.1.1 For Cell Culture Maintenance, 1× Media. Minimum and reproducible degree of cytotoxicity (for example, an
Essential Medium (MEM) is prepared by mixing 90 mL of aqueous solution of phenol (0.45 6 0.05% by volume), or
Eagle’s MEM (with Earle’s salts, without L-glutamine), ad-
other material producing a known cytotoxic response, for
justing the solution to pH of 7.15, and adding 5 to 10 mL of example, latex rubber).
fetalbovineserum,and1mLof100×nonessentialaminoacids
8.2.1 Use an aqueous solution of phenol to give a diffuse
(L-glutamine). reaction of cellular degeneration and sloughing; a latex rubber
6.1.1.1 Opened containers of prepared MEM may be stored will give a zone of toxicity.
8.2.2 Takecarewhenpreparingaqueoussolutionsofphenol
at a temperature of 2 to 8°C for periods of not more than two
weeks. Glutamine is omitted from this formulation to maxi- to ensure the homogeneity of the solution since phase separa-
tions may occur.
mize the shelf life. Immediately before use, 1 mL of
L-glutamine solution (see 6.1.3) is added to each 100 mL of 8.2.3 Latexrubberisawidelyusedcontrolmaterialthathas
MEM. demonstrated reproducible results in several laboratories.
6.1.1.2 Antibiotics, such as penicillin G10000 I.U.⁄mL,
9. General Technique
andstreptomycin10000I.U./mL,maybeaddedtothemedium
toreducetheincidenceofbacterialcontamination.Use1mLof
9.1 Useaseptictechniquethroughoutthisassaytominimize
antibioticper100-mLmedia.Careshallbetakentoensurethat
microbial contamination.
the antibiotics do not have an adverse effect on the viability of
NOTE 4—Mouth pipetting should not be used to transfer cells, medium,
the cell cultures.
or reagents.
6.1.2 For Agar Media Overlay, to prepare 2× Media
9.2 Warm all solutions and materials to a temperature of 37
(100-mL final volume). Twice concentrated (2×) MEM is
6 2°C before being placed in contact with cells.
prepared by mixing 20 mLof 10× Eagle’s MEM (with Earle’s
SaltswithoutL-glutamine),0.22-gsodiumbicarbonate(buffer) 9.3 Wash all glass vessels thoroughly with a cleaning
and sterile distilled water to bring to 70mL. Adjust the pH to solution and rinse thoroughly with copious amounts of deion-
7.15. Add 20-mL fetal bovine serum and 2 mL of 100× ized water.
nonessential amino acid (L-glutamine). Bring to final volume
9.4 Clean all work surfaces with a disinfectant solution
(100 mL) with sterile distilled water. Filter sterilize the 2×
before use.
media. Mix with equal amounts of sterilized 3% agar nobel to
9.5 Record the culture history of the cells.
give the final concentration of the media as 1×.
6.1.3 L-Glutamine Solution (Lyophilized), 29.2 mg/mL. Re- 9.6 Stock cultures should be periodically screened for my-
hydrate with sterile distilled water. (Store frozen.) coplasma contamination.
F895 − 11 (2016)
10. Specimen Preparation 11.5 Add a sufficient volume of trypsin solution (0.1%) to
the flask to cover the cell monolayer (approximately 5 mL).
10.1 Sterilize all specimens by a method appropriate to the
end use of the device.
11.6 Incubate for 5 to 10 min to suspend the cells.
10.2 Whereadeviceissufficientlysmall(see10.3and10.4)
11.7 Transfer the cell suspension to a centrifuge tube.
to fit into the culture dish leaving an adequate margin of cells
11.8 Centrifuge at 1300 xg for 6 min.
for evaluation, use the entire device as a specimen.
11.9 Discard the supernatant.
10.3 Cutlargesolidmaterialsanddevicesincrosssec
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.