ASTM_E_2525_-_08_2013.pdf

Designation:E252508(Reapproved 2013)Standard Test Method forEvaluation of the Effect of Nanoparticulate Materials on theFormation of Mouse Granulocyte-Macrophage Colonies1This standard is issued under the fixed designation E2525;the number immediately following the designation indicates the year 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 protocol for quantitativeanalysis of the effect of nanoparticulate materials in physi-ologic solution on granulocyte-macrophage colony-formingunits.1.2 This test method employs murine bone marrow he-matopoietic stem cells which proliferate and differentiate toform discrete cell clusters or colonies which are counted.1.3 This test method is part of the in vitro preclinicalcharacterization cascade for nanoparticulate materials for sys-temic administration in medical applications.1.4 The values stated in SI units are to be regarded asstandard.No other units of measurement are 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 prior to use.2.Referenced Documents2.1 ASTM Standards:2F1903 Practice for Testing For Biological Responses toParticles In Vitro2.2 ANSI Standard:3ANSI/AAMIST72 BacterialEndotoxinsTestMethodologies,Routine Monitoring,and Alternatives toBatch Testing3.Terminology3.1 Abbreviations:3.1.1 BMbone marrow3.1.2 CFU-GMcolony forming unit of granulocyte andmacrophage3.1.3 Cisplatinpositive control3.1.4 DMSOdimethyl sulfoxide3.1.5 DPBSDulbeccos phosphate buffered saline3.1.6 FBSfetal bovine serum3.1.7 IMDMIscoves media3.1.8 LPSipopolysaccharide3.1.9 Physiologic Solutionisotonic,pH 7.2 6 0.24.Summary of Test Method4.1 The effect of nanoparticulate materials on the formationof granulocyte and macrophage colonies is assessed.Bonemarrow cells are obtained from mice and cultured in stimula-tory media.The number of colony forming units followingcontact with nanoparticles is counted and compared to baselineand positive control.This determines if the nanoparticulatematerial in physiologic solution is stimulatory or inhibitory tobone marrow stem cells.Aseptic procedures are necessary.5.Significance and Use5.1 Stem cells of hematopoietic origin are pluripotential andmay be particularly sensitive to the effects of stimulation bynanoparticulate materials.5.2 The effect of particles on macrophage responses has anextensive history and can be assessed by Practice F1903.Thetest method described here will assess the effect on stem cellswhich can be progenitor cells to the macrophage line.6.Reagents 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 where1This test method is under the jurisdiction of ASTM Committee E56 onNanotechnology and is the direct responsibility of 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 E2525 08.DOI:10.1520/E2525-08R13.2For 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 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.Copyright ASTM International,100 Barr Harbor Drive,PO Box C700,West Conshohocken,PA 19428-2959.United States1 such specifications are available.4Other 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:6.2.1 MethoCult medium,StemCell Technologies Inc cat.#03534.6.2.2 Fetal Bovine Serum prescreened for hematopoieticstem cells,StemCell Technologies Inc cat.#06200.6.2.3 IMDM with 2%FBS,StemCell Technologies Inccat.#07700.6.2.4 Sterile distilled water.6.2.5 Cisplatin,(positive control)Sigma cat#P4394.6.2.6 Sterile Ca2+/MG2+-free DPBS,(negative control)Sigma cat.#D8537.NOTE1The source of the reagents is shown for information purposesonly to aid laboratories initiating this procedure.Equivalent reagents fromother suppliers may be used.6.3 EquipmentAseptic procedures are necessary and careshould be used in acquiring sterile equipment as needed.6.3.1 Pipettes covering the range of 0.05 to 10 mL.6.3.2 35-mm culture dishes prescreened to support stem cellgrowth and differentiation,