1、Designation: F895 11 (Reapproved 2016)Standard Test Method forAgar Diffusion Cell Culture Screening for Cytotoxicity1This standard is issued under the fixed designation F895; the number immediately following the designation indicates the year of originaladoption or, in the case of revision, the year
2、 of last revision.Anumber in parentheses indicates the year of last reapproval.Asuperscriptepsilon () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method is appropriate for materials in a varietyof shapes and for materials that are not necessarily sterile
3、. Thistest method would be appropriate in situations in which theamount of material is limited. For example, small devices orpowders could be placed on the agar and the presence of a zoneof inhibition of cell growth could be examined.1.1.1 This test method is not appropriate for leachables thatdo no
4、t diffuse through agar or agarose.1.1.2 While the agar layer can act as a cushion to protect thecells from the specimen, there may be materials that aresufficiently heavy to compress the agar and prevent diffusion orto cause mechanical damage to the cells. This test methodwould not be appropriate fo
5、r these materials.1.2 The L-929 cell line was chosen because it has asignificant history of use in assays of this type. This is notintended to imply that its use is preferred, only that the L-929is an established cell line, well characterized and readilyavailable, that has demonstrated reproducible
6、results in severallaboratories.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
7、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:2F748 Practice for Selecting Generic Biological Test Methodsfor Materials and Devices2.2 ATCC Document:American Type C
8、ulture Collection, (ATCC) Catalogue ofStrains II3USP Negative Control Plastic Reference Standard43. Summary of Test Method3.1 Cell cultures are grown to a monolayer in culture dishes.The medium is aspirated and replaced with an agar-containingmedium that is allowed to solidify. Test control articles
9、 areplaced on the agar surface to evaluate the cytotoxic propertiesof a given material or device. Toxic components in the testarticle can diffuse into the culture medium, forming a concen-tration gradient and adversely affecting cells at varying dis-tances from the test article. This method is well
10、suited forlow-density materials (film, paper, and so forth), powders,liquids, and high-density materials that could physically dam-age the cells if placed in direct contact with the cell monolayer.4. Significance and Use4.1 This test method is useful for assessing the cytotoxicpotential of new mater
11、ials and formulations and as part of aquality control program for established medical devices andcomponents.4.2 This test method assumes that assessment of cytotoxic-ity provides useful information to aid in predicting the potentialclinical applications in humans. Cell culture methods haveshown good
12、 correlation with animal assays and are frequentlymore sensitive to cytotoxic agents.4.3 This cell culture test method is suitable for incorporationinto specifications and standards for materials to be used in theconstruction of medical devices that are to be implanted intothe human body or placed i
13、n contact with tissue fluids or bloodon a long-term basis.4.4 Some biomaterials with a history of safe clinical use inmedical devices are cytotoxic. This test method does not implythat all biomaterials must pass this assay to be considered safefor clinical use (Practice F748).5. Apparatus5.1 The fol
14、lowing apparatus shall be used:1This test method is under the jurisdiction ofASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved April 1, 2016. Published June 2016. Originally
15、approved in 1984. Last previous edition approved in 2011 as F895 11. DOI:10.1520/F0895-11R16.2For 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
16、Summary page onthe ASTM website.3Fourth edition, 1983, is available from American Type Culture Collection,12031 Parklawn Dr., Rockville, MD 10892. Library of Congress No. 76-640122.4U.S. Pharmacopeia, current edition, Rockville, MD.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, We
17、st Conshohocken, PA 19428-2959. United States15.2 Incubator, which maintains the cultures at 37 6 2C, 56 1%CO2, and greater than 90 % relative humidity.5.3 Water Bath, capable of maintaining a temperature of 376 2C and 45 6 2C.5.4 Microscope, with inverted phase contrast optics andmagnifications of
18、40, 100, and 200.5.5 Clinical Centrifuge, capable of attaining 1000 xg.5.6 Sterile, Disposable 150-cm2Tissue Culture Flasks.5.7 Sterile, Tissue Culture Dishes, 35 mm in diameter and10 mm deep.NOTE 1Plastic dishes are recommended because they provide a flatsurface that promotes the formation of a uni
19、form monolayer of cells.5.8 Sterile, Disposable, Centrifuge Tubes.5.9 Sterile Pipettes, 1, 5, and 10 mL.5.10 Filter Disks, 10 mm in diameter for evaluation ofliquids.NOTE 2MilliporeAP2501000 filter disks have been found satisfactoryfor use in cytotoxicity evaluations because they elicit no cytopathi
20、c effect.Other filter disks that do not elicit a cytopathic effect may also be used.NOTE 3A laminar flow work area capable of filtering out 99.99 % ofall particles greater than 0.3 m in diameter, or a Class 100 clean roommay be necessary to prevent contamination of cultures.6. Reagents6.1 The follow
21、ing reagents shall be used:6.1.1 For Cell Culture Maintenance, 1 Media. MinimumEssential Medium (MEM) is prepared by mixing 90 mL ofEagles MEM (with Earles salts, without L-glutamine), ad-justing the solution to pH of 7.15, and adding 5 to 10 mL offetal bovine serum, and 1 mLof 100 nonessential amin
22、o acids(L-glutamine).6.1.1.1 Opened containers of prepared MEM may be storedat a temperature of 2 to 8C for periods of not more than twoweeks. Glutamine is omitted from this formulation to maxi-mize the shelf life. Immediately before use, 1 mL ofL-glutamine solution (see 6.1.3) is added to each 100
23、mL ofMEM.6.1.1.2 Antibiotics, such as penicillin G10 000 I.U.mL,and streptomycin 10 000 I.U./mL, may be added to the mediumto reduce the incidence of bacterial contamination. Use 1 mLofantibiotic per 100-mL media. Care shall be taken to ensure thatthe antibiotics do not have an adverse effect on the
24、 viability ofthe cell cultures.6.1.2 For Agar Media Overlay, to prepare 2 Media(100-mL final volume). Twice concentrated (2) MEM isprepared by mixing 20 mL of 10 Eagles MEM (with EarlesSalts without L-glutamine), 0.22-g sodium bicarbonate (buffer)and sterile distilled water to bring to 70 mL. Adjust
25、 the pH to7.15. Add 20-mL fetal bovine serum and 2 mL of 100nonessential amino acid (L-glutamine). Bring to final volume(100 mL) with sterile distilled water. Filter sterilize the 2media. Mix with equal amounts of sterilized 3 % agar nobel togive the final concentration of the media as 1.6.1.3 L-Glu
26、tamine Solution (Lyophilized), 29.2 mg/mL. Re-hydrate with sterile distilled water. (Store frozen.)6.1.4 Hanks Balanced Salt Solution, calcium- andmagnesium-free (store at room temperature).6.1.5 Trypsin, 0.1 % solution in Hanks balanced salt solu-tion or calcium- and magnesium-free, phosphate-buffe
27、red sa-line (store frozen).6.1.6 Water, sterile, deionized, or distilled water should beused.6.1.7 Noble Agar, 3%.6.1.8 Neutral Red Stain, 0.01 % by weight in phosphate-buffered saline.6.2 All reagents shall be tissue-culture grade or equivalent.6.3 Reagents shall be reconstituted in accordance with
28、 themanufacturers directions, using aseptic technique.7. Cell Culture7.1 Cell cultures used in this assay shall be the ATCC, CCLI NCTC clone 929 strain (clone of Strain L, mouse connectivetissue) designated L-929. Other suitable validated cell linesmay be considered.8. Control Materials8.1 Prepare n
29、egative control specimens in accordance withSection 10 from a material that consistently elicits negligiblecellular response in this assay (for example, USP NegativeControl Plastic Reference Standard).8.2 Prepare positive control specimens in accordance withSection 10 from a material that consistent
30、ly elicits a moderateand reproducible degree of cytotoxicity (for example, anaqueous solution of phenol (0.45 6 0.05 % by volume), orother material producing a known cytotoxic response, forexample, latex rubber).8.2.1 Use an aqueous solution of phenol to give a diffusereaction of cellular degenerati
31、on and sloughing; a latex rubberwill give a zone of toxicity.8.2.2 Take care when preparing aqueous solutions of phenolto ensure the homogeneity of the solution since phase separa-tions may occur.8.2.3 Latex rubber is a widely used control material that hasdemonstrated reproducible results in severa
32、l laboratories.9. General Technique9.1 Use aseptic technique throughout this assay to minimizemicrobial contamination.NOTE 4Mouth pipetting should not be used to transfer cells, medium,or reagents.9.2 Warm all solutions and materials to a temperature of 376 2C before being placed in contact with cel
33、ls.9.3 Wash all glass vessels thoroughly with a cleaningsolution and rinse thoroughly with copious amounts of deion-ized water.9.4 Clean all work surfaces with a disinfectant solutionbefore use.9.5 Record the culture history of the cells.9.6 Stock cultures should be periodically screened for my-copl
34、asma contamination.F895 11 (2016)210. Specimen Preparation10.1 Sterilize all specimens by a method appropriate to theend use of the device.10.2 Where a device is sufficiently small (see 10.3 and 10.4)to fit into the culture dish leaving an adequate margin of cellsfor evaluation, use the entire devic
35、e as a specimen.10.3 Cut large solid materials and devices in cross section toobtain a flat surface having an area of 100 to 250 mm2to beplaced in direct contact with the agar surface.10.4 Prepare specimens of rod or tubing or of rod- ortube-shaped devices as follows:10.4.1 Where the diameter is les
36、s than 6.4 mm, cut 5 to 15mm in length.10.4.2 Where the diameter is 6.4 to 15 mm, cut 2 to 8 mmin length.10.4.3 Where the diameter exceeds 15 mm, prepare crosssections as described in 10.3.10.5 Obtain specimens from larger medical items fromlocations with relatively large cross sections to expose in
37、teriormaterial.10.6 If a device is constructed of two or more materials thatare intended to contact body fluids or tissues, either cut the testspecimen from the materials interface or test separate speci-mens of each material or both.10.7 Prepare specimens for evaluating the cytotoxicity ofliquids o
38、r extracts by saturating a sterile filter disk and allowingthe excess liquid to drain off while maintaining asepsis. Use thesaturated filter disk as a test specimen.NOTE 5When ethylene oxide or other chemical sterilants are used,adequate aeration time to permit dissipation of residues which mayadver
39、sely affect the results recorded in this assay should be determined.NOTE 6In general, specimens should be cleaned to remove anyresidues from specimen preparation and sterilized after they have been cutto size. If the specimen is very hard (for example, ceramics), care shouldbe taken to remove the re
40、sidues that may be left on the freshly cut surfaceby the cutting tool. When evaluating the cytotoxic potential of medicalmaterials or devices that are contained in the final sterile package,resterilization, further processing, or delay between the time of openingthe package and starting the test mus
41、t be avoided. With small items, theentire content of the sterile package may be used as the test specimen.When the size of the sterile packaged item is too large, an appropriate,representative, small-sized specimen must be obtained. The application ofthis assay to items in the final sterile package
42、is limited to items that aresmall or can be cut and reshaped using aseptic technique.10.8 Absorbant materials tested in this method shall beprewetted with culture medium to prevent loss of water fromthe agar and subsequent cellular damage.11. Cell Culture Maintenance11.1 Use the following procedures
43、 to maintain the cells byserial subculture:11.2 Aspirate the medium from a 150-cm2cell culture flaskcontaining a near-confluent monolayer.11.3 Rinse the cells with a sufficient volume (for example, 5to 10 mL) of Hanks balanced salt solution to remove residualserum.11.4 Aspirate the rinse solution.11
44、.5 Add a sufficient volume of trypsin solution (0.1 %) tothe flask to cover the cell monolayer (approximately 5 mL).11.6 Incubate for 5 to 10 min to suspend the cells.11.7 Transfer the cell suspension to a centrifuge tube.11.8 Centrifuge at 1300 xg for 6 min.11.9 Discard the supernatant.11.10 Resusp
45、end the cells in 10 6 0.1 mL of fresh mediumand mix the suspension thoroughly.11.11 Distribute the cell suspension equally among each oftwo to eight 150-cm2tissue culture flasks.11.12 Add a sufficient volume of fresh medium so that eachflask will contain approximately 50 mL.11.13 Change the medium e
46、very two to three days until themonolayer is nearly confluent, then repeat Steps 11.2 11.12.12. Cell Layer Preparation12.1 Prepare confluent cell monolayers as follows:12.2 Follow Steps 11.1 11.9 to prepare a cell suspension.12.3 Add 2.0 6 0.1 mL of medium to each culture dish.12.4 Using a sterile 1
47、0-mL serological pipette, add five toseven drops of cell suspension to each dish. Rotate the dishesto ensure an even distribution of cells.12.5 Incubate until a near-confluent monolayer has formed,as observed by microscopic examination.NOTE 7The formation of a near-confluent monolayer usually requir
48、esthree to five days. By counting cells with a hemacytometer (to ensure theconcentration of the inoculum), the time required for monolayer formu-lation may be regulated. A cell concentration of 1.3 105cells/mL willgive a consistent time of 24 h.12.6 If the cell suspension remains unused after Step 1
49、2.3,asubculture may be prepared by adding 9 mL of fresh mediumto each millilitre of cell suspension in a cell culture flask witha surface area of approximately 3 cm2for each millilitre ofdiluted cells and incubating it until a near-confluent monolayerhas formed, as determined by microscopic examination.13. Test Procedure13.1 Perform the agar diffusion cytotoxicity assay as fol-lows:13.2 Microscopically examine the cell cultures and rejectany in which the cell monolayer is not of correct confluency orthe cells show signs of granulation or sloughi
