In vitro cytotoxicity tests. UNE-EN ISO 10993-5: 2009. Biological evaluation of medical devices, Part 5: In vitro cytotoxicity tests.
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This standard describes the procedure to detect in vitro cytotoxicity of medical devices. The test can be performed with an extract of the device or with the entire device in direct contact with the cells. With the entire device, an indirect application can also be made by diffusion in agarose or by diffusion through a filter.
The devices must be sterile, or they must be sterilized in the laboratory according to the manufacturer´s guidelines, as they must be contacted with sterile cell cultures. The manufacturer´s recommendations for the extraction solution should be provided in case the test should be performed with an extract of the device, and conditions that may degrade it to be avoided. If there is any preference for the use of a specific cell line, it should be indicated. The most commonly used cells available are Vero cells, BHK-21, WI-38, 3T3 cells, ... Unless the client indicates another choice, the test will be performed with Vero cells.
The test must be carried out in triplicate, and must include the corresponding positive and negative controls. The monolayers used will be in a state of partial confluence with 24 to 48 hours of culture and after exposure to the device in any of its forms, will be followed for 24 and 48 hours.
To evaluate the effect can be chosen between a qualitative or a quantitative method. In the qualitative method a microscopic exam of the cultures is performed giving a graduation of the effects (0 / 1 / 2 / 3 / 4), indicating grades 2 to 4 different intensity of product cytotoxicity (Annex B of the standard). For the quantitative method, the standard offers three alternatives: the reduction of uptake of neutral red dye (NRU: Neutral Red Uptake) (Annex A of the standard); the reduction of MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromid] leading to the formation of formazan (Annex C of the standard) or the reduction of XTT [2,3-bis] (2-methoxy-4-nitro-5-sulfophenyl) -5 - [(phenylamino) carbonyl] -2H-tetrazolium hydroxide] leading to the formation of formazan (Annex D of the standard). Any of the three alternatives, allow to calculate the cell death rate, which when it is higher than 30%, indicates cytotoxicity. In IVAMI, the method with neutral red is usually used.