1、Designation: F895 11Standard 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 of last revision.
2、 A number in parentheses indicates the year of last reapproval. A superscriptepsilon () indicates an editorial change since the last revision or reapproval.This standard has been approved for use by agencies of the Department of Defense.1. Scope1.1 This test method is appropriate for materials in a
3、varietyof shapes and for materials that are not necessarily sterile. 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
4、.1.1.1 This test method is not appropriate for leachables thatdo not 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 mechanic
5、al damage to the cells. This test methodwould not be appropriate for 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 char
6、acterized and readilyavailable, that has demonstrated reproducible 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 an
7、y, 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:2F748 Practice for Selecting Generic Biological Tes
8、t Meth-ods for Materials and Devices2.2 ATCC Document:American Type Culture 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 aga
9、r-containingmedium that is allowed to solidify. Test control articles 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 ce
10、lls at varying dis-tances from the test article. This method is well 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 te
11、st method is useful for assessing the cytotoxicpotential of new materials 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 potentia
12、lclinical applications in humans. Cell culture methods haveshown good 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 me
13、dical devices that are to be implanted intothe human body or placed in 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 co
14、nsidered safefor clinical use (Practice F748).5. Apparatus5.1 The following apparatus shall be used:1This test method is under the jurisdiction of ASTM Committee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.C
15、urrent edition approved Oct. 1, 2011. Published October 2011. Originallyapproved in 1984. Last previous edition approved in 2006 as F895 84 (2006).DOI: 10.1520/F0895-11.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annua
16、l Book of ASTMStandards volume information, refer to the standards Document 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, Rockvi
17、lle, MD.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5.2 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
18、 2C.5.4 Microscope, with inverted phase contrast optics andmagnifications of 40, 100, and 2003.5.5 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 recom
19、mended because they provide a flatsurface that promotes the formation of a uniform 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 2Millipore AP2501000 filter disks have been found s
20、atisfac-tory for use in cytotoxicity evaluations because they elicit no cytopathiceffect. Other filter disks that do not elicit a cytopathic effect may also beused.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 roo
21、mmay be necessary to prevent contamination of cultures.6. Reagents6.1 The following reagents shall be used:6.1.1 For Cell Culture Maintenance,13 Media. MinimumEssential Medium (MEM) is prepared by mixing 90-mLEagles MEM (with Earles salts, without L-glutamine), ad-justing the solution to pH of 7.15,
22、 and adding 5 to 10 mL offetal bovine serum, and 1-mL 1003 nonessential amino 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. Immediate
23、ly before use, 1 mL ofL-glutamine solution (see 6.1.3) is added to each 100 mL ofMEM.6.1.1.2 Antibiotics, such as penicillin G10 000 I.U./mL, andstreptomycin 10 000 I.U./mL, may be added to the medium toreduce the incidence of bacterial contamination. Use 1 mL ofantibiotic per 100-mL media. Care sha
24、ll be taken to ensure thatthe antibiotics do not have an adverse effect on the viability ofthe cell cultures.6.1.2 For Agar Media Overlay, to prepare 23 Media(100-mL final volume). Twice concentrated (23) MEM isprepared by mixing 20 mL of 103 Eagles MEM (with EarlesSalts without L-glutamine), 0.22-g
25、 sodium bicarbonate (buffer)and sterile distilled water to bring to 70 % volume (70 mL).Adjust the pH to 7.15. Add 20-mL fetal bovine serum and2-mL 1003 nonessential amino acid (L-glutamine). Bring tofinal volume (100 mL) with sterile distilled water. Filtersterilize the 23 media. Mix with equal amo
26、unts of sterilized3 % agar nobel to give the final concentration of the media as13.6.1.3 L-Glutamine Solution (Lyophilized), 29.2 mg/mL.Rehydrate 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
27、 % solution in Hanks balanced salt solu-tion or calcium- and magnesium-free, phosphate-buffered 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 b
28、e tissue-culture grade or equivalent.6.3 Reagents shall be reconstituted in accordance with 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-
29、929. Other suitable validated cell linesmay be considered.8. Control Materials8.1 Prepare negative 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 Prep
30、are positive control specimens in accordance withSection 10 from a material that consistently 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 rubbe
31、r).8.2.1 Use an aqueous solution of phenol to give a diffusereaction of cellular degeneration 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 r
32、ubber is a widely used control material that hasdemonstrated reproducible results in several 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
33、 solutions and materials to a temperature of 376 2C before being placed in contact with cells.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
34、culture history of the cells.9.6 Stock cultures should be periodically screened for my-coplasma contamination.10. Specimen Preparation10.1 Sterilize all specimens by a method appropriate to theend use of the device.F895 11210.2 Where a device is sufficiently small (see 10.3 and 10.4)to fit into the
35、culture dish leaving an adequate margin of cellsfor evaluation, use the entire device 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
36、 tubing or of rod- ortube-shaped devices as follows:10.4.1 Where the diameter is less 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
37、larger medical items fromlocations with relatively large cross sections to expose interiormaterial.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
38、 material or both.10.7 Prepare specimens for evaluating the cytotoxicity ofliquids or 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 sterila
39、nts are used,adequate aeration time should be determined to permit dissipation ofresidues which may adversely affect the results recorded in this assay.NOTE 6In general, specimens should be cleaned to remove anyresidues from specimen preparation and sterilized after they have been cutto size. If the
40、 specimen is very hard (for example, ceramics), care shouldbe taken to remove the residues 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 p
41、rocessing, or delay between the time of openingthe package and starting the test must 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 m
42、ust be obtained. The application ofthis assay to items in the final sterile package 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 subs
43、equent cellular damage.11. Cell Culture Maintenance11.1 Use the following procedures 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 Hank
44、s balanced salt solution to remove residualserum .11.4 Aspirate the rinse solution.11.5 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 centrifug
45、e tube.11.8 Centrifuge at 1300 xg for 6 min.11.9 Discard the supernatant.11.10 Resuspend 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 fres
46、h medium so that eachflask will contain approximately 50 mL.11.13 Change the medium every 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 s
47、uspension.12.3 Add 2.0 6 0.1 mL of medium to each culture dish.12.4 Using a sterile 10-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 mic
48、roscopic examination.NOTE 7The formation of a near-confluent monolayer usually requiresthree 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 3 105cells/mL wil
49、lgive a consistent time of 24 h.12.6 If the cell suspension remains unused after Step 12.3,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
