1、Designation: F 1027 86 (Reapproved 2007)Standard Practice forAssessment of Tissue and Cell Compatibility of OrofacialProsthetic Materials and Devices1This standard is issued under the fixed designation F 1027; 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 (e) 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 mate
3、rials 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
4、compared.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 F 748.1.5 This
5、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 safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2
6、.1 ASTM Standards:2D 883 Terminology Relating to PlasticsF 604 Specification for Silicone Elastomers Used in Medi-cal Applications3F 703 Specification for Implantable Breast ProsthesesF 748 Practice for Selecting Generic Biological Test Meth-ods for Materials and DevicesF 813 Practice for Direct Con
7、tact Cell Culture Evaluation ofMaterials for Medical Devices3. Terminology3.1 Nomenclature relating to the physical, mechanical, andchemical characteristics of plastics shall be in accordance withTerminology D 883.3.2 The nomenclature and glossary of terms related to tissueculturing shall conform to
8、 that of the Tissue Culture Associa-tion (1).43.3 For other definitions used in this practice, seeAnnexA1.4. Summary of Practice4.1 Primary human orofacial tissue or cells and establishedhuman cell lines are cultured in Medium A3 or any mediumsupporting primary cell growth with homologous processedh
9、uman 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 cell layer.NOTE 1One or more replicates of 4.1.1 may be necessary.4.1.2 Primary control wherein no material contacts the celllayer.
10、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 (2).4.1.4 Negative control wherein the cell layer is contacted bypolystyrene used in tissue culture labware.4.2 The test cultur
11、e 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 Holmes alphagrowth factor (AGF), confluency is usually achieved in slightly more than5 days.NOTE 3For first passage cells from HED
12、 cultures, confluency isusually achieved between 10 to 20 days with an upper limit of 30 days.5. Significance and Use5.1 This practice is useful for assessing the cytotoxicpotential both when evaluating new materials or formulations1This practice is under the jurisdiction ofASTM Committee F04 on Med
13、ical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Feb. 1, 2007. Published February 2007. Originallyapproved in 1986. Last previous edition approved in 2002 as F 1027 86 (2002).2For referenced ASTM s
14、tandards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Withdrawn.4The boldface numbers in parentheses refer to the list of references at
15、the end ofthis practice.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.for 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 cyt
16、otoxic poten-tial of materials intended for the fabrication 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 Th
17、is practice incorporates procedures to monitor thequality 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 tis
18、sues to materials and devices used fororofacial prostheses.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 sourc
19、e of supply by catalog numberor trade designation of the 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
20、 m or larger.6.5 Water Purification System, with filtration capability fororganic contaminants, capable of producing water with resis-tivity of 18 MV-cm or greater.6.6 Inverted Stage Microscope, with phase contrast optics.6.7 Bright Field Microscope, or a photomicroscope withmagnification to 2003.7.
21、 Reagents7.1 Medium A3Chemically defined mediumA3 describedby 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 lyo
22、philized form at 3to 5C. A solution may be prepared as needed 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
23、The human serum shall be processed in accordance withthe method 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
24、 Factors9.1 Alpha Growth Factor (AGF)AGF, separated from thedialyzed human serum as described inAnnexA3, 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
25、for the 7 to 30 day test period when added to a chemically 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 pr
26、ocedural details for uniformity of the testingsystem and for 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 cel
27、l, as well as themucosal (nasal, maxillo, and so forth), 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 p
28、rimarycell line.12. Preparation of Specimens12.1 Aseptic 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 sq
29、uare or triangular geometries, 10 to 15mm on a side.12.3.2 Test specimens shall be sterilized by the method usedin the preparation of the finished device.12.3.3 The test arrangement must provide total immersionand immobilization of specimens (see Fig. 1).Apair of slots iscut in the specimen and a su
30、itably cleaned cover glass (9 by 50mm, No. 1) is threaded 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 assembl
31、ed test material-microslip issuspected, it may be autoclaved before insertion into a sterile culture flask.12.4 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
32、in numerous texts and handbooks. In thisconnection, see the work by John Paul (5) as one of severaltexts dealing with prevention of aerial and fluid contaminationin cell culture practices.13. Preparation of (ECL) Cultures13.1 The reference cell line (see 10.1) shall be routinelymaintained as stock c
33、ultures, either in completely chemicallyF 1027 86 (2007)2defined 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,
34、and Fri-days. Cultures are checked microscopically at the time andobserved for any morphological changes or contamination,delayed or incidental.13.3 Cell stock cultures of established cell line are main-tained not only as source of cells for the biocompatibilityassessment but also as a means to asce
35、rtain and verify thequality and titer of the production 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 A3med
36、ium containing 10 % processed human serum.14.2 Dissect 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 forme
37、d and confirmed microscopically.14.4 Trypsinize with fresh, 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
38、 first passage cells for the biocompatibilitytest (see 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
39、 often entrapped in oral mucosaltissues as contaminants, 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 inamounts needed in 17.3. Store any unused portion in glycerol
40、or dimethyl sulphoxide (DMSO) at 70C for new sets 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 e
41、ffectiveness using:15.1.1 Initial low density cell 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.15.1.4 Grown to confluent monolayer with a schedule ofthree fre
42、sh 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 to inpopulation doubling ti
43、me, leading to monolayer confluency (III), andultimately to 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 confluency in the above AGF 0to
44、 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 cell growth as observed vis
45、ibly or by anNOTE 1Test specimens less than 1 mm in thickness tend to float. The figure depicts one means of maintaining submerged contact between specimensand cell cultures.NOTE 2Dimensions and configuration of the hole, serving for initial cell seeding, may be optionally modified and appropriately
46、 specified.FIG. 1 Arrangement for Submersing and Immobilizing SpecimensF 1027 86 (2007)3appropriate 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
47、 sheetings are Teflon FEP fluorocarbon, 10 mil(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 mo
48、st of the nutrient media,and (c) exceptional clarity 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 F 813, section8.2.2, or specially compounded at
49、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 (PracticeF 813, Section 8.7.1), its aqueous solubility and hence leachability is toorapid. For suitable, less soluble alternatives, use any of its aryl substitutedchloro or nitro derivatives listed in the Registry.16.3 Prosthetic material used in the device shall be in thechemically converted form, appropriately polymerized or oth-erwise consolidated to the final fabricated
