ASTM F813-2007 Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices《直接接触细胞培养评估医疗器械材料的标准实施规程》.pdf

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1、Designation: F 813 07Standard Practice forDirect Contact Cell Culture Evaluation of Materials forMedical Devices1This standard is issued under the fixed designation F 813; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of

2、 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.1. Scope1.1 This practice covers a reference method of direct contactcell culture testing which may be used in evaluating thecyt

3、otoxic potential of materials for use in the construction ofmedical materials and devices.1.2 This practice may be used either directly to evaluatematerials or as a reference against which other cytotoxicity testmethods may be compared.1.3 This is one of a series of reference test methods for theass

4、essment of cytotoxic potential, employing different tech-niques.1.4 Assessment of cytotoxicity is one of several testsemployed in determining the biological response to a material,as recommended in Practice F 748.1.5 The L-929 cell line was chosen because it has asignificant history of use in assays

5、 of this type. This is notintended to imply that its use is preferred; only that the L-929is a well-characterized, readily available, established cell linethat has demonstrated reproducible results in several laborato-ries.1.6 Since the test sample is not removed at the time ofmicroscopic evaluation

6、 and underlying cells may be affectedby the specific gravity of the test sample, this practice is limitedto evaluation of cells outside the perimeter of the overlying testsample.1.7 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is therespons

7、ibility 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:2F 619 Practice for Extraction of Medical PlasticsF 748 Practice for Selecting Generic Biologic

8、al Test Meth-ods for Materials and DevicesF 895 Test Method for Agar Diffusion Cell Culture Screen-ing for CytotoxicityF 1027 Practice for Assessment of Tissue and Cell Compat-ibility of Orofacial Prosthetic Materials and Devices2.2 Other Documents:The American Type Culture Collection (ATCC), Catalo

9、gueof Strains II3USP Negative Control Plastic Reference Standard43. Summary of Practice3.1 Cell cultures are grown to a confluent monolayer inculture dishes. The growth medium is aspirated and replen-ished to provide a resting, confluent cell layer. Test and controlspecimens are placed in direct con

10、tact with the cell layer toprovide an accelerated assessment of the presence or absenceof a cytotoxic effect from a given material or device. SeePractice F 1027 for definitions.4. Significance and Use4.1 This practice is useful for assessing cytotoxic potentialboth when evaluating new materials or f

11、ormulations forpossible use in medical applications, and as part of a qualitycontrol program for established medical materials and medicaldevices.4.2 This practice assumes that assessment of cytotoxicitypotential provides one method for predicting the potential forcytotoxic or necrotic reactions to

12、medical materials and devicesduring clinical applications to humans. In general, cell culturetesting methods have shown good correlation with animalassays and are frequently more sensitive to toxic moieties.4.3 This cell culture test method is suitable for adoption inspecifications and standards for

13、 materials for use in theconstruction of medical devices that are intended to be im-planted in the human body or placed in contact with tissue,tissue fluids, or blood on a long-term basis. However, careshould be taken when testing materials that are resorbable to besure the method is applicable.1Thi

14、s practice is under the jurisdiction ofASTM Committee F04 on Medical andSurgical Materials and Devices and is the direct responsibility of SubcommitteeF04.16 on Biocompatibility Test Methods.Current edition approved Feb. 1, 2007. Published March 2007. Originallyapproved in 2001. Last previous editio

15、n approved in 2001 as F 813 01.2For 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.3American Type Culture Collect

16、ion, P.O. Box 1549, Manassas, VA 201084U.S. Pharmacopeia, Vol 24, Rand McNally, Taunton, MA, 1994, pp.16521653. Use latest publication to ensure current cumulative revisions are used.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.4.

17、4 Since cells in this direct contact test method are notprotected by an overlying agarose layer, they are more suscep-tible to potential mechanical damage imparted by the overlyingtest sample. Investigators wishing to evaluate the cytotoxicresponse of cells underlying the test sample should consider

18、agarose-based methods similar to Test Method F 895. Alterna-tively, dependent on sample characteristics, extraction methodssuch as Practice F 619 may also be considered.5. Apparatus5.1 The following apparatus shall be used:5.2 Incubator, to maintain a temperature of 37 6 2C and 4to 6 % CO2with great

19、er than 90 % relative humidity.5.3 Tissue Culture Grade Culture Dishes, that are sterile and35 mm in diameter by 10 mm deep.NOTE 1Plastic dishes are recommended because they provide a flatsurface that contributes to the formation of a uniform cell monolayer.5.4 Disposable, Sterile, Centrifuge Tubes.

20、5.5 Inverted Optical Microscope, with magnifications of403, 1003, and 2003.5.6 Clinical Centrifuge, capable of attaining 1300xg.5.7 Filter Disks10 mm in diameter (for evaluation ofliquids).NOTE 2Millipore AP2501000 filter disks have been found satisfac-tory for use in cytotoxicity evaluations becaus

21、e they elicit no cytopathiceffect. Other filter disks that do not elicit a cytopathic effect may also beused.5.8 Water Bath, capable of maintaining a temperature of 376 2C.NOTE 3A laminar flow work area capable of filtering out 99.99 % ofall particles greater than 0.5 m in diameter, or a class 100 c

22、lean room maybe necessary to prevent contamination of cultures.6. Reagents6.1 The following reagents shall be used:6.1.1 Minimum Essential Medium (MEM), prepared with-out L-glutamine and augmented by the addition of Earles saltsand 510 % fetal bovine serum.NOTE 4Glutamine is omitted from this formul

23、ation in order tomaximize the shelf life of the medium. Immediately before use, 5 mL ofL-glutamine solution (see 6.1.2) are added to each 500 mL of MEM.NOTE 5Opened containers of MEM may be stored at a temperature of2 to 8C for periods of not more than one week.NOTE 6Antibiotics, such as penicillin

24、G 10,000 I.U./ml and strepto-mycin 10,000 I.U./ml, may be added to the medium (1 ml of antibiotic per100 ml of media) to reduce the incidence of bacterial contamination. Thismay, however, have an adverse effect on the viability of the cell cultures.6.1.2 L-glutamine Solution, 29.2 mg/mL of sterile w

25、ater.6.1.3 Hanks Solution, calcium-and magnesium-free (storeat room temperature).6.1.4 Trypsin, 0.1 % solution in Hanks solution or calcium-and magnesium-free, phosphate-buffered saline (store frozen).6.1.5 Water, distilled, deionized, and sterile, with a mini-mum resistivity of 1 MVcm.6.2 All reage

26、nts shall be tissue-culture grade or equivalent.6.3 Reagents shall be reconstituted in accordance with themanufacturers directions, using aseptic technique.6.4 Reagents shall be stored in accordance with the manu-facturers directions unless otherwise indicated in 6.1.7. Cell Cultures7.1 Cell culture

27、s used in this assay should be the ATCC,CCL 1 NCTC clone 929 strain (clone of Strain L, mouseconnective tissue) designated L-929. Other suitable validatedcell lines may be considered. Cells should be tested periodi-cally for Mycoplasma contamination.8. Control Materials8.1 Prepare negative control s

28、pecimens 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 consistently elicits a pred

29、ict-able, moderate degree of cytotoxicity.8.2.1 Use aqueous phenol (0.45 6 0.05 % by volume) as apositive control for a diffuse reaction of cellular degenerationand sloughing. Take care to ensure that the preparation ishomogenous.8.2.2 Latex rubber has been used as a positive polymericcontrol for a

30、zone of inhibition.9. General Technique9.1 Use the aseptic technique throughout this assay tominimize microbial contamination.NOTE 7Mouth pipetting should not be employed to transfer cells,medium, or reagents.9.2 Warm all solutions and materials to a temperature of 376 2C before placing in contact w

31、ith cells.10. Preparation of Specimens10.1 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 device as a specimen.10.3

32、 Cut large solid materials and devices in cross-sectionto obtain a flat surface having an area of 100 to 250 mm2to beplaced in direct contact with the cell monolayer.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

33、 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 cross-sections as described in 10.3.10.5 Obtain specimens from larger medical items fromlocations with relatively large cross-sections in order to exposeinterior ma

34、terial.10.6 If a device is constructed of two or more materials, cuteither the test specimen from the materials interface or testseparate specimens from each material.10.7 Prepare specimens for evaluating the cytotoxicity ofliquids or extracts by saturating a sterile filter disk and allowingF813072t

35、he excess liquid to drain off while maintaining asepsis. Use thesaturated filter disk as a test specimen.NOTE 8When ethylene oxide or other chemical sterilants are used,adequate aeration time should be allowed, to permit dissipation of residueswhich may adversely affect the results recorded in this

36、assay.NOTE 9In general, specimens should be cleaned to remove anyresidues from specimen preparation, and sterilized after they have beencut to size. If the large solid materials are very hard, like ceramics, whichrequire cutting with metal or diamond saws, care should be taken toremove any contamina

37、tion from the metal blade or from the metal bondingthe diamonds to the blade. When evaluating the cytotoxicity potential ofmedical materials or devices that are contained in the final sterile package,resterilization, further processing, or delay between the time of openingthe package and starting th

38、e 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 must be obtained. The application ofthis assay to items in the final sterile

39、 package is limited to items that aresmall or can be cut and reshaped using aseptic technique.NOTE 10The size and shape of test specimens may vary considerably,providing they physically fit into the culture dish. This test is intendedonly to provide a qualitative assessment of cytotoxicity potential

40、 with noquantitative measurement of cytotoxicity magnitude. Thus specimen sizeis not considered important.11. Preparation of Cell LayerPrepare confluent cell monolayers as follows:11.1 Aspirate the medium from a 150 cm2-cell culture flaskcontaining a near-confluent cell monolayer.11.2 Rinse the cell

41、s with 5 6 0.5 mL of Hanks solution.11.3 Aspirate the rinse solution.11.4 Add 5 6 0.5 mL of trypsin solution (0.1 %) to theflask.11.5 Incubate for 5 to 10 min to suspend the cells.11.6 Dilute the suspension sufficiently to reduce the effect ofthe trypsin or:11.7 Transfer the cell suspension to a cen

42、trifuge tube andcentrifuge at 1300 g for 6 min and discard the supernatant.11.8 Dilute or suspend the cells in 10 6 0.1 mL of freshmedium, and mix the suspension thoroughly.11.9 Add 2.0 6 0.1 mL of medium to each culture dish.11.10 Using a sterile 10-mL serological pipette, add 5 to 7drops of cell s

43、uspension to each dish.11.11 Incubate until a near-confluent monolayer has formed,as observed by microscopic examination. During handling thecell culture dishes and during transport to and from theincubator, agitation of the medium shall be avoided.NOTE 11The formation of a near-confluent monolayer

44、usually re-quires 3 to 5 days. By counting cells with a hemacytometer (to ensure theconcentration of the inoculum) the time required for monolayer formationmay be regulated. A cell concentration of 1.3 3 105cells/mL will give aconsistent time of 24 h. It should be recognized, however, that thisproce

45、dure may increase the risk of bacterial contamination.11.12 If cell suspension remains unused after step 11.10,asubculture may be prepared by adding 9 mL of fresh mediumto each millilitre of each suspension in a cell culture flask witha surface area of approximately 3 cm2for each millilitre ofdilute

46、d cells and incubating it until a near-confluent monolayerhas formed, as determined by microscopic examination.12. Test Procedure12.1 Perform the direct-contact cytotoxicity assay as fol-lows:12.2 Microscopically examine the cell cultures and rejectany in which the cell monolayer is not of correct c

47、onfluency orthe cells show signs of granulation or sloughing.12.3 Aspirate the medium from all the acceptable culturesand replace it with 1.5 to 2 mL of fresh medium.12.4 Place a single test or control specimen in each dish indirect contact with the cell monolayer. Prepare triplicatecultures for eac

48、h test material and both positive and negativecontrols. The materials should be added to the culture gentlyand agitation shall be avoided.NOTE 12Low-density materials that tend to float in the medium maybe held in contact with the cell monolayers by placing a piece of negativecontrol on top to weigh

49、 them down.12.5 Incubate all cultures for 24 6 1h.12.6 Examine each culture microscopically.NOTE 13The use of an histologic stain such as 2 % crystal violet in20 % ethanol is helpful in assessing the cell cultures.12.7 Quantitative or semi-quantitative assays such as MTT,LDH, and neutral red may be considered if validated againstthe evaluation of results described in section 13.13. Evaluation of Results13.1 A cell culture shall be deemed to show a cytotoxiceffect if microscopic examination (see 12.6) reveals the fol-lowing:13.1.1 Malformation, degeneration