1、Designation: E 2526 08Standard Test Method forEvaluation of Cytotoxicity of Nanoparticulate Materials inPorcine Kidney Cells and Human Hepatocarcinoma Cells1This standard is issued under the fixed designation E 2526; the number immediately following the designation indicates the year oforiginal adop
2、tion or, 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 test method provides a methodology to assess thecytotoxicity of su
3、spensions 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 test method co
4、nsists 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 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is
5、 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 Documents2.1 ASTM Standards:2F 813 Practice for Direct Contact Cell Culture Evaluation ofMaterials for Medical D
6、evicesF 895 Test Method for Agar Diffusion Cell Culture Screen-ing for CytotoxicityF 1877 Practice for Characterization of ParticlesF 1903 Practice for Testing For Biological Responses toParticles in vitro2.2 ISO Standard:ISO 10993-5 Biological Evaluation of Medical Devices:Part 5 Tests for in vitro
7、 Cytotoxicity33. Terminology3.1 Abbreviations:3.1.1 APAPacetaminophen- positive control3.1.2 DMSOdimethyl sulfoxide3.1.3 DMEMDulbelccos modified eagles media3.1.4 Hep G2human hepatocarcinoma cells3.1.5 LDHlactic dehydrogenase3.1.6 LLC-PK1porcine proximal tubule cells3.1.7 LPSlipopolysacchride, bacte
8、rial endotoxin3.1.8 MTT3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide3.1.9 Physiologic solutionisotonic with a pH 7.2 6 0.24. Summary of Test Method4.1 Nanoparticulate test materials in suspension in cellculture media and appropriate controls are added to cellcultures. The release of LD
9、H indicates membrane damage andthe diminution of MTT reduction indicates loss of cell viability.These are quantitative indicators of cytotoxicity. Aseptic pro-cedures are required.5. Significance and Use5.1 Assessing the propensity of a nanomaterial to causecytotoxicity to the cells of a target orga
10、n can assist inpreclinical development.5.2 The standard historical cytotoxicity testing of materialsand extracts of materials has used fibroblasts and is welldocumented in Practice F 813, Test Method F 895, andISO 10993-5. The use of macrophages and micron size par-ticles has also provided informati
11、on on cytotoxicity andstimulation using Practice F 1903.5.3 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 This test method may not be predictive of eventsoccur
12、ring 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 nanomaterials. Meaningful comparison of unrelatednanoma
13、terials is not possible without additional characteriza-tion of physicochemical properties of each individual nanoma-terial in the assay matrix.1This test method is under the jurisdiction of ASTM Committee E56 onNanotechnology and is the direct responsibility of Subcommittee E56.02 onCharacterizatio
14、n: Physical, Chemical, and Toxicological Properties.Current edition approved Feb. 1, 2008. Published February 2008.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to t
15、he 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.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.6. R
16、eagents and Materials6.1 Purity of ReagentsReagent grade chemicals shall beused in all tests. Unless otherwise indicated, it is intended thatall reagents conform to the specifications of the Committee onAnalytical Reagents of the American Chemical Society wheresuch specifications are available.4Othe
17、r grades may be used,provided it is first ascertained that the reagent is of sufficientlyhigh purity to 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)
18、.6.2.1 MTT (3-(4,5-dimethylthiazolyl-2)-2,5- diphenyltetra-zolium bromide).6.2.2 Acetaminophen.6.2.3 Dimethyl 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 t
19、est method)*6.2.9 96 well flat bottom cell culture plates.6.2.10 RPMI 1640.6.2.11 L-glutamine.6.2.12 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 C
20、entrifuge set at 700-800 g.6.4.3 Cell Culture Microscope.NOTE 1Commercial sources are indicated for informational purposesonly to aid laboratories initiating these test procedures. This does notindicate endorsement by ASTM. Other equivalent sources may beavailable.7. Experimental Procedure7.1 Asepti
21、c precautions are required.7.2 Positive Control Preparation:7.2.1 LLC-PK1 Acetaminophen (APAP) positive 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
22、medium.This is the positive control for the LDH assay.7.3 MTT Assay Reagents:7.3.1 MTT solution-5mg/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 Ki
23、t Reagents:7.4.1 Reconstitute catalyst in 1 mL dH20 for 10 min andvortex (stable for 2 weeks at 4C).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 t
24、heirinstructions.7.5 Cell Culture:7.5.1 LLC-PK1 Cell Preparation:7.5.1.1 Harvest cells from flasks 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
25、Coulter typecounter or hemocytometer.7.5.1.3 Dilute cells to a density of 2.5 3 105cells/mol inM199 (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)
26、.The format indicates no cells in rows D hepatocytes; kidney cells; nanoparticlesASTM 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 va
27、lidity of any such patent rights, and the riskof infringement of such rights, are entirely their own 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 co
28、mments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel that your comments hav
29、e not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at 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
30、 copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org).5Alley, M.C., et al, Cancer Res., 48, 1988, pp. 589-601.6Decker, T., and Lohmann-Matthes, M. L., J. Immunol Methods, 15, 1988, pp.61-69.7Korzeniewski, C., and Callewaert, D. M., J. Immunol Methods, 64, 1983, pp.313-320.E2526086
