1、Designation: E2526 08 (Reapproved 2013)Standard Test Method forEvaluation of Cytotoxicity of Nanoparticulate Materials inPorcine Kidney Cells and Human Hepatocarcinoma Cells1This standard is issued under the fixed designation E2526; the number immediately following the designation indicates the year
2、 oforiginal adoption or, 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 test method provides a methodology to assess thecyt
3、otoxicity of suspensions of nanoparticulate materials inporcine proximal tubule cells (LLC-PK1) and human hepato-carcinoma cells (Hep G2) which represents potential targetorgans following systemic administration1.2 This test method is part of the in vitro preclinicalcharacterization cascade.1.3 This
4、 test method consists of a protocol utilizing twomethods for estimation of cytotoxicity, 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT)reduction and lactate dehydrogenase (LDH) release.1.4 The values stated in SI units are to be regarded asstandard. No other units of measurement a
5、re included in thisstandard.1.5 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 safety and health practices and determine the applica-bility of regulatory limitations
6、prior to use.2. Referenced Documents2.1 ASTM Standards:2F813 Practice for Direct Contact Cell Culture Evaluation ofMaterials for Medical DevicesF895 Test Method forAgar Diffusion Cell Culture Screeningfor CytotoxicityF1877 Practice for Characterization of ParticlesF1903 Practice for Testing For Biol
7、ogical Responses toParticles In Vitro2.2 ISO Standard:3ISO 10993-5 Biological Evaluation of Medical Devices: Part5 Tests for in vitro Cytotoxicity3. Terminology3.1 Abbreviations:3.1.1 APAPacetaminophen- positive control3.1.2 DMSOdimethyl sulfoxide3.1.3 DMEMDulbelccos modified eagles media3.1.4 Hep G
8、2human hepatocarcinoma cells3.1.5 LDHlactic dehydrogenase3.1.6 LLC-PK1porcine proximal tubule cells3.1.7 LPSlipopolysacchride, bacterial endotoxin3.1.8 MTT3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetra-zolium bromide3.1.9 Physiologic solutionisotonic with a pH 7.2 6 0.24. Summary of Test Method4.1 Na
9、noparticulate test materials in suspension in cellculture media and appropriate controls are added to cellcultures. The release of LDH indicates membrane damage andthe diminution of MTTreduction indicates loss of cell viability.These are quantitative indicators of cytotoxicity. Aseptic pro-cedures a
10、re required.5. Significance and Use5.1 Assessing the propensity of a nanomaterial to causecytotoxicity to the cells of a target organ can assist inpreclinical development.5.2 The standard historical cytotoxicity testing of materialsand extracts of materials has used fibroblasts and is welldocumented
11、 in Practice F813, Test Method F895, and ISO10993-5. The use of macrophages and micron size particles hasalso provided information on cytotoxicity and stimulationusing Practice F1903.1This test method is under the jurisdiction of ASTM Committee E56 onNanotechnology and is the direct responsibility o
12、f Subcommittee E56.03 onEnvironment, Health, and Safety.Current edition approved Sept. 1, 2013. Published September 2013. Originallyapproved in 2008. Last previous edition approved in 2008 as E2526 08. DOI:10.1520/E2526-08R13.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orco
13、ntact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from American National Standards Institute (ANSI), 25 W. 43rd St.,4th Floor, New York, NY 10036, http:/www.ansi.org.Copyrigh
14、t ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States15.3 This test method adds to the cytotoxicity test protocolsby using target organ cells. Two quantitative assays measuringLDH leakage and MTT reduction are used to estimate cyto-toxicity.5.4 Thi
15、s test method may not be predictive of eventsoccurring in all types of nanomaterial applications and the useris cautioned to consider the appropriateness of the test forvarious types of nanomaterial applications. This procedureshould only be used to compare the cytoxicity of a series ofrelated nanom
16、aterials. Meaningful comparison of unrelatednanomaterials is not possible without additional characteriza-tion of physicochemical properties of each individual nanoma-terial in the assay matrix.6. Reagents and Materials6.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless o
17、therwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.4Other grades may be used,provided it is first ascertained that the reagent is of sufficientlyhigh purity t
18、o permit its use without lessening the accuracy ofthe determination.6.2 Reagents and supplies (aseptic procedures are neededand care should be taken to use sterile reagents and supplies asnecessary).6.2.1 MTT (3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetra-zolium bromide).6.2.2 Acetaminophen.6.2.3 D
19、imethyl sulfoxide.6.2.4 Glycine.6.2.5 Sodium Chloride.6.2.6 Medium 199 Cell Culture Media.6.2.7 Triton X 100.6.2.8 LDH-Cytotoxicity Assay Kit (Biovision Cat. # K311-400 was used in developing this test method)*.6.2.9 96 well flat bottom cell culture plates.6.2.10 RPMI 1640.6.2.11 L-glutamine.6.2.12
20、Fetal bovine serum (FBS).6.3 Cell Lines:6.3.1 LLC- PK1 (porcine proximal tubule cell) (ATCC#CL-101)*.6.3.2 Hep G2 (human hepatocarcinoma)(ATCC # HB-8065)*.6.4 Equipment:6.4.1 Plate reader.6.4.2 Plate Centrifuge set at 700-800 g.6.4.3 Cell Culture Microscope.NOTE 1Commercial sources are indicated for
21、 informational purposesonly to aid laboratories initiating these test procedures. This does notindicate endorsement by ASTM. Other equivalent sources may beavailable.7. Experimental Procedure7.1 Aseptic precautions are required.7.2 Positive Control Preparation:7.2.1 LLC-PK1 Acetaminophen (APAP) posi
22、tive control:25 mM APAP in M199 cell culture media.7.2.2 Hepatocyte Acetaminophen (APAP) positive control:20 mM APAP in RPMI 1640 cell culture media.7.2.3 Triton X100 is diluted to 1 % in cell culture medium.This is the positive control for the LDH assay.7.3 MTT Assay Reagents:7.3.1 MTT solution-5mg
23、/mL MTT in PBS, store for up toone month at 4C in the dark.7.3.2 Glycine Buffer-0.1M glycine (MW 75.07), 0.1 MNaCl (MW 58.44), pH 10.5, store at room temperature.7.4 Biovision LDH-Cytotoxicity Assay Kit Reagents:7.4.1 Reconstitute catalyst in 1 mL dH20 for 10 min andvortex (stable for 2 weeks at 4C)
24、.7.4.2 Reaction mixture (for one 96-well plate):Add 250 mLof reconstituted, catalyst solution to 11.25 mL of dye solution(stable for 2 weeks at 4C).7.4.3 For other LDH Cytotoxicity assay kits, follow theirinstructions.7.5 Cell Culture:7.5.1 LLC-PK1 Cell Preparation:7.5.1.1 Harvest cells from flasks
25、prepared from cryopre-served cells according to the instructions from the supplier(limit passages to 20). An example of the appearance of thecells is in Fig. 1.7.5.1.2 Count cell concentration using a Coulter type coun-ter or hemocytometer.7.5.1.3 Dilute cells to a density of 2.5 105cells/mol inM199
26、 (3 % FBS) cell culture media.7.5.1.4 4 Plate 100 L cells/well as per plate format de-scribed in Fig. 3 for 4 plates (time zero, 6 hour sampleexposure, 24 hour sample exposure, 48 hour sample exposure).The format indicates no cells in rows D hepatocytes; kidney cells; nanoparticlesASTM International
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28、n responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be ad
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30、t the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585
31、 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).5Alley, M.C., et al., Cancer Research, Vol 48, 1988, pp. 589601.6Decker, T., and Lohmann-Matthes, M. L., Journal of Immunological Methods,Vol 15, 1988, pp. 6169.7Korzeniewski, C., and Callewaert, D. M., Journal of Immunological Methods,Vol 4, 1983, pp. 313320.E2526 08 (2013)6
