YY/T 0993-2015Biological evaluation of medical devices—Nanomaterial:In vitro cytotoxicity tests(MTT assay and LDH assay) (English PDF)

3.1 Terms and definitions

The following terms and definitions defined in GB/T 16886 apply to this document.

3.1.1 Nanomaterial

The material structure is at least one dimension in three dimensions in the nanometer scale, or consists of nanostructured units and has special properties. material.

3.1.2 Nanomaterial medical device

A medical device that combines or contains nanomaterials.

3.2 Abbreviations

The following abbreviations apply to this

document. APAP. Acetaminophen (acetaminophen)

DMSO. dimethylsulfoxide

FBS. fetal bovine serum (fetalbovineserum)

L929. mouse fibroblast (mousefibroblastcel)

LDH. lactate dehydrogenase (lactatedehydrogenase)

MEM. culture solution (modifiedeaglesmedium)

MTT. 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyl-2hydro-tetrazolium bromide [3-(4,5-Dimethyl-2-thiazolyl)-2.5 -di- phenyl-2H-tetrazoliumbromide]

PBS. phosphate buffered saline (phosphatebufferedsaline)

4.1 General

The test sample can be selected from nanomaterials (for the preparation of raw materials containing nanomaterials medical devices) dilutions and nanomaterial medical devices.

Leach solution. The material should be fully physicochemically characterized prior to testing in order to reasonably interpret the biological response. Such as the size of the particles Small and surface chemistry can cause different test results. Positive and negative controls should be included for each test.

The reagents, consumables and equipment used in the test are detailed in Appendix A.

Note: With the progress of international nanotoxicology research and the improvement of cognitive level, it is encouraged to choose a more appropriate yang according to the intended use and characteristics of the test sample. Sex control and negative control.

4.2 Preparation of sample diluent or extract

4.2.1 Sample diluent

Nanomaterial Diluent. The nanomaterial is directly diluted in the cell culture medium to prepare different concentrations of the diluent.

4.2.2 Sample extract

4.2.2.1 Principle of extraction

The extract should be prepared in accordance with the requirements of GB/T 16886.12.

a) Nanomaterial medical device according to the surface area ratio required by GB/T 16886.12 (the part containing nanomaterials) The machine is placed in the leaching medium for leaching;

b) The quality ratio can also be selected according to the material characteristics.

4.2.2.2 Leaching medium

The selection of the leaching medium can be referred to the requirements of GB/T 16886.5.

Note 1. Different leaching media may affect the surface characteristics of the nanoparticles, such as. particle size, particle aggregation characteristics, surface charge changes, proteins And the adsorption of other substances, the release of ions, etc., thereby affecting the cytotoxic reaction of nanomaterials. Therefore, the choice of extraction medium is very important, Explain the reasons for the choice.

Note 2. Based on current cognitive levels, it is recommended to use serum-free cell culture fluid as the extraction medium for nanomaterials. With the international nanotoxicology research The progress and level of cognition are encouraged to select a more suitable leaching medium based on the intended use of the test sample and the nature of the nanomaterial itself.

4.2.2.3 Extraction conditions

Can refer to the requirements of GB/T 16886.5.

According to the current cognitive level, the recommended extraction conditions are. (37 ± 2) ° C for not less than 24h.

In the case where it is not necessary, the extract does not require filtration, and the nanoparticle- and ion-containing extracts leached from the nanomaterial medical device For testing; if filtering, centrifugation, or other disposal methods are used, they should be stated in the final report. The pH adjustment of the extract should also be The report states.

Note 1. It is advisable to avoid treatment of the leachate, for example pH adjustment.

Note 2. With the progress of international nanotoxicology research and the improvement of cognitive level, it is encouraged to choose more suitable according to the intended use and characteristics of the test sample. Leaching conditions.

4.2.3 Sterility requirements

The test sample diluent or extract should be sterile.

4.3 Preparation of control samples

4.3.1 Positive control

Acetaminophen (APAP) solution. Prepare a solution of appropriate concentration with a culture solution (recommended concentration 25mmol/L, filter-sterilized) After use), used for positive control.

Note: Cadmium oxide nanoparticles are capable of inducing toxic reactions in many test systems at relatively low doses and are similar to other nanoparticles.

The particle size, therefore, is considered to be a candidate for a positive control of the nanoparticles.

Polyethylene glycol octyl phenyl ether solution (Triton-X-100). Prepare a solution of the appropriate concentration with the culture solution (recommended concentration is 1%, filtered For use after sterilizing, a positive control for the LDH test.

4.3.2 Negative control

A suitable negative control material was selected and a negative control was prepared according to GB/T 16886.5.

4.4 Reagent preparation

4.4.1 MTT test

4.4.1.1 MTT solution. Prepare 5mg/mL with PBS and store at 4°C in the dark for no more than one month.

4.4.1.2 Glycine buffer

0.1 mol/L glycine (Mr75.07), 0.1 mol/L sodium chloride (Mr58.44). With 0.1mol/L The sodium hydroxide solution was adjusted to pH 10.5 and stored at room temperature.

4.4.2 LDH test

LDH cytotoxicity test kit (containing lactate dehydrogenase recombinant catalyst, dye solution). Prepare reagents according to the instructions in the kit.

Note: When selecting different brands of kits, it is advisable to verify the sensitivity and sensitivity with a positive control before the test.

7.1 Overview

In this test, the extract of nanomaterial dilution or nanomaterial medical device is contacted with cultured cells through MTT and LDH.

The test method evaluated the toxic effects on cell mitochondria and the toxic effects on cell membranes, respectively.

The reagents, consumables and equipment used in the test are detailed in Appendix A.

This test is to obtain two test results in one test.

2.5-diphenyl-2H-tetrazolium bromide, MTT] is a yellow water-soluble tetrazolium dye that can be killed by living cells.

Originally purple non-water soluble formazan. The MTT assay uses dimethyl sulfoxide (DMSO) to dissolve formazan and splits through a microplate.

The photometer (microplate reader) quantifies the hyperthyroidism, compares the absorbance values of the test group and the control group, and evaluates the cytotoxicity of the test sample.

b) LDH test. lactated dehydrogenase (LDH) is a cytoplasmic enzyme that is released after cell lysis.

Extracellular. Therefore, the LDH assay can be used to detect the integrity of cell membranes. LDH test principle.

1) LDH oxidizes lactic acid to form pyruvate;

2) conversion of pyruvate and tetrazolium salt to formamidine;

3) The formazan dye is soluble in water and can be detected by a microplate spectrophotometer.

7.2 Test procedure

7.2.1 Preparing cells

The cells can be prepared by referring to GB/T 16886.5. Or according to the cell supplier recommended method.

Note: This procedure and all of the following test procedures are performed under aseptic conditions.

7.2.2 Cell seeding

Dilute the cell suspension with cell culture medium (containing 10% fetal bovine serum) and adjust the concentration to 1 x 10^5 cells/mL in each well of a 96-well plate. Inoculate 100 µL.

5 replicate wells per cell or concentration (or no less than 3 replicate wells). See Appendix B for cell seeding and loading.

7.2.3 Cell culture

Incubate for 24 h at 37 ° C, 5% carbon dioxide, humidity >= 90% (cell growth to 70% ~ 80% confluent state).

7.2.4 Test sample and control sample loading

7.2.4.1 If the test sample is a dilution of the nanomaterial raw material, or a sample that can be dispersed in the cell culture fluid, then each nanomaterial Or the sample should be tested at 5 to 7 different concentrations (diluted within the dose-response relationship and verify that the test system is valid).

When the test sample is a nanomaterial raw material, the highest concentration of the nanoparticles to be dispensed should be within its maximum solubility range. For non-soluble Metal nanoparticles, according to the pretest, choose the maximum toxic concentration as the initial concentration (such as. cell growth inhibition rate is about 80%);

A small concentration should achieve a cell growth inhibition rate of about 10%.

7.2.4.2 If the test sample is a leach solution containing a nanomaterial medical device, and the content of the nanomaterial in the extract is very low (cytotoxicity)

Less than or equal to grade II), only need to test the stock solution of the extract without dilution; but if the content of nanomaterials in the extract is high (cytotoxicity)

Greater than or equal to grade III), it is advisable to dilute 5 to 7 different concentrations. Add 10 S to each of the different dilutions of the extract Test sample.

Note: It is advisable to avoid treatment of the leachate, for example to adjust the pH. Instructions should be given if pH adjustment is required.

The 96-well plates of the grown cells were taken out, and 100 µL of the test sample and the control sample were added in the order of the template arrangement. Continue to train after loading Raise for 24 hours.

Note: Each experimental group is equipped with a cell-free loading control, which means that there is no cell, only the culture solution, and the control hole of the same test sample is added, and the measured absorbance is measured.

The value is used as the background value. In the final calculation, the actual sample measurement value should be deducted from the background value of the cell-free sample, thereby subtracting the absorbance of some nanoparticles. The measured interference.

7.2.5 Testing

7.2.5.1 MTT test

7.2.5.1.1 Remove the 96-well plate from the incubator, discard the liquid ready for use as a Triton-X-100 positive control well, and add 100 µL.

1% Triton-X-100 (prepared in 4.3.1). Leave at room temperature for 10 min and centrifuge at 700 g for 3 min.

7.2.5.1.2 Aspirate 50 µL of liquid from each well and transfer to another 96-well plate (transferred according to the sample distribution of the original template), temporarily put 4 ° C Save and perform the LDH test as soon as possible (see 7.2.5.2).

7.2.5.1.3 Discard all liquid in the original 96-well plate and add.200 µL of fresh medium to each well.

7.2.5.1.4 Add 50 µL of MTT solution (final concentration 1 mg/mL) to each well and incubate for 4 h at 37 °C.

7.2.5.1.5 Remove the 96-well plate and centrifuge at 700g for 3min.

7.2.5.1.6 Aspirate the culture medium and MTT solution.

7.2.5.1.8 Add 25 µL of glycine buffer to each well and mix on a shaker for 10 min.

7.2.5.1.9 Detection by microplate reader, detection wavelength 570nm, reference wavelength 680nm or other results according to the test system verification results wavelength.

7.2.5.1.7 Add.200 µL DMSO to each well

7.2.5.2 LDH test

7.2.5.2.1 Remove 96-well plates (see 7.2.5.1.2) that have been transferred out of 50 µL of liquid, and add 50 µL of reaction mixture to each well. (See 4.4.2), mix on a vortex mixer.

7.2.5.2.2 Incubate for 30 min at room temperature in the dark.

7.2.5.2.3 Detection with a microplate reader, detection wavelength 490nm, reference wavelength 680nm or other results according to the test system verification results wavelength.

8.1 Data Recording

Five wells were replicated for each of the test samples, positive control, negative control, and solvent control. All test results should be recorded in tabular form.

In the calculation, the absorbance of each well needs to be deducted from the absorbance of its corresponding cell-free sample group, and then the values of the 5 wells are added together to calculate The mean value of surgery.

8.2 Calculation method

8.2.1 MTT test

ODB× 100% (1) In the formula. C --- relative cell activity, %; ODA---the average absorbance of the test sample group; ODB---the average absorbance of the solvent control group.

8.2.2 LDH test

ODa-ODb ODc-ODb× 100% (2) In the formula. T ---LDH total release rate; %; ODa---the average absorbance of the test sample group; ODb---the average absorbance of the solvent control group; The average absorbance of the ODc---Triton-X-100 control group.

The mean, standard deviation (SD) and coefficient of variation (CV) of the absorbance values of each positive control, negative control, and test sample need to be calculated.

8.3 Analysis of results

8.3.1 When the test sample is a nanomaterial raw material, the concentration-response curve is drawn from the MTT test data and the LDH test data.

For the resulting concentration-response curve, a nonlinear fit to the equation of the reaction curve is required, and the IC50 is calculated from the obtained equation.

Note: IC50 in this standard refers to the concentration of test sample and LDH when the inhibition rate of relative cell activity (relative proliferation rate) reaches 50% in the MTT test results.

The test sample concentration at which the relative release rate of LDH in the test was 50%.

8.3.2 When the test sample is an extract containing a nanomaterial medical device, directly report the calculation results of 8.2.1 and 8.2.2 (including parallel samples or The mean value of the replicated holes ± standard deviation).

9.1 Test quality control

a) Positive and negative controls should be provided for each test;

b) The OD570 of the solvent control group (without any treatment) should be greater than or equal to 0.2 to confirm that the inoculated 1 x 10^4 cells/well is No in the normal double-proliferation long-term during the 2d test.

Note: Any excluded values (except for outliers) should also be stated in the report.

A.1 Reagent

A.1.1 3-(4,5-Dimethyl-2-thiazole)-2,5-diphenyl-2 hydrogen-tetrazolium bromide (MTT).

A.1.11 Double antibody (PS)

A.1.10 Phosphate buffer (PBS)

A.1.9 LDH-cytotoxicity test kit

A.1.8 Fetal bovine serum (FBS)

A.1.7 Cell culture fluid (MEM)

A.1.6 Trinitro-toluene X-100 (Triton-X-100)

A.1.5 Sodium chloride (NaCl)

A.1.4 Glycine (Glycine)

A.1.3 Dimethyl sulfoxide (DMSO)

A.1.2 Acetaminophen (APAP)

A.2 Consumables

A.2.1 96-well flat-bottom cell culture plate.

A.2.3 Culture flasks

A.2.2 Cell cryotubes

A.3 Equipment

A.3.7 Sampler

A.3.3 CO2 cell culture incubator

A.3.1 Microplate spectrophotometer (microplate reader)

A.3.2 Centrifuges that can be placed in 96-well plates.

A.3.4 Constant temperature water bath

A.3.5 Invert the microscope. A.3.6 Shakers for 96-well plates.