1、Designation: E2525 08 (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
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 protocol for quantitativeana
3、lysis 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 t
4、est 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
5、 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 Stand
6、ards:2F1903 Practice for Testing For Biological Responses toParticles In Vitro2.2 ANSI Standard:3ANSI/ AAMI ST72 Bacterial EndotoxinsTestMethodologies, Routine Monitoring, and Alternatives toBatch Testing3. Terminology3.1 Abbreviations:3.1.1 BMbone marrow3.1.2 CFU-GMcolony forming unit of granulocyt
7、e 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 nanoparticulat
8、e 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
9、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 bynanoparticu
10、late 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 ReagentsReag
11、ent 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
12、 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 A
13、STM 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 100
14、36, http:/www.ansi.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1such specifications are available.4Other grades may be used,provided it is first ascertained that the reagent is of sufficientlyhigh purity to permit its use witho
15、ut 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
16、.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.NOTE 1The source of the reagents is shown for information purposesonly to aid laboratories initiating this procedure. Equivalent reagents fromother
17、 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, StemCell Technologies Inc cat
18、.#27100.6.3.3 Blunt-end 16-gauge needles, StemCell TechnologiesInc cat.# 03534.6.3.4 100-mm Petri dishes.6.3.5 Plastic beakers.6.3.6 Polypropylene tubes 50 and 15-mL.6.3.7 Centrifuge.6.3.8 Refrigerator, 2 to 8C.6.3.9 Freezer, 20C.6.3.10 Cell culture incubator with 5 % CO2and 95 %humidity.6.3.11 CO2e
19、uthanasia box, or appropriate equipment ap-proved by institution.6.3.12 Scissors for tissue dissection.6.3.13 Forceps.6.3.14 Biohazard safety cabinet approved for level II han-dling of biological material.6.3.15 Inverted microscope.6.3.16 Vortex.6.3.17 Hemocytometer.6.4 AnimalsMice of the strain C56
20、BL/6, males or females8 to 12 weeks old, are used. Use of the pooled cells derivedfrom at least two (2) animals is highly recommended.7. Procedure7.1 Aseptic Precautions are required.7.2 Reagent and Control Preparation:7.2.1 MethoCult MediumThe MethoCult medium is sup-plied in 100-mL size batches. I
21、t is recommended by manufac-turer that the medium be thawed at room temperature or in arefrigerator overnight, vortexed to mix the ingredients, then leftat a room temperature for approximately 5 min to allow airbubbles to dissipate. If using another source of media, followthe instructions indicated
22、by the supplier. Use a 16-gaugeblunt-end needle to dispense 3-mL aliquots of the MethoCultmedium into sterile 15-mL tubes. Store the aliquoted mediumat a nominal temperature of 20C. Before the test thaw therequired number of tubes at room temperature for approxi-mately 20 min and keep on ice prior t
23、o use. Repeated freezingand thawing should be avoided.7.2.2 50 mM Cisplatin (Positive Control)Cisplatin is sup-plied in a lyophilized form. Reconstitute the lyophilizedpowder by adding the appropriate amount of DMSO to make astock solution with nominal concentration of 50 mM. Preparesmall aliquots a
24、nd store at a nominal temperature of 80 C.Prior to use in the assay, thaw an aliquot of the stock solutionat room temperature and dilute in IMDM supplemented with2 % FBS to bring the Cisplatin concentration to 2 mM. Onehundred fifty (150) L of this intermediate solution is thenadded to 3 mL of cultu
25、re medium. Final concentration ofCisplatin in the positive control sample is 100 M.7.3 Preparation of Study Samples:7.3.1 This assay requires 1200 L of nanoparticles, 150 Lsamples in duplicate for each of 4 concentrations. The nano-particles subjected to the biological test environment shouldhave be
26、en characterized as appropriate to allow adequate datainterpretation and to help provide information to predictbiological responses. For example, lot-to-lot variations inparticle size and surface characteristics of the particles couldresult in different assay results. For this assay, the particlessh
27、all be provided in physiologic solution (isotonic with pH7.2 6 0.2) and this solution shall be defined. The preparationshall be sterile and the level of LPS provided or determined bythe testing laboratory. ANSI/AAMI ST72 may be helpful. Thenumber of particles/mL and mg/mL shall be indicated.7.3.2 Th
28、e test sample shall be used at the highest concen-tration possible and at three serial one to five (1:5) dilutions.The highest possible concentration is that at which the particlesappear evenly dispersed in the liquid. If the concentration forits intended use is known, this may serve as the highestc
29、oncentration to be tested and at least three dilutions made.7.4 Isolation and Counting of Bone Marrow Cells:7.4.1 Position the euthanized mouse on its back and rinsefur thoroughly with 70 % alcohol.7.4.2 Cut a slit in the fur just below the rib cage withoutcutting the peritoneal membrane.7.4.3 Firml
30、y grasp skin and peal back to expose hind limbs.7.4.4 Using sterile sharp dissecting scissors cut the kneejoint in the center. Cut through ligaments and excess tissue.7.4.5 Grasp femur with forceps and cut femur near hip joint.7.4.6 Free tibia by cutting near the ankle joint.7.4.7 Trim the ends of t
31、he long bones to expose the interiormarrow shaft. Put bones in a sterile Petri dish or in sterileculture medium and place on ice. Bones should be collectedfrom multiple animals.4Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For Suggestions on
32、the testing of reagents notlisted by the American Chemical Society, see Analar Standards for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.E2525 08 (2013)27.4.8 Using a 3-cc syrin
33、ge with 21 or 22-gauge needle, drawup to 1 to 3 mL of cold Iscoves MDM supplemented with 2 %FBS.7.4.9 Insert bevel of needle into marrow shaft and flushmarrow into 15-mL tube. Repeat this procedure for all bones.The same medium can be used to isolate marrow from 1 to 3animals. The medullary canal sh
34、ould appear white once all themarrow has been expelled.7.4.10 Keep the needle below the surface of the medium andgently draw the cell suspension up and down with 3-cc syringeand 21-gauge needle 3 to 4 times to make a single cellsuspension without introducing air bubbles.7.4.11 Keep cells in medium o
35、n ice until use.7.4.12 Perform a nucleated cell count. Dilute an aliquotsample of the cells with 3 % acetic acid with methylene blue1:50 (20 L cells + 980 L 3 % acetic acid methylene blue) or1:100 (10 L cells + 990 L 3 % acetic acid methylene blue).Then use either hemocytometer or automatic cell cou
36、nter (thatcounts nucleated live cells) to determine the number of viablenucleated cells. An average cell count is expected to be12107for femur and 0.61 107for tibia from each mouse.7.4.13 If cell viability and count are acceptable providing atleast 106viable cells proceed to the next step.7.5 Experi
37、mental Procedure:7.5.1 Label lids of 35-mm culture dishes at the edge using apermanent fine felt marker. There will be two 35-mm culturedishes for each control and test article.7.5.2 Thaw the aliquoted tubes of MethoCult medium atroom temperature or in a refrigerator overnight.7.5.3 Vortex tubes to
38、ensure all components are thoroughlymixed.7.5.4 Dilute bone marrow cells isolated according to theprocedure described in section 6 with Iscoves medium supple-mented with 2 % FBS to4105cells per mL.7.5.5 Add 150 L of cell suspension and 150 L of eitherIscoves medium with 2 % FBS (baseline), PBS (nega
39、tivecontrol), Cisplatin (positive control), or nanoparticles (testsample) to 3 mL of MethoCult medium.7.5.6 Vortex tubes to ensure all cells and medium compo-nents are mixed thoroughly.7.5.7 Let the tubes stand for 5 min to allow bubbles todissipate.7.5.8 Label two 35-mm Petri dishes for each sample
40、:baseline, negative control, positive control, and the test articles.Each sample is thus tested in duplicate.7.5.9 Attach a 16-gauge blunt-ended needle to a 3-ccsyringe, place the needle below the surface of the solutionprepared in 7.5.5. Draw up approximately 1 mL, then gentlydepress the plunger an
41、d expel medium back into the tube.Repeat this step until no air space is visible.7.5.10 With this syringe and needle, draw up the solutionand dispense 1.1 mLinto each of two 35-mm dishes. Distributethe cell suspension evenly by gently tilting and rotating eachdish. Cover the 35-mm dishes and place t
42、he two replicates intoa 100-mm Petri dish. Place a third 35-mm dish into the100-mm Petri dish and fill this 35-mm dish with 3 mLof sterilewater and do no cover this dish. This will provide a humiditychamber. Place the lid on the 100-mm Petri dish.7.5.11 Repeat 7.5.10 for all samples and place the cu
43、lturesin an incubator maintained at 37C, 5 % CO2and 95 %humidity.7.5.12 Incubate for 12 days. On the 12th day remove dishesfrom incubator, identify and count colonies as described below.Representative values of CFU-GM for C57BL6 mice at 8 to 12weeks of age range from 64 6 16.7.6 Count the CFU-GM Col
44、onies:7.6.1 This count includes the CFU-granulocyte (CFU-G),CFU-macrophage (CFU-M) and CFU-granulocyte macro-phage (CFU-GM). Count the colonies containing at least 30CFU-G, CFU-M or both cell types (CFU-GM). CFU-GMcolonies often contain multiple clusters and appear as densecore surrounded by cells.
45、The monocytic lineage cells are largecells with an oval to round shape and appear to have a grainyor grey center. The granulocytic lineage cells are round, bright,and are much smaller and more uniform in size than macro-phages. It is easy to see individual cells of a CFU-GM colony,especially in the
46、periphery of the colony.7.6.2 See Fig. 1 for the following colony examples. Colo-nies seen on Figures A and B are CFU-GM. Colonies onFigures C and D are CFU-M. Figure E demonstrates anexample of single CFU-G colony. Few CFU-G coloniesgrowing together are shown on Figure F.8. Calculation or Interpret
47、ation of Results8.1 Determine the mean and standard deviation and thePercent Coefficient of Variation for each control or testaccording to the following formula:%CV5 SD/Mean 3100% (1)8.2 Percent CFU Inhibition is calculated as follows:% CFU 2 Inhibition 5Baseline CFU 2 GM 2 Test CFU 2 GM!Baseline CF
48、U 2 GM3100% (2)9. Report9.1 %CV for each control and test sample should less than30 %.9.1.1 If the positive control or negative control fail to meetacceptance criterion described in 9.1 the assay should berepeated.9.1.2 If the assay meets the acceptable criteria but a testsample fails to meet accept
49、ance criterion described in 9.1 thistest sample should be re-analyzed.9.1.3 If two duplicates of the same test sample demonstrateresults different by more then 20 %, this sample should bereanalyzed.9.1.4 Determine if the results of the test sample are differentfrom the media control and indicate if the test sample isstimulatory or inhibitory.9.1.5 Determine if there is a dose response.10. Precision and Bias10.1 Precision and bias have not been determined for thistest method and will be determined within 5 years of publica-tion of the st
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