ASTM F1027-86(2002)

NOTICE: This standard has either been superseded and replaced by a new version or withdrawn.
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Designation: F 1027 – 86 (Reapproved 2002)
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 (e) indicates an editorial change since the last revision or reapproval.
1. Scope 3. Terminology
1.1 This practice describes a procedure to assess the cyto- 3.1 Nomenclature relating to the physical, mechanical, and
toxic potential of materials for use in the construction of chemical characteristics of plastics shall be in accordance with
medical materials and devices using human excised donor Terminology D883.
(HED) tissues and their derived primary cells taken from the 3.2 Thenomenclatureandglossaryoftermsrelatedtotissue
orofacial region. culturing shall conform to that of the Tissue Culture Associa-
1.2 This practice may be used either directly to evaluate tion (1).
materials or as a reference against which other cytotoxicity 3.3 Forotherdefinitionsusedinthispractice,seeAnnexA1.
methods may be compared.
4. Summary of Practice
1.3 This practice is one of a series of reference methods for
4.1 Primary human orofacial tissue or cells and established
assessment of cytotoxic potential, employing different tech-
niques. human cell lines are cultured in Medium A3 or any medium
supporting primary cell growth with homologous processed
1.4 Assessment of cytotoxicity is one of several procedures
employed in determining the biological response to a material, human serum or serum components in cell culture flasks or
appropriate containers. The following series of cultures is set
as recommended in Practice F748.
1.5 This standard does not purport to address all of the up:
4.1.1 Test material placed in contact with the cell layer.
safety concerns, if any, associated with its use. It is the
responsibility of the user of this standard to establish appro-
NOTE 1—One or more replicates of 4.1.1 may be necessary.
priate safety and health practices and determine the applica-
4.1.2 Primary control wherein no material contacts the cell
bility of regulatory limitations prior to use.
layer.
2. Referenced Documents 4.1.3 Positive control wherein the cell layer is contacted by
amaterialelicitingaknowncytotoxicresponse,suchasatoxic
2.1 ASTM Standards:
2 chemical published in the Toxic Substances List (2).
D883 Terminology Relating to Plastics
4.1.4 Negativecontrolwhereinthecelllayeriscontactedby
F604 Specification for Silicone Elastomers Used in Medi-
3 polystyrene used in tissue culture labware.
cal Applications
4.2 The test culture shall be observed daily for growth and
F703 Specification for Implantable Breast Prostheses
signs of toxicity. The test shall be terminated upon the
F748 Practice for Selecting Generic Biological Test Meth-
attainment of confluency.
ods for Materials and Devices
F813 PracticeforDirectContactCellCultureEvaluationof
NOTE 2—For an established cell line cultured with Holmes alpha
Materials for Medical Devices growthfactor(AGF),confluencyisusuallyachievedinslightlymorethan
5 days.
NOTE 3—For first passage cells from HED cultures, confluency is
usually achieved between 10 to 20 days with an upper limit of 30 days.
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
Surgical Materials and Devices and is the direct responsibility of Subcommittee
F04.16 on Biocompatibility Test Methods .
Current edition approved Sept. 26, 1986. Published November 1986.
Annual Book of ASTM Standards, Vol 08.01.
3 5
Discontinued; See 2000 Annual Book of ASTM Standards, Vol 13.01. Theboldfacenumbersinparenthesesrefertothelistofreferencesattheendof
Annual Book of ASTM Standards, Vol 13.01. this practice.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.
F 1027 – 86 (2002)
5. Significance and Use 8. Human Serum
5.1 This practice is useful for assessing the cytotoxic 8.1 Thehumanserumshallbeprocessedinaccordancewith
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-
5.2 This practice is used for assessing the cytotoxic poten-
hibitants with exclusion up to molecular weight of 3500 Daltons.
tial of materials intended for the fabrication of inserts or
9. Cell Growth Factors
implants in the orofacial region.
5.3 This practice is restricted to normal nontransformed,
9.1 Alpha Growth Factor (AGF)—AGF, separated from the
human orofacial tissues using cells cultured in human serum
dialyzedhumanserumasdescribedinAnnexA3,shallbeused
factors and does not depend upon cells and serum from
as needed to enhance cell growth.
nonhuman sources.
NOTE 6—Initially designated alpha-1-protein (4), AGF can be used in
5.4 This practice incorporates procedures to monitor the
placeofwholeserumtomaintainthereferenceestablishedcellline(ECL)
quality of ingredient materials and the uniformity of the
cultures for the 7 to 30 test period when added to a chemically defined
production process for formulating stock compositions.
medium (See 7.1).
5.5 This practice may be useful to determine the effects of
10. Reference ECL Cells
age and radiation, and the state of carcinogenicity on the
sensitivity of HED tissues to materials and devices used for
10.1 Human non-transformed established cell line (ECL)
orofacial prosthesis.
cell obtainable from a repository source, such as theAmerican
Type CultureAssociation (ATCC), shall be used as a reference
6. Apparatus
to monitor the procedural details for uniformity of the testing
system and for indication of quality and reliability of culture
6.1 Incubator,capableofmaintainingatemperatureof37 6
medium, human serum preparation, and quality of selected
1°C and an atmosphere of 95% air and 5% CO with at least
growth factors.
90% relative humidity.
6.2 Plastic and Glassware, that is specified by chemical
NOTE 7—For interlaboratory comparison of these procedural details,
type and is traceable to source of supply by catalog number or the clinically accessible gingival orofacial tissue cell, as well as the
mucosal (nasal, maxillo, and so forth), shall be selected and appropriately
trade designation of the manufacturer or vendor.
designated.
6.3 Laminar Flow Cabinet, that meets the Class 100 clean
room requirements of the U. S. Federal Standard209B or the
11. (HED) Cells
National Standard Foundation Standard NSF 49.
11.1 Human tissues of the orofacial region, obtained from
6.4 Fluid Filters, capable of removing 95% of particles
patientdonors,shallbeculturedasexplantsuntilsufficientcell
0.22 µm or greater.
densityisattainedforsucceedingpassagesintoavalidprimary
6.5 Water Purification System, with filtration capability for
cell line.
organic contaminants, capable of producing water with resis-
tivity of 18 MV-cm or greater.
12. Preparation of Specimens
6.6 Inverted Stage Microscope, with phase contrast optics.
12.1 Aseptic techniques shall be used throughout the pro-
6.7 Bright Field Microscope, or a photomicroscope with
cedure.
magnification to 2003.
12.2 Warmallsolutionsandmaterialstoatemperatureof37
6 2°C before placing in contact with cells.
7. Reagents
12.3 Specimens:
7.1 Medium A3—ChemicallydefinedmediumA3described
12.3.1 Test materials shall range from 0.1 to 1 mm in
by Holmes (3).
thickness, cut into square or triangular geometries, 10 to 15
NOTE 4—Other chemically defined media shall be acceptable provided
mm on a side.
the test human cell adapts within 1 to 3 days to a steady growth rate from
12.3.2 Testspecimensshallbesterilizedbythemethodused
low cell density for a period of 7 to 30 days.
in the preparation of the finished device.
12.3.3 The test arrangement must provide total immersion
7.2 Trypsin 0.25 % Solution, stored in lyophilized form at 3
andimmobilizationofspecimens(seeFig.1).Apairofslotsis
to 5°C. A solution may be prepared as needed and used at
cutinthespecimenandasuitablycleanedcoverglass(9by50
37°C.
mm, No. 1) is threaded through the slots. One or more round
7.3 Insulin, 6.6 U/100 mL, used as supplement for primary
holes (3 mm in diameter) are pre-cut in the center of the
cell and cell line cultures.
specimen.Thisprovidesanareaofhighleachingconcentration
7.4 Miscellaneous Fixatives, dehydrating solutions, stains,
as well as a focus for a photomicrographic record.
and so forth, for making permanent record microscopic slides.
NOTE 8—If contamination of the assembled test material-microslip is
suspected,itmaybeautoclavedbeforeinsertionintoasterilecultureflask.
12.4 Conventional practices of maintaining contaminant
Commercially available as HolmesA3 medium from Grand Island Biologicals
(GIBCO) Laboratories, 3175 Staley Rd., Grand Island, NY, 14072. free working conditions shall be applied in handling tissues,
F 1027 – 86 (2002)
NOTE 1—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
NOTE 9—Othermethodsofenzymetreatmentmaybeutilizedprovided
cells, and glassware with well-established techniques as al-
the outcome of the assay has been substantially equivalent.
ready prescribed in numerous texts and handbooks. In this
connection, see the work by John Paul (5) as one of several
14.5 Place the trypsinized cells in sterile culture flasks to
texts dealing with prevention of aerial and fluid contamination
prepare a stock of first passage cells for the biocompatibility
in cell culture practices.
test (see Section 17).
14.6 Check the first passage cultured cells for native con-
13. Preparation of (ECL) Cultures
taminationbyvirus,bacteria,andpleuropneumonia-likeorgan-
isms(PPLO).Discardifpresentandidentifiedandreplacewith
13.1 The reference cell line (see 10.1) shall be routinely
new donor explants.
maintained as stock cultures, either in completely chemically
defined A3 medium or in Annex A3 medium containing the
NOTE 10—Such microorganisms are often entrapped in oral mucosal
alpha growth factor (AGF), or in A3 medium supplemented
tissues as contaminants which could compromise the validity of the test
with 10% processed human serum.
result by imposing foreign, nonspecific cytotoxicity in the procedure in
13.2 Medium changes are made every 48 to 72 h or on a Section 17.
triweekly schedule, such as Mondays, Wednesdays, and Fri-
14.7 Harvest the cultured propagated cells for use in
days. Cultures are checked microscopically at the time and
amounts needed in 17.3. Store any unused portion in glycerol
observed for any morphological changes or contamination,
ordimethylsulphoxide(DMSO)at−70°Cfornewsetsoftests,
delayed or incidental.
using the procedure described in Chapter XIX of Ref. 5.
13.3 Cell stock cultures of established cell line are main-
tained not only as source of cells for the biocompatibility 15. Ascertaining Minimum Effective Titer of Growth
assessment but also as a means to ascertain and verify the Factor
quality and titer of the production lots of processed human
15.1 The selected reference human ECL cell shall be
serum and preparation lots of separated AGF.
adapted to grow in the Holmes A3 medium or in a culturing
medium of equivalent effectiveness using:
14. Preparation and Maintenance of Primary Human 3 4
15.1.1 Initial low density cell level of 10 to 10 cells/mL,
Cells
15.1.2 One percent processed (Annex A2) human serum,
14.1 Place the human excised donor tissue (explant) ascep-
15.1.3 A series of alpha growth factor comprising 0, 0.1,
tically onto a sterile petri dish holding gauze covered withA3
1.0, and 10 µg (dry basis) per millilitre of media.
medium containing 10% of processed human serum.
15.1.4 Grown to confluent monolayer with a schedule of
14.2 Dissect explant immediately into small pieces, cut into
three fresh media maintenance replenishments per week as
1 to 2 mm thin slices of approximately 2 to 4 mm dimension.
indicated in 13.2.
14.3 Incubate at 37 61°Cina5%CO and 95% air
NOTE 11—It is essential to recognize the various phases of cell growth,
atmosphereinquadruplicateseriesofculturingcontainersuntil
which includes adaptation (I), usually with decreasing cell population for
a monolayer is formed and confirmed microscopically.
one or more days, followed by log growth (II), usually referred to in
14.4 Trypsinize with fresh, ready prepared 0.25% trypsin
population doubling time, leading to monolayer confluency (III), and
solution. ultimately to decline (IV) in cell population by reason of senescence or
F 1027 – 86 (2002)
toxicity.Inthisconnection,appropriate,periodiccellcountstoconfluency
16.4.2 Density or specific gravity.
(IV) can be applied as described by Lontz (6).
16.4.3 Thickness (mm).
15.2 Following the attained confluency in the aboveAGF 0 16.4.4 Weight (mg).
to 10 µg/mL range, the minimal supplementation by AGF is 16.4.5 Surface area (m ).
noted for use in the ensuing procedure for biocompatibility of 16.4.6 Volume (mL).
prosthetic material samples (Section 17). 16.4.7 Surface/volume ratio.
16.5 Incaseoflowdensitytestmaterialspecimensthattend
16. Reference Control and Materials
to float on the culture medium and away from the developing,
16.1 The negative control shall be a material that consis- culturing monolayer, submerge the test specimen with an
tently does not inhibit cell growth as observed visibly or by an appropriate weighting, such as depicted in Fig. 1.
appropriate increase in cell count during growth to confluency.
The following material may be used:
17. Assessment of Biocompatibility of Prosthetic Material
16.1.1 USP Negative Control Plastic Reference Standard
17.1 Conduct concurrently the biocompatibility assessment
(7).
of the prosthetic material samples by using the three combina-
tions (a, b, and c) of cell type, medium and serum options
TABLE 1 Test Cell Type, Medium, and Serum Options
shown in Table 1.
Human Serum (P) 17.2 Conduct the biocompatibility assessment of the pros-
Cell Type Medium
Maximum Level(*), %
thetic material sample in accordance with the format shown in
(A) Human Orofacial (primary) A3 with 6.60 10
Table 2.
Medium
17.3 Suspend the stock human primary cells, (see 14.7
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