1、Designation: F1903 10Standard Practice forTesting For Biological Responses to Particles In Vitro1This standard is issued under the fixed designation F1903; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision.
2、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 covers the production of wear debris anddegradation products from implanted materials that may lead toa cascade of biol
3、ogical responses resulting in damage toadjacent and remote tissues. In order to ascertain the role ofparticles in stimulating such responses, the nature of theresponses, and the consequences of the responses, establishedprotocols are needed. This is an emerging, rapidly developingarea and the inform
4、ation gained from standard protocols isnecessary to interpret responses and to determine if there iscorrelation with the in vivo responses. Since there are manypossible and established ways of determining responses, asingle standard protocol is not stated. However, well describedprotocols are needed
5、 to compare results from different inves-tigators using the same materials and to compare biologicalresponses for evaluating (ranking) different materials. Forlaboratories without established protocols, recommendationsare given and indicated with an asterisk*.1.2 Since the purpose of these studies i
6、s to predict theresponse in humans, the use of human cells would providemuch information. However, in this practice, the use ofnon-human and non-primate cells is described. If the usershould wish to employ cell lines from humans, cell lines areavailable from ATCC and most of the information and reco
7、m-mendations will still apply.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of t
8、his standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2F619 Practice for Extraction of Medical PlasticsF748 Practice for Selecting Generic Biological Test Meth-ods for Mater
9、ials and Devices3. Summary of Practice3.1 Biological responses to particles may be evaluated usingspecimens from animals being tested according to the PracticeF748 matrix for irritation and sensitivity, or for implantation.Blood, organs, or tissues from the animals may be used.3.2 Biological respons
10、es to particles may be evaluated usingmaterials or extracts according to Practice F619. These mate-rials or extracts may be used for in vivo tests or for the in vitrotests. Particles generated by other methods may also be used.3.3 The purpose of this practice is to assess the response ofcells in dir
11、ect contact with particles and, therefore, this practiceis primarily intended to cover the testing of particles placedinto culture with the cells. This practice should be equallyappropriate for the testing of the response to nanoparticlesplaced in culture, if particles of that size are the particles
12、 ofinterest.4. Significance and Use4.1 This practice is to be used to help assess the biocom-patibility of materials used in medical devices. It is designed totest the effect of particles from the materials on macrophages.The use of nonhuman, nonprimate cells is recommended in thispractice. For labo
13、ratories equipped and approved to work withhuman blood and tissue, the use of these same protocols wouldbe advantageous for development of understanding of theinteraction of cells and particles.4.2 The appropriateness of the methods should be carefullyconsidered by the user since not all materials o
14、r applicationsneed be tested by this practice.4.3 Abbreviations:4.3.1 LPSlipopolysaccharide (endotoxin).4.3.2 LALLimulus Amebocyte Lysate.1This practice is under the jurisdiction ofASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16
15、on Biocompatibility Test Methods.Current edition approved June 1, 2010. Published June 2010. Originallyapproved in 1998. Last previous edition approved in 2003 as F1903 98 (2003).DOI: 10.1520/F1903-10.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Servi
16、ce at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.4.3.3 ATCCAmerican Type Culture Collection.4.
17、3.4 FCS (FBS)Fetal Calf Serum.4.3.5 NCSNewborn Calf Serum.4.3.6 PBSPhosphate Buffered Saline.4.3.7 HANKSA balanced salt solution.4.3.8 MMPSMatrix Metallo Proteases.4.3.9 RPMI 1640Specific Growth Medium (Roswell ParkMemorial Institute).4.3.10 HEPESA buffering salt.5. Responses from Cells Grown In Vit
18、ro5.1 ParticlesDefine the nature of the particles used:5.1.1 Source,5.1.2 Chemistry,5.1.3 Size (mean and range),5.1.4 Shape,5.1.5 Method of sterilization,5.1.6 If the presence of bacterial lipopolysaccharide (LPS)was determined, specify how this was done and the sensitivityof the method. (LAL testin
19、g with a sensitivity of at least 0.06EU is recommended),5.1.7 Concentration of particles used as weight or number orsurface area/106cells, and5.1.8 Surface charge (if known).5.1.9 Since some particles may have a density less than thatof the culture medium being used, careful consideration shouldbe g
20、iven to determining appropriate procedures for ensuringthat the particles and cells are in contact. Techniques in whichcells are grown on a substrate and then the substrate is invertedin culture may be appropriate.5.2 CellsDefine the nature of the cells used:5.2.1 Established Cell Line (if not, go t
21、o 5.2.2)The use ofestablished cell lines provides a known cell type with areproducible response. Although the correlation with the invivo system may not be known at this time, careful studies withestablished cell lines could eventually allow determination ofcorrelation between in vivo and in vitro s
22、ystems.5.2.1.1 Source of cell and identifying number or code,5.2.1.2 Nature of the cell (for example, macrophage), and5.2.1.3 Special attributes of the cell line (for example,nonphagocytic),5.2.1.4 *ATCC murine macrophages such as RAW 264.7,J774A, P388D1, or IC-21 are recommended.5.2.2 Primary Isola
23、te (if not, go to 1.2.1):5.2.2.1 Source of cell including species and location (forexample, murine, alveolar),5.2.2.2 Nature of the cell (for example, macrophage),5.2.2.3 Mechanism of isolation (for example, lavage), and5.2.2.4 Specify if stimulant used and if so which one (forexample, mineral oil).
24、5.2.2.5 *Mouse (specify strain, age, and sex used) perito-neal exudate cells are recommended with a mild stimulant suchas nutrient broth.5.3 Culture Conditions:5.3.1 Specify source and type of medium. If not a commer-cial source, list ingredients and sources of ingredients.5.3.2 Specify source and t
25、ype of serum, and whether it washeat inactivated. If the presence of LPS was determined,specify method and sensitivity of the method.5.3.3 Specify culture conditions (for example, 37C, hu-midified, 5 % CO2incubator).5.3.4 Specify time of termination of culture or sampling ofculture medium.5.3.5 If c
26、ell counts were determined specify as to when andhow. If estimates of cell number/mL specify when and how.5.3.5.1 *Medium and serum specified by the supplier of thecells are recommended. Generally RPMI 1640 with heatinactivated 10 % newborn or fetal calf serum are recom-mended. LPS levels are genera
27、lly provided or available fromthe distributor. Recommended culture conditions are 37C,with 5 % CO2, in a humidified incubator. Cell counts at thetime of initial plating and at the termination of the culture arerecommended using a hemocytometer with monolayer cellsresuspended by trypsin solution (not
28、 recommended for mac-rophages), washing with Ca and Mg free PBS or Hanks, orscraping in 1 mL of Ca and Mg free PBS or Hanks. Theaddition of trypan blue is helpful. The supernatant of themedium from macrophages exposed to particles for specifiedtime periods is assayed.5.3.6 Controls:5.3.6.1 Cells not
29、 stimulated with particles should be main-tained at the same time under the same conditions.5.3.6.2 Polystyrene particles, spherical, size range 1 to 5m, should be used as a reference control.5.3.6.3 LPS is a known stimulant and is a good positivecontrol. A concentration between 0.25 and 1 ng/mL of
30、culturemedium is sufficient.5.3.6.4 Culture medium is a recommended diluent for theassays.5.4 Products or Response DeterminedOne or more of thefollowing:5.4.1 Cell viability can be determined by any of severalmethods:5.4.1.1 From the total cell counts and viable cell count withtrypan blue dye exclus
31、ion,5.4.1.2 From flow cytometry with vital stains,5.4.1.3 From uptake of DNA precursors, and5.4.1.4 From total DNA or RNA in the culture.5.4.2 Soluble Cell Products Elaborated:5.4.2.1 Reliable reagents or kits are available to detectproducts. The products are classified into three classes: growthfac
32、tors such as TNFa, TGF beta, and PGE2, interleukins suchas IL-1, IL-1 receptor antagonist, IL-1 beta, and IL-6, andreactive oxygen species such as superoxide or nitric oxide(NO) (usually measured as nitrite produced using the Griessreagent). The release of at least two products, which are fromdiffer
33、ent classes, should be determined.5.4.2.2 The time in culture should be specified since theseproducts peak at different times (for example, TNFa peaks by24 h, NO peaks around 72 h, the others generally peak at 48 to72 h). The detection of NO and TNFa are strongly recom-mended and the assay procedure
34、s are described in some detailin this recommended practice.5.4.3 Assay for Nitric Oxide:5.4.3.1 The production of NO is most conveniently mea-sured by detection of the stable endproduct nitrite. This reflectsthe action of NO on arginine. The technique is simple,F1903 102inexpensive and quantitative.
35、 The reagent used to quantitatenitrite is the Griess reagent. This is prepared by dissolving 100mg of Sulfanilamide and 10 mg of N-(1-naphthyl)ethylenediamine in 2.5 % phosphoric acid. Assay ofculture medium: 100 uL of the culture medium is placed in a96-well microtiter plate and then 100 uL of the
36、Griess reagentis added. This is incubated at room temperature for 10 min andthe optical density determined at 540 nm wavelength. Astandard curve is prepared with sodium nitrite in water. It isrecommended that two-fold serial dilutions be prepared en-compassing the range of 32 to 0.125 nmoles/mL. The
37、 standardcurve is prepared using 100 uL of the dilutions of sodiumnitrite and 100 uL of the Griess reagent.5.4.4 Assay for TNFa:5.4.4.1 Kits for assay of TNFa using immunological assaysare available from supply houses including, but not limited to,R growth factors; interleukins; macroph-ages; nitric
38、 oxide; particles; TNFaF1903 103APPENDIXES(Nonmandatory Information)X1. RATIONALEX1.1 The primary purpose of this practice is to describemethodology to determine the biological response to particlesusing in vitro cellular responses.X1.2 It is well recognized that the biological responses toparticles
39、 could be different from those to solid materials. Theinteraction of the particles with phagocytic cells, notablymacrophages, is a key to the final biological response.X1.3 The interaction of macrophages with particles hasbeen an active research area for many years. Many investiga-tors have develope
40、d procedures for doing these studies. Thisdocument is intended to delineate the information necessary sothat the results from these various studies can be interpretedand to describe methodology appropriate for those investiga-tors developing such studies.X1.4 The interaction of phagocytic cells with
41、 particles willlead to the production of various soluble mediators which mayinfluence the progression of the biological response and theimmune response. It is unknown at this time which of theseresponses are favorable and which are unfavorable to the host.Similarly it is unknown whether quantitative
42、 evaluation ofthese factors is predictive of the biological response. Studiessuch as the ones described here are needed to determine theimportance of these responses in biocompatibility and biocom-patibility testing of materials.X1.5 The level of LAL for device evaluation is commonly0.5 EU/m/L . LAL
43、 testing with a sensitivity of 0.06 EU/mL isrecommended in this practice for evaluating in vitro responsesto particles since levels of LPS above 0.06 EU/mL may bestimulatory to macrophages. This will permit distinguishingcellular responses to particles from cellular responses to LPS.X1.6 Polystyrene
44、 (PS) particles were chosen as a referencecontrol for this standard practice so that testing results fromdifferent laboratories of from different cell lines or assaysystems could be compared and normalized to PS. Theanticipated responses of the cells to these particles and pro-duction of various cyt
45、okines and other growth factors areunknown. They may stimulate some factors and not others.These particles are not device material applicable and serve asa neutral territory, commercially available, well characterizedparticulate material for normalization of results.X2. ADDITIONAL REFERENCESGreen, L
46、.C., DA Wagner, J Glowoski, PL Skipper, JSWishnok, SR Tannenbaum, “Analysis of Nitrate, Nitrite, and15N Nitrate in Biological Fluids,” Anal. Biochem. 126:131,1982.Flick DA, GE Gifford, “Comparison of In Vitro Cell Cyto-toxicity Assays for Tumor Necrosis Factor,” J. ImmunolMethods 68: 167, 1984.Chape
47、kar, MS, TG Zaremba, RK Kuester, VM Hitchins,“Synergistic Industion of Tumor Necrosis Factor a by Bacte-rial Lipopolysaccharide and Lipoteichoic Acid in Combinationwith Polytetrafluoroethylene Particles in a Murine MacrophageCell Line RAW 264.7,” J. Biomed. Mater. Res. 31: 1996, pp251-256.St. John K
48、.R., (ed), Particulate Debris from Medical Im-plants: Mechanisms of Formation and Biological Conse-quences, STP 1144, ASTM, Philadelphia, PA 1992.Wright, T.M., Goodman, S.B., (eds), Implant Wear: TheFuture of Total Joint Replacement, American Academy ofOrthopaedic Surgeons, Rosemont, IL 1996.Ruff, M
49、.R., Gifford, G.E., “Tumor Necrosis Factor,” Lym-phokines 2: 1991, pp. 235-272.Bonfield, T.L., Anderson, J.M., “Functional Versus Quanti-tative Comparison of IL-1 Beta from Monocytes/Macrophageson Biomedical Polymers,” J. Biomed. Mater. Res. 27:, 1993,pp. 1195-1199.ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are e
