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Delivery: <= 4 days. True-PDF full-copy in English will be manually translated and delivered via email. GB/T 41915-2022: Nanotechnologies - In vitro MTS assay for measuring the cytotoxic effect of nanoparticles Status: Valid
Basic dataStandard ID: GB/T 41915-2022 (GB/T41915-2022)Description (Translated English): Nanotechnologies - In vitro MTS assay for measuring the cytotoxic effect of nanoparticles Sector / Industry: National Standard (Recommended) Classification of Chinese Standard: C04 Classification of International Standard: 07.120 Word Count Estimation: 34,336 Date of Issue: 2022-10-14 Date of Implementation: 2023-05-01 Issuing agency(ies): State Administration for Market Regulation, China National Standardization Administration GB/T 41915-2022: Nanotechnologies - In vitro MTS assay for measuring the cytotoxic effect of nanoparticles---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order. Nanotechnologies -- In vitro MTS assay for measuring the cytotoxic effect of nanoparticles ICS 07.120 CCSC04 National Standards of People's Republic of China Nanotechnology MTS method for determination of nanometers Cytotoxicity of particles (ISO 19007.2018, IDT) Published on 2022-10-12 2023-05-01 Implementation State Administration for Market Regulation Released by the National Standardization Administration directory Preface III Introduction IV 1 Scope 1 2 Normative references 1 3 Terms and Definitions 1 4 Symbols and Abbreviations 2 5 Material 2 5.1 Cell Lines 2 5.2 Detection 3 5.3 Control 3 6 Instrument 3 7 Preparation of Nanoparticle Test Samples 3 8 Preparation 4 8.1 Overview 4 8.2 Medium 4 8.3 Preparation of Cell Stock 4 8.4 Validation of cell growth status 4 8.5 Verifying Reader Consistency 5 8.6 Preparing the Controlled Experiment 5 8.6.1 Description of Controlled Experiment 5 8.6.2 Preparation of CdSO4 stock solution (10mmol/L) 5 8.6.3 Preparation of Nanoparticle Control Suspension 5 8.7 Precise dispensing 6 9 Characterizing the effect of nanoparticles on cell viability 6 9.1 Overview 6 9.2 Preparing cell culture plates 6 9.3 Preparing the Nanoparticle Loading Plate 7 9.4 Exposure of cells to nanoparticles in cell culture medium 9 9.5 Treatment of cells with MTS reagent 9 9.6 Measuring the absorbance of formazan 9 10 Cell Viability Analysis9 11 Interpretation of test results 10 Appendix A (normative) Alternative cell lines and assays11 Appendix B (informative) Case. MTS method based on A549 cell line (EMPA-NIST draft) 12 Appendix C (informative) Case. MTS method based on Raw264.7 cell line (IANH draft) 19 Reference 24 forewordThis document is in accordance with the provisions of GB/T 1.1-2020 "Guidelines for Standardization Work Part 1.Structure and Drafting Rules of Standardization Documents" drafted. This document is equivalent to ISO 19007.2018 "Determination of cytotoxicity of nanoparticles by nanotechnology MTS method". The following minimal editorial changes have been made to this document. --- Modified the error numbers of the original texts B.2.2.3 and C.2.1.2.3 (see Appendix B and Appendix C); --- Adjust some of the contents in C.2.1.2.1 and C.2.1.2.3.12 to "Note"; --- References reordered. Please note that some content of this document may be patented. The issuing agency of this document assumes no responsibility for identifying patents. This document is proposed by the Chinese Academy of Sciences. This document is under the jurisdiction of the National Nanotechnology Standardization Technical Committee (SAC/TC279). This document is drafted by. National Nanoscience Center, Shanghai University of Science and Technology, Jinshi Packaging (Jiaxing) Co., Ltd., China Food and Drug Administration Research Institute. The main drafters of this document. Wu Xiaochun, Ji Yinglu, Li Yan, Zhao Guotu, Qing Qianzhong, Li Haiyun, Wen Hairuo, Fan Huizhen.IntroductionWith the continuous development of new materials, products and applications, the field of nanotechnology continues to develop rapidly. At the same time, some of the Concerns about potential risks to human health and the environment posed by rice-based materials are also on the rise. At present, a large number of related researches are being carried out internationally research to better understand and quantify these potential risks. In addition, the surface coating of nanoparticles during processing or in the final product Chemicals may also affect the interaction between nanoparticles and cells. Especially considering the large specific surface area of nanoparticles, this The impact may be more pronounced. Cells are the basic unit of life. Monitoring the biological response of model cells following nanoparticle exposure promises to deepen our understanding of the role of nanoparticles patterns”, and to determine which of these factors may require further study for subsequent risk assessment. In.2008, some international research teams found that some published nanomaterial toxicity research results could not be replicated in different laboratories. Therefore, there is a need to develop accurate and reproducible nanotoxicological testing methods. International Consortium for Nano Environmental Health and Safety (NanoEHS) Coordination (IANH) came into being to develop test protocols that can accurately assess the toxicity of nanoparticles and their effects on cells, and these test methods The case can be repeated in any laboratory. IANH has passed several common cytotoxicity tests to determine the particle size distribution in liquid suspension and nanoparticle size distribution. The in vitro interaction of rice materials with cells was compared in experiments (Appendix A). The alliance identified a number of factors that would increase data volatility elements, and developed techniques to reduce data volatility. National Institute of Environmental Health Sciences (NIEHS) NanoGo)-funded research further evaluated some of the testing protocols, especially 3-(4,5-dimethyl-2-thiazolyl)-5-(3-carboxymethoxy Detection scheme for phenyl)-2-(4-sulfophenyl)-2H-tetrazole (MTS) [4]. A third team extended IANH's approach and adopted a unified design The multi-well plate of the meter was used for experiments, which improved the consistency of the analysis results (Appendix B) [5]. Of particular importance is the fact that the above projects are based on multiple real Interlaboratory testing to identify sources of uncertainty and improve test methods. This document uses the MTS method to assess the viability of cells in vitro [6]. The method utilizes the formazan (absorbing The peak is located at 490 nm) to produce a color reaction. Typically, the change in absorbance is proportional to the number of cells. However, during the detection process Changed reductase activity or insufficient reagents may also lead to color reaction, resulting in inaccuracy between absorbance and cell viability (such as cell number). than relationship. Adding the MTS reagent directly to the cell culture plate allows a rapid assessment of the potential endotoxicity of nanoparticles. due to nanoparticles Particles may potentially interfere with colorimetric analysis, so before giving final test results, it is not advisable to conduct a control experiment of the interaction between the two. very important. Direct observation of treated cells under the microscope is an orthogonal method for validating MTS assay results. The regulations set forth in this document The normalized protocol is only used for adherent cells and can also be used for suspension cells after modification. Toxicity testing of nanoparticle effects on individual cell lines in this method is a primary test. The standard approach presented in this document is based on the above 3 MTS analysis protocols. The differences between the experimental systems are shown in Table 1. Table 1 Summary of studies used to develop standardized MTS assay protocols Sponsoring Institution Cell Line a Nanoparticles for Testing b Positive and Negative Control Materials Are Centrifuged IANH Raw264.7 PS-NP, CeO2 CdSO4, no particle exposure no NanoGo BEAS-2B, RLE-6TN and THP-1 ZnO, TiO2, MWCNT No particle exposure Yes EMPA-NISTc A549 PS-NP CdCl2, no particle exposure no Name in the aATCC cell bank. b PS-NP are positively charged PS nanoparticles, CeO2 is ceria, ZnO is zinc oxide, TiO2 is titanium dioxide, MWCNT is multi-walled carbon nanoparticle meter tube. cEMPA is the Swiss National Laboratory for Materials Science and Technology. Because of these differences, optional steps proposed by 3 laboratory studies were included in the standard method. In addition to the MTS method [6], there are other methods that can be used to measure cell viability, such as 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl bromide Tetrazolium (MTT) method[7], 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) method [8], lactate dehydrogenase (LDH) method [9], trypan blue exclusion method [10] and neutral red assay [11]. The MTS method has carried out multiple comparison experiments. The MTS method is a modified The advanced MTT method can provide a simple and high-throughput detection of cell viability[4,12]. After MTS is reduced by functional enzymes in living cells, the solution is Optical density increases. Control experiments are required to determine baseline optical densities of cell viability in untreated cells and to verify that cells respond to known nontoxic nanoparticles particles, toxic chemicals, and toxic nanoparticles have expected responses [13]. In addition, determine whether nanoparticles interfere with the optical readout of the analysis and can It is important to invalidate the assessment of nanoparticle cytotoxic responses [14]. Notably, the MTS method described here is one of many commercial methods used to assess the cytotoxicity of nanomaterials. Although this The document does not address the LDH method for assessing plasma membrane integrity, the ATP method for assessing energy metabolism, and the BrdU method for DNA synthesis, etc. Combined with the MTS method, the comprehensive effect of nanoparticles on cells can be more comprehensively evaluated. Nanotechnology MTS method for determination of nanometers Cytotoxicity of particles1 ScopeThis document describes the MTS method for evaluating the effect of nanoobjects and their aggregates and aggregates (NOAA) on cell viability. this Methods include operational requirements and control experiments to determine and analyze the uncertainty of test results. This document applies to 96-well plates.2 Normative referencesThe contents of the following documents constitute essential provisions of this document through normative references in the text. Among them, dated citations documents, only the version corresponding to that date applies to this document; for undated references, the latest edition (including all amendments) applies to this document. ISO /T S80004-2 Nanotechnology Terminology Part 2.Nano-objects (Nanotechnologies-Vocabulary- Part 2.Nano-objects)3 Terms and DefinitionsTerms and definitions defined in ISO /T S80004-2 and the following apply to this document. 3.1 culture vessel culturevessels This document uses a 96-well tissue culture grade plate. Note 1.As long as it meets the requirements of tissue culture grade and is suitable for mammalian cell culture, other well plates (such as 384-well plate, 24-well plate and 6-well plate) can also be used Used interchangeably. Note 2.If other sizes of well plates are used in this document, it may Make necessary adjustments. [Source. GB/T 16886.5-2017, 3.1] 3.2 dispersion system A microscopic heterogeneous system in which a discontinuous phase (solid, liquid or gas. discontinuous phase) is dispersed in a continuous phase that differs in composition or state. Note. If the solid particles are dispersed in a liquid, the dispersion system is called a suspension. If a dispersion system consists of two or more liquid phases, it is called an emulsion liquid. Suspensions consist of solid and liquid phases dispersed in a continuous liquid phase. 3.3 endotoxin Part of the outer membrane of the extracellular envelope of Gram-negative bacteria. Note. The main active ingredient is lipopolysaccharide (LPS). [Source. GB/T 41309-2022, 2.3] ...... |
