ASTM F895-25 琼脂扩散细胞培养法细胞毒性筛选的标准试验方法
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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:F895-25
Standard Test Method for
Agar Diffusion Cell Culture Screening for Cytotoxicity¹
This standard is issued under the fixed designation F895;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(8)indicates an editorial change since the last revision or reapproval.
1.Scope
1.1 This qualitative test method is appropriate for materials
in a variety of shapes.This test method would be appropriate
in situations in which the amount of material is limited.For
example,small devices could be placed on the agar and the
presence of a zone of inhibition of cell growth could be
examined,if the chemicals from the test sample can diffuse
through the agar layer.
1.1.1 This test method is not appropriate for chemicals that
do not diffuse through agar or agarose,or chemicals that
interact with the agar or agarose layer.
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,including units officially
accepted for use with SI,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 appro-
priate safety,health,and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
1.5 This international standard was developed in accor-
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the
Development of International Standards,Guides and Recom-
mendations issued by the World Trade Organization Technical
Barriers to Trade(TBT)Committee.
2.Referenced Documents
2.1 ASTM Standards:²
F748 Practice for Selecting Generic Biological Test Methods
for Materials and Devices
2.2 ATCC Document:
American Type Culture Collection,(ATCC)Catalogue of
Strains II³
USP Negative Control Plastic Reference Standard⁴
3.Summary of Test Method
3.1 Cell cultures are grown to a monolayer in culture dishes.
The medium is aspirated and replaced with an agar-containing
medium that is allowed to solidify.Test articles are placed on
the agar surface to evaluate the cytotoxic properties of a given
material or device.If the toxic components/chemicals from the
test article can diffuse through the agar layer they could
adversely affect the cells underneath the agar layer.This
method is well suited for low-density materials(film,paper,
and so forth),liquids,and high-density materials that could
physically damage the cells if placed in direct contact with the
cell monolayer.
4.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 cytotoxic-
ity provides useful information to aid in predicting the potential
for cytotoxicity including necrotic reactions to medical mate-
rials and devices during clinical applications in humans.Cell
culture methods have shown good correlation with animal
assays when only chemical toxicities are being considered and
are frequently more sensitive to cytotoxic agents.
4.3 Some biomaterials with a history of safe clinical use in
medical devices are cytotoxic.This test method does not imply
This test method is under the jurisdiction of ASTM Committe F04 on Medical
and Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.16 on Biocompatibility Test Methods.
Current edition approved Jan.1,2025.Published January 2025.Originally
approved in 1984.Last previous edition approved in 2016 as F895-11(2016).
DOI:10.1520/F0895-25.
2For referenced ASTM standards,visit the ASTM website,www.astm.org,or
contact ASTM Customer Service at www.astm.org/contact.For Annual Book of
ASTM Standards volume information,refer to the standard's Document Summary
page on the ASTM website.
³Fourth edition,1983,is available from American Type Culture Collection,
12031 Parklawn Dr.,RockvilleMD 10892.Library of Congress No.76-640122.
4U.SPharmacopeia,current edition,Rockville,MD.
Copyright OASTM Intermational,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States
2
F895-25
that all biomaterials must pass this assay to be considered safe
for clinical use(Practice F748).
5.Apparatus
5.1 The following apparatus shall be used:
5.2 Incubator;which maintains the cultures at 37±2℃,5
±1%CO₂,and greater than 90 %relative humidity.
5.3 Water Bath,capable of maintaining a temperature of 37
±2℃ and 45±2℃.
5.4 Microscope,with inverted phase contrast optics and
magnifications of 40,100,and 200×.
5.5 Clinical Centrifuge,capable of attaining 1000 xg.
5.6 Sterile,Disposable 150 cm²Tissue Culture Flasks
5.7 Sterile Tissue Culture Dishes,35 mm in diameter and
10 mm deep.
NoTE 1—Plastic dishes are recommended because they provide a flat
surface that promotes the formation of a uniform monolayer ofcells.
5.8 Sterile,Disposable,Centrifuge Tubes.
5.9 Sterile Pipettes,1,5,and 10 mL.
5.10 Filter Disks,10 mm in diameter for evaluation of
liquids.
NoTE 2—Millipore AP2501000 filter disks have been found satisfactory
for use in cytotoxicity evaluations because they elicit no cytopathic effect.
Other filter disks that do not elicit a cytopathic effect may also be used.
NoTE 3—A laminar flow work area capable offiltering out 99.99%of
all particles greater than 0.3 μm in diameter,or a Class 100 clean room
may be necessary to prevent contamination ofcultures.
6.Reagents
6.1 The following reagents shall be used:
6.1.1 For Cell Culture Maintenance,1×media.Minimum
Essential Medium(MEM)is prepared by mixing 90 mL of
Eagle's MEM(with Earle's salts,without L-glutamine),ad-
justing the solution to pH of 7.2,and adding 5 to 10 mL of fetal
bovine serum,and 1 mL of 100×nonessential amino acids
(L-glutamine).
6.1.1.1 Opened containers of prepared MEM may be stored
at a temperature of 2 to 8℃ for periods of not more than two
weeks.However,Glutamine should be omitted from this
formulation to maximize the shelf life.Immediately before use,
1 mL of L-glutamine solution(see 6.1.3)is added to each
100 mL of MEM.
6.1.1.2 Antibiotics,such as penicillin G10000I.U./mL,and
streptomycin 10000 IU./mL,may be added to the medium to
reduce the incidence of bacterial contamination.Use 1mL of
antibiotic per 100 mL media.Care shall be taken to ensure that
the antibiotics do not have an adverse effect on the viability of
the cell cultures.
6.1.2 For Agar Media Overlay,to prepare 2×media
(100 mL final volume).Twice concentrated (2×)MEM is
prepared by mixing 20 mL of 10×Eagle's MEM(with Earle's
Salts without L-glutamine),0.22g sodium bicarbonate
(buffer),and sterile distilled water to bring to 70 mL.Adjust
the pH to 7.2.Add 20 mL fetal bovine serum and 2 mL of 100×
nonessential amino acid (L-glutamine).Bring to final volume
(100 mL)with sterile distilled water.Filter sterilize the 2×
media.Mix with equal amounts of sterilized 3%agar nobel to
give the final concentration of the media as 1x.
6.1.3 L-Glutamine Solution(Lyophilized),29.2 mg/mL.Re-
hydrate with sterile distilled water.(Store frozen.)
6.1.4 Hanks'Balanced Salt Solution,calcium and magne-
sium free (store at room temperature).
6.1.5 Trypsin,0.1%solution in Hanks'balanced salt solu-
tion or calcium and magnesium-free,phosphate-buffered saline
(store frozen).
6.1.6 Water;sterile,deionized,or distilled water should be
used.
6.1.7 Noble Agar;3%.
6.1.8 Neutral Red Stain,0.01%by weight in phosphate-
buffered saline.
6.2 All reagents shall be tissue culture grade or equivalent.
6.3 Reagents shall be reconstituted in accordance with the
manufacturer's directions,using aseptic technique.
7.Cell Culture
7.1 Cell cultures used in this assay shall be the ATCC,CCL
I NCTC clone 929 strain(clone of Strain L,mouse connective
tissue)designated L-929.Other suitable validated cell lines
may be considered.Cells should be tested periodically for
mycoplasma contamination.A passage or doubling limit
should be established within the laboratory with supportive
data to prevent the use of aged cells in this practice,which may
exhibit phenotypic abnormalities that could affect morphologic
assessment.
8.Control Materials
8.1 Prepare negative control specimens in accordance with
Section 10 from a material that consistently elicits negligible
cellular response in this assay(for example,USP Negative
Control Plastic Reference Standard).
8.2 Prepare positive control specimens in accordance with
Section 10 from a material that consistently elicits a cytotoxic
response(for example,latex rubber).
8.2.1 Latex rubber is a widely used positive control material
that has demonstrated reproducible results in several laborato-
ries and creates a zone of toxicity.
9.General Technique
9.1 Use aseptic technique throughout this assay to minimize
microbial contamination.
NoTE 4—Mouth pipetting should not be used to transfercells,medium,
or reagents.
9.2 Warm all solutions and materials to a temperature of 37
±2℃ before being placed in contact with cells.
9.3 Wash all glass vessels thoroughly with a cleaning
solution and rinse thoroughly with copious amounts of deion-
ized water.
9.4 Clean all work surfaces with a disinfectant solution
before use.
9.5 Record the culture history of the cells.
9.6 Stock cultures should be periodically screened for my-
coplasma contamination.
F895-25
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10.Specimen Preparation
10.1 Sterilize all specimens by a method appropriate to the
end use of the device.
10.2 Where a device is sufficiently small(see 10.3 and 10.4)
to fit into the culture dish leaving an adequate margin of cells
for evaluation,use the entire device as a specimen.
10.3 Cut large solid materials and devices in cross section to
obtain a flat surface having an area of 100 to 250 mm²to be
placed in direct contact with the agar surface.
10.4 Uneven items and tubing or rod-shaped materials and
devices may be cut into cross sections and placed side to side
to provide a flat test surface that covers an area of 100 mm²on
the agar surface.
10.5 Materials or devices with irregular or complex surface
areas such as resins,pellets,or molding with porous or
complex shapes may be placed on the agar layer so that it
covers an area of 100 mm²on the agar layer as uniformly as
possible.
10.6 Obtain specimens from larger medical items from
locations with relatively large cross sections to expose interior
material.
10.7 If a device is constructed of two or more materials that
are intended to contact body fluids or tissues,either cut the test
specimen from the materials'interface or test separate speci-
mens of each material or both.
10.8 Prepare specimens for evaluating the cytotoxicity of
liquids or extracts by saturating a sterile filter disk and allowing
the excess liquid to drain off while maintaining asepsis.Use the
saturated filter disk as a test specimen.
NoTE 5—When ethylene oxide or other chemical sterilants are used,
adequate aeration time should be allowed to permit dissipation ofresidues
which may adversely affect the results recorded in this assay.The aeration
time should be based on the time established by the sterilization validation
procedure for the dissipation ofthe residues.
NoTE 6—In general,the specimens should be prepared using aseptic
technique.If the specimen is very hard(for example,ceramics),care
should be taken to remove the residues that may be left on the freshly cut
surface by the cutting tool.When evaluating the cytotoxic potential of
medical materials or devices that are contained in the final sterilepackage,
resterilization,further processing,or delay between the time of opening
the package and starting the test must be avoided.With small items,the
entire content ofthe sterile package may be used as the test specimen.
When the size of the sterile packaged item is too large,an appropriate,
representative,small-sized specimen must be obtained.The application of
this assay to items in the final sterile package is limited to items that are
small or can be cut and reshaped using aseptic technique.
10.9 Absorbent materials tested in this method shall be
pre-wetted with culture medium to prevent loss of water from
the agar and subsequent cellular damage.
11.4 Aspirate the rinse solution.
11.5 Add a sufficient volume of trypsin solution(0.1%)to
the flask to cover the cell monolayer(approximately 5 mL).
11.6 Incubate for 5 to 10 min to dissociate the cells.
11.7 Transfer the cell suspension to a centrifuge tube.
11.8 Centrifuge at 1300 xg for 6 min.
11.9 Discard the supernatant.
11.10 Resuspend the cells in 10±0.1 mL of fresh medium
and mix the suspension thoroughly.
11.11 Distribute the cell suspension equally among each of
two to eight 150 cm²tissue culture flasks.
11.12 Add a sufficient volume of fresh medium so that each
flask will contain approximately 50 mL.
11.13 Change the medium every two to three days until the
monolayer is nearly confluent,then repeat Steps 11.2-11.12.
12.Cell Layer Preparation
12.1 Prepare confluent cell monolayers as follows:
12.2 Follow Steps 11.1-11.9 to prepare a cell suspension.
12.3 Add 2.0±0.1 mL of medium to each culture dish.
12.4 Using a sterile 10 mL serological pipette,add five to
seven drops of cell suspension to each dish.Rotate the dishes
to ensure an even distribution of cells.
12.5 Incubate until a near-confluent monolayer has formed,
as observed by microscopic examination.
NoTE 7—The formation of a near-confluent monolayer usually requires
three to five days.By counting cells with a hemacytometer(to ensure the
concentration ofthe inoculum),the time required for monolayer formation
may be regulated.A cell concentration of approximately 1×10⁵cells/mL,
plated at 2 mL per 35mm dish,will generally result in a near confluent
monolayer within four days.Plating densities and time to near confluence
should be established by each laboratory performing the test method.
12.6 If the cell suspension remains unused after Step 12.3,a
subculture may be prepared by adding 9mL of fresh medium
to each milliliter of cell suspension in a cell culture flask with
a surface area of approximately 3 cm²for each milliliter of
diluted cells and incubating it until a near-confluent monolayer
has formed,as determined by microscopic examination.
13.Test Procedure
13.1 Perform the agar diffusion cytotoxicity assay as fol-
lows:
13.2 Microscopically examine the cell cultures and reject
11.Cell Culture Maintenance
11.1 Use the following procedures to maintain the cells by
serial subculture:
11.2 Aspirate the medium from a 150 cm²cell culture flask
containing a near-confluent monolayer.
11.3 Rinse the cells with a sufficient volume (for example,5
to 10 mL)of Hanks'balanced salt solution to remove residual
serum.
any in which the cell monolayer is not of correct confluency or
the cells show signs of granulation or sloughing.
13.3 Autoclave 3%Nobel Agar for 15 min at 121℃.
13.4 Place the autoclaved agar into a 45℃ water bath and
allow it to cool to 45℃.
13.5 Place 2×MEM into a 45℃ water bath and allow it to
warm to 45℃.Do not allow the medium to remain at 45℃
longer than 1 h.
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1ThisinternationalstandardwasdevelopedinaccordancewithinternationallyrecognizedprinciplesonstandardizationestablishedintheDecisiononPrinciplesfortheDevelopmentofInternationalStandards,GuidesandRecommendationsissuedbytheWorldTradeOrganizationTechnicalBarrierstoTrade(TBT)Committee.Designation:F895-25S...
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