ASTM F1027-86(1995)e1

e1
Designation: F 1027 – 86 (Reapproved 1995)
Standard Practice for
Assessment of Tissue and Cell Compatibility of Orofacial
Prosthetic Materials and Devices
This standard is issued under the fixed designation F 1027; 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 (e) indicates an editorial change since the last revision or reapproval.
e NOTE—Editorial changes were made throughout November 1995.
1. Scope 3.2 The nomenclature and glossary of terms related to tissue
culturing shall conform to that of the Tissue Culture Associa-
1.1 This practice describes a procedure to assess the cyto-
tion (1).
toxic potential of materials for use in the construction of
3.3 For other definitions used in this practice, see Annex A1.
medical materials and devices using human excised donor
(HED) tissues and their derived primary cells taken from the
4. Summary of Practice
orofacial region.
4.1 Primary human orofacial tissue or cells and established
1.2 This practice may be used either directly to evaluate
human cell lines are cultured in Medium A3 or any medium
materials or as a reference against which other cytotoxicity
supporting primary cell growth with homologous processed
methods may be compared.
human serum or serum components in cell culture flasks or
1.3 This practice is one of a series of reference methods for
appropriate containers. The following series of cultures is set
assessment of cytotoxic potential, employing different tech-
up:
niques.
4.1.1 Test material placed in contact with the cell layer.
1.4 Assessment of cytotoxicity is one of several procedures
employed in determining the biological response to a material,
NOTE 1—One or more replicates of 4.1.1 may be necessary.
as recommended in Practice F 748.
4.1.2 Primary control wherein no material contacts the cell
1.5 This standard does not purport to address all of the
layer.
safety concerns, if any, associated with its use. It is the
4.1.3 Positive control wherein the cell layer is contacted by
responsibility of the user of this standard to establish appro-
a material eliciting a known cytotoxic response, such as a toxic
priate safety and health practices and determine the applica-
chemical published in the Toxic Substances List (2).
bility of regulatory limitations prior to use.
4.1.4 Negative control wherein the cell layer is contacted by
polystyrene used in tissue culture labware.
2. Referenced Documents
4.2 The test culture shall be observed daily for growth and
2.1 ASTM Standards:
signs of toxicity. The test shall be terminated upon the
D 883 Terminology Relating to Plastics
attainment of confluency.
F 604 Specification for Silicone Elastomers Used in Medi-
NOTE 2—For an established cell line cultured with Holmes alpha
cal Applications
growth factor (AGF), confluency is usually achieved in slightly more than
F 703 Specification for Implantable Breast Prostheses
5 days.
F 748 Practice for Selecting Generic Biological Test Meth-
NOTE 3—For first passage cells from HED cultures, confluency is
ods for Materials and Devices
usually achieved between 10 to 20 days with an upper limit of 30 days.
F 813 Practice for Direct Contact Cell Culture Evaluation of
Materials for Medical Devices 5. Significance and Use
5.1 This practice is useful for assessing the cytotoxic
3. Terminology
potential both when evaluating new materials or formulations
3.1 Nomenclature relating to the physical, mechanical, and
for possible use in medical applications, and as part of a quality
chemical characteristics of plastics shall conform to Definitions
control program for established medical devices.
D 883.
5.2 This practice is used for assessing the cytotoxic poten-
tial of materials intended for the fabrication of inserts or
1 implants in the orofacial region.
This practice is under the jurisdiction of ASTM Committee F-4 on Medical and
5.3 This practice is restricted to normal nontransformed,
Surgical Materials and Devicesand is the direct responsibility of Subcommittee
F04.16 on Biocompatibility.
Current edition approved Sept. 26, 1986. Published November 1986.
2 4
Annual Book of ASTM Standards, Vol 08.01. The boldface numbers in parentheses refer to the list of references at the end
Annual Book of ASTM Standards, Vol 13.01. of this practice.
Copyright © ASTM, 100 Barr Harbor Drive, West Conshohocken, PA 19428-2959, United States.
F 1027
NOTE 6—Initially designated alpha-1-protein (4), AGF can be used in
human orofacial tissues using cells cultured in human serum
place of whole serum to maintain the reference established cell line (ECL)
factors and does not depend upon cells and serum from
cultures for the 7 to 30 test period when added to a chemically defined
nonhuman sources.
medium (7.1).
5.4 This practice incorporates procedures to monitor the
quality of ingredient materials and the uniformity of the
10. Reference ECL Cells
production process for formulating stock compositions.
10.1 Human non-transformed established cell line (ECL)
5.5 This practice may be useful to determine the effects of
cell obtainable from a repository source, such as the American
age and radiation, and the state of carcinogenicity on the
Type Culture Association (ATCC), shall be used as a reference
sensitivity of HED tissues to materials and devices used for
to monitor the procedural details for uniformity of the testing
orofacial prosthesis.
system and for indication of quality and reliability of culture
medium, human serum preparation, and quality of selected
6. Apparatus
growth factors.
6.1 Incubator, capable of maintaining a temperature of 37 6
NOTE 7—For interlaboratory comparison of these procedural details,
1°C and an atmosphere of 95 % air and 5 % CO with at least
the clinically accessible gingival orofacial tissue cell, as well as the
90 % relative humidity.
mucosal (nasal, maxillo, etc.), shall be selected and appropriately desig-
6.2 Plastic and Glassware, that is specified by chemical
nated.
type and is traceable to 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 Standard 209B or the
patient donors, shall be cultured as explants until sufficient cell
National Standard Foundation Standard NSF 49.
density is attained for succeeding passages into a valid primary
6.4 Fluid Filters, capable of removing 95 % of particles
cell line.
0.22 μm or greater.
6.5 Water Purification System, with filtration capability for
12. Preparation of Specimens
organic contaminants, capable of producing water with resis-
12.1 Aseptic techniques shall be used throughout the pro-
tivity of 18 MV-cm or greater.
cedure.
6.6 Inverted Stage Microscope, with phase contrast optics.
12.2 Warm all solutions and materials to a temperature of 37
6.7 Bright Field Microscope, or a photomicroscope with
6 2°C before placing in contact with cells.
magnification to 2003.
12.3 Specimens:
12.3.1 Test materials shall range from 0.1 to 1 mm in
7. Reagents
thickness, cut into square or triangular geometries, 10 to 15
7.1 Medium A3—Chemically defined medium A3 described
mm on a side.
by Holmes (3).
12.3.2 Test specimens shall be sterilized by the method used
NOTE 4—Other chemically defined media shall be acceptable provided in the preparation of the finished device.
the test human cell adapts within 1 to 3 days to a steady growth rate from
12.3.3 The test arrangement must provide total immersion
low cell density for a period of 7 to 30 days.
and immobilization of specimens (see Fig. 1). A pair of slots is
cut in the specimen and a suitably cleaned cover glass (9 by 50
7.2 Trypsin 0.25 % Solution, stored in lyophilized form at 3
to 5°C. A solution may be prepared as needed and used at mm, No. 1) is threaded through the slots. One or more round
holes (3 mm in diameter) are pre-cut in the center of the
37°C.
7.3 Insulin, 6.6 U/100 mL, used as supplement for primary specimen. This provides an area of high leaching concentration
as well as a focus for a photomicrographic record.
cell and cell line cultures.
7.4 Miscellaneous Fixatives, dehydrating solutions, stains,
NOTE 8—If contamination of the assembled test material-microslip is
etc. for making permanent record microscopic slides.
suspected, it may be autoclaved before insertion into a sterile culture flask.
12.4 Conventional practices of maintaining contaminant
8. Human Serum
free working conditions shall be applied in handling tissues,
8.1 The human serum shall be processed in accordance with
cells, and glassware with well-established techniques as al-
the method described in Annex A2.
ready prescribed in numerous texts and handbooks. In this
NOTE 5—The dialysis treatment serves to remove suspect toxicants,
connection, see the work by John Paul (5) as one of several
ingested medication, unneeded adventitiae, and unidentified growth in-
texts dealing with prevention of aerial and fluid contamination
hibitants with exclusion up to molecular weight of 3500 Daltons.
in cell culture practices.
9. Cell Growth Factors
13. Preparation of (ECL) Cultures
9.1 Alpha Growth Factor (AGF)—AGF, separated from the
13.1 The reference cell line (see 10.1) shall be routinely
dialyzed human serum as described in Annex A3, shall be used
maintained as stock cultures, either in completely chemically
as needed to enhance cell growth.
defined A3 medium or in Annex A3 medium containing the
alpha growth factor (AGF), or in A3 medium supplemented
with 10 % processed human serum.
Commercially available as Holmes A3 medium from Grand Island Biologicals
(GIBCO) Laboratories, 3175 Staley Rd., Grand Island, NY, 14072. 13.2 Medium changes are made every 48 to 72 h or on a
F 1027
NOTE 1—Test specimens less than 1 mm in thickness tend to float. The figure depicts one means of maintaining submerged contact between specimens
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
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, or dimethyl sulphoxide (DMSO) at −70°C for new sets of tests,
delayed or incidental. using the procedure described in Chapter XIX of Ref. 5.
13.3 Cell stock cultures of established cell line are main-
15. Ascertaining Minimum Effective Titer of Growth
tained not only as source of cells for the biocompatibility
Factor
assessment but also as a means to ascertain and verify the
15.1 The selected reference human ECL cell shall be
quality and titer of the production lots of processed human
adapted to grow in the Holmes A3 medium or in a culturing
serum and preparation lots of separated AGF.
medium of equivalent effectiveness using:
3 4
14. Preparation and Maintenance of Primary Human
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,
15.1.3 A series of alpha growth factor comprising 0, 0.1,
14.1 Place the human excised donor tissue (explant) ascep-
1.0, and 10 μg (dry basis) per millilitre of media.
tically onto a sterile petri dish holding gauze covered with A3
15.1.4 Grown to confluent monolayer with a schedule of
medium containing 10 % of processed human serum.
three fresh media maintenance replenishments per week as
14.2 Dissect explant immediately into small pieces, cut into
indicated in 13.2.
1 to 2 mm thin slices of approximately 2 to 4 mm dimension.
14.3 Incubate at 37 6 1°C ina5%CO and 95 % air
NOTE 11—It is essential to recognize the various phases of cell growth,
atmosphere in quadruplicate series of culturing containers until
which includes adaptation (I), usually with decreasing cell population for
one or more days, followed by log growth (II), usually referred to in
a monolayer is formed and confirmed microscopically.
population doubling time, leading to monolayer confluency (III), and
14.4 Trypsinize with fresh, ready prepared 0.25 % trypsin
ultimately to decline (IV) in cell population by reason of senescence or
solution.
toxicity. In this connection, appropriate, periodic cell counts to confluency
NOTE 9—Other methods of enzyme treatment may be utilized provided (IV) can be applied as described by Lontz (6).
the outcome of the assay has been substantially equivalent.
15.2 Following the attained confluency in the above AGF 0
14.5 Place the trypsinized cells in sterile culture flasks to
to 10 μg/mL range, the minimal supplementation by AGF is
prepare a stock of first passage cells for the biocompatibility
noted for use in the ensuing procedure for biocompatibility of
test (see Section 17).
prosthetic material samples (Section 17).
14.6 Check the first passage cultured cells for native con-
16. Reference Control and Materials
tamination by virus, bacteria, and pleuropneumonia-like organ-
isms (PPLO). Discard if present and identified and replace with
16.1 The negative control shall be a material that consis-
new donor explants. tently does not inhibit cell growth as observed visibly or by an
appropriate increase in cell count during growth to confluency.
NOTE 10—Such microorganisms are often entrapped in oral mucosal
The following material may be used:
tissues as contaminants which could compromise the validity of the test
16.1.1 USP Negative Control Plastic Reference Standard
result by imposing foreign, nonspecific cytotoxicity in the procedure in
Section 17. (7).
F 1027
TABLE 1 Test Cell Type, Medium, and Serum Options
17.2 Conduct the biocompatibility assessment of the pros-
Human Serum (P) thetic material sample in accordance with the format shown in
Cell Type Medium
Maximum Level(*), %
Table 2.
(A) Human Orofacial (primary) A3 with 6.60 10
17.3 Suspend the stock human primary cells, (see 14.7),
Medium
dislodged either by scraping or by trypsin treatment, into fresh
A
(B) Established Cell Line A3 (Replaced by 0.1
A3, or equivalent medium, with optimal additives of serum,
to 10 % AGF)
(C) Established Cell Line A3 10
insulin, and growth factor described in 17.1, Table 1.
A
The combinations (B) and (C) of this table are used to ensure cell viability and
17.4 Place an aliquot portion adjusted to a level of 10 to
procedural consistency.
10 cells/mL, determined by counting using a hemocytometer,
each of the quadruplicate series of Table 2, with the appropriate
A3 medium or its equivalent.
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