1、 + $% measurements of cell damage; measurements of cell growth; measurements of specific aspects of cellular metabolism. There are several means of producing results in each of these four categories. The investigator should be aware of the test categories and into which category a particular techniq
2、ue fits, in order that comparisons be able to be made with other results on similar devices or materials both at the intra- and interlaboratory level. Examples of quantitative test protocols are given in annexes. Guidance for the interpretation of the results is given in this part of ISO 10993. BS E
3、N ISO 10993-5:2009INTERNATIONAL STANDARD ISO 10993-5:2009(E) ISO 2009 All rights reserved 1Biological evaluation of medical devices Part 5: Tests for in vitro cytotoxicity 1 Scope This part of ISO 10993 describes test methods to assess the in vitro cytotoxicity of medical devices. These methods spec
4、ify the incubation of cultured cells in contact with a device and/or extracts of a device either directly or through diffusion. These methods are designed to determine the biological response of mammalian cells in vitro using appropriate biological parameters. 2 Normative references The following re
5、ferenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 10993-1, Biological evaluation of medical devices Part 1: Eva
6、luation and testing within a risk management system ISO 10993-12, Biological evaluation of medical devices Part 12: Sample preparation and reference materials 3 Terms and definitions For the purposes of this document, the terms and definitions given in ISO 10993-1 and the following apply. 3.1 cultur
7、e vessels vessels appropriate for cell culture including glass petri dishes, plastic culture flasks or plastic multiwells and microtitre plates NOTE These can be used interchangeably in these methods provided that they meet the requirements of tissue culture grade and are suitable for use with mamma
8、lian cells. 3.2 positive control material material which, when tested in accordance with this part of ISO 10993, provides a reproducible cytotoxic response BS EN ISO 10993-5:2009ISO 10993-5:2009(E) 2 ISO 2009 All rights reservedNOTE The purpose of the positive control is to demonstrate an appropriat
9、e test system response. For example, an organotin-stabilized polyurethane1)has been used as positive control for solid materials and extracts. Dilutions of phenol, for example, have been used as a positive control for extracts. In addition to a material, pure chemicals can also be used to demonstrat
10、e the performance of the test system. 3.3 blank extraction vehicle not containing the test sample, retained in a vessel identical to that which holds the test sample and subjected to conditions identical to those to which the test sample is subjected during its extraction NOTE The purpose of the bla
11、nk is to evaluate the possible confounding effects due to the extraction vessel, vehicle and extraction process. 3.4 negative control material material which, when tested in accordance with this part of ISO 10993, does not produce a cytotoxic response NOTE The purpose of the negative control is to d
12、emonstrate background response of the cells. For example, high-density polyethylene2)for synthetic polymers, and aluminium oxide ceramic rods for dental material have been used as negative controls. 3.5 test sample material, device, device portion, component, extract or portion thereof that is subje
13、cted to biological or chemical testing or evaluation 3.6 subconfluency approximately 80 % confluency, i.e. the end of the logarithmic phase of growth 4 Sample and control preparation 4.1 General The test shall be performed on a) an extract of the test sample and/or b) the test sample itself. Sample
14、preparation shall be in accordance with ISO 10993-12. Negative and positive controls shall be included in each assay. 1) The ZDEC and ZDBC polyurethanes are available from the Food and Drug Safety Center, Hatano Research Institute, Ochiai 729-5, Hadanoshi, Kanagawa 257, Japan. 2) High-density polyet
15、hylene can be obtained from the U.S. Pharmacopeia (Rockville, MD, USA) and from the Food and Drug Safety Center, Hatano Research Institute (Ochiai 729-5, Hadanoshi, Kanagawa 257, Japan). The information given in 1) and 2) is for the convenience of the user of this part of ISO 10993 and does not cons
16、titute an endorsement by ISO of these products. Equivalent products may be used if they can be shown to lead to the same results. BS EN ISO 10993-5:2009ISO 10993-5:2009(E) ISO 2009 All rights reserved 34.2 Preparation of liquid extracts of material 4.2.1 Principles of extraction Extracting condition
17、s should attempt to simulate or exaggerate the clinical use conditions so as to determine the potential toxicological hazard without causing significant changes in the test sample, such as fusion, melting or any alteration of the chemical structure, unless this is expected during clinical applicatio
18、n. Due to the nature of certain materials (e.g. biodegradable materials), alteration of the chemical structure can occur during the extraction procedure. NOTE The concentration of any endogenous or extraneous substances in the extract, and hence the amount exposed to the test cells, depends on the i
19、nterfacial area, the extraction volume, pH, chemical solubility, diffusion rate, osmolarity, agitation, temperature, time and other factors. For devices that involve mixing two or more components in the patient to arrive at the final device (for example bone cement), the final device should not be w
20、ashed prior to extraction. Washing the test sample can reduce or remove residuals present on the device. If the test sample is to be used in a sterile environment, a sterilized test sample should be used to extract chemical constituents. 4.2.2 Extraction vehicle The choice of the extraction vehicle(
21、s) taking into account the chemical characteristics of the test sample shall be justified and documented. For mammalian cell assays one or more of the following vehicles shall be used: a) culture medium with serum; b) physiological saline solution; c) other suitable vehicle. The choice of vehicle sh
22、ould reflect the aim of the extraction. Consideration shall be given to the use of both a polar and a non-polar vehicle. Culture medium with serum is the preferred extraction vehicle. The use of culture medium with serum is preferred for extraction because of its ability to support cellular growth a
23、s well as extract both polar and non-polar substances. In addition to culture medium with serum, use of medium without serum should be considered in order to specifically extract polar substances (e.g. ionic compounds). Other suitable vehicles include purified water and dimethyl sulfoxide (DMSO). DM
24、SO is cytotoxic in selected assay systems at greater than 0,5 % (volume fraction). The cellular exposure concentration of extractables in DMSO will be lower due to the greater dilution as compared to extraction in culture medium with serum. NOTE 1 Different types of serum (e.g. foetal, bovine/calf s
25、erum, newborn calf serum) might be used and the choice of the serum is dependent on the cell type. NOTE 2 It is important to recognise that serum/proteins are known to bind, to some extent, extractables. 4.2.3 Extraction conditions 4.2.3.1 The extraction shall be performed in sterile, chemically ine
26、rt, closed containers by using aseptic techniques, in accordance with ISO 10993-12. 4.2.3.2 With the exception of circumstances given below, the extraction shall be conducted under one of the following conditions and shall be applied according to the device characteristics and specific conditions fo
27、r use: a) (24 r 2) h at (37 r 1) C; b) (72 r 2) h at (50 r 2) C; c) (24 r 2) h at (70 r 2) C; d) (1 r 0,2) h at (121 r 2) C. BS EN ISO 10993-5:2009ISO 10993-5:2009(E) 4 ISO 2009 All rights reservedExtraction conditions described above, which have been used to provide a measure of the hazard potentia
28、l for risk estimation of the device or material, are based on historical precedent. Other conditions, e.g. prolonged or shortened extraction times at 37 C, which simulate the extraction that occurs during clinical use or provide an adequate measure of the hazard potential, may be used, but shall be
29、justified and documented. For medical devices that are in short-term contact (no greater than 4 h cumulative contact duration) with intact skin or mucosa and that are not implanted, this may include extraction times of less than 24 h but no less than 4 h, as given in a) to c). Cell culture medium wi
30、th serum should only be used in accordance with a) because extraction temperatures greater than (37 r 1) C can adversely impact chemistry and/or stability of the serum and other constituents in the culture medium. For polymeric test samples, the extraction temperature should not exceed the glass tra
31、nsition temperature as the higher temperature can change the extractant composition. 4.2.3.3 If the extract is filtered, centrifuged or processed by other methods prior to being applied to the cells, these details shall be recorded in the final report along with a rationale for the additional steps
32、(see Clause 9). Any pH adjustment of the extract shall be reported. Manipulation of the extract, such as by pH adjustment, should be avoided because it could influence the result. 4.3 Preparation of material for direct-contact tests 4.3.1 Form of test samples Materials that have various shapes, size
33、s or physical states (i.e. liquid, gels, solids, etc.) may be tested without modification in the cytotoxicity assays. The preferred test sample of a solid material should have at least one flat surface. If not, adjustments shall be made to achieve flat surfaces. 4.3.2 Sterility of test samples 4.3.2
34、.1 Sterility of the test sample shall be taken into account. 4.3.2.2 Test samples from sterilized devices shall be handled aseptically throughout the test procedure. 4.3.2.3 Test samples from devices that are normally supplied non-sterile but are sterilized before use shall be sterilized by the meth
35、od recommended by the manufacturer and handled aseptically throughout the test procedure. The effect of sterilization methods or agents on the device should be considered in defining the preparation of the test sample prior to use in the test system. 4.3.2.4 Test samples from devices not required to
36、 be sterile in use shall be used as supplied and handled aseptically throughout the test procedure. It may be justifiable to sterilize the test material in order to avoid microbial contamination of the cell culture; however, the sterilization process shall not alter the properties of the test materi
37、al. If non-sterile test samples are used, they should be checked for bacterial contamination because the contamination can lead to a false assessment of cytotoxicity. 4.3.3 Liquid test samples Liquid test samples shall be tested by either a) direct deposition or b) deposition on a biologically inert
38、 absorbent matrix. Filter discs have been found to be suitable for use as inert absorbent matrices. BS EN ISO 10993-5:2009ISO 10993-5:2009(E) ISO 2009 All rights reserved 54.3.4 Absorbent test samples If appropriate, test samples that are absorbent shall be soaked with culture medium prior to testin
39、g to prevent adsorption of the culture medium in the testing vessel. 4.4 Preparation of controls Controls should be selected so that they can be prepared by the same procedure as the test sample. 5 Cell lines Established cell lines are preferred and where used shall be obtained from recognised repos
40、itories3). Where specific sensitivity is required, primary cell cultures, cell lines and organotypic cultures obtained directly from living tissues shall only be used if reproducibility and accuracy of the response can be demonstrated. If a stock culture of a cell line is stored, storage shall be at
41、 80 C or below in the corresponding culture medium but containing a cryoprotectant, e.g. dimethylsulfoxide or glycerol. Long-term storage (several months up to many years) is only possible at 130 C or below. Only cells free from mycoplasma shall be used for the test. Before use, stock cultures shoul
42、d be tested for the absence of mycoplasma. It is important to check cells regularly (e.g. morphology, doubling time, modal chromosome number) because sensitivity in tests can vary with passage number. Good cell culture practices should be used. See Reference 5. 6 Culture medium The culture medium sh
43、all be sterile. The culture medium with or without serum shall meet the growth requirements of the selected cell line. Antibiotics may be included in the medium provided that they do not adversely affect the assays. Storage conditions shall be validated. NOTE The stability of the culture medium vari
44、es with the composition and storage conditions. The culture medium shall be maintained at a pH of between 7,2 and 7,4. 3) For example, cell lines American Type Culture Collection CCL 1 (NCTC clone 929), CCL 163 (Balb/3T3 clone A31), CCL 171 (MRC-5) and CCL 75 (WI-38), CCL 81 (Vero) and CCL 10 BHK-21
45、 (C-13) and V-79 379A are endorsed by ISO experts to be suitable. This information is given for the convenience of the user of this part of ISO 10993 and does not constitute an endorsement by ISO of the products named. Other cell lines may be used if they can be shown to lead to the same or more rel
46、evant results. BS EN ISO 10993-5:2009ISO 10993-5:2009(E) 6 ISO 2009 All rights reserved7 Preparation of cell stock culture Using the chosen cell line and culture medium, prepare sufficient cells to complete the test. If the cells are to be grown from cultures taken from storage, remove the cryoprote
47、ctant, if present. Subculture the cells at least once before use. When subculturing cells, remove and resuspend the cells by enzymatic and/or mechanical disaggregation using a method appropriate for the cell line. 8 Test procedures 8.1 Number of replicates A minimum of three replicates shall be used
48、 for test samples and controls. 8.2 Test on extracts 8.2.1 This test allows both qualitative and quantitative assessment of cytotoxicity. 8.2.2 Pipette an aliquot of the continuously stirred cell suspension into each of a sufficient number of vessels for exposure to the extracts. Distribute the cell
49、s evenly over the surface of each vessel by gentle rotation. 8.2.3 Incubate the cultures at (37 r 1) C in air with or without carbon dioxide as appropriate for the buffer system chosen for the culture medium. The test should be performed on a subconfluent monolayer or on freshly suspended cells. In the colony-forming assay only an appropriate low cell density shall be used. 8.2.4 Verify the subconfluency and the morphology of the cultures with a microscope before starting the test. In exceptional cases, exponenti
