ASTM F1027-86(2007)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: F1027 − 86(Reapproved 2007)
Standard Practice for
Assessment of Tissue and Cell Compatibility of Orofacial
Prosthetic Materials and Devices
This standard is issued under the fixed designation F1027; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope F813Practice for Direct Contact Cell Culture Evaluation of
Materials for Medical Devices
1.1 This practice describes a procedure to assess the cyto-
toxic potential of materials for use in the construction of
3. Terminology
medical materials and devices using human excised donor
(HED) tissues and their derived primary cells taken from the
3.1 Nomenclature relating to the physical, mechanical, and
orofacial region.
chemical characteristics of plastics shall be in accordance with
Terminology D883.
1.2 This practice may be used either directly to evaluate
materials or as a reference against which other cytotoxicity
3.2 Thenomenclatureandglossaryoftermsrelatedtotissue
methods may be compared.
culturing shall conform to that of the Tissue Culture Associa-
tion (1).
1.3 This practice is one of a series of reference methods for
assessment of cytotoxic potential, employing different tech-
3.3 Forotherdefinitionsusedinthispractice,seeAnnexA1.
niques.
4. Summary of Practice
1.4 Assessment of cytotoxicity is one of several procedures
employed in determining the biological response to a material,
4.1 Primary human orofacial tissue or cells and established
as recommended in Practice F748.
human cell lines are cultured in Medium A3 or any medium
1.5 This standard does not purport to address all of the supporting primary cell growth with homologous processed
human serum or serum components in cell culture flasks or
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro- appropriate containers. The following series of cultures is set
up:
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use. 4.1.1 Test material placed in contact with the cell layer.
NOTE 1—One or more replicates of 4.1.1 may be necessary.
2. Referenced Documents
4.1.2 Primary control wherein no material contacts the cell
2.1 ASTM Standards:
layer.
D883Terminology Relating to Plastics
4.1.3 Positive control wherein the cell layer is contacted by
F604Specification for Silicone Elastomers Used in Medical
amaterialelicitingaknowncytotoxicresponse,suchasatoxic
Applications (Withdrawn 2001)
chemical published in the Toxic Substances List (2).
F703Specification for Implantable Breast Prostheses
4.1.4 Negativecontrolwhereinthecelllayeriscontactedby
F748PracticeforSelectingGenericBiologicalTestMethods
polystyrene used in tissue culture labware.
for Materials and Devices
4.2 The test culture shall be observed daily for growth and
signs of toxicity. The test shall be terminated upon the
attainment of confluency.
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
Surgical Materials and Devicesand is the direct responsibility of Subcommittee
NOTE 2—For an established cell line cultured with Holmes alpha
F04.16 on Biocompatibility Test Methods.
growthfactor(AGF),confluencyisusuallyachievedinslightlymorethan
Current edition approved Feb. 1, 2007. Published February 2007. Originally
5 days.
approved in 1986. Last previous edition approved in 2002 as F1027–86 (2002).
NOTE 3—For first passage cells from HED cultures, confluency is
DOI: 10.1520/F1027-86R07.
usually achieved between 10 to 20 days with an upper limit of 30 days.
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
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
3 4
The last approved version of this historical standard is referenced on Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
www.astm.org. this practice.
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F1027 − 86 (2007)
5. Significance and Use 8. Human Serum
8.1 Thehumanserumshallbeprocessedinaccordancewith
5.1 This practice is useful for assessing the cytotoxic
the method described in Annex A2.
potential both when evaluating new materials or formulations
forpossibleuseinmedicalapplications,andaspartofaquality
NOTE 5—The dialysis treatment serves to remove suspect toxicants,
control program for established medical devices.
ingested medication, unneeded adventitiae, and unidentified growth in-
hibitants with exclusion up to a molecular weight of 3500 Daltons.
5.2 This practice is used for assessing the cytotoxic poten-
tial of materials intended for the fabrication of inserts or
9. Cell Growth Factors
implants in the orofacial region.
9.1 Alpha Growth Factor (AGF)—AGF, separated from the
5.3 This practice is restricted to normal non-transformed,
dialyzedhumanserumasdescribedinAnnexA3,shallbeused
human orofacial tissues using cells cultured in human serum
as needed to enhance cell growth.
factors and does not depend upon cells and serum from
NOTE 6—Initially designated alpha-1-protein (4), AGF can be used in
non-human sources.
placeofwholeserumtomaintainthereferenceestablishedcellline(ECL)
culturesforthe7to30daytestperiodwhenaddedtoachemicallydefined
5.4 This practice incorporates procedures to monitor the
medium (See 7.1).
quality of ingredient materials and the uniformity of the
production process for formulating stock compositions.
10. Reference ECL Cells
5.5 This practice may be useful to determine the effects of
10.1 Human non-transformed established cell line (ECL)
age and radiation, and the state of carcinogenicity on the
cell obtainable from a repository source, such as theAmerican
sensitivity of HED tissues to materials and devices used for
Type CultureAssociation (ATCC), shall be used as a reference
orofacial prostheses.
to monitor the procedural details for uniformity of the testing
system and for indication of quality and reliability of culture
6. Apparatus
medium, human serum preparation, and quality of selected
6.1 Incubator,capableofmaintainingatemperatureof37 6 growth factors.
1°C and an atmosphere of 95% air and 5% CO with at least
NOTE 7—For interlaboratory comparison of these procedural details,
90% relative humidity.
the clinically accessible gingival orofacial tissue cell, as well as the
mucosal (nasal, maxillo, and so forth), shall be selected and appropriately
6.2 Plastic and Glassware, that is specified by chemical
designated.
type and is traceable to its source of supply by catalog number
or trade designation of the manufacturer or vendor.
11. (HED) Cells
6.3 Laminar Flow Cabinet, that meets the Class 100 clean
11.1 Human tissues of the orofacial region, obtained from
room requirements of the U. S. Federal Standard209B or the
patientdonors,shallbeculturedasexplantsuntilsufficientcell
National Standard Foundation Standard NSF 49.
densityisattainedforsucceedingpassagesintoavalidprimary
cell line.
6.4 Fluid Filters, capable of removing 95% of particles
0.22 µm or larger.
12. Preparation of Specimens
6.5 Water Purification System, with filtration capability for
12.1 Aseptic techniques shall be used throughout the pro-
organic contaminants, capable of producing water with resis-
cedure.
tivity of 18 MΩ-cm or greater.
12.2 Warmallsolutionsandmaterialstoatemperatureof37
6.6 Inverted Stage Microscope, with phase contrast optics.
6 2°C before placing in contact with cells.
6.7 Bright Field Microscope, or a photomicroscope with
12.3 Specimens:
magnification to 200×.
12.3.1 Test materials shall range from 0.1 to 1 mm in
thickness, cut into square or triangular geometries, 10 to 15
7. Reagents
mm on a side.
12.3.2 Testspecimensshallbesterilizedbythemethodused
7.1 Medium A3—Chemically defined mediumA3 described
in the preparation of the finished device.
by Holmes (3).
12.3.3 The test arrangement must provide total immersion
NOTE 4—Other chemically defined media shall be acceptable provided
andimmobilizationofspecimens(seeFig.1).Apairofslotsis
the test human cell adapts within 1 to 3 days to a steady growth rate from
cutinthespecimenandasuitablycleanedcoverglass(9by50
low cell density for a period of 7 to 30 days.
mm, No. 1) is threaded through the slots. One or more round
7.2 Trypsin 0.25 % Solution, stored in lyophilized form at 3
holes (3 mm in diameter) are pre-cut in the center of the
to 5°C. A solution may be prepared as needed and used at
specimen.Thisprovidesanareaofhighleachingconcentration
37°C.
as well as a focus for a photomicrographic record.
7.3 Insulin, 6.6 U/100 mL, used as supplement for primary
NOTE 8—If contamination of the assembled test material-microslip is
cell and cell line cultures.
suspected,itmaybeautoclavedbeforeinsertionintoasterilecultureflask.
7.4 Miscellaneous Fixatives, dehydrating solutions, stains, 12.4 Conventional practices of maintaining contaminant-
and so forth, for making permanent record microscopic slides. free working conditions shall be applied in handling tissues,
F1027 − 86 (2007)
NOTE1—Testspecimenslessthan1mminthicknesstendtofloat.Thefiguredepictsonemeansofmaintainingsubmergedcontactbetweenspecimens
and cell cultures.
NOTE 2—Dimensions and configuration of the hole, serving for initial cell seeding, may be optionally modified and appropriately specified.
FIG. 1 Arrangement for Submersing and Immobilizing Specimens
cells, and glassware with well-established techniques as al- 14.4 Trypsinize with fresh, ready prepared 0.25% trypsin
ready prescribed in numerous texts and handbooks. In this solution.
connection, see the work by John Paul (5) as one of several
NOTE 9—Other methods of enzyme treatment may be utilized provided
texts dealing with prevention of aerial and fluid contamination
the outcome of the assay has been substantially equivalent.
in cell culture practices.
14.5 Place the trypsinized cells in sterile culture flasks to
prepare a stock of first passage cells for the biocompatibility
13. Preparation of (ECL) Cultures
test (see Section 17).
13.1 The reference cell line (see 10.1) shall be routinely
14.6 Check the first passage cultured cells for native con-
maintained as stock cultures, either in completely chemically
taminationbyvirus,bacteria,andpleuropneumonia-likeorgan-
defined A3 medium or in Annex A3 medium containing the
isms(PPLO).Discardifpresentandidentifiedandreplacewith
alpha growth factor (AGF), or in A3 medium supplemented
new donor explants.
with 10% processed human serum.
NOTE 10—Such microorganisms are often entrapped in oral mucosal
13.2 Medium changes are made every 48 to 72 h or on a
tissues as contaminants, which could compromise the validity of the test
triweekly schedule, such as Mondays, Wednesdays, and Fri-
result by imposing foreign, nonspecific cytotoxicity in the procedure in
days. Cultures are checked microscopically at the time and
Section 17.
observed for any morphological changes or contamination,
14.7 Harvest the cultured propagated cells for use in
delayed or incidental.
amounts needed in 17.3. Store any unused portion in glycerol
13.3 Cell stock cultures of established cell line are main-
ordimethylsulphoxide(DMSO)at−70°Cfornewsetsoftests,
tained not only as source of cells for the biocompatibility
using the procedure described in Chapter XIX of Ref. 5.
assessment but also as a means to ascertain and verify the
quality and titer of the production lots of processed human
15. Ascertaining Minimum Effective Titer of Growth
serum and preparation lots of separated AGF.
Factor
15.1 The selected reference human ECL cell shall be
14. Preparation and Maintenance of Primary Human
adapted to grow in the Holmes A3 medium or in a culturing
Cells
medium of equivalent effectiveness using:
14.1 Place the human excised donor tissue (explant) ascep-
3 4
15.1.1 Initial low density cell level of 10 to 10 cells/mL,
tically onto a sterile petri dish holding gauze covered withA3
15.1.2 One percent processed (Annex A2) human serum,
medium containing 10% processed human serum.
15.1.3 A series of alpha growth factor comprising 0, 0.1,
14.2 Dissect explant immediately into small pieces, cut into 1.0, and 10 µg (dry basis) per millilitre of media.
1 to 2 mm thin slices approximately 2 to 4 mm in cross-
15.1.4 Grown to confluent monolayer with a schedule of
sectional area.
three fresh media maintenance replenishments per week as
indicated in 13.2.
14.3 Incubate at 37 61°Cina5%CO and 95% air
atmosphereinquadruplicateseriesofculturingcontainersuntil
NOTE 11—It is essential to recognize the various phases of cell growth,
a monolayer is formed and confirmed microscopically. which includes adaptation (I), usually with decreasing cell population for
F1027 − 86 (2007)
one or more days, followed by log growth (II), usually referred to in
16.4.4 Weight (mg).
population doubling time, leading to monolayer confluency (III), and
16.4.5 Surface area (m ).
ultimately to decline (IV) in cell population by reason of senescence or
16.4.6 Volume (mL).
toxicity.Inthisconnection,appropriate,periodiccellcountstoconfluency
16.4.7 Surface/volume ratio.
(IV) can be applied as described by Lontz (6).
16.5 Incaseoflow-densitytestmaterialspecimensthattend
15.2 Following the attained confluency in the aboveAGF 0
to float on the culture medium and away from the developing,
to 10 µg/mL range, the minimal supplementation by AGF is
culturing monolayer, submerge the test specimen with an
noted for use in the ensuing procedure for biocompatibility of
appropriate weighting, such as depicted in Fig. 1.
prosthetic material samples (Section 17).
16. Reference Control and Materials
17. Assessment of Biocompatibility of Prosthetic Material
16.1 The negative control shall be a material that consis-
17.1 Conduct concurrently the biocompatibility assessment
tently does not inhibit cell growth as observed visibly or by an
of the prosthetic material samples by using the three combina-
appropriate increase in cell count during growth to confluency.
tions (a, b, and c) of cell type, medium and serum options
The following material may be used:
shown in Table 1.
16.1.1 USP Negative Control Plastic Reference Standard
17.2 Conduct the biocompatibility assessment of the pros-
(7).
thetic material sample in accordance with the format shown in
16.1.2 Fluorocarbon film or sheeting.
Table 2.
NOTE 12—Satisfactory sheetings are Teflon FEP fluorocarbon, 10 mil
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