1、Designation: F 895 84 (Reapproved 2006)Standard Test Method forAgar Diffusion Cell Culture Screening for Cytotoxicity1This standard is issued under the fixed designation F 895; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the ye
2、ar 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.This standard has been approved for use by agencies of the Department of Defense.1. Scope1.1 This test method is appropriat
3、e for materials in a 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 gro
4、wth could be examined.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 diffusio
5、n orto cause mechanical 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
6、 cell line, well characterized and readilyavailable, that has demonstrated reproducible results in severallaboratories.1.3 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-pri
7、ate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2F 748 Practice for Selecting Generic Biological Test Meth-ods for Materials and Devices2.2 ATCC Document:American Type Culture Collection, (ATCC) Catalog
8、ue 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 areplaced on the agar surface to
9、 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 suited forlow-density materials (f
10、ilm, 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 materials and formulations and as part
11、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 correlation with animal assays an
12、d 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 in contact with tissue fluids or bl
13、oodon 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 F 748).1This test method is under the jurisdiction of ASTM Comm
14、ittee F04 on Medicaland Surgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved March 1, 2006. Published April 2006. Originallyapproved in 1984. Last previous edition approved in 2001 as F 895 84 (2001)e1.2For re
15、ferenced 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.3Fourth edition, 1983, is available from American Type Culture Colle
16、ction,12031 Parklawn Dr., Rockville, MD 10892. Library of Congress No. 76-640122.4U.S. Pharmacopeia, current edition, Rockville, MD.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5. Apparatus5.1 The following apparatus shall be used
17、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 2C.5.4 Microscope, with inverted phase contrast optics andmagnifications of 40, 100, and 2003.5.5 Clinical Centrifuge, ca
18、pable 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 uniform monolayer of cells.5.8 Sterile, Disposa
19、ble, 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 satisfac-tory for use in cytotoxicity evaluations because they elicit no cytopathiceffect. Other filter disks that do not e
20、licit 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 roommay be necessary to prevent contamination of cultures.6. Reagents6.1 The following reagents shall be used:6.1.1 For Cell
21、Culture Maintenance,13 Media. MinimumEssential Medium (MEM) is prepared by mixing 90-mLEagles MEM (with Earles salts, without L-glutamine), adjustsolution to pH 7.15, add 5- to 10-mL fetal bovine serum, and1-mL 1003 nonessential amino acids (L-glutamine).6.1.1.1 Opened containers of prepared MEM may
22、 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 mL ofMEM.6.1.1.2 Antibiotics, such as penicillin G10 000 I.U./mL,
23、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 shall 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
24、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 sodium bicarbonate (buffer)and sterile distilled water to bring to 70 % volume (70 mL).Adjust pH to 7.15. Add 20-mL fetal bovine serum and 2-
25、mL1003 nonessential amino acid (L-glutamine). Bring to finalvolume (100 mL) with sterile distilled water. Filter sterilize the23 media. Mix with equal amounts of sterilized 3 % agarnobel to give the final concentration of the media as 13.6.1.3 L-Glutamine Solution (Lyophilized), 29.2 mg/mL.Rehydrate
26、 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-buffered sa-line (store frozen).6.1.6 Water, sterile, deio
27、nized, 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 themanufacturers directions, using aseptic technique.
28、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 negative control specimens in accordance withSection 10
29、 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 consistently elicits a moderateand reproducible degree of cytoto
30、xicity (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 degeneration and sloughing; a latex rubberwill give a zone of to
31、xicity.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 several laboratories.9. General Technique9.1 Use aseptic tec
32、hnique 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 material to a temperature of 376 2C before being placed in contact with cells.9.3 Wash all glass vessels thoroughly with a cleanin
33、gsolution 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-coplasma contamination.10. Specimen Preparation10.1 Sterili
34、ze all specimens by a method appropriate to theend use of the device.F 895 84 (2006)210.2 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 device as a specimen.10.3 Cut large solid materials and dev
35、ices 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 less than 6.4 mm, cut 5 to 15mm in length.10.4.2 Where th
36、e 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 interiormaterial.10.6 If a device is constructed of two
37、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 or extracts by saturating a sterile filter disk and all
38、owingthe 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 should be determined to permit dissipation ofresidues which may adversely affect the results recorded
39、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 specimen is very hard (for example, ceramics), care shouldbe taken to remove the residues that may be left on the freshly cut surfaceby t
40、he 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 must be avoided. With small items, theentire content of t
41、he 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 is limited to items that aresmall or can be cut and re
42、shaped using aseptic technique.10.8 Absorbant materials tested in this method must beprewetted with culture medium to prevent loss of water fromthe agar and subsequent cellular damage.11. Cell Culture Maintenance11.1 Use the following procedures to maintain the cells byserial subculture:11.2 Aspirat
43、e the medium from a 150-cm2cell culture flaskcontaining a near-confluent monolayer.11.3 Rinse the cells with a sufficient volume of Hanksbalanced salt solution to remove residual serum (for example,5 to 10 mL).11.4 Aspirate the rinse solution.11.5 Add a sufficient volume of trypsin solution (0.1 %)
44、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 Resuspend the cells in 10 6 0.1 mL of fresh mediumand mix the
45、 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 every two to three days until themonolayer is nearly con
46、fluent, 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 10-mL serological pipette, add five toseven drops of cel
47、l suspension to each dish. Rotate the dishesto ensure 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 requiresthree to five days. By counting cells with a hemacytomet
48、er (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 willgive a consistent time of 24 h.12.6 If cell suspension remains unused after Step 12.3,asubculture may be prepared by adding 9 mL of fresh medi
49、umto 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 sloughing.13.3 Autoclave 3 % Nobel Agar for 15 min at 121C.13.4 Place the autoclave
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