In-vitro test method of gaseous substance

Abstract

The invention belongs to the technical field of biology, and discloses an in-vitro test method for gaseous substances, which comprises the following steps: selecting a sealable air bag as an airtight contamination device, and sterilizing the air bag by using a high-pressure sterilization technology; air in the sealing bag is extracted to form a vacuum state, and gas substances prepared in advance are guided into the sealing bag; and controlling the exposure time of the tested strain or cell in the gas substance in the sealing bag, and evaluating the exposed strain or cell. According to the scheme, in-vitro contamination exposure of gaseous substances can be achieved, sterile conditions and dosage gradient requirements required by tests can be met, the toxicity of the gaseous substances can be evaluated more safely and effectively, and a new tool is provided for drug development, chemical safety evaluation and environment monitoring.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to an in vitro test method for gaseous substances. Background Art

[0002] In the process of drug development, chemical manufacturing and research on new food ingredients, the safety assessment of new substances is an essential part. Among them, in vitro genotoxicity testing and cytotoxicity testing are two key evaluation methods used to evaluate the potential risks that new substances may cause to humans or the environment.

[0003] The bacterial reverse mutation test is a commonly used genetic toxicity test method used to evaluate whether a chemical substance may cause DNA damage. By detecting the ability of the test substance to produce mutations in certain special mutants of the test strain, even if the strain mutates from histidine / tryptophan dependent to prototrophic, it is judged whether the test substance is a mutagen. The principle of this test is that the histidine auxotrophic strain of Salmonella typhimurium cannot synthesize histidine, so only a few spontaneously reversed mutant bacteria grow on a medium lacking histidine; the tryptophan auxotrophic strain of Escherichia coli cannot synthesize tryptophan, so only a few spontaneously reversed mutant bacteria grow on a medium lacking tryptophan. If a mutagen is present, the auxotrophic bacteria will reverse to a prototrophic type, so they can grow and form colonies, and the test substance can be judged as a mutagen.

[0004] In vitro cytotoxicity tests are used to evaluate the effects of chemicals on cell growth and survival. The commonly used MTT method is to evaluate cell survival by detecting the metabolic activity of cells.

[0005] At present, basically similar test methods are introduced according to the standards of various industries. However, when encountering unconventional gases (such as certain chemical gases) for detection during the test, although the use of appropriate poisoning methods is mentioned, the specific operation details are often not described in detail. Therefore, an in vitro test method for gaseous substances is proposed. Summary of the invention

[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose an in vitro test method for gaseous substances, which can more safely and effectively evaluate the toxicity of gaseous substances and provide a new tool for drug development, chemical safety assessment and environmental monitoring.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] An in vitro test method for a gaseous substance comprises the following steps:

[0009] Select a sealable air bag as the closed poisoning device and sterilize the air bag using high-pressure sterilization technology;

[0010] The air in the sealed bag is extracted to form a vacuum state, and the pre-prepared gas substance is introduced into the sealed bag;

[0011] The exposure time of the tested strains or cells in the sealed bag to the gaseous substance is controlled, and the strains or cells after exposure are evaluated.

[0012] As a further solution of the present invention: the volume of the closed air bag is 2L-40L, and the volume of the sealed bag is adjusted according to the duration of the contamination.

[0013] As a further solution of the present invention: the closable air bag is provided with an air valve with a rubber stopper and an air valve with a knob, the air valve with a rubber stopper is used to be connected to a vacuum pumping device, and the air valve with a knob is used to be connected to a sterile pipeline system for introducing gaseous substances, and the sterile pipeline system is provided with a filter and a flow meter.

[0014] As a further embodiment of the present invention: the gaseous substance includes compressed gas, sample gas, oxygen or carbon dioxide.

[0015] As a further aspect of the present invention: the high pressure sterilization treatment comprises the use of steam or chemical sterilants.

[0016] Compared with the prior art, the advantages of the present invention are:

[0017] This scheme can achieve toxic exposure of gaseous substances and meet the sterile conditions and dose gradient requirements required for the test. It can more safely and effectively evaluate the toxicity of gaseous substances and provide a new tool for drug development, chemical safety assessment and environmental monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the present invention. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention; it is obvious that the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments, and all other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making creative work are within the scope of protection of the present invention.

[0020] See also Figure 1 .

[0021] 1. Cytotoxicity Test

[0022] The following steps are taken as examples (except for the poisoning operation steps, the remaining test operations can be adjusted accordingly with reference to the current national standards or technical specifications):

[0023] 1. Test system:

[0024] Primary cells or cell lines with clear sources should be selected, and the presence or absence of mycoplasma contamination can be identified upon request.

[0025] 2. Preparation of test samples:

[0026] If the test sample is a gas at room temperature, a closable sampling bag with a volume of 2L to 40L (adjusted according to the duration of exposure) can be selected as a closed exposure device for the cytotoxicity test. After high-pressure sterilization and vacuum treatment, the prepared test sample is introduced into the sampling bag. The test sample is exposed to the gaseous state under closed and sterile conditions, and the exposure dose is designed according to the contact time and concentration of the cells.

[0027] 3. Cell Preparation:

[0028] The cells that grew vigorously for 48 h to 72 h were selected for the experiment on the same day. The adherent cells in the cell culture flask were digested with trypsin and the cell density was adjusted to 1 × 10 5 / mL. The cell suspension was inoculated in a 96-well cell culture plate. There were 4 groups, including blank control, positive control, negative control and test sample. Each group had one culture plate (blank control and positive control could share one culture plate). Each plate had 6 to 8 wells. Each well was inoculated with 100 μl of cell suspension. The plate was placed in a CO2 incubator at 37°C for 24 hours to allow the cells to adhere to the wall and grow.

[0029] 4. Experimental Grouping:

[0030] The experiment included a blank control group, a positive control group, a negative control group and a test sample group.

[0031] The blank control group was not placed in the sampling bag, and no complete culture medium of the test sample was added;

[0032] The positive control group was not placed in the sampling bag, but was added with complete culture medium containing 10% DMSO;

[0033] The negative control group was placed in a sterile sampling bag and sterile air without test samples (sterile air + 5% carbon dioxide) was added;

[0034] The test sample group is placed in a sterile sampling bag, and a certain volume of test sample + oxygen + sterile air + 5% carbon dioxide (calculated based on the oxygen content of 21% in the air) is added.

[0035] 5. Toxic treatment:

[0036] Remove the upper liquid from each well. Add complete culture medium to the blank control group, negative control group and test sample group, 100 μL / well; add complete culture medium containing 10% dimethyl sulfoxide to the positive control group; 100 μL per well, place in CO2 incubator and continue to culture for 48 hours. At the same time, the differences between the groups are as follows:

[0037] The blank control group and the positive control group were not placed in the sampling bag;

[0038] The negative control group was placed in a sterile sampling bag without test sample gas, and the rest was the same as the test sample group;

[0039] The test sample is placed in a sterile sampling bag containing the highest concentration of the test sample gas and other mixed gases.

[0040] The cell culture plates of the negative control group and the test sample group were placed in sterile sampling bags. The top cover of the cell culture plate was opened, and the sealed sterile sampling bag was evacuated. Then the mixed gas was introduced and kept in contact at 37°C for 4 to 48 hours. Then the sterile sampling bag was opened, the top cover was closed, and the sterile sampling bag was placed in a CO2 incubator at 37°C for a total of 4+44h to 48h.

[0041] 6. Cell Observation

[0042] The cell culture plate was taken out and the cell morphology of each well in the cell culture plate was observed under an inverted microscope to determine the cell inoculation system error and the growth characteristics of each group of cells.

[0043] After checking the cell morphology of the cell culture plate, carefully remove the culture medium. Add 50 μL of MTT solution to each experimental well, and incubate the plate in a 37°C incubator for another 2 hours. Then discard the MTT solution and add 100 μL of isopropanol solution to each well. Shake the cell culture plate for 10 minutes, measure the absorbance value at 570nm and 630nm wavelengths on a microplate reader, and calculate the relative proliferation rate (RGR) of the cells.

[0044] 7. Evaluation and interpretation of results:

[0045] 1) Criteria for determining the effectiveness of the test system

[0046] The cells in the blank control group grew well; the cells in the negative control group grew well, with an average OD value of ≥ 0.2 and a survival rate of the positive control group of < 30%, indicating that the test system was effective.

[0047] 2. Test result judgment criteria

[0048] If the survival rate of the test substance group drops below 70% of the negative control, it is considered to be potentially cytotoxic and is judged to be positive; otherwise, it is judged to be negative.

[0049] 2. Bacterial reverse mutation test

[0050] The following steps are examples (except for the poisoning operation steps, the remaining test operations can be adjusted accordingly with reference to the current national standards or technical specifications):

[0051] 1. Strain selection: Five test strains of histidine-deficient Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA1535 were selected as the test system. The strains were derived from Molecular Toxicity, INC., and after being identified as qualified for histidine synthesis deletion mutation, cell wall lipopolysaccharide rfa deletion mutation, excision repair system uvrB deletion mutation (except TA102), ampicillin resistance R factor (except TA1535), and tetracycline resistance factor (only TA102), they were frozen in a -80℃ low-temperature refrigerator for use.

[0052] 2. Metabolic activation system: The metabolic activation system uses the demitochondrial component (S9) of the liver of male SD rats induced by enzyme inducers (sodium phenobarbital and β-naphthoflavone), and supplements auxiliary factors to form an S9 mixed solution, wherein the S9 mixed solution is composed of: 10% S9 (v / v), 5mM glucose-6-phosphate, 4mM coenzyme II solution (β-NADP+), 8mM MgCl2, 33mM KCl, and is supplemented with phosphate buffer (0.2mol / L, pH 7.4). Prepare before use.

[0053] 3. Preparation of strains and culture media: After amplification, histidine-deficient Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 were added with glycerol and stored at ultra-low temperature. They were thawed at room temperature before use and used directly. The bottom and top culture media were prepared according to the instructions of the kit, wherein the bottom culture media contained 1.5% agar (w / v, weight / volume ratio), glucose and phosphate buffer. The top culture media contained 0.6% agar (w / v) and 0.5% sodium chloride (w / v), and supplemented with L-histidine and D-biotin.

[0054] 4. Preparation of contaminated gas bags: Select a closable sampling bag (volume of 40L) as the closed contamination device for the bacterial reverse mutation test. After high-pressure sterilization at 121°C and vacuum treatment, the test sample is in a gaseous state under closed and sterile conditions, and the contamination dose is designed according to the contact time and concentration of bacteria.

[0055] 5. Toxic treatment: Use the plate incorporation method, place the centrifuge tubes with 2mL of the top culture medium in a 45℃ water bath for preheating, add 0.1mL of saline (or 0.1mL of positive control), 0.5mL of PBS (without metabolic activation system, -S9), and 0.1mL of bacterial solution one by one, mix well, quickly pour into the bottom culture medium, rotate the plate to make it evenly distributed, and lay flat until solidified. Each group of plates is placed in a sterile sampling bag, the top cover of the plate is opened, the plate is facing up, and after the sealed sterile sampling bag is evacuated, a dose concentration gas is introduced, and the contact is maintained at 37℃ for 4h, then the sterile sampling bag is opened, the plate is closed and inverted, and the sealed sampling bag with the same dose concentration gas is continued to be cultured at 37℃ for 48h for contact infection, and then the reversed colony count is performed. At the same time, a blank control group, a negative control group, and a positive control group are set up. Three parallel plates are set up for each dose concentration group.

[0056] 6. Test grouping: The test includes positive control, blank control, negative control and test sample concentration groups. The blank control group is not placed in a sterile sampling bag, but directly inverted and cultured at 37℃ for 48h. The positive control group is not placed in a sterile sampling bag, but the positive agent is added and directly inverted and cultured at 37℃ for 48h. The negative control group is placed in a sterile sampling bag, and only sterile air without test samples is introduced.

[0057] Gas addition volume:

[0058]

[0059]

[0060] 7. Result determination: The validity of the test system is determined when the average number of reverse mutant colonies in the positive control group is significantly higher than that in the blank / solvent control group or is 2 times (TA97a, TA98, TA100, TA102) or 3 times (TA1535) or more than the average number of reverse mutant colonies in the blank / solvent control group. The results of the test samples are determined to be valid under the premise that the test system is determined to be valid. The standards are as follows:

[0061] 1) At least one strain meets the following criteria: for TA97a, TA98, TA100, and TA102 strains, the number of reverse mutant colonies is 2 times (or more) that of the blank control with or without metabolic activation; for the TA1535 test strain, the number of induced reverse mutant colonies is 3 times (or more) that of the respective negative control groups; and there is a concentration-dependent increase and / or a repeatable increase in one or more dose groups, the result is judged as positive.

[0062] 2) If the above-mentioned multiple ratio relationship is not achieved for any strain and there is no concentration-dependent and / or reproducible increase, the result can be judged as negative.

[0063] Conclusion: Under the experimental conditions, gaseous samples did not show mutagenic effects on strains TA97a, TA98, TA100, TA102 and TA1535 under the two test conditions with or without metabolic activation. Under this test method, no influence on the colony due to operation was observed, and this test method can be applied to bacterial reverse mutation tests for gaseous or volatile substances.

[0064] The above is only a preferred specific implementation of the present invention; however, the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solution and its improved conception within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An in vitro test method for gaseous substances, characterized in that: The following steps are involved: Select a sealable air bag as the closed poisoning device and sterilize the air bag using high pressure sterilization technology; The air in the sealed bag is extracted to form a vacuum state, and the pre-prepared gas substance is introduced into the sealed bag; The exposure time of the tested strains or cells in the gaseous substance in the sealed bag is controlled, and the strains or cells after exposure are evaluated.

2. The in vitro test method for a gaseous substance according to claim 1, characterized in that: The volume of the closed air bag is 2L-40L, and the volume of the sealed bag is adjusted according to the duration of the contamination.

3. The in vitro test method for gaseous substances according to claim 1, characterized in that: The closable air bag is provided with an air valve with a rubber plug and an air valve with a knob. The air valve with a rubber plug is used to connect with a vacuum pumping device, and the air valve with a knob is used to connect with a sterile pipeline system for introducing gaseous substances. The sterile pipeline system is provided with a filter and a flow meter.

4. The in vitro test method for gaseous substances according to claim 3, characterized in that: The gas substance includes compressed gas, sample gas, oxygen or carbon dioxide.

5. The in vitro test method for gaseous substances according to claim 1, characterized in that: The autoclaving process involves the use of steam or chemical sterilants.

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