ASTM F813-07(2012)
(Practice)Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices
Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices
SIGNIFICANCE AND USE
4.1 This practice is useful for assessing cytotoxic potential both when evaluating new materials or formulations for possible use in medical applications, and as part of a quality control program for established medical materials and medical devices.
4.2 This practice assumes that assessment of cytotoxicity potential provides one method for predicting the potential for cytotoxic or necrotic reactions to medical materials and devices during clinical applications to humans. In general, cell culture testing methods have shown good correlation with animal assays and are frequently more sensitive to toxic moieties.
4.3 This cell culture test method is suitable for adoption in specifications and standards for materials for use in the construction of medical devices that are intended to be implanted in the human body or placed in contact with tissue, tissue fluids, or blood on a long-term basis. However, care should be taken when testing materials that are resorbable to be sure the method is applicable.
4.4 Since cells in this direct contact test method are not protected by an overlying agarose layer, they are more susceptible to potential mechanical damage imparted by the overlying test sample. Investigators wishing to evaluate the cytotoxic response of cells underlying the test sample should consider agarose-based methods similar to Test Method F895. Alternatively, depending on sample characteristics, extraction methods such as Practice F619 may also be considered.
SCOPE
1.1 This practice covers a reference method of direct contact cell culture testing which may be used in evaluating the cytotoxic potential of materials for use in the construction of medical materials and devices.
1.2 This practice may be used either directly to evaluate materials or as a reference against which other cytotoxicity test methods may be compared.
1.3 This is one of a series of reference test methods for the assessment of cytotoxic potential, employing different techniques.
1.4 Assessment of cytotoxicity is one of several tests employed in determining the biological response to a material, as recommended in Practice F748.
1.5 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 a well-characterized, readily available, established cell line that has demonstrated reproducible results in several laboratories.
1.6 Since the test sample is not removed at the time of microscopic evaluation and underlying cells may be affected by the specific gravity of the test sample, this practice is limited to evaluation of cells outside the perimeter of the overlying test sample.
1.7 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.8 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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Standards Content (Sample)
NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
Contact ASTM International (www.astm.org) for the latest information
Designation: F813 − 07 (Reapproved 2012)
Standard Practice for
Direct Contact Cell Culture Evaluation of Materials for
Medical Devices
ThisstandardisissuedunderthefixeddesignationF813;thenumberimmediatelyfollowingthedesignationindicatestheyearoforiginal
adoptionor,inthecaseofrevision,theyearoflastrevision.Anumberinparenthesesindicatestheyearoflastreapproval.Asuperscript
epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
1.1 Thispracticecoversareferencemethodofdirectcontact 2.1 ASTM Standards:
cell culture testing which may be used in evaluating the F619Practice for Extraction of Medical Plastics
cytotoxic potential of materials for use in the construction of F748PracticeforSelectingGenericBiologicalTestMethods
medical materials and devices. for Materials and Devices
F895TestMethodforAgarDiffusionCellCultureScreening
1.2 This practice may be used either directly to evaluate
for Cytotoxicity
materialsorasareferenceagainstwhichothercytotoxicitytest
F1027Practice for Assessment of Tissue and Cell Compat-
methods may be compared.
ibility of Orofacial Prosthetic Materials and Devices
1.3 This is one of a series of reference test methods for the
2.2 Other Documents:
assessment of cytotoxic potential, employing different tech-
The American Type Culture Collection (ATCC), Catalogue
niques.
of Strains II
1.4 Assessment of cytotoxicity is one of several tests USP Negative Control Plastic Reference Standard
employed in determining the biological response to a material,
3. Summary of Practice
as recommended in Practice F748.
3.1 Cell cultures are grown to a confluent monolayer in
1.5 The L-929 cell line was chosen because it has a
culture dishes. The growth medium is aspirated and replen-
significant history of use in assays of this type. This is not
ishedtoprovidearesting,confluentcelllayer.Testandcontrol
intended to imply that its use is preferred; only that the L-929
specimens are placed in direct contact with the cell layer to
is a well-characterized, readily available, established cell line
provide an accelerated assessment of the presence or absence
that has demonstrated reproducible results in several laborato-
of a cytotoxic effect from a given material or device. See
ries.
Practice F1027 for definitions.
1.6 Since the test sample is not removed at the time of
microscopic evaluation and underlying cells may be affected
4. Significance and Use
bythespecificgravityofthetestsample,thispracticeislimited
4.1 This practice is useful for assessing cytotoxic potential
toevaluationofcellsoutsidetheperimeteroftheoverlyingtest
both when evaluating new materials or formulations for
sample.
possible use in medical applications, and as part of a quality
1.7 The values stated in SI units are to be regarded as control program for established medical materials and medical
standard. No other units of measurement are included in this
devices.
standard.
4.2 This practice assumes that assessment of cytotoxicity
1.8 This standard does not purport to address all of the
potential provides one method for predicting the potential for
safety concerns, if any, associated with its use. It is the
cytotoxicornecroticreactionstomedicalmaterialsanddevices
responsibility of the user of this standard to establish appro-
during clinical applications to humans. In general, cell culture
priate safety and health practices and determine the applica-
testing methods have shown good correlation with animal
bility of regulatory limitations prior to use.
assays and are frequently more sensitive to toxic moieties.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Surgical Materials and Devices and is the direct responsibility of Subcommittee Standards volume information, refer to the standard’s Document Summary page on
F04.16 on Biocompatibility Test Methods. the ASTM website.
Current edition approved Oct. 1, 2012. Published November 2012. Originally American Type Culture Collection, P.O. Box 1549, Manassas, VA 20108.
approved in 2001. Last previous edition approved in 2007 as F813–07. DOI: U.S. Pharmacopeia, Vol 24, Rand McNally, Taunton, MA, 1994, pp.
10.1520/F0813-07R12. 1652–1653. Use latest publication to ensure current cumulative revisions are used.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F813 − 07 (2012)
4.3 This cell culture test method is suitable for adoption in 6.1.4 Trypsin,0.1%solutioninHanks’solutionorcalcium-
specifications and standards for materials for use in the and magnesium-free, phosphate-buffered saline (store frozen).
construction of medical devices that are intended to be im- 6.1.5 Water, distilled, deionized, and sterile, with a mini-
planted in the human body or placed in contact with tissue, mum resistivity of 1 MΩ·cm.
tissue fluids, or blood on a long-term basis. However, care
6.2 All reagents shall be tissue-culture grade or equivalent.
shouldbetakenwhentestingmaterialsthatareresorbabletobe
6.3 Reagents shall be reconstituted in accordance with the
sure the method is applicable.
manufacturer’s directions, using aseptic technique.
4.4 Since cells in this direct contact test method are not
6.4 Reagents shall be stored in accordance with the manu-
protected by an overlying agarose layer, they are more suscep-
facturer’s directions unless otherwise indicated in 6.1.
tibletopotentialmechanicaldamageimpartedbytheoverlying
test sample. Investigators wishing to evaluate the cytotoxic
7. Cell Cultures
response of cells underlying the test sample should consider
7.1 Cell cultures used in this assay should be the ATCC,
agarose-based methods similar to Test Method F895.
CCL 1 NCTC clone 929 strain (clone of Strain L, mouse
Alternatively, depending on sample characteristics, extraction
connective tissue) designated L-929. Other suitable validated
methods such as Practice F619 may also be considered.
cell lines may be considered. Cells should be tested periodi-
5. Apparatus cally for Mycoplasma contamination.
5.1 The following apparatus shall be used:
8. Control Materials
5.2 Incubator, to maintain a temperature of 37 6 2°C and 4
8.1 Prepare negative control specimens in accordance with
to 6% CO with greater than 90% relative humidity.
Section 10 from a material that consistently elicits negligible
5.3 Tissue Culture Grade Culture Dishes,thataresterileand
cellular response in this assay (for example, USP Negative
35 mm in diameter by 10 mm deep.
Control Plastic Reference Standard).
NOTE 1—Plastic dishes are recommended because they provide a flat
8.2 Prepare positive control specimens in accordance with
surface that contributes to the formation of a uniform cell monolayer.
Section 10 from a material that consistently elicits a
5.4 Disposable, Sterile, Centrifuge Tubes. predictable, moderate degree of cytotoxicity.
8.2.1 Use aqueous phenol (0.45 6 0.05% by volume) as a
5.5 Inverted Optical Microscope, with magnifications of
positive control for a diffuse reaction of cellular degeneration
40×, 100×, and 200×.
and sloughing. Take care to ensure that the preparation is
5.6 Clinical Centrifuge, capable of attaining 1300xg.
homogenous.
8.2.2 Latex rubber has been used as a positive polymeric
5.7 Filter Disks—10 mm in diameter (for evaluation of
liquids). control for a zone of inhibition.
NOTE2—MilliporeAP2501000filterdiskshavebeenfoundsatisfactory
9. General Technique
foruseincytotoxicityevaluationsbecausetheyelicitnocytopathiceffect.
Other filter disks that do not elicit a cytopathic effect may also be used.
9.1 Use the aseptic technique throughout this assay to
minimize microbial contamination.
5.8 Water Bath, capable of maintaining a temperature of 37
6 2°C.
NOTE 7—Mouth pipetting should not be employed to transfer cells,
medium, or reagents.
NOTE 3—Alaminar flow work area capable of filtering out 99.99% of
allparticlesgreaterthan0.5µmindiameter,oraclass100cleanroommay 9.2 Warm all solutions and materials to a temperature of 37
be necessary to prevent contamination of cultures.
6 2°C before placing in contact with cells.
6. Reagents
10. Preparation of Specimens
6.1 The following reagents shall be used:
10.1 Sterilize all specimens by a method appropriate to the
6.1.1 Minimum Essential Medium(MEM),preparedwithout
end use of the device.
L-glutamineandaugmentedbytheadditionofEarle’ssaltsand
10.2 Whereadeviceissufficientlysmall(see10.3and10.4)
5–10% fetal bovine serum.
to fit into the culture dish leaving an adequate margin of cells
NOTE 4—Glutamine is omitted from this formulation in order to
for evaluation, use the entire device as a specimen.
maximize the shelf life of the medium. Immediately before use, 5 mL of
10.3 Cutlargesolidmaterialsanddevicesincrosssectionto
L-glutamine solution (see 6.1.2) are added to each 500 mL of MEM.
NOTE5—OpenedcontainersofMEMmaybestoredatatemperatureof
obtain a flat surface having an area of 100 to 250 mm to be
2 to 8°C for periods of not more than one week.
placed in direct contact with the cell monolayer.
NOTE 6—Antibiotics, such as penicillin G10,000 I.U./ml and strepto-
10.4 Prepare specimens of rod or tubing or of rod- or
mycin10,000I.U./ml,maybeaddedtothemedium(1mlofantibioticper
100 ml of media) to reduce the incidence of bacterial contamination.This
tube-shaped devices as follows:
may, however, have an adverse effect on the viability of the cell cultures.
10.4.1 Where the diameter is less than 6.4 mm, cut 5 to 15
6.1.2 L-glutamine Solution, 29.2 mg/mL of sterile water. mm in length.
6.1.3 Hanks’ Solution, calcium-and magnesium-free (store 10.4.2 Where the diameter is 6.4 to 15 mm, cut 2 to 8 mm
at room temperature). in length.
F813 − 07 (2012)
NOTE 11—The formation of a near-confluent monolayer usually re-
10.4.3 Where the diameter exceeds 15 mm, prepare cross-
quires 3 to 5 days. By counting cells with a hemacytometer (to ensure the
sections as described in 10.3.
concentrationoftheinoculum)thetimerequiredformonolayerformation
10.5 Obtain specimens from larger medical items from
may be regulated. A cell concentration of 1.3×10 cells/mL will give a
consistent time of 24 h. It should be recognized, however, that this
locationswithrelativelylargecrosssectionsinordertoexpose
procedure may increase the risk of bacterial contamination.
interior material.
11.12 If cell suspension remains unused after step 11.10,a
10.6 If a d
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