CN113774106A - Method for detecting genotoxicity by using 3D hepatocyte in-vitro micronucleus cytomics - Google Patents

Abstract

The invention discloses a method for detecting genotoxicity by using 3D hepatocyte in-vitro micronucleus cytomics. The method comprises the following steps: (1) a 3D hepatic cell model is constructed in vitro by adopting a non-support culture method combining hanging drop and ultra-low adsorption culture and taking a commonly used humanized hepatic cell HepG2 as a cell material; (2) micronucleus cytomics assays were performed on the compounds using a 3D hepatocyte model, analyzing the genotoxicity mechanism of the compounds. The in vitro detection method can analyze multiple genotoxicity mechanisms of the compound, improve the capability of in vitro cell models for predicting the genetic toxicity of the drug and provide information related to human body tests.

Description

Method for detecting genotoxicity by using 3D hepatocyte in-vitro micronucleus cytomics

Technical Field

The invention belongs to the field of genotoxicity detection, and particularly relates to a method for detecting genotoxicity by using 3D (three-dimensional) hepatocyte in-vitro micronucleus cytomics.

Background

Current 3D cell culture techniques have great potential for development in toxicology studies. As the scientific research field pays more and more attention to animal ethics, the 3D cell culture technology meets the requirements of 'reduction, substitution and optimization' of 3R principle. And because the species difference exists between the experimental animals (such as rodents) used for evaluating the drug toxicity and the human beings, the humanized cell model is used for evaluating the drug toxicity, so that more reference data related to the human beings can be provided in the early stage of drug screening, the research and development time is shortened, and the research and development cost is reduced. 3D cell models that have been used for genotoxicity testing include 3D skin models, 3D liver models, 3D airway models, and the like. Because the medicine generally enters a human body through oral administration and injection and is metabolized in the liver, the 3D liver cell model is developed, the metabolism of the medicine in the body can be simulated in vitro, the in vitro genotoxicity screening requirements of most medicines are met, and a huge development prospect is displayed.

Compared with the CBMN method in the published literature, the invention adopts a cytokininis-block micronucleous (CBMN-cyt) method aiming at capturing a plurality of molecular events causing chromosome damage and chromosome instability. CBMN only counts binuclear micronuclei, while CBMN-cyt method needs to count micronuclei, nuclear bud and nuclear bridge of binuclear cells.

Micronucleus (MN) is derived from chromosome fragments or whole chromosomes that lag behind in the late stages of cell division. The cytokinin-block micronucleus (CBMN) assay is the first method of micronucleus determination in human or mammalian cells in vitro, in which the micronucleus rate in the first dividing cell can be identified by observing the binuclear cells after the cell division is blocked with Cytochalasin B (Cytochalasin B, Cyto-B). The CBMN method has become one of the standard methods for the genetic toxicology testing of human and mammalian cells due to its reliability and good reproducibility. With the study of the mechanisms of the relevant DNA damage biomarkers micronucleus, nuclear matrix bridges (NPB) and nuclear buds (Nbud), cell death and apoptosis, this approach eventually evolved into a cytokinesis-block microcytome (CBMN-cycle) approach aimed at capturing all these DNA damage events. The advantage and highlight of CBMN-cyt detection is that it allows simultaneous detection of multiple molecular events leading to chromosome damage and chromosome instability.

MN, NPB and Nbud are common nuclear abnormalities in cancer, representing a common chromosome-unstable cellular phenotype. Chromosomal instability leads to changes in the genetic quantity of cells and has the potential to evolve and mutate rapidly, resulting in a variety of abnormal genotypes (Wenhaing, et al, J.Newcastle disease, 2016,25(7): 787-793.6). MN is mainly derived from either non-centromeric chromosome fragments or whole chromosomes that fail to be contained in the daughter nuclei at the end of mitosis (MILLER B, et al. Mutat Res,1998,410(1): 81-116). MN can sensitively detect DNA damage and repair and chromosome fragment and chromosome loss (Wenhaaif, etc. different liver cell lines micronucleus cytomics research, the seventh national toxicology major of the Chinese toxicology society and the eighth Hubei science and technology forum argument set). The mechanism of Nbdu is not clear, but Nbdu generation is associated with DNA damage and repair, and is a biological marker of the process by which amplified DNA is eliminated and/or the DNA repair complex (FENECH M, et al. NPB is a biomarker of double centromere chromosomes caused by DNA strand breakage errors and double centromere chromosomes caused by telomere end fusion (Wenhahua, et al. Microcytomics research of different liver cell lines, seventh national toxicology conference of China toxicology society and eighth Hubei science and technology forum argument).

Mitomycin C (MMC) is an antineoplastic drug and positive in vitro micronucleus test. It acts as an alkylating agent to cross-link complementary DNA copies, thereby affecting nucleic acid synthesis and function (FENECH M, et al.

The currently known detection scheme is the CBMN-cyt scheme of human peripheral blood lymphocytes established by Fenech (FENECH M. Nature Protocols,2007,2(5): 1084-1104). The CBMN-cyt method has only a small number of applications in HepG2 cells, but lacks a standardized protocol for testing. The existing 3D hepatocyte genotoxicity detection model is established by adopting a pendant drop method to culture A three-dimensional in vitro HepG2 cells over coherent model for genotoxicity studies, and is not suitable for long-term culture and subsequent reagent addition to carry out genotoxicity detection because nutrient substances are reduced and a culture medium cannot be replaced.

Conventional micronuclear cytomics detection indicators include counting apoptotic and necrotic cell numbers to reflect cytotoxicity. The detection index is easily interfered by the necrotic core gradually formed inside the 3D cell sphere in the forming process. Furthermore, studies have shown that apoptotic and necrotic cells do not show dose-related increases under the action of positive compounds and do not respond well to cytotoxicity (MAES A, et al. Folia biol (Praha),2012,58(5): 215-220).

The invention uses MMC as a positive compound, takes the established 3D hepatocyte model as an experimental model, tries to establish a 3D hepatocyte micronucleus cytomics method, and compares the result with the CBMN-cyt detection result of the 2D hepatocyte model.

The invention establishes a 3D hepatocyte micronucleus cytomics method to count only mononuclear, binuclear and multinuclear cells, and reflects the toxicity of the compound to the cells by calculating CPBI, RI and cell growth inhibition rate. The cytotoxicity index 100-RI of CBMN-cyt is equivalent to the cell growth inhibition rate and can be used as the index of cell proliferation toxicity. Therefore, the invention tries to establish a 3D hepatocyte micronucleus cytomics detection method by taking the established 3D hepatocyte spheroids as an experimental model, has the problem of large difference with the human test result, and provides the genetic toxicity mechanism information related to the compound.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for detecting genotoxicity by using 3D hepatocyte in-vitro micronucleus cytomics, aiming at the defects of poor correlation between a method for detecting in-vitro cytogenetic toxicity and human body tests in the prior art and the like. The method for detecting in vitro cytogenetic toxicity is more approximate to the effect of the compound in vivo and can provide various mechanism information.

In order to study the response of cells to physical and biochemical stimuli, 2D cell culture has become widely accepted by the scientific community as a commonly used in vitro model, and the understanding of the drug action of the present invention is greatly enhanced. However, there is increasing evidence that 2D cells in some cases have a large difference in morphology and function from cells in vivo. In vitro toxicology studies require replication of a portion of normal tissue function for evaluation of the effects of exogenous factors (e.g., drugs, cosmetics, pollutants, oxidative environment) on cellular function and carcinogenic risk. Therefore, the inventor creatively uses the 3D liver cell model and the micronucleus cytomics to establish a new genotoxicity evaluation method, solves the technical problem in the field and promotes the development of the in vitro genotoxicity detection method.

To better detect the genotoxicity of compounds, providing information on the mechanisms associated with DNA damage, the inventors chose to use micronuclear cytomics for detection. An advantage and highlight of micronuclear cytomic detection is that it allows simultaneous detection of multiple molecular events leading to chromosome damage and chromosome instability.

The invention mainly solves the technical problems by the following technical means: the invention provides a method for detecting genotoxicity by using 3D hepatocyte in-vitro micronucleus cytomics, which comprises the following steps: the method comprises the following steps of 3D cell culture, compound treatment, sheet preparation, dyeing and counting of one or more of micronucleus, nuclear buds and nuclear bridges of binuclear cells by a cytokinesis method, wherein the 3D cell culture adopts a scaffold-free culture method combining a hanging drop method and an ultra-low adsorption culture method, and cell spheres cultured in a hanging drop manner for 2-4 days are inoculated into an ultra-low adsorption 96-well plate and continuously cultured until the 5-8 th day.

In a specific embodiment, the scaffold-free culture method inoculates cell spheres cultured in hanging drops for 3 days into an ultra-low adsorption 96-well plate to continue culturing until day 7.

In a specific embodiment, the cells to be detected in the 3D cell culture are humanized hepatocyte HepG2 cells.

In a specific embodiment, the 3D cell culture comprises:

(1) subjecting the cells in logarithmic growth phase to trypsinization;

(2) diluting the cell suspension after digestion to a corresponding density, e.g. 2.5X 10⁵/mL；

(3) Inoculating the cell suspension at 20 μ L/drop into the inside of an inverted 100mm culture dish lid;

(4) culturing the cell suspension, and forming a cell sphere with initial inoculation number inside the hanging drop;

(5) and (4) transferring the cell spheres obtained in the step (4) to a U-shaped bottom 96-well plate for continuous culture, and replacing the culture medium every other day.

In a specific embodiment, the initial inoculation number in step (4) is 5000 cells/droplet, and the culture time in step (5) is 5-8 days. Preferably, the culture time is 7 days.

In one embodiment, the compound treatment comprises: sample adding, cleaning, digesting and centrifuging; preferably, the treatment time of digestion is 6-8 min.

In a specific embodiment, the flaking and staining comprises: hypotonic treatment, fixation and staining; preferably, the time of the hypotonic treatment is 3-5 min.

In one embodiment, the compound-treated positive control is an adduct formed by crosslinking and formation of DNA; preferably, the positive control is mitomycin C, CAS number 50-07-7.

In a specific embodiment, the genotoxicity refers to a toxic effect caused by physical and chemical factors in the environment acting on an organism to cause various damages to its genetic materials at the chromosome level, the molecular level and the base level; preferably, the genotoxicity is that of a chemical substance.

In a specific embodiment, the chemical substance is a genotoxic compound requiring metabolic activation, an aneuploid inducer, a suspected genotoxic compound, or a non-genotoxic compound; preferably, the chemical substances are benzopyrene, cyclophosphamide, colchicine, (-) -epigallocatechin gallate, furfuryl thioacetate and amiodarone.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows:

the invention successfully applies the 3D hepatocyte model to micronucleus cytology detection, establishes a 3D hepatocyte in-vitro micronucleus cytomics detection method and provides more genotoxicity information related to human body tests. The method can use the humanized liver cell HepG2 as a genetic toxicity detection material, avoids the problem that the species difference can not effectively extrapolate the test data when the genetic toxicity detection is carried out by using microorganisms or animal cell strains, and can detect the substances which need to be metabolized and activated to generate the genetic toxicity under the condition of not adding exogenous metabolic enzyme S9.

(1) The method has the advantages that the step of culturing the 3D hepatic cells is improved, and the 3D cell spheres are an aggregate with a compact structure, so that cells cannot be completely digested by adding pancreatin for 2-3 min conventionally, the digestion time needs to be prolonged, but the digestion step is not too long, otherwise, excessive digestion is caused, cell membranes are damaged, the cell state is affected, and the step of vortex is added. The invention can ensure that the cell spheres can be fully digested into single cell suspension, can avoid the influence of cell membrane rupture on the subsequent tabletting process due to overlong digestion time, and finally ensures the accuracy and the effectiveness of the detection method.

(2) Hypotonic time affects the quality of the final slide, hypotonic time is insufficient, cytoplasm does not expand, and cytoplasm is darker after staining and is not greatly distinguished from cell nucleus. And two cell nucleuses can not be completely separated, so that the judgment of the binuclear micronucleus, the nuclear buds and the nuclear bridge is influenced. Hypotonic conditions are too long, the cytoplasm is over-inflated, and the cell membrane is ruptured. Only the nucleus after the cell membrane rupture can be seen under the mirror, and the scoring of the picture cannot be carried out. The hypotonic time of the present invention can ensure that the cells are fully expanded and the cells are not broken due to excessive hypotonic.

(3) The invention inoculates the cell spheres cultured by hanging drop for 3 days into an ultra-low absorption (ULA) 96-well plate for continuous culture, can realize the purpose of prolonging the culture time, and adds compounds for subsequent genotoxicity detection. The culture mode of the support-free sphere is simple and easy to standardize, and the accessibility, repeatability, stability and high-throughput detection capability of 3D hepatocyte genotoxicity evaluation are greatly enhanced. By selecting proper culture conditions, the 3D hepatocyte spheroids can have molecular phenotype and function superior to those of 2D cells, and are more suitable for in vitro genotoxicity evaluation.

Drawings

FIG. 1 is a micronucleus cytomics observation index morphological feature.

FIG. 2 shows spheres (10X 20) of 2D HepG2 cells and 3D HepG2 cells cultured for 3 days.

FIG. 3 is a comparison of the urea concentration in the supernatant of the 2D model and the 3D cell model at different incubation times; wherein

n＝5；**：P≤0.01。

FIG. 4 is a comparison of albumin concentrations in supernatants of 3D models versus 3D cell models at different incubation times; wherein

n＝5；**：P≤0.01。

FIG. 5 is a real-time fluorescent quantitative PCR analysis of metabolic enzymes in 2D and 3D cultures; wherein

n＝3；*：P≤0.05；**：P≤0.01；***:P≤0.001。

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Main reagent, culture medium and preparation of reagent

High glucose DMEM medium (Gibco, usa); fetal bovine serum (BI, usa); 0.25% pancreatin-EDTA digest (Gibco, USA); 100 × streptomycin mixture (Invitrogen, usa); phosphate Buffered Saline (PBS) (Solebao, China); DMSO (Sigma, usa); MMC (Sigma, usa); Cyto-B (Sigma, USA); giemsa dye liquor kit (solibao, china); potassium chloride (national chemical group, chemical reagents limited, china); methanol (national chemical group, chemical reagents limited, china); glacial acetic acid (national chemical group, chemical agents limited, china); BaP, COL, CPA, amiodarone (sigma, usa); EGCG, furfuryl thioacetate (mclin, china); urea kit, albumin (BCG method) kit (BioAssay Systems, usa); miRNeasy Mini extraction kit (QIAGEN, usa); evo M-MLV reverse transcription reagent premix, ROX Reference Dye solution (20. mu.M), SYBR Green Pro Taq HS premix type qPCR kit (Ecoyor, China); primers (Thermo corporation, usa); fetal Bovine Serum (FBS) (BI, usa).

HepG2 cells were purchased from the cell resource center of Shanghai Biotech, Chinese academy of sciences. The cells were stored in liquid nitrogen and 3-10 passages were used for subsequent experiments after resuscitation.

Cyto B stock solution: quantitative Cyto B was weighed and dissolved in DMSO, and sonicated until dissolved. The stock solution concentration is 5mg/mL, and the stock solution is stored at-20 ℃ for one month and at-80 ℃ for six months.

Hypotonic solution: weighing 2.796g of potassium chloride in a beaker, adding a proper amount of deionized water, uniformly mixing until the potassium chloride is completely dissolved, supplementing the volume to 500mL, uniformly mixing, transferring the solution into a glass bottle, marking, and storing at room temperature for later use.

Fixing liquid: the required volume is prepared according to the proportion of methanol to glacial acetic acid which is 3:1, and the preparation is carried out on site.

Giemsa application liquid: and diluting the Giemsa concentrated solution with a Giemsa diluent at a ratio of 1:9 just before dyeing to prepare a Giemsa application solution.

Main instrument and consumable

CLM-170A-B carbon dioxide incubator (ESCO corporation, Singapore); 500-II-A/B3 biosafety cabinet (Shanghai Ruiyang clarification plant Co., Ltd., China); CX21 optical microscope (olympus, japan); cytometric instrument (Chemometec A/S, Denmark); ST16R centrifuge (Thermo Fisher, usa); IMT-2-21 inverted microscope (Olympus, Japan); pipettors and associated tips (Eppendorf, germany); ultra low adsorption U-bottom 96-well plates (Corning, usa).

The test compounds and information on the compounds and formulation methods are shown in tables 1 and 2, respectively.

Table 1 test compound information and formulation method

Table 2 information on compounds and methods of preparation

Positive control

The judgment of sensitivity is explained in the test system of the present invention. The three indexes of the invention represent different genotoxicity mechanisms respectively and are evaluated independently. Detection of any of the three indicators represents a possible genotoxic insult.

Example 1 cell culture and Compound treatment

The CBMN-cyt assay was performed according to the method recommended by the guidelines of the in vitro mammalian cell micronucleus test (OECD TG487,2016) (FENECH M. Nature Protocols,2007,2(5): 1084-1104).

1.12D cell culture and treatment

HepG2 cells in logarithmic growth phase were trypsinized to prepare single cell suspensions and cultured at 1X 10⁵The density of each well is inoculated in 6-well cell culture plate, the temperature is 37 +/-1 ℃, and the CO content is 5 +/-1 percent₂And (5) humidifying and culturing. After 24h, the original medium was aspirated off, and the prepared medium containing the compound and having a final concentration of 5. mu.g/mL cyto-B was added for cultureAnd (4) liquid. Each treatment group treated 2 wells in parallel per condition, loading volume 4 mL. Meanwhile, DMEM medium containing solvent and cyto-B with final concentration of 5 mug/mL is set as a control group. Negative and positive control administration preparations were allowed to react with the cells for about 48h (> 1.5 doubling cycles), respectively, and the culture medium was discarded. After the treatment, the cells were digested and prepared into cell suspension, and 2D cell suspension per well was collected in a 15mL centrifuge tube, centrifuged (1000 rpm. times.5 min), and the supernatant was discarded.

1.23D cell culture and treatment

Diluting the cell suspension to the corresponding density of 2.5X 10⁵and/mL. 10mL of PBS buffer was added to the bottom of a 100mm dish. Using a multichannel pipettor, the cell suspension at the above cell density was seeded at 20. mu.L/drop into the inside of inverted 100mm dish lids, each of which was seeded with 50 drops. And slightly turning the dish cover to place the dish bottom, transferring the dish containing the hanging drops into a carbon dioxide incubator, and horizontally placing the single layer. After 3 days of culture, cell spheres with an initial seeding number of 5000 cells/droplet were formed inside the hanging drop. And on day 3, transferring the hanging drop inner spheres to an ultra-low adsorption U-shaped bottom 96-well plate for continuous culture, and replacing half of the culture medium every other day until day 7. The experimental result shows that the initial inoculation number of the cell spheres is 5000 cells/drop, the liver specificity index and the metabolic enzyme gene expression quantity of the cell spheres cultured for 7 days are the highest, and the cell spheres are the optimal 3D liver cell model culture conditions.

1.3 measurement of albumin and Urea content

(1) And (6) collecting the supernatant. Selecting 2.5X 10⁵Each mL-1 cell suspension was used as an initial cell seeding density to inoculate cell pellets, the cells were cultured in the culture format of 1.2, and cell samples and supernatants were collected on specific days (3, 7, 10, 14 days). The 2D cell sample is inoculated in a standard flat-bottom 6-well plate by using the cell suspension with the same density, and the cell sample and the supernatant are collected after 3 days of conventional culture. Each cell culture sample was subjected to a total change 24h before sampling (reagents would bind to albumin in bovine serum and should be treated with serum-free medium for 24h), and after 24h, the supernatant was aspirated and frozen at-20 ℃ for subsequent detection.

(2) Digesting and counting. The remaining cell spheres (n-12) were collected in a 2mL EP tube, and after the spheres had settled to the bottom of the tube naturally, 1mL PBS was added to rinse twice and the supernatant was discarded. Subsequently 500 μ L of pancreatin digestion (37 ℃, 3min) was added, followed by vortexing to lyse the spheres as small as possible into small clumps, digestion was continued (37 ℃, 3min) and then 1mL of complete medium was added to stop digestion, and the total number of cells was counted to estimate the number of cells at each time point. The remaining supernatant in the 6-well plate was aspirated away, the 2D cells were digested, centrifuged, resuspended, made into a cell suspension and the total number of cells counted for estimation of cell number.

(3) And (4) measuring the urea content. And (5) detecting the urea content according to the instruction of the urea detection kit. Mixing the solution A and the solution B at normal temperature in a ratio of 1:1 to prepare working solution. 5 mu L of 5 mg. dL-1 urea standard substance, deionized water and a sample to be detected are put into a 96-hole plate, 200 mu L/hole working solution is added into each hole, and the incubation is carried out for 50min at room temperature. The absorbance value at 430nm was obtained using an IX6 fluorescence microplate reader. And the calculation is carried out according to the following calculation formula, and the data are normalized to 6 multiplied by 10⁴And (4) cells.

Concentration calculation formula:

OD samples, OD blank and OD standards are the absorbance of the sample, blank control and standard, respectively, and n is the dilution factor. When the urea sample was tested, [ STD ] ═ 5.

(4) And (4) measuring the content of albumin. Detecting the content of albumin according to the specification of the albumin detection kit. Before use, the reagents were allowed to stand at room temperature and shaken gently. The standards of different concentrations were diluted with distilled water. A clear flat-bottomed 96-well plate was taken, 15. mu.L of diluted standards (0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 5.0 mg. dL-1) and samples were placed in different wells, 200. mu.L of the reagent was added to each well, and after incubation for 5min, absorbance values at 620nm were obtained using an IX6 fluorometer. A standard curve was generated using absorbance values of standards and blanks to determine albumin concentration in samples, and data was normalized to 6X 10⁴And (4) cells.

1.4 measurement of expression amounts of I-phase and II-phase metabolizing enzyme genes

(1) Collecting a sample: 2D flat growth HepG2 cells cultured in T25 culture flask and 3D cell balls cultured for 3 and 7 days were digested, and the number of cells was determined (not more than 1X 10)⁷). The cell suspension was transferred to a centrifuge tube and centrifuged at 1000rpm × 5min and the supernatant was aspirated. The cell pellet was thoroughly loosened by flicking the bottom of the tube and the cells lysed by adding an appropriate volume of Buffer RLT in miRNeasy Mini extraction kit. The cell lysate can be stored at-70 ℃ for several months.

(2) Extraction of total RNA: the collected frozen lysate samples were incubated in a water bath at 37 ℃ until completely thawed and the salts dissolved, total RNA isolated using miRNeasy Mini extraction kit and stored on ice.

(3) An RT reaction solution is prepared by using an Evo M-MLV reverse transcription reagent premix and 500ng of Total RNA according to a proportion, and is transferred into an eight-connected tube, wherein the reverse transcription system is 10 mu L. The reverse transcription reaction condition is 15min at 37 ℃; 5sec at 85 ℃; 4 ℃ is prepared.

(4) Using SYBR

Premixing a Premix Pro Taq HS qPCR Kit reagent, a primer and a cDNA product prepared by the previous reaction in proportion, and reacting on a Step One fluorescent quantitative PCR instrument under the reaction conditions that: 95 ℃ for 30sec (1 cycle); 95 ℃ 5sec, 60 ℃ 30sec (40 cycles); and establishing a PCR product dissolution curve.

(5) Data were recorded using a 7500Fast Real-Time PCR system (Applied Biosystems, usa) with 3 replicate wells per sample. According to the RT-PCR detection result, the gene expression of the 2D cell sample is taken as a control, and delta C is adopted_TThe method calculates the relative expression quantity of the gene to be detected of each object to be detected relative to the reference gene beta-actin.

1.5 statistical analysis

The results of urea, albumin and RT-PCR all adopt an independent sample t test method, a 2D cell model group is taken as a control group, and the values of 3D cell spheres under different culture days are compared with the control group.

TABLE 3 phase I and phase II metabolising enzyme gene primer sequences

Microscopic observations of 3D hepatocyte spheroids and conventionally cultured cells showed that 2D and 3D cells exhibited different cell morphologies after 3 days of continuous culture (see fig. 2). HepG2 cells in conventional culture grew flat and had a distinct cell morphology. HepG2 cells cultured in hanging drops for 3 days form a compact cell sphere by self-aggregation under the action of gravity, and the cell morphology is not obvious.

Comparison of differences in liver-specific function of HepG2 cells from different culture regimes using albumin production and urea levels as detection indices, values normalized to 1X 10⁶The number of individual cells to eliminate the differences in cell number between samples. The supernatant albumin concentration of 3D cultured cells was significantly increased compared to 2D cells by measurement of supernatant samples from 3 to 14 days, with the highest value at day 7 (see fig. 3). At the same time, the urea content of the 3D sphere supernatant reached a maximum on day 7 (see fig. 4). Since 3D cell balls with the culture time of 3 days and 7 days show better cell functions and the culture time is shorter, 3D cell balls with the culture time of 3 days and 7 days are further selected to be compared with the expression quantity of the I, II-phase metabolic enzyme gene of 2D cells, and the proper 3D cell culture time is selected.

Low levels of drug metabolizing enzymes in HepG2 cells may result in undetectable substances requiring metabolic activation. The real-time fluorescent quantitative PCR analysis showed that the expression level of different I, II phase metabolic enzymes in 3D HepG2 spheroids was generally higher than that of 2D cultured HepG2 cells (see fig. 5). 7500Fast Real-Time PCR System analyzes the level of transcription of phase I metabolizing enzyme mRNA during the culture of spheroids. The 3D HepG2 cells cultured for 3 days have obviously increased CYP1A1 and UGT1A6 gene expression, and the CYP1A2, CYP3A4, CYP2E1, CYP2B6, UGT1A1 and 2D cells have no obvious difference. The expression levels of CYP1A1, CYP2C9 and CYP2D6 of 3D HepG2 cells cultured for 7 days are remarkably increased compared with 2D cells, and the expression levels of CYP1A2, CYP3A4, CYP2B6 and CYP2E1 are consistent with those of 2D cells. The mRNA level of the phase II xenobiotic metabolic enzyme UGT1A6 of the 3D HepG2 cells cultured for 7 days is remarkably increased compared with that of the 2D cells, and the expression level of UGT1A1 is slightly higher than that of the 2D cells.

The ULA96 well plates containing the 3D cell spheres were removed from the incubator on day 7, the original medium was aspirated as far as possible, and different concentrations of positive compound and final concentration of 5 μ g/mL cyto-B in each well were added. Each treatment group was treated in parallel with 12 wells for each condition, and the loading volume was 100. mu.L. Meanwhile, DMEM medium containing solvent and cyto-B with final concentration of 5 mug/mL is set as a control group. The negative and positive control drug preparations were allowed to react with the cells for about 48 hours, respectively, and the culture medium was discarded and the spheres in 6 wells of each group were collected in 15mL centrifuge tubes, which were collected in two tubes. Washed 2 times with PBS and the supernatant discarded. Adding 300 μ L of pancreatin for digestion for 6min, blowing to remove cell mass, adding 1mL of complete culture medium to stop digestion, adding 1mL of pancreatin in EP tube to resuspend cell spheres, and placing in a 37 deg.C incubator for digestion for 3 min. At this point the cell spheres were initially digested and placed on a vortexer to vortex, allowing the intact cell spheres to lyse into smaller cell clumps. And then continuously placing the mixture in an incubator at 37 ℃ for continuously digesting for 3-5 min, and then continuously placing the mixture on a vortex instrument for vortex or blowing the mixture by using a pipette until no obvious cell mass exists. After the cell spheres had been completely digested into a single cell suspension, they were centrifuged (1000 rpm. times.5 min) and the supernatant was discarded. As shown in Table 4, the experimental results show that 6-8 min is the optimal digestion time of the 3D cell spheres, digestion within the time can ensure that the cell spheres can be fully digested into single cell suspension, the influence of cell membrane rupture on the subsequent sheet making process due to overlong digestion time can be avoided, and the accuracy and the effectiveness of the detection method are finally ensured. Compound information and formulation methods are shown in table 2.

TABLE 4 Effect of digestion time on cell suspension preparation

1.3 flaking and staining

Hypotonic treatment: after centrifugation the supernatant was discarded as much as possible and the tube was flicked and the cells were resuspended in the remaining small volume of medium to ensure that the cells did not form clumps. Slowly adding 1mL of hypotonic solution at a pre-temperature of 37 ℃ into the resuspended cells, shaking the resuspended cells while adding the hypotonic solution, and performing hypotonic treatment at room temperature for 3-5 minutes.

Fixing: 3mL of the fixative was added dropwise slowly per set, and after the fixation was completed, the mixture was centrifuged (1000 rpm. times.5 min), and the supernatant was discarded. Each centrifuge tube was further added dropwise with about 3 mL/piece of the fixative followed by centrifugation (1000 rpm. times.5 min) and the supernatant discarded. And (4) dripping: and (2) leaving about 0.1-0.2 mL of fixing solution in the centrifugal tube, slightly and uniformly mixing to prepare a cell suspension for preparing a drop slide, strictly cleaning the glass slide, holding the glass slide by one hand to be slightly inclined, sucking a small amount of uniformly mixed cell suspension by the other hand, dropping 2-4 drops to one end of the glass slide to enable the glass slide to be naturally and uniformly dispersed downwards, drying at room temperature, and placing the prepared chromosome slide at room temperature or an incubator for drying to facilitate dyeing. The slides were completely air dried for at least 30 minutes, possibly overnight.

Dyeing: the drop preparations were prepared and numbered. Immersing the fixed slide specimen into Giemsa application liquid, and dyeing for 25-30 min; and (4) washing the dyed specimen with deionized water, and naturally drying at room temperature.

As shown in Table 5, 3 to 5min is the optimum hypotonic time. The hypotonic time ensures that the cells are not only fully expanded, but also not too hypotonic to rupture.

TABLE 5 Effect of hypotonic time on stained slides

1.4 microscopic examination index and statistical analysis

Microscopic examination index

The numbers of mononuclear cells, binuclear cells and polynuclear cells among 500 cells were counted to calculate the Index of Proliferation of cytokininism-Block Proliferation (CBPI) and Replication Index (RI). CBPI represents the average number of cell cycles per cell during cyto-B exposure and can be used to calculate cell proliferation. RI represents the relative number of nuclei in the treated and control cultures and can be used to calculate the rate of cytostatic (Cytostasis).

Cytostasis(％)＝100-100{(CBPIT-1)/(CBPIC-1)}

CBPI ═ number (monocyte number +2 × number of binuclear cells +3 × number of multinucleated cells)/(total cell number)

T-treatment group C-control group

b. The percentage of MN, Nbaud and NPB present in each 1000 binuclear cells was scored according to the Fenech method (FENECH M, et al. Mutagenesis,2010,26(1):125- & 132).

Statistical analysis

Using SPSS.18, the Fisher exact test method was used to compare the MN ‰, Nbds ‰ and NPBs ‰ of the chemically treated group with the vehicle control group for significance (P ≦ 0.05).

The results show that (1) MN permillage, Nbdpermillage and NPB permillage induced by the test article are obviously increased, and concentration dependence exists;

(2) MN thousandths, Nbdl thousandths and NPB thousandths induced by a certain test point are obviously increased and can be repeated.

If the above is met, the positive result can be judged, otherwise, the negative result is judged. Statistical methods are not the only criteria for evaluating positive results, taking into account biological significance.

The results are shown in fig. 1, the concentration groups of MN ‰, 0.05, and 0.1 μ g/mL in the 2D model group at 4 treatment concentrations were significantly increased (p ≦ 0.05), and had dose-dependence, while NPB ‰ was significantly different from 0.025-0.1 μ g/mL, but had no dose-dependence (see table 5). The cell growth inhibition rate (Cytostasis) and RI of each concentration of 3D hepatocyte micronucleus cytomics were comparable to those of the 2D hepatocyte model group. Under the treatment concentration of 4 MMCs in the 3D liver cell model group, both MN per mill and Nboud per mill are obviously increased (p is less than or equal to 0.05), and each concentration group of NPB per mill is not obviously increased (see table 6). The lowest detection concentration of MN ‰ and Nbd ‰ of the 3D model group MMC is 0.0125 μ g/mL, and the lowest detection concentration of the 2D model group MMC is 0.025 μ g/mL.

The results are shown in tables 6 and 7, respectively.

TABLE 6 MMC 2D cell micronucleus cytomics assay results

*: p is less than or equal to 0.05. CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: a nuclear bridge; NPB: nuclear bridge

TABLE 7 MMC 3D cell micronucleus cytomics assay results

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: nuclear bridge

This example shows that after treatment with positive compound MMC, 2D and 3D cultured HepG2 cell models can detect significant increases in micronuclear cytomics indicators within a certain cytotoxicity range.

And comparing the CBMN-cyt detection results of the MMC of the 2D hepatocyte model and the 3D hepatocyte model. In the aspect of cell proliferation inhibition detection, the cytotoxicity index 100-RI of 2D liver cell and 3D liver cell models and the cell growth inhibition rate are increased in concentration correlation, and the suggestion is that MMC causes cell damage of similar degree to 2D liver cell and 3D liver cell spheres. In the aspect of genetic toxicity detection, the minimum detection concentration of MN ‰ and Nbdl ‰ of the MMC in the 3D model group is 0.0125 μ g/mL, the minimum detection concentration of the MMC in the 2D model group is 0.025 μ g/mL, NPB positive is not detected in the concentration range of the 3D cell model, NPB positive is detected in the 2D cell model but has no dose correlation, and the value of NPB falls within the negative reference range of 0-10 ‰ of CBMN-cyt test of peripheral blood lymphocytes (FECH M, et al. Suggesting that MMC may not cause genotoxic damage by inducing DNA strand break errors and telomere end fusion in HepG2 cells. Similar to the results of micronuclear cytomics studies of existing HepG2 and derived cell lines (DIAMOND L, et al. Carcinogenesis,1980,1(10): 871-.

After treatment with a positive compound MMC, the 3D hepatocyte model can detect an increase in the statistical significance of micronuclear cytomics indicators within a certain cytotoxicity range. The CBMN-cyt method can be successfully applied to HepG2 liver spheres, and a 3D hepatocyte micronucleus cytomics method is initially established. Compared with MMC micronucleus cytomics results of the 3D model and the 2D model, detection sensitivity of the 3D hepatocyte model MN and Nbdd is higher than that of the 2D hepatocyte model. The compound detection range of 3D hepatocyte micronucleus cytomics is further expanded, and the method is further verified by using genotoxic compounds, suspected genotoxic compounds and negative compounds with different mechanisms.

Example 23 validation of hepatocyte micronucleus cytomics assay method

The invention selects two genetic toxicity compounds benzopyrene (BaP) and Cyclophosphamide (CPA) which need to be metabolized and activated, an aneuploid inducer Colchicine (COL), two suspicious genetic toxicity compounds (-) -epigallocatechin gallate (EGCG) and furfuryl thioacetate and a non-genetic toxicity compound amiodarone according to the recommendation of an in vitro mammalian cell micronucleus test (OECD TG487,2016), verifies the established 3D hepatocyte micronucleus cytomic test method, and explores the applicability of the method for compounds with different mechanisms.

In this example, 6 representative substances with different genotoxicity mechanisms were selected to preliminarily verify the in vitro 3D hepatocyte micronucleus cytomics approach, and the experimental results were compared with those of micronucleus cytomics under the conventional cell culture approach.

Benzopyrene (BaP) is a known indirect mutagen and the result is positive in MN test. BaP as a precancerogen belongs to polycyclic aromatic hydrocarbon compounds, and can be activated by CYPs (1A1 and/or 3A4) and microsome epoxide hydrolysate in turn to generate genetic toxicity. When HepG2 cells are exposed to BaP, different BaP metabolites (BaP-4, 5-diol; BaP-7, 8-diol; BaP-9,10-diol), some quinone compounds and various hydroxyl metabolites are produced; the major DNA adducts are generated by the BaP-7,8-diol-9.10-epoxide (BPDE) reaction and are the final carcinogenic intermediates (KIRCHNER S, ZELLER A. mutation Research/Genetic homology and Environmental Mutagenesis,2010,702(2): 193-198).

Colchicine (COL) is a typical aneuploid inducer with the characteristics of typical non-carcinogenic mutagenic agents. Positive in vivo and in vitro micronucleus assays. It causes numerical aberration of chromosomes due to metaphase arrest caused by inhibition of tubulin polymerization. In vitro micronucleus assays, colchicine both specifically induced micronucleus increase in CHL cells and human lymphocytes, L5178Y cells (KNASMulER S, et al. mutation research,1998,402(1-2): 185).

Cyclophosphamide (CPA) must be metabolically activated in vivo to exert cytotoxic effects. In vivo, about 70-80% of CPA is metabolized to generate intermediate products through CYP2B6, CYP3A4, CYP3A5, CYP2C9 and CYP2C19, and the intermediate products are easy to generate phosphoramide mustard and acrolein through non-enzymatic elimination reaction, thereby generating genetic toxicity (Chenling Yan et al. pharmaceutical science, 2014,49(7): 971-976.).

(-) -epigallocatechin gallate (EGCG) is the main component of green tea extract, and positive results are shown in L5178Y tk +/-mouse lymphocyte in vitro mammalian gene mutation test through oxidative stress reaction, and negative results are shown in vivo micronucleus study (UEHARA T, et al. toxicology,2008,250(1): 15-26). According to the invention, EGCG is used as a model compound with non-in-vivo genetic toxicity and positive in-vitro genetic toxicity detection, and whether the in-vitro 3D liver cell model has better prediction capability on suspicious genetic toxic substances is further evaluated. The experimental result shows that in the 2D cell culture, MN of the 62.5-500 mu g/mL dose group has a positive result. It has been reported that EGCG or catechins (a family of chemicals to which EGCG belongs) produce H under in vitro culture conditions₂O₂Therefore, it can be a positive reaction in vitro genotoxicity test (JOHNSON M K, LOO G. Mutat Res,2000,459(3): 211-.

Furfuryl thioacetate is a commonly used food flavor. Furfuryl thioacetate was found to be cytotoxic (< 80% relative cell density) and genotoxic in the Blue Screen assay; ames test is negative result; the in vitro human peripheral blood lymphocyte micronucleus test is positive in the dose groups of 100 mug/mL (-S9,3h) and 43 mug/mL (-S9,24 h); mice were negative in micronucleus experiments (API AM, et al. food and Chemical biology, 2020,144: 111615.). Taken together with in vitro and in vivo experimental data, furfuryl thioacetate can be considered as a non-genotoxic compound.

Amiodarone is a widely used antiarrhythmic drug for the treatment of the most common arrhythmia type of atrial fibrillation, a non-genotoxic hepatotoxic drug (UEHARA T, et al. toxicology,2008,250(1):15-26.)

2.1 Micronucleated cytomics detection of benzopyrenes

The results of micronuclear cytomics assay of benzopyrene are shown in tables 8 and 9.

Compared with a solvent control group, the MN per thousand of the 2D liver cell model group at each treatment concentration is obviously increased, the Nbdc per thousand of the 2D liver cell model group at the concentrations of 10.09 and 20.19 mu g/mL is obviously increased (p is less than or equal to 0.05), and no obvious cytotoxicity is shown in BaP within the range (see table 8).

Compared with the solvent control group, the 3D cell model group does not show obvious cytotoxicity, MN thousandths and Nbdthousandths are obviously increased (p is less than or equal to 0.05) under the treatment concentrations of 2.52, 5.05, 10.09 and 20.19 mu g/mL, and NPB thousandths are obviously increased only under the concentration of 10.09 mu g/mL, but dose correlation does not occur (see table 9). Under the same dosage, MN per mill of the 3D cell model group is higher than that of the corresponding 2D cell model group, and the index sensitivity of the 3D cell model Nbdc per mill is higher than that of the 2D cell model group.

TABLE 8 2D cell micronucleus cytomics assay results for BaP

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: nuclear bridge

TABLE 9 3D cell micronucleus cytomics assay results for BaP

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: nuclear bridge

Both 2D and 3D cell models can detect the genetic toxicity of BaP through the index of MN. Compared with the experimental result of the 2D cell model, the MN rate of the 3D cell model experiment is generally higher than that of the 2D cell model, and the significant increase of Nbdd can be detected at a lower concentration, so that the related information of BaP genetic toxicity is provided.

2.2 micronucloeomics detection of colchicine

The results of micronuclear cytomics assay of colchicine are shown in tables 10 and 11.

RI and cytostatic rates of the 2D cell model group showed that cytotoxicity of COL increased with increasing concentration. Compared with the solvent control group, MN per thousand and Nbdc per thousand of the 2D cell model group are increased firstly under the treatment concentration of 0.0025-0.01 mu g/mL, and are reduced under the treatment concentration of 0.02 mu g/mL (see table 10).

Compared with a solvent control group, the 3D cell model group has obviously increased MN thousandths, Nbdthousandths and NPB thousandths of 0.0025-0.02 mu g/mL (p is less than or equal to 0.05), and has concentration-dependent cytotoxicity (see table 11). The NPB ‰ of the positive control group is significantly different, but falls within the recommended negative range of 0-10 ‰ (LORGE, et al. mutation Research/Genetic diagnosis and Environmental mutation, 2006,607(1): 13-36). Compared with a 2D hepatocyte model group, the 3D hepatocyte micronucleus cytomics method has higher sensitivity in detecting COL, and can detect the genotoxicity of COL through MN, Nbdu and NPB in a larger concentration range.

TABLE 10 COL 2D cell micronucleus cytomics assay results

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: nuclear bridge

TABLE 11 COL 3D cell micronucleus cytomics assay results

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: nucleoplasm

The 2D results in this example show that MN% and Nbud% increase first at a concentration of 0.0025-0.01 μ g/mL and then decrease at a high dose of 0.02 μ g/mL compared to the solvent control. This may be due to cell loss due to mitotic arrest. In addition, since COL induces mitotic arrest, RI indices etc. may over-reflect this strong cytostatic activity (especially over a longer treatment time of 48 h) compared to acute cell death. Thus, aneuploidy inducers may inhibit micronuclei formation through mitosis in a high toxic concentration range.

In detecting COLs, inappropriate concentration intervals may reduce the sensitivity to MN of COL, and too large concentration intervals may miss the maximum or even weaker concentrations, so that appropriate concentration intervals are critical to the detection of COL. In the 3D hepatocyte model, RI and cell growth inhibition rate indexes show that a hepatocyte spheroid has a certain resistance effect on cell proliferation inhibition toxicity of COL, and genetic toxicity of COL can be detected by MN, Nbdd and NPB within the range of 0.0025-0.02 mu g/mL.

2.3 Microcytomics assay for cyclophosphamide

The results of the micronucleocytomic detection of cyclophosphamide are shown in tables 12 and 13.

The RI and cytostatic rates of the 2D cell model group showed a concentration-dependent increase in CPA cytotoxicity. Compared with a solvent control group, MN thousandths under the treatment concentration of 312.5-25000 mu g/mL are obviously increased (p is less than or equal to 0.05), and Nbdthousandths and NPB thousandths of 312.5 and 2500 mu g/mL are obviously increased without obvious concentration correlation (see table 12).

Compared with a solvent control group, the micronucleus related index of the CPA group is remarkably increased by MN per thousand within the dosage range of 312.5-2500 mug/mL; within the dosage range of 625-2500 mu g/mL, Nbou per mill is obviously increased, and NPB per mill is not obviously increased (see Table 13). On the index of Nboud ‰, the 3D cell model is more sensitive than the 2D cell model, and has dose-dependence.

Table 12 CPA 2D cell micronucleus cytomics assay results

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: nuclear bridge

Table 13 CPA 3D cell micronucleus cytomics assay results

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: nuclear bridge

In the embodiment, MN ‰ is obviously increased (p is less than or equal to 0.05) under the treatment concentration of 312.5-2500 [ mu ] g/mL. In the 3D liver cell group, the MN per thousand is obviously increased (p is less than or equal to 0.05) under the treatment concentration of 312.5-2500 mu g/mL. The micronucleus induction rates of the 2D and 3D HepG2 cell models were relatively similar, probably because the CYP2B6 and CYP3a4 metabolic enzyme genes that induce CPA metabolism to produce intermediates did not show enhanced expression in the 3D hepatocyte model. The 3D hepatocyte model group is 625-2500 mu g/mL Nboud per mill remarkably increased, has a certain dose correlation, and is sensitive compared with a 2D model.

2.4-Epigallocatechin gallate Microcytomic assay

The results of micronucloemic detection of (-) -epigallocatechin gallate are shown in tables 14 and 15.

RI and cell growth inhibition rate of the 2D cell model group show that EGCG has obvious cytotoxicity to 2D liver cells within the concentration range of 62.5-500 mug/mL. Compared with a solvent control group, MN per thousand of each concentration group is obviously increased (p is less than or equal to 0.05); 250. nboud per mill of the 500 μ g/mL dose group was significantly elevated; no significant difference was found in the NPB ‰ concentration groups (see table 14).

The RI and cytostatic rate of the 3D cell model group showed that EGCG had significant cytotoxicity on 3D hepatocytes only in the 500 μ g/mL dose group, MN ‰, Nbud ‰ of the 250, 500 μ g/mL dose groups were significantly increased, and NPB ‰ of each concentration group was not significantly increased (see table 15).

TABLE 14 2D cell micronucleus cytomics assay results for EGCG

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: nuclear bridge

TABLE 15 3D cell micronucleus cytomics assay results for EGCG

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: nuclear bridge

The concentration groups of MN thousandths, Nbdthousandths and NPB thousandths are not obviously increased in the dosage range of 62.5 and 125 mu g/mL by the 3D hepatocyte micronucleus cytomics method. Cytotoxicity and genetic toxicity appeared in the 250, 500. mu.g/mL dose groups. Genotoxicity occurred in both the 2D and 3D cell model groups within about 20% of the same cytotoxicity range.

2.5 micronucleus cytomics assay of furfuryl thioacetate

The results of micronuclear cytomics assay of furfuryl thioacetate are shown in tables 16 and 17.

The RI and cell growth inhibition rate of the 2D cell model group show that the furfuryl thioacetate has no obvious cytotoxicity to the 2D liver cells within the concentration range of 125-1000 mug/mL, and compared with a solvent control group, no significant difference is found among MN thousandths, Nbdthousandths and NPB thousandths of each dosage group (see Table 16). No obvious cytotoxicity or genetic toxicity is generated when 125-1000 mu g/mL of furfuryl thioacetate acts on 3D liver cells (see Table 17).

TABLE 16 2D cell micronucleus cytomics assay results for furfuryl thioacetate

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: nuclear bridge

TABLE 17 3D cell micronucleus cytomics assay results for furfuryl thioacetate

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: cell growth inhibition ratio; MN: a microkernel; nbdd: nuclear bud; NPB: nuclear bridge

In this example, neither the 2D HepG2 cell model nor the 3DHepG2 cell model detected cytotoxicity and genotoxicity of furfuryl thioacetate at the highest dose in the dose range of 1000. mu.g/mL.

2.6 micronuclear cytomics assay of amiodarone

The results of micronuclear cytomics assay of amiodarone are shown in tables 18 and 19.

The RI and cytostatic rate of the 2D cell model group showed that amiodarone acted on 2D hepatocytes with concentration-related cytotoxicity within the highest dose of 20.19. mu.g/mL. Compared with a solvent control group, no significant difference is found among MN ‰, Nbdc ‰, and NPB ‰ of amiodarone dosage groups (see Table 18). The cytotoxicity was lower for the 3D cell model group at the same concentration compared to the 2D cell model. No significant increase in genotoxicity was detected for both the 2D and 3D cell model groups (see table 19).

TABLE 18 results of 2D cell micronucleus cytomics assay of amiodarone

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: nuclear bridge

TABLE 19 results of 3D cell micronucleus cytomics assay of amiodarone

*: p is less than or equal to 0.05; CBPI: index of cytokinesis arrest; RI: a replication index; cytostasis: the rate of inhibition of cell growth; MN: a microkernel; nbdd: nuclear bud; NPB: nuclear bridge

In this example, both 2D and 3D cytomicronucleus cytotoxicity indicators RI, cytostatic rates showed concentration-dependent changes in the 51.6 μ g/mL dose range. Unlike the uniform exposure of single-layer tiled HepG2 cells, when hepatocyte spheroids were exposed to amiodarone overall, the exposure level of the internal cells was lower due to transport of material inside and outside the spheroids. In addition, 3D cells have the functions of ECM formation and whole intercellular substance information exchange after long-term culture, and can better resist the toxic effect of external substances. The 2D HepG2 cell model and the 3D HepG2 cell model of the invention do not detect the genetic toxicity of amiodarone.

TABLE 202D and 3D summary of hepatocyte micronucleus cytomics results

Note: the number represents the lowest detected concentration (. mu.g.mL)^-1) (ii) a n.t.: no detectable, or biological significance, was found in the concentration range of this study.

The invention uses positive compounds BaP, COL and CPA with different mechanisms, suspicious genotoxic compounds EGCG, furfuryl thioacetate and non-genotoxic compounds amiodarone to verify the 3D hepatocyte micronucleus cytomics method. The results that BaP, COL and CPA are positive compounds, furfuryl thioacetate and amiodarone are negative compounds, the EGCG detects the genetic toxicity in a high-concentration group, and the detected concentration is higher than that of 2D hepatocyte micronucleus cytomics are successfully detected. The 3D hepatocyte model improves the threshold value of EGCG in-vitro micronucleus cytomics detection concentration. And by integrating the detection results of all substances, compared with the conventional micronucleus cytomics, the detection sensitivity of the index Nbdu can be doubled.

Sequence listing

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<120> method for detecting genotoxicity by using 3D hepatocyte in-vitro micronucleus cytomics

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Claims (10)

1. A method for detecting genotoxicity using 3D hepatocyte in vitro micronucleus cytomics, comprising: the method is characterized in that the 3D cell culture adopts a scaffold-free culture method combining a hanging drop method and an ultra-low adsorption culture method, and cell spheres cultured in a hanging drop manner for 2-4 days are inoculated into an ultra-low adsorption 96-well plate and continuously cultured until the 5 th-8 th day.

2. The method of claim 1, wherein the scaffold-free culture method inoculates cell spheres cultured in hanging drops for 3 days into an ultra-low adsorption 96-well plate to continue culturing until day 7.

3. The method of claim 1, wherein the cells to be detected in the 3D cell culture are humanized hepatocyte HepG2 cells.

4. The method of any one of claims 1 to 3, wherein the 3D cell culture comprises:

(1) subjecting the cells in logarithmic growth phase to trypsinization;

(2) diluting the cell suspension after digestion to a corresponding density, e.g. 2.5X 10⁵/mL；

(3) Inoculating the cell suspension at 20 μ L/drop into the inside of an inverted 100mm culture dish lid;

(4) culturing the cell suspension, and forming a cell sphere with initial inoculation number inside the hanging drop;

(5) and (4) transferring the cell spheres obtained in the step (4) to a U-shaped bottom 96-well plate for continuous culture, and replacing the culture medium every other day.

5. The method of claim 4, wherein the initial inoculation number in step (4) is 5000 cells/droplet, and the culture time in step (5) is 5 to 8 days; preferably, the culture time is 7 days.

6. The method of any one of claims 1 to 3, wherein the compound treatment comprises: sample adding, cleaning, digesting and centrifuging; preferably, the treatment time of digestion is 6-8 min.

7. The method according to any one of claims 1 to 3, wherein the flaking and staining comprises: hypotonic treatment, fixation and staining; preferably, the time of the hypotonic treatment is 3-5 min.

8. The method of any of claims 1 to 3, wherein the compound-treated positive control is an adduct formed by crosslinking and formation of DNA; preferably, the positive control is mitomycin C, CAS number 50-07-7.

9. The method according to any one of claims 1 to 3, wherein the genotoxicity refers to a toxic effect caused by physical and chemical factors in the environment acting on an organism to cause various damages on genetic materials thereof at a chromosome level, a molecular level and a base level; preferably, the genotoxicity is that of a chemical substance.

10. The method of claim 9, wherein the chemical substance is a genotoxic compound requiring metabolic activation, an aneuploid inducer, a suspected genotoxic compound, or a non-genotoxic compound; preferably, the chemical substances are benzopyrene, cyclophosphamide, colchicine, (-) -epigallocatechin gallate, furfuryl thioacetate and amiodarone.

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