ASTM F813-2007(2012) Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices《医疗器械材料直接接触细胞培养评价的标准实施规程》.pdf

《ASTM F813-2007(2012) Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices《医疗器械材料直接接触细胞培养评价的标准实施规程》.pdf》由会员分享，可在线阅读，更多相关《ASTM F813-2007(2012) Standard Practice for Direct Contact Cell Culture Evaluation of Materials for Medical Devices《医疗器械材料直接接触细胞培养评价的标准实施规程》.pdf（5页珍藏版）》请在麦多课文档分享上搜索。

1、Designation: F813 07 (Reapproved 2012)Standard Practice forDirect Contact Cell Culture Evaluation of Materials forMedical Devices1This standard is issued under the fixed designation F813; the number immediately following the designation indicates the year of originaladoption or, in the case of revis

2、ion, the year 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 practice covers a reference method of direct contactcell culture testing which may be used in evalua

3、ting thecytotoxic 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

4、 for theassessment 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 F748.1.5 The L-929 cell line was chosen because it has asignificant history of use

5、 in assays 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

6、evaluation 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 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in t

7、hisstandard.1.8 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-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2.

8、Referenced Documents2.1 ASTM Standards:2F619 Practice for Extraction of Medical PlasticsF748 Practice for Selecting Generic Biological Test Methodsfor Materials and DevicesF895 Test Method forAgar Diffusion Cell Culture Screeningfor CytotoxicityF1027 Practice for Assessment of Tissue and Cell Compat

9、-ibility of Orofacial Prosthetic Materials and Devices2.2 Other Documents:The American Type Culture Collection (ATCC), Catalogueof Strains II3USP Negative Control Plastic Reference Standard43. Summary of Practice3.1 Cell cultures are grown to a confluent monolayer inculture dishes. The growth medium

10、 is aspirated and replen-ished to provide a resting, confluent cell layer. Test and controlspecimens are placed in direct contact with the cell layer toprovide an accelerated assessment of the presence or absenceof a cytotoxic effect from a given material or device. SeePractice F1027 for definitions

11、.4. Significance and Use4.1 This practice is useful for assessing cytotoxic potentialboth when evaluating new materials or formulations forpossible use in medical applications, and as part of a qualitycontrol program for established medical materials and medicaldevices.4.2 This practice assumes that

12、 assessment of cytotoxicitypotential provides one method for predicting the potential forcytotoxic or necrotic reactions to medical materials and devicesduring clinical applications to humans. In general, cell culturetesting methods have shown good correlation with animalassays and are frequently mo

13、re sensitive to toxic moieties.1This 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 Oct. 1, 2012. Published November 2012. Originally

14、approved in 2001. Last previous edition approved in 2007 as F813 07. DOI:10.1520/F0813-07R12.2For 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

15、Summary page onthe ASTM website.3American Type Culture Collection, 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.Copyright ASTM International, 100 Barr Harbor Drive, PO

16、 Box C700, West Conshohocken, PA 19428-2959. United States14.3 This cell culture test method is suitable for adoption inspecifications and standards for 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,tissu

17、e fluids, or blood on a long-term basis. However, careshould be taken when testing materials that are resorbable to besure the method is applicable.4.4 Since cells in this direct contact test method are notprotected by an overlying agarose layer, they are more suscep-tible to potential mechanical da

18、mage imparted by the overlyingtest sample. Investigators wishing to evaluate the cytotoxicresponse of cells underlying the test sample should consideragarose-based methods similar to Test Method F895.Alternatively, depending on sample characteristics, extractionmethods such as Practice F619 may also

19、 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 greater 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 recom

20、mended because they provide a flatsurface that contributes to the formation of a uniform cell monolayer.5.4 Disposable, Sterile, Centrifuge Tubes.5.5 Inverted Optical Microscope, with magnifications of40, 100, and 200.5.6 Clinical Centrifuge, capable of attaining 1300xg.5.7 Filter Disks10 mm in diam

21、eter (for evaluation ofliquids).NOTE 2MilliporeAP2501000 filter disks have been found satisfactoryfor use in cytotoxicity evaluations because they elicit no cytopathic effect.Other filter disks that do not elicit a cytopathic effect may also be used.5.8 Water Bath, capable of maintaining a temperatu

22、re 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 clean room maybe necessary to prevent contamination of cultures.6. Reagents6.1 The following reagents shall be used:6.1.1 Minimum Essential Medium (MEM), prepar

23、ed withoutL-glutamine and augmented by the addition of Earles salts and510 % fetal bovine serum.NOTE 4Glutamine is omitted from this formulation 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 5Ope

24、ned 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 G10,000 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 contami

25、nation. 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 water.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, ph

26、osphate-buffered saline (store frozen).6.1.5 Water, distilled, deionized, and sterile, with a mini-mum resistivity of 1 Mcm.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.6.4 Rea

27、gents shall be stored in accordance with the manu-facturers directions unless otherwise indicated in 6.1.7. Cell Cultures7.1 Cell cultures 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 line

28、s may be considered. Cells should be tested periodi-cally for Mycoplasma contamination.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

29、 Reference Standard).8.2 Prepare positive control specimens in accordance withSection 10 from a material that consistently elicits apredictable, 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

30、sloughing. Take care to ensure that the preparation ishomogenous.8.2.2 Latex rubber has been used as a positive polymericcontrol for a zone of inhibition.9. General Technique9.1 Use the aseptic technique throughout this assay tominimize microbial contamination.NOTE 7Mouth pipetting should not be emp

31、loyed to transfer cells,medium, or reagents.9.2 Warm all solutions and materials to a temperature of 376 2C before placing in contact with 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

32、 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 devices in cross section toobtain a flat surface having an area of 100 to 250 mm2to beplaced in direct contact with the cell monolayer

33、.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 the diameter is 6.4 to 15 mm, cut 2 to 8 mmin length.F813 07 (2012)210.4.3 Where the diameter exceeds 15 mm, prepare cross-sections

34、 as described in 10.3.10.5 Obtain specimens from larger medical items fromlocations with relatively large cross sections in order to exposeinterior material.10.6 If a device is constructed of two or more materials, cuteither the test specimen from the materials interface or testseparate specimens fr

35、om each material.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 8When ethylene oxide or other chemical sterilan

36、ts are used,adequate aeration time should be allowed, to permit dissipation of residueswhich may adversely affect the results recorded in this assay.NOTE 9In general, specimens should be cleaned to remove anyresidues from specimen preparation, and sterilized after they have beencut to size. If the l

37、arge solid materials are very hard, like ceramics, whichrequire cutting with metal or diamond saws, care should be taken toremove any contamination from the metal blade or from the metal bondingthe diamonds to the blade. When evaluating the cytotoxicity potential ofmedical materials or devices that

38、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 the sterile package may be used as the test specimen.When the size of the sterile packaged item

39、 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 reshaped using aseptic technique.NOTE 10The size and shape of test specimens may vary considerab

40、ly,providing they physically fit into the culture dish. This test is intendedonly to provide a qualitative assessment of cytotoxicity potential with noquantitative measurement of cytotoxicity magnitude. Thus specimen sizeis not considered important.11. Preparation of Cell LayerPrepare confluent cell

41、 monolayers as follows:11.1 Aspirate the medium from a 150 cm2-cell culture flaskcontaining a near-confluent cell monolayer.11.2 Rinse the cells 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 mi

42、n 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 centrifuge 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 suspen

43、sion 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 suspension to each dish.11.11 Incubate until a near-confluent monolayer has formed,as observed by microscopic examination. During handling thecell culture dis

44、hes and during transport to and from theincubator, agitation of the medium shall be avoided.NOTE 11The formation of a near-confluent monolayer usually re-quires 3 to 5 days. By counting cells with a hemacytometer (to ensure theconcentration of the inoculum) the time required for monolayer formationm

45、ay be regulated. A cell concentration of 1.3 105cells/mL will give aconsistent time of 24 h. It should be recognized, however, that thisprocedure 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 fr

46、esh mediumto each millilitre of each 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.12. Test Procedure12.1 Perform the direct-contac

47、t cytotoxicity assay as fol-lows:12.2 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.12.3 Aspirate the medium from all the acceptable culturesand replace it with 1.5 to 2 mL of fresh

48、medium.12.4 Place a single test or control specimen in each dish indirect contact with the cell monolayer. Prepare triplicatecultures for each test material and both positive and negativecontrols. The materials should be added to the culture gentlyand agitation shall be avoided.NOTE 12Low-density ma

49、terials 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 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