1、Designation: F1027 86 (Reapproved 2012)Standard Practice forAssessment of Tissue and Cell Compatibility of OrofacialProsthetic Materials and Devices1This standard is issued under the fixed designation F1027; the number immediately following the designation indicates the year oforiginal adoption or,
2、in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This practice describes a procedure to assess the cyto-toxic potential of materia
3、ls for use in the construction ofmedical materials and devices using human excised donor(HED) tissues and their derived primary cells taken from theorofacial region.1.2 This practice may be used either directly to evaluatematerials or as a reference against which other cytotoxicitymethods may be com
4、pared.1.3 This practice is one of a series of reference methods forassessment of cytotoxic potential, employing different tech-niques.1.4 Assessment of cytotoxicity is one of several proceduresemployed in determining the biological response to a material,as recommended in Practice F748.1.5 The value
5、s stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.6 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safe
6、ty and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D883 Terminology Relating to PlasticsF604 Specification for Silicone Elastomers Used in MedicalApplications (Withdrawn 2001)3F703 Specification for Implantable B
7、reast ProsthesesF748 Practice for Selecting Generic Biological Test Methodsfor Materials and DevicesF813 Practice for Direct Contact Cell Culture Evaluation ofMaterials for Medical Devices3. Terminology3.1 Nomenclature relating to the physical, mechanical, andchemical characteristics of plastics sha
8、ll be in accordance withTerminology D883.3.2 The nomenclature and glossary of terms related to tissueculturing shall conform to that of the Tissue Culture Associa-tion (1).43.3 For other definitions used in this practice, see AnnexA1.4. Summary of Practice4.1 Primary human orofacial tissue or cells
9、and establishedhuman cell lines are cultured in Medium A3 or any mediumsupporting primary cell growth with homologous processedhuman serum or serum components in cell culture flasks orappropriate containers. The following series of cultures is setup:4.1.1 Test material placed in contact with the cel
10、l layer.NOTE 1One or more replicates of 4.1.1 may be necessary.4.1.2 Primary control wherein no material contacts the celllayer.4.1.3 Positive control wherein the cell layer is contacted bya material eliciting a known cytotoxic response, such as a toxicchemical published in the Toxic Substances List
11、 (2).4.1.4 Negative control wherein the cell layer is contacted bypolystyrene used in tissue culture labware.4.2 The test culture shall be observed daily for growth andsigns of toxicity. The test shall be terminated upon theattainment of confluency.NOTE 2For an established cell line cultured with Ho
12、lmes alphagrowth factor (AGF), confluency is usually achieved in slightly more than5 days.1This practice is under the jurisdiction ofASTM Committee F04 on Medical andSurgical Materials and Devicesand is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition
13、approved Oct. 1, 2012. Published October 2012. Originallyapproved in 1986. Last previous edition approved in 2007 as F1027 86 (2007).DOI: 10.1520/F1027-86R12.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of A
14、STMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3The last approved version of this historical standard is referenced onwww.astm.org.4The boldface numbers in parentheses refer to the list of references at the end ofthis practice.Copyright ASTM Internat
15、ional, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1NOTE 3For first passage cells from human excised donor (HED)cultures, confluency is usually achieved between 10 to 20 days with anupper limit of 30 days.5. Significance and Use5.1 This practice is useful for a
16、ssessing the cytotoxicpotential both when evaluating new materials or formulationsfor possible use in medical applications, and as part of a qualitycontrol program for established medical devices.5.2 This practice is used for assessing the cytotoxic poten-tial of materials intended for the fabricati
17、on of inserts orimplants in the orofacial region.5.3 This practice is restricted to normal non-transformed,human orofacial tissues using cells cultured in human serumfactors and does not depend upon cells and serum fromnon-human sources.5.4 This practice incorporates procedures to monitor thequality
18、 of ingredient materials and the uniformity of theproduction process for formulating stock compositions.5.5 This practice may be useful to determine the effects ofage and radiation, and the state of carcinogenicity on thesensitivity of HED tissues to materials and devices used fororofacial prosthese
19、s.6. Apparatus6.1 Incubator, capable of maintaining a temperature of 37 61C and an atmosphere of 95 % air and 5 % CO2with at least90 % relative humidity.6.2 Plastic and Glassware, that is specified by chemicaltype and is traceable to its source of supply by catalog numberor trade designation of the
20、manufacturer or vendor.6.3 Laminar Flow Cabinet, that meets the Class 100 cleanroom requirements of the U. S. Federal Standard 209B or theNational Standard Foundation Standard NSF 49.6.4 Fluid Filters, capable of removing 95 % of particles0.22 m or larger.6.5 Water Purification System, with filtrati
21、on capability fororganic contaminants, capable of producing water with resis-tivity of 18 M-cm or greater.6.6 Inverted Stage Microscope, with phase contrast optics.6.7 Bright Field Microscope, or a photomicroscope withmagnification to 200.7. Reagents7.1 Medium A3Chemically defined medium A3 describe
22、dby Holmes (3).NOTE 4Other chemically defined media shall be acceptable providedthe test human cell adapts within 1 to 3 days to a steady growth rate fromlow cell density for a period of 7 to 30 days.7.2 Trypsin 0.25 % Solution, stored in lyophilized form at 3to 5C. A solution may be prepared as nee
23、ded and used at37C.7.3 Insulin, 6.6 U/100 mL, used as supplement for primarycell and cell line cultures.7.4 Miscellaneous Fixatives, dehydrating solutions, stains,and so forth, for making permanent record microscopic slides.8. Human Serum8.1 The human serum shall be processed in accordance withthe m
24、ethod described in Annex A2.NOTE 5The dialysis treatment serves to remove suspect toxicants,ingested medication, unneeded adventitiae, and unidentified growth in-hibitants with exclusion up to a molecular weight of 3500 Daltons.9. Cell Growth Factors9.1 Alpha Growth Factor (AGF)AGF, separated from t
25、hedialyzed human serum as described in AnnexA3, shall be usedas needed to enhance cell growth.NOTE 6Initially designated alpha-1-protein (4), AGF can be used inplace of whole serum to maintain the reference established cell line (ECL)cultures for the 7 to 30 day test period when added to a chemicall
26、y definedmedium (See 7.1).10. Reference ECL Cells10.1 Human non-transformed established cell line (ECL)cell obtainable from a repository source, such as the AmericanType Culture Association (ATCC), shall be used as a referenceto monitor the procedural details for uniformity of the testingsystem and
27、for an indication of quality and reliability of culturemedium, human serum preparation, and quality of selectedgrowth factors.NOTE 7For interlaboratory comparison of these procedural details,the clinically accessible gingival orofacial tissue cell, as well as themucosal (nasal, maxillo, and so forth
28、), shall be selected and appropriatelydesignated.11. (HED) Cells11.1 Human tissues of the orofacial region, obtained frompatient donors, shall be cultured as explants until sufficient celldensity is attained for succeeding passages into a valid primarycell line.12. Preparation of Specimens12.1 Asept
29、ic techniques shall be used throughout the pro-cedure.12.2 Warm all solutions and materials to a temperature of 376 2C before placing in contact with cells.12.3 Specimens:12.3.1 Test materials shall range from 0.1 to 1 mm inthickness, cut into square or triangular geometries, 10 to 15mm on a side.12
30、.3.2 Test specimens shall be sterilized by the method usedin the preparation of the finished device.12.3.3 The test arrangement shall provide total immersionand immobilization of specimens (see Fig. 1).Apair of slots iscut in the specimen and a suitably cleaned cover glass (9 by 50mm, No. 1) is thre
31、aded through the slots. One or more roundholes (3 mm in diameter) are pre-cut in the center of thespecimen. This provides an area of high leaching concentrationas well as a focus for a photomicrographic record.NOTE 8If contamination of the assembled test material-microslip issuspected, it may be aut
32、oclaved before insertion into a sterile culture flask.F1027 86 (2012)212.4 Conventional practices of maintaining contaminant-free working conditions shall be applied in handling tissues,cells, and glassware with well-established techniques as al-ready prescribed in numerous texts and handbooks. In t
33、hisconnection, see the work by Paul (5) as one of several textsdealing with prevention of aerial and fluid contamination incell culture practices.13. Preparation of (ECL) Cultures13.1 The reference cell line (see 10.1) shall be routinelymaintained as stock cultures, either in completely chemicallyde
34、fined A3 medium or in Annex A3 medium containing thealpha growth factor (AGF), or in A3 medium supplementedwith 10 % processed human serum.13.2 Medium changes are made every 48 to 72 h or on atriweekly schedule, such as Mondays, Wednesdays, and Fri-days. Cultures are checked microscopically at the t
35、ime andobserved for any morphological changes or contamination,delayed or incidental.13.3 Cell stock cultures of an established cell line aremaintained not only as a source of cells for the biocompatibil-ity assessment but also as a means to ascertain and verify thequality and titer of the productio
36、n lots of processed humanserum and preparation lots of separated AGF.14. Preparation and Maintenance of Primary HumanCells14.1 Place the human excised donor tissue (explant) ascep-tically onto a sterile petri dish holding gauze covered with A3medium containing 10 % processed human serum.14.2 Dissect
37、 explant immediately into small pieces, cut into1 to 2 mm thin slices approximately 2 to 4 mm2in cross-sectional area.14.3 Incubate at 37 6 1Cina5%CO2and 95 % airatmosphere in quadruplicate series of culturing containers untila monolayer is formed and confirmed microscopically.14.4 Trypsinize with f
38、resh, ready prepared 0.25 % trypsinsolution.NOTE 9Other methods of enzyme treatment may be utilized providedthe outcome of the assay has been substantially equivalent.14.5 Place the trypsinized cells in sterile culture flasks toprepare a stock of first passage cells for the biocompatibilitytest (see
39、 Section 17).14.6 Check the first passage cultured cells for native con-tamination by virus, bacteria, and pleuropneumonia-like organ-isms (PPLO). Discard if present and identified and replace withnew donor explants.NOTE 10Such microorganisms are often entrapped in oral mucosaltissues as contaminant
40、s, which could compromise the validity of the testresult by imposing foreign, nonspecific cytotoxicity in the procedure inSection 17.14.7 Harvest the cultured propagated cells for use in theamounts needed in 17.3. Store any unused portion in glycerolor dimethyl sulphoxide (DMSO) at 70C for new sets
41、of tests,using the procedure described in Chapter XIX of Ref. (5).15. Ascertaining Minimum Effective Titer of GrowthFactor15.1 The selected reference human ECL cell shall beadapted to grow in the Holmes A3 medium or in a culturingmedium of equivalent effectiveness using:15.1.1 Initial low density ce
42、ll level of 103to 104cells/mL,15.1.2 One percent processed (Annex A2) human serum,15.1.3 A series of alpha growth factor comprising 0, 0.1,1.0, and 10 g (dry basis) per millilitre of media.NOTE 1Test specimens less than 1 mm in thickness tend to float. The figure depicts one means of maintaining sub
43、merged contact between specimensand cell cultures.NOTE 2Dimensions and configuration of the hole, serving for initial cell seeding, may be optionally modified and appropriately specified.FIG. 1 Arrangement for Submersing and Immobilizing SpecimensF1027 86 (2012)315.1.4 Grown to confluent monolayer w
44、ith a schedule ofthree fresh media maintenance replenishments per week asindicated in 13.2.NOTE 11It is essential to recognize the various phases of cell growth,which includes adaptation (I), usually with decreasing cell population forone or more days, followed by log growth (II), usually referred t
45、o inpopulation doubling time, leading to monolayer confluency (III), andultimately to a decline (IV) in cell population by reason of senescence ortoxicity. In this connection, appropriate, periodic cell counts to confluency(IV) can be applied as described by Lontz (6).15.2 Following the attained con
46、fluency in the above AGF 0to 10 g/mL range, the minimal supplementation by AGF isnoted for use in the ensuing procedure for biocompatibility ofprosthetic material samples (Section 17).16. Reference Control and Materials16.1 The negative control shall be a material that consis-tently does not inhibit
47、 cell growth as observed visibly or by anappropriate increase in cell count during growth to confluency.The following material may be used:16.1.1 USP Negative Control Plastic Reference Standard(7).16.1.2 Fluorocarbon film or sheeting.NOTE 12Satisfactory sheetings are Teflon FEP fluorocarbon, 10 mil(
48、0.010 in.) in thickness and a copolymer of tetrafluorethylene andhexafluoropropylene of commercial prominence; This grade of fluorocar-bon polymer is uniquely useful because of (a) exceptional chemicalinertness, (b) high specific gravity, higher than most of the nutrient media,and (c) exceptional cl
49、arity for viewing cell structure.16.1.3 Polystyrene culturing flask used in the test procedure.16.2 The positive control shall be a material as required inother cell culture test methods, such as Practice F813, section8.2.2, or specially compounded at a level of 1 %, mixed inRTV grade of commercial silicone with known toxic agent,such as listed in Registry of Toxic Substance (2).NOTE 13Although phenol is a common reference toxicant (PracticeF813, Section 8.7.1), its aqueous solubility and hence leachability is toorapid. For suitable, less soluble alter
