ASTM F1027-86(2012)
(Practice)Standard Practice for Assessment of Tissue and Cell Compatibility of Orofacial Prosthetic Materials and Devices
Standard Practice for Assessment of Tissue and Cell Compatibility of Orofacial Prosthetic Materials and Devices
SIGNIFICANCE AND USE
5.1 This practice is useful for assessing the 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 devices.
5.2 This practice is used for assessing the cytotoxic potential of materials intended for the fabrication of inserts or implants in the orofacial region.
5.3 This practice is restricted to normal non-transformed, human orofacial tissues using cells cultured in human serum factors and does not depend upon cells and serum from non-human sources.
5.4 This practice incorporates procedures to monitor the quality of ingredient materials and the uniformity of the production process for formulating stock compositions.
5.5 This practice may be useful to determine the effects of age and radiation, and the state of carcinogenicity on the sensitivity of HED tissues to materials and devices used for orofacial prostheses.
SCOPE
1.1 This practice describes a procedure to assess the cytotoxic potential of materials for use in the construction of medical materials and devices using human excised donor (HED) tissues and their derived primary cells taken from the orofacial region.
1.2 This practice may be used either directly to evaluate materials or as a reference against which other cytotoxicity methods may be compared.
1.3 This practice is one of a series of reference methods for assessment of cytotoxic potential, employing different techniques.
1.4 Assessment of cytotoxicity is one of several procedures employed in determining the biological response to a material, as recommended in Practice F748.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 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)
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Designation: F1027 − 86 (Reapproved 2012)
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 F703Specification for Implantable Breast Prostheses
F748PracticeforSelectingGenericBiologicalTestMethods
1.1 This practice describes a procedure to assess the cyto-
for Materials and Devices
toxic potential of materials for use in the construction of
F813Practice for Direct Contact Cell Culture Evaluation of
medical materials and devices using human excised donor
Materials for Medical Devices
(HED) tissues and their derived primary cells taken from the
orofacial region.
3. Terminology
1.2 This practice may be used either directly to evaluate
3.1 Nomenclature relating to the physical, mechanical, and
materials or as a reference against which other cytotoxicity
chemical characteristics of plastics shall be in accordance with
methods may be compared.
Terminology D883.
1.3 This practice is one of a series of reference methods for
3.2 Thenomenclatureandglossaryoftermsrelatedtotissue
assessment of cytotoxic potential, employing different tech-
culturing shall conform to that of the Tissue Culture Associa-
niques. 4
tion (1).
1.4 Assessment of cytotoxicity is one of several procedures
3.3 Forotherdefinitionsusedinthispractice,seeAnnexA1.
employed in determining the biological response to a material,
as recommended in Practice F748. 4. Summary of Practice
1.5 The values stated in SI units are to be regarded as
4.1 Primary human orofacial tissue or cells and established
standard. No other units of measurement are included in this human cell lines are cultured in Medium A3 or any medium
standard.
supporting primary cell growth with homologous processed
human serum or serum components in cell culture flasks or
1.6 This standard does not purport to address all of the
appropriate containers. The following series of cultures is set
safety concerns, if any, associated with its use. It is the
up:
responsibility of the user of this standard to establish appro-
4.1.1 Test material placed in contact with the cell layer.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
NOTE 1—One or more replicates of 4.1.1 may be necessary.
4.1.2 Primary control wherein no material contacts the cell
2. Referenced Documents
layer.
2.1 ASTM Standards:
4.1.3 Positive control wherein the cell layer is contacted by
D883Terminology Relating to Plastics
amaterialelicitingaknowncytotoxicresponse,suchasatoxic
F604Specification for Silicone Elastomers Used in Medical
chemical published in the Toxic Substances List (2).
Applications (Withdrawn 2001)
4.1.4 Negativecontrolwhereinthecelllayeriscontactedby
polystyrene used in tissue culture labware.
4.2 The test culture shall be observed daily for growth and
ThispracticeisunderthejurisdictionofASTMCommitteeF04onMedicaland
signs of toxicity. The test shall be terminated upon the
Surgical Materials and Devicesand is the direct responsibility of Subcommittee
F04.16 on Biocompatibility Test Methods. attainment of confluency.
Current edition approved Oct. 1, 2012. Published October 2012. Originally
NOTE 2—For an established cell line cultured with Holmes alpha
approved in 1986. Last previous edition approved in 2007 as F1027–86 (2007).
growthfactor(AGF),confluencyisusuallyachievedinslightlymorethan
DOI: 10.1520/F1027-86R12.
5 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 NOTE 3—For first passage cells from human excised donor (HED)
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.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F1027 − 86 (2012)
cultures, confluency is usually achieved between 10 to 20 days with an
8. Human Serum
upper limit of 30 days.
8.1 Thehumanserumshallbeprocessedinaccordancewith
5. Significance and Use
the method described in Annex A2.
5.1 This practice is useful for assessing the cytotoxic
NOTE 5—The dialysis treatment serves to remove suspect toxicants,
potential both when evaluating new materials or formulations ingested medication, unneeded adventitiae, and unidentified growth in-
hibitants with exclusion up to a molecular weight of 3500 Daltons.
forpossibleuseinmedicalapplications,andaspartofaquality
control program for established medical devices.
9. Cell Growth Factors
5.2 This practice is used for assessing the cytotoxic poten-
9.1 Alpha Growth Factor (AGF)—AGF, separated from the
tial of materials intended for the fabrication of inserts or
dialyzedhumanserumasdescribedinAnnexA3,shallbeused
implants in the orofacial region.
as needed to enhance cell growth.
5.3 This practice is restricted to normal non-transformed,
NOTE 6—Initially designated alpha-1-protein (4), AGF can be used in
human orofacial tissues using cells cultured in human serum
placeofwholeserumtomaintainthereferenceestablishedcellline(ECL)
factors and does not depend upon cells and serum from
culturesforthe7to30daytestperiodwhenaddedtoachemicallydefined
non-human sources.
medium (See 7.1).
5.4 This practice incorporates procedures to monitor the
10. Reference ECL Cells
quality of ingredient materials and the uniformity of the
10.1 Human non-transformed established cell line (ECL)
production process for formulating stock compositions.
cell obtainable from a repository source, such as theAmerican
5.5 This practice may be useful to determine the effects of
Type CultureAssociation (ATCC), shall be used as a reference
age and radiation, and the state of carcinogenicity on the
to monitor the procedural details for uniformity of the testing
sensitivity of HED tissues to materials and devices used for
systemandforanindicationofqualityandreliabilityofculture
orofacial prostheses.
medium, human serum preparation, and quality of selected
6. Apparatus growth factors.
6.1 Incubator,capableofmaintainingatemperatureof37 6
NOTE 7—For interlaboratory comparison of these procedural details,
the clinically accessible gingival orofacial tissue cell, as well as the
1°C and an atmosphere of 95% air and 5% CO with at least
mucosal (nasal, maxillo, and so forth), shall be selected and appropriately
90% relative humidity.
designated.
6.2 Plastic and Glassware, that is specified by chemical
11. (HED) Cells
type and is traceable to its source of supply by catalog number
or trade designation of the manufacturer or vendor.
11.1 Human tissues of the orofacial region, obtained from
patientdonors,shallbeculturedasexplantsuntilsufficientcell
6.3 Laminar Flow Cabinet, that meets the Class 100 clean
densityisattainedforsucceedingpassagesintoavalidprimary
room requirements of the U. S. Federal Standard209B or the
cell line.
National Standard Foundation Standard NSF 49.
6.4 Fluid Filters, capable of removing 95% of particles
12. Preparation of Specimens
0.22 µm or larger.
12.1 Aseptic techniques shall be used throughout the pro-
6.5 Water Purification System, with filtration capability for
cedure.
organic contaminants, capable of producing water with resis-
12.2 Warmallsolutionsandmaterialstoatemperatureof37
tivity of 18 MΩ-cm or greater.
6 2°C before placing in contact with cells.
6.6 Inverted Stage Microscope, with phase contrast optics.
12.3 Specimens:
6.7 Bright Field Microscope, or a photomicroscope with
12.3.1 Test materials shall range from 0.1 to 1 mm in
magnification to 200×.
thickness, cut into square or triangular geometries, 10 to 15
mm on a side.
7. Reagents
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 shall 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 (2012)
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 Paul (5) as one of several texts
NOTE 9—Other methods of enzyme treatment may be utilized provided
dealing with prevention of aerial and fluid contamination in
the outcome of the assay has been substantially equivalent.
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 the
delayed or incidental.
amounts needed in 17.3. Store any unused portion in glycerol
13.3 Cell stock cultures of an established cell line are
ordimethylsulphoxide(DMSO)at−70°Cfornewsetsoftests,
maintained not only as a source of cells for the biocompatibil-
using the procedure described in Chapter XIX of Ref. (5).
ity 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 (2012)
one or more days, followed by log growth (II), usually referred to in
16.4.3 Thickness (mm).
population doubling time, leading to monolayer confluency (III), and
16.4.4 Weight (mg).
ultimately to a decline (IV) in cell population by reason of senescence or
16.4.5 Surface area (m ).
toxicity.Inthisconnection,appropriate,periodiccellcountstoconfluency
16.4.6 Volume (mL).
(IV) can be applied as described by Lontz (6).
16.4.7 Surface/volume ratio.
15.2 Following the attained confluency in the aboveAGF 0
16.5 Incaseoflow-densitytestmaterialspecimensthattend
to 10 µg/mL range, the minimal supplementation by AGF is
to float on the culture medium and away from the developing,
noted for use in the ensuing procedure for biocompatibility of
culturing monolayer, submerge the test specimen with an
prosthetic material samples (Section 17).
appropriate weighting, such as depicted in Fig. 1.
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
appropriate increase in cell count during growth to confluency. of the prosthetic material samples by using the three combina-
The following material may be used: tions (a, b, and c) of cell type, medium and serum options
16.1.1 USP Negative Control Plastic Reference Standard shown in Table 1.
(7).
17.2 Conduct the biocompatibility assessment of the pros-
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