CN113774106B - Method for detecting genotoxicity by utilizing 3D liver cell in-vitro micronucleus cytology - Google Patents

Abstract

The invention discloses a method for detecting genetic toxicity by utilizing 3D liver cell in-vitro micronucleus cytology. The method comprises the following steps: (1) A bracket-free culture method combining hanging drop and ultralow adsorption culture is adopted, and a 3D liver cell model is constructed in vitro by taking common humanized liver cells HepG2 as a cell material; (2) Micronuclear cytologic assays were performed on compounds using 3D hepatocyte models to analyze the genotoxic mechanisms of the compounds. The in vitro detection method can analyze various genotoxicity mechanisms of the compound, improve the capability of in vitro cell models for predicting the genotoxicity of the drug, and provide information related to human body experiments.

Description

Method for detecting genotoxicity by utilizing 3D liver cell in-vitro micronucleus cytology

Technical Field

The invention belongs to the field of genotoxicity detection, and particularly relates to a method for detecting genotoxicity by utilizing 3D hepatocytes in vitro micronucleus cytology.

Background

Current 3D cell culture techniques have great potential for development in toxicology studies. As the scientific research field is increasingly paid attention to animal ethics, the 3D cell culture technology is used to meet the requirements of the 3R principle of reduction, substitution and optimization. And because of the species difference between experimental animals (such as rodents) and human beings which are commonly used for evaluating the drug toxicity, 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 screening stage of the drug, the research and development time is shortened, and the research and development cost is reduced. The 3D cell model that has been used for the genotoxicity test at present includes a 3D skin model, a 3D liver model, a 3D airway model, and the like. Because the drugs are usually taken orally and injected into human bodies to be metabolized in livers, the 3D liver cell model is developed, so that the metabolism of the drugs in vivo can be simulated in vitro, the in vitro genotoxicity screening requirements of most of the drugs are met, and a huge development prospect is shown.

In contrast to the CBMN method in published literature, the present invention employs a cytokinesis-blocking micronuclear cytohistology (CBMN-cyt) method aimed at capturing a variety of molecular events leading to chromosomal damage and chromosomal instability. CBMN counts only binuclear micronuclei, whereas CBMN-cyt requires counting micronuclei, nuclear buds, nuclear bridges of binuclear cells.

Micronuclei (MN) originate from chromosome fragments or whole chromosomes that lag the late phase of cell division. The cytokinesis-blocking micronucleus (CBMN) assay is the method of choice for micronucleus determination in human or mammalian cells in vitro, in which the micronucleus rate in cells that divide for the first time can be identified by observing binuclear cells after blocking cell division with Cytochalasin B (Cytochalasin B, cyto-B). The CBMN method has become one of the standard methods for genetic toxicology detection of human and mammalian cells due to its reliability and good reproducibility. With the study of the mechanisms of the related DNA damage biomarkers micronuclei, nuclear bridges (nucleoplasmic bridges, NPB) and nuclear buds (Nbud), cell death and apoptosis, this approach eventually evolved to a cytokinesis-block micronucleus cytome (CBMN-cyto) approach aimed at capturing all of these DNA damage events. The advantage and bright spot 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 nuclear abnormalities common in cancer, representing a common chromosome-unstable cellular phenotype. Chromosomal instability can lead to changes in the genome of the cell and has the potential to rapidly evolve and mutate, thereby forming a variety of abnormal genotypes (Wen Hai, et al, J.China J.New drug, 2016,25 (7): 787-793.6). MN is derived mainly from a chromosome fragment without centromeres or from the whole chromosome which is not contained in the daughter nucleus 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 fragments and chromosome loss (Wen Hai. The research of micronucleus cyto-histology of different liver cell lines: seventh national toxicology Congress of China society of toxicology and eighth Hubei scientific forum discussion). The mechanism of Nbbd is not known, but Nbbd production is associated with DNA damage and repair, and is a biological marker of amplified DNA elimination processes and or DNA repair complexes (FENECH M, et al Mutagenesis,2010,26 (1): 125-132). NPB is a biomarker of double-centromere chromosome caused by DNA strand breakage error and double-centromere chromosome caused by telomere end fusion (Wen Hai, et al, research on micronuclear cytology of different liver cell lines, the seventh national toxicology conference of the Chinese society of toxicology and the eighth Hubei scientific forum).

Mitomycin C (MMC) is an anti-tumor drug and positive in vitro micronucleus test. It acts as an alkylating agent to crosslink complementary DNA replicators, thereby affecting nucleic acid synthesis and function (FENECH M, et al Mutagenesis,2010,26 (1): 125-132).

The currently known detection protocol is the human peripheral blood lymphocyte CBMN-cyt protocol 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 standardized protocols. The existing 3D hepatocyte genetic toxicity detection model is cultured by adopting a hanging drop method (A three-dimensional in vitro HepG2 cells liver spheroid model for genotoxicity studies), and the method is not suitable for long-term culture and subsequent reagent addition for genetic toxicity detection because nutrient substances are reduced and culture mediums cannot be replaced.

Conventional micronuclear cytology assays involve counting apoptotic and necrotic cell numbers to reflect cytotoxicity. Because the inside of the 3D cell sphere gradually forms a necrosis core in the forming process, the detection index is easy to interfere. And it has been demonstrated that apoptotic and necrotic cells do not respond well to cytotoxicity by positive compounds without dose-dependent increase (MAES A, et al Folia Biol (Praha), 2012,58 (5): 215-220).

According to the invention, MMC is used as a positive compound, an established 3D liver cell model is used as an experimental model, an attempt is made to establish a 3D liver cell micronucleus cytology method, and the result is compared with the CBMN-cyt detection result of a 2D cell model.

The invention establishes a 3D liver cell micronucleus cytology method to count only single-nucleus, double-nucleus and polynuclear cells, and the toxicity of the compound to the cells is reflected 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 an index of cell proliferation toxicity. Therefore, the invention takes the established 3D hepatocyte spheroid as an experimental model, tries to establish a 3D hepatocyte micronucleus cytology detection method, has a great difference with the human body test result, and provides the genotoxicity mechanism information related to the compound.

Disclosure of Invention

The invention aims to solve the technical problems that in the prior art, a method for detecting the in-vitro cytogenomics of 3D liver cells is poor in correlation with human body tests and the like. The method for detecting in vitro cytogenotoxicity is closer 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 been widely accepted by the scientific community as a commonly used in vitro model and greatly enhanced the understanding of the effects of the present invention on drugs. However, there is growing evidence that in some cases 2D cells have a large gap in morphology and function from cells in vivo. In vitro toxicology studies require replication of part of the function of normal tissues for evaluation of the effects of exogenous factors (e.g. drugs, cosmetics, pollutants, oxidative environment) on cellular function and risk of carcinogenesis. Therefore, the inventor creatively uses the combination of the 3D liver cell model and micronucleus cytology to establish a novel genetic toxicity evaluation method, solves the technical problems in the field and promotes the development of an in vitro genetic toxicity detection method.

To better detect the genotoxicity of compounds and to provide information on the mechanisms associated with DNA damage, the inventors have chosen to use micronuclear cytology methods for detection. The advantage and bright spot of micronuclear cytology detection is that it allows simultaneous detection of multiple molecular events that lead to chromosomal damage and chromosomal instability.

The invention mainly solves the technical problems by the following technical means: the invention provides a method for detecting genetic toxicity by utilizing 3D liver cell in-vitro micronucleus cytology, which comprises the following steps: the method comprises the steps of 3D cell culture, compound treatment, tabletting, staining and counting one or more of micronuclei, nuclear buds and nuclear bridges of binuclear cells by a cytohistology method of cytokinesis, wherein the 3D cell culture adopts a bracket-free culture method combining a hanging drop method and an ultralow adsorption culture method, and the bracket-free culture method inoculates cell spheres which are cultured by hanging drops for 2-4 days into an ultralow adsorption 96-well plate for continuous culture until the 5 th-8 th day.

In a specific embodiment, the stentless culture method inoculates cell spheres cultured in hanging drop to 3 days into ultra low adsorption 96 well plates for continued culture 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 cells in the logarithmic growth phase to pancreatin digestion;

(2) Diluting the digested cell suspension to a corresponding density, e.g. 2.5X10 ⁵ /mL；

(3) Inoculating the cell suspension at 20 μl/drop into the inside of an inverted 100mm dish cover;

(4) Culturing the cell suspension, and forming cell spheres with initial inoculation number inside the hanging drop;

(5) Transferring the cell spheres in the step (4) into a U-shaped bottom 96-well plate subjected to ultralow adsorption culture for continuous culture, and changing the culture medium every other day.

In a specific embodiment, the initial seed number in step (4) is 5000 cells/drop and the incubation time in step (5) is 5 to 8 days. Preferably, the incubation time is 7 days.

In a specific embodiment, the compound treatment comprises: sample adding, cleaning, digestion and centrifugation; preferably, the digestion is carried out for a period of time ranging from 6 to 8 minutes.

In a specific embodiment, the tabletting and staining comprises: hypotonic treatment, fixing and dyeing; preferably, the hypotonic treatment is carried out for 3-5 min.

In a specific embodiment, the positive control treated with the compound is an adduct formed by DNA cross-linking; preferably, the positive control is mitomycin C with a CAS number of 50-07-7.

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

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

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.

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

The invention has the positive progress effects that:

the invention successfully applies the 3D liver cell model to micronucleus cytology detection, and establishes a 3D liver cell in-vitro micronucleus cytology detection method so as to provide more genetic toxicity information related to human body tests. The method can use the humanized liver cell HepG2 as a genotoxicity detection material, avoids that the experiment data can not be effectively extrapolated due to the species difference in the genotoxicity detection by using microorganisms or animal cell lines, and can detect the substances which are required to be metabolically activated to generate the genotoxicity under the condition of not adding exogenous metabolic enzyme S9.

(1) The step of 3D liver cell culture is improved, because 3D cell spheres are an aggregate with a compact structure, the cells cannot be completely digested after being added with pancreatin for 2-3 min, and the digestion time is required to be prolonged, but the digestion step is not too long, otherwise, the cells are excessively digested, the cell membranes are damaged, the cell state is influenced, and the vortex is increased. The invention can ensure that the cell spheres can be fully digested into single cell suspension, and can avoid the influence of cell membrane rupture on the subsequent tabletting process caused by overlong digestion time, thereby finally ensuring the accuracy and the effectiveness of the detection method.

(2) The hypotonic time affects the quality of the final slide, the hypotonic time is insufficient, cytoplasm does not expand, and the color of the cytoplasm after dyeing is darker and is not greatly distinguished from the nucleus. And the two cell nuclei can not be completely separated, so that the judgment of binuclear micronuclei, nuclear buds and nuclear plasma bridges is affected. Hypotonic time is too long, cytoplasmic swelling is excessive, and the envelope breaks. Only the nuclei after rupture of the envelope were visible under the mirror and no reading scoring was performed. The hypotonic time of the invention can ensure that cells are fully expanded and can also ensure that the cells cannot be broken due to excessive hypotonic.

(3) According to the invention, cell spheres cultured in hanging drops for 3 days are inoculated into an ultra-low adsorption (ULA) 96-well plate for continuous culture, so that the prolonged culture time can be realized, and the compound is added for subsequent genotoxicity detection. The bracket-free sphere culture mode is simple and easy to standardize, and the accessibility, repeatability and stability of the 3D hepatocyte genetic toxicity evaluation and the capability of high-throughput detection are greatly enhanced. By selecting proper culture conditions, the 3D hepatocyte spheroids have molecular phenotypes and functions superior to those of 2D cells, and are more suitable for in vitro genetic toxicity evaluation.

Drawings

FIG. 1 shows morphological characteristics of micronucleus cytology observation indexes.

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

FIG. 3 is a 2D model and 3D of different incubation timesComparing the concentration of urea in the supernatant of the cell model; wherein the method comprises the steps ofn＝5；**：P≤0.01。

FIG. 4 shows albumin concentration comparison of 3D model and 3D cell model supernatant at different culture times; wherein the method comprises the steps ofn＝5；**：P≤0.01。

FIG. 5 is a real-time fluorescent quantitative PCR analysis of metabolic enzymes in 2D and 3D cultures; wherein the method comprises the steps ofn＝3；*：P≤0.05；**：P≤0.01；***:P≤0.001。

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Preparation of main reagents, medium and reagents

High sugar DMEM medium (Gibco, usa); fetal bovine serum (BI, usa); 0.25% pancreatin-EDTA digest (Gibco, usa); 100 XStreptomyces lividans mix (Invitrogen, USA); phosphate-Buffered Saline (PBS) (solebao, china); DMSO (Sigma, usa); MMC (Sigma, USA); cyto-B (Sigma, USA); giemsa dye liquor kit (Soy pal, china); potassium chloride (national pharmaceutical group chemical reagent limited, china); methanol (national pharmaceutical group chemical agent limited, china); glacial acetic acid (national pharmaceutical group chemical reagent limited, china); baP, COL, CPA amiodarone (sigma, usa); EGCG, furfuryl thioacetate (micin, china); urea kits, albumin (BCG) kits (BioAssay Systems, usa); miRNeasy Mini miniextraction kit (QIAGEN, usa); evo M-MLV reverse transcription reagent premix, ROX Reference Dye dye liquor (20. Mu.M), SYBR Green Pro Taq HS premix qPCR kit (Ai Kerui organism, china); primers (Thermo company, usa); fetal bovine serum (Fetal Bovine Serum, FBS) (BI, united states).

HepG2 cells were purchased from the Shanghai department of science center of China. Cells were stored in liquid nitrogen and used for subsequent experiments 3-10 passages after resuscitation.

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

Hypotonic solution: 2.796g of potassium chloride is weighed into a beaker, a proper amount of deionized water is added, the mixture is uniformly mixed until the deionized water is completely dissolved, the volume is supplemented to 500mL, the solution is transferred into a glass bottle after uniform mixing, and the glass bottle is well marked and stored at room temperature for standby.

Fixing solution: the required volumes were prepared in the ratio methanol to glacial acetic acid=3:1, ready-to-use.

Giemsa application liquid: just prior to staining, the Giemsa concentrate was diluted 1:9 with Giemsa diluent to prepare Giemsa application fluid.

Main instrument and consumable

CLM-170A-B carbon dioxide incubator (ESCO corporation, singapore); 500-II-A/B3 biosafety cabinet (Shanghai Ruiyan purifying equipment Co., ltd., china); CX21 optical microscope (olympus, japan); cell counter (chememetec a/S company, denmark); ST16R centrifuge (Thermo Fisher, usa); IMT-2-21 inverted microscope (olympus, japan); pipette and associated tip (Eppendorf, germany); ultra low adsorption U-bottom 96 well plates (Corning Inc., USA).

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

Table 1 test compound information and method of formulation

Table 2 compound information and method of formulation

* Positive control

The determination of sensitivity is described in the test system of the present invention. The three indexes of the invention respectively represent different genotoxic mechanisms and are evaluated independently. Any one of the three indicators detected represents a possible genotoxic lesion.

EXAMPLE 1 cell culture and Compound treatment

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

1.1 2D cell culture and treatment

HepG2 cells in logarithmic growth phase were digested with pancreatin to prepare single cell suspension and the suspension was used in 1X 10 ⁵ Density of individual/well inoculated in 6 well cell culture plates at 37 ℃ + -1deg.C, 5+ -1% CO ₂ And (5) humidifying and culturing. After 24h, the original medium was aspirated, and the prepared medium containing the compound and having a final concentration of 5. Mu.g/mL cyto-B was added. For each treatment group, 2 wells were treated in parallel and the loading volume was 4mL. And DMEM medium containing 5 mug/mL cyto-B final concentration as control group. The negative and positive control formulations were each allowed to act on the cells for about 48 hours (> 1.5 doubling cycles) and the culture was discarded. After the treatment, the cells were digested and prepared as a cell suspension, and the 2D cell suspension per well was collected in a 15mL centrifuge tube, centrifuged (1000 rpm. Times.5 min), and the supernatant was discarded.

1.2 3D cell culture and processing

Diluting the digested cell suspension to a corresponding density of 2.5X10 ⁵ /mL. 10mL of PBS buffer was previously added to the bottom of a 100mm cell culture dish. The cell suspension of the above cell density was seeded at 20. Mu.L/drop into the inside of an inverted 100mm dish of 50 drops each, using a multichannel pipette. Gently lightThe dish cover is turned over and placed at the bottom of the dish, the dish containing hanging drops is transferred into a carbon dioxide incubator, and the monolayer is placed horizontally. After 3 days of culture, cell spheres with an initial seeding number of 5000 cells/drop were formed inside the hanging drop. On day 3, transferring the inner sphere of the hanging drop into a 96-well plate with an ultra-low adsorption U-shaped bottom for continuous culture, and changing half of the culture medium every other day until day 7. Experimental results show that the initial inoculation number of the cell spheres is 5000 cells/drop, and the cell spheres cultured for 7 days have the highest liver specificity index and metabolic enzyme gene expression, so that the cell spheres are the optimal culture conditions of the 3D liver cell model.

1.3 Albumin and Urea content determination

(1) Collecting supernatant. Selecting 2.5X10 ⁵ Cell suspensions of individual mL-1 were used as initial cell seeding densities to seed cell pellets, cultured in a 1.2 culture format, and cell samples and supernatants were collected on specific days (3, 7, 10, 14 days). The invention uses cell suspension with the same density to inoculate in a standard flat bottom 6-hole plate, and collects cell samples and supernatant after conventional culture for 3 days. Each cell culture sample was subjected to a total change of fluid 24 hours prior to sampling (the reagents would bind to albumin in the bovine serum, and a serum-free medium should be used for 24 hours), after 24 hours the supernatant was aspirated and frozen at-20 ℃ for subsequent detection.

(2) Digestion and counting. The remaining cell spheres (n=12) were collected in a 2mL EP tube and, after the spheres had settled naturally to the bottom of the tube, washed twice with 1mL PBS and the supernatant discarded. Subsequently, 500. Mu.L of pancreatin was added for digestion (37 ℃,3 min), followed by vortexing to break the spheres into small clumps as much as possible, and after continued digestion (37 ℃,3 min), 1mL of complete medium was added to terminate digestion, and the total number of cells was counted for estimating the number of cells at each time point. The remaining supernatant from the 6-well plate was aspirated, 2D cells were digested, centrifuged, resuspended, and the total number of cells counted to estimate the number of cells.

(3) And (5) measuring urea content. And detecting the urea content according to the specification of the urea detection kit. And mixing the solution A and the solution B which are placed at normal temperature according to a ratio of 1:1 to prepare the working solution. 5 mu L of 5mg dL-1 urea standard substance, deionized water and a sample to be tested are placed in a 96-well plate, 200 mu L/well working solution is added into each well,incubate at room temperature for 50min. Absorbance values at 430nm were obtained using an IX6 fluorescence microplate reader. And calculated according to the following calculation formula, the data is normalized to 6×10 ⁴ Individual cells.

Concentration calculation formula:

OD samples, OD blanks and OD standards are the absorbance of the sample, blank control and standard, respectively, and n is the dilution factor. When urea samples were measured low [ STD ] =5.

(4) And (5) measuring the albumin content. The albumin content is detected according to the instruction of the albumin detection kit. Before use, the reagent was left to room temperature and gently swirled. Standards of different concentrations were diluted with distilled water. A96-well plate with a transparent flat bottom was used, and 15. Mu.L of diluted standard (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 reagent was added to each well, and after incubation for 5min, absorbance values at 620nm were obtained using an IX6 fluorescence microplate reader. Standard curves were made using absorbance values of standard and blank to determine albumin concentration of samples, data normalized to 6 x 10 ⁴ Individual cells.

1.4 phase I and phase II Metabolic enzyme Gene expression determination

(1) Collecting a sample: digestion of 2D tiled HepG2 cells and 3D cell pellets cultured for 3 and 7 days in T25 flasks and determination of cell number (not more than 1X 10) ⁷ ). The cell suspension was transferred to a centrifuge tube and after centrifugation at 1000rpm x 5min the supernatant was aspirated. Cell pellet was thoroughly loosened by flicking the bottom of the tube and cells were lysed by addition of Buffer RLT in appropriate volume in miRNeasy Mini miniextraction kit. Cell lysates can be stored for several months at-70 ℃.

(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, and total RNA was isolated using miRNeasy Mini extraction kit and stored on ice.

(3) An RT reaction was prepared using Evo M-MLV reverse transcription reagent premix and 500ng Total RNA in proportion and transferred to an octal tube with a reverse transcription system of 10. Mu.L. The reverse transcription reaction condition is that the temperature is 37 ℃ for 15min; 5sec at 85 ℃;4 ℃.

(4) Using SYBRPremix Pro Taq HS qPCR Kit reagent, primer and cDNA product prepared by the reaction in the previous Step are premixed in proportion, and react on a Step One fluorescence quantitative PCR instrument under the following reaction conditions: 30sec (1 cycle) at 95 ℃;95℃for 5sec,60℃for 30sec (40 cycles); and (5) establishing a PCR product dissolution curve.

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

1.5 statistical analysis

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

TABLE 3 primer sequences for phase I and phase II Metabolic enzymes

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

Comparing liver specificity of HepG2 cells of different culture modes using albumin production and urea level as detection indicatorsDifferentiation of sexual function, the values were normalized to 1×10 ⁶ The amount of individual cells to eliminate differences in cell number between samples. By measuring the supernatant samples from days 3 to 14, the serum albumin concentration on the 3D-cultured cells was significantly increased compared to the 2D cells, with the highest value on day 7 (see fig. 3). At the same time, the urea content of the 3D sphere supernatant reached a maximum value on day 7 (see fig. 4). Since 3D cell spheres with 3 and 7 days of culture time showed better cell functions and the culture time was shorter, 3D cell spheres with 3 and 7 days were further selected to compare with the expression level of the metabolic enzyme gene in I, II phase of 2D cells, and a proper 3D cell culture time was selected.

Low levels of drug metabolizing enzymes in HepG2 cells may result in undetectable substances requiring metabolic activation. The results of the real-time fluorescent quantitative PCR analysis showed that the expression levels of the different I, II phase metabolic enzymes in 3D HepG2 spheroids were generally higher than in 2D cultured HepG2 cells (see fig. 5). 7500Fast Real-Time PCR system analyzes the transcription level of phase I metabolizing enzyme mRNA during spheroid culture. The 3D HepG2 cells with 3 days of culture have remarkably increased CYP1A1 and UGT1A6 gene expression, and have no remarkable difference between CYP1A2, CYP3A4, CYP2E1, CYP2B6 and UGT1A1 and 2D cells. The 3D HepG2 cells with 7 days of culture had significantly higher expression levels of CYP1A1, CYP2C9, and CYP2D6 than the 2D cells, and the 2D cells had consistent expression levels of CYP1A2, CYP3A4, CYP2B6, and CYP2E 1. The mRNA level of the phase II heterologous biological metabolizing enzyme UGT1A6 of 3D HepG2 cells cultured for 7 days was significantly increased compared to 2D cells, with UGT1A1 expression levels slightly higher than for 2D cells.

ULA 96-well plates containing 3D cell pellets were removed from the incubator at day 7, and the original medium was aspirated as much as possible, and different concentrations of positive compound and 5. Mu.g/mL of cyto-B final culture medium were added to each well. The wells were treated in a total of 12 wells per treatment group and the loading volume was 100. Mu.L for each condition. And DMEM medium containing 5 mug/mL cyto-B final concentration as control group. After the negative and positive control preparations were allowed to act on the cells for about 48 hours, the culture medium was discarded and the spheres from each group of 6 wells were collected in a 15mL centrifuge tube and the two tubes were collected. Wash 2 times with PBS and discard the supernatant. The cells were digested by adding 300. Mu.L of pancreatin for 6min, blown to no obvious cell mass, stopped by adding 1mL of complete medium, resuspended in cell spheres by adding 1mL of pancreatin to the EP tube, and digested for 3min in an incubator at 37 ℃. At this point, the cell spheres are initially digested and vortexed on a vortexing device to lyse the whole cell spheres into smaller cell clusters. Then the mixture is continuously placed in a 37 ℃ incubator for continuous digestion for 3 to 5 minutes, and then is continuously placed on a vortex instrument for vortex or is blown by a pipetting gun until no obvious cell mass exists. After the cell spheres have been completely digested into single cell suspensions, they are centrifuged (1000 rpm. Times.5 min) and the supernatant is discarded. As shown in Table 4, the experimental results show that 6-8 min is the preferred digestion time of the 3D cell spheres, and digestion within the time can ensure that the cell spheres can be fully digested into single cell suspension, avoid the influence of rupture of cell membranes on the subsequent tabletting process due to overlong digestion time, and finally ensure the accuracy and the effectiveness of the detection method. The information and preparation method of the compounds are shown in Table 2.

TABLE 4 Effect of digestion time on cell suspension preparation

1.3 tabletting and staining

Hypotonic treatment: after centrifugation, the supernatant was discarded and the tube was flicked off as much as possible, and the cells were resuspended with a small remaining amount of medium to ensure that the cells did not form clumps. Slowly adding 1 mL/branch of hypotonic solution with the temperature of 37 ℃ to the resuspended cells, vibrating while adding, and standing at room temperature for hypotonic for 3-5 minutes.

Fixing: 3mL of the fixing solution was slowly added dropwise to the mixture for fixation, and after the completion of fixation, the mixture was centrifuged (1000 rpm. Times.5 min), and the supernatant was discarded. Each of the tubes was further solidified dropwise with a fixed solution of about 3 mL/branch followed by centrifugation (1000 rpm. Times.5 min), and the supernatant was discarded. Dropping tablets: the fixed liquid is left in the centrifuge tube by about 0.1-0.2 mL, the mixture is gently mixed to prepare cell suspension, the glass slide is required to be strictly cleaned, the glass slide is slightly inclined, the other hand is used for sucking a small amount of the uniformly mixed cell suspension, 2-4 drops are dropped to one end of the glass slide, the glass slide is naturally and uniformly dispersed downwards, then the glass slide is dried at room temperature, and the prepared chromosome glass is placed at room temperature or an incubator for drying, thereby being beneficial to dyeing. The slides were allowed to air dry completely for at least 30 minutes, and allowed to sit overnight.

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

As shown in Table 5, 3 to 5 minutes is the optimal hypotonic time. The hypotonic time can ensure that cells are fully inflated and can also ensure that the cells cannot be broken due to excessive hypotonic.

TABLE 5 Effect of hypotonic time on stained slides

1.4 microscopic index and statistical analysis

Microscopic index

The number of monocytes, dinuclear cells and polynuclear cells in 500 cells was counted, and the Cytokinesis proliferation index (Cytokinesis-Block Proliferation Index, CBPI) and replication index (Replicative Index, RI) were calculated. 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 culture and control culture and can be used to calculate the cell growth inhibition (Cytostasis).

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

Cbpi= (number of monocytes+2×number of binuclear cells+3×number of polynuclear cells)/(total number of cells)

T=treatment group c=control group

b. Every 1000 binuclear cells showed thousandths of MN, nbaud and NPB, and scored according to the Fenech method (FENECH M, et al Mutagenesis,2010,26 (1): 125-132).

Statistical analysis

Using spss.18, MN thousandths, nbuds thousandths and NPBs thousandths of the chemically treated group were compared for significance (p.ltoreq.0.05) with the vehicle control group using Fisher's exact test method.

The result shows that (1) MN thousandth, nboud thousandth and NPB thousandth induced by the test sample are obviously increased, and the concentration dependence exists;

(2) MN thousandth, nbud thousandth and NPB thousandth induced by a test point are obviously increased and are repeatable.

And if the result meets one of the above, the result is positive, otherwise, the result is negative. Statistical methods are not the only criteria for evaluating positive results, while taking biological significance into account.

As a result, as shown in FIG. 1, the 2D model group showed a significant increase in MN ∈0.05, nbu ∈0.1 μg/mL concentration group (p.ltoreq.0.05) at 4 treatment concentrations, a significant difference in NPB ∈0.025-0.1 μg/mL, but no dose dependence (see Table 5). Cell growth inhibition (Cytostasis) and RI at various concentrations in 3D hepatocyte micronucleus cytology were comparable to those in the 2D hepatocyte model. MN and Nbud per mill at 4 MMC treatment concentrations in the 3D liver cell model group were both significantly elevated (p.ltoreq.0.05), and NPB per mill concentrations were not significantly elevated (see table 6). The minimum detection concentration of MN milland Nbaud millof MMC in the 3D model group was 0.0125. Mu.g/mL, and the minimum detection concentration in the 2D model group was 0.025. Mu.g/mL.

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

Table 6 MMC 2D cell micronucleus cytology detection results

* : p is less than or equal to 0.05.CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: a nuclear mass bridge; NPB: nuclear mass bridge

Table 7 MMC 3D cell micronucleus cytology test results

* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass bridge

This example shows that after treatment with positive compound MMC, 2D and 3D cultured HepG2 cell models can detect significant increases in micronuclear cytology index over a range of cytotoxicity.

The 2D hepatocyte model was compared with the CBMN-cyt detection results of MMC of the 3D hepatocyte model. In the aspect of cell proliferation inhibition detection, the cytotoxicity index 100-RI and the cell growth inhibition rate of the 2D liver cells and the 3D liver cell model are increased in concentration correlation, so that the MMC is indicated to cause similar cell damage to the 2D liver cells and the 3D liver cell spheres. In terms of genotoxicity detection, the minimum detection concentration of MN%o and Nbaud%o of MMC in the 3D model group was 0.0125. Mu.g/mL, the minimum detection concentration in the 2D model group was 0.025. Mu.g/mL, NPB positivity was not detected in this concentration range in the 3D cell model, but was not dose-dependent in the 2D cell model, and the NPB values were within the negative reference range of 0-10%o for peripheral blood lymphocyte CBMN-cyt assay (FENECH M, et al Mutagenesis,2010,26 (1): 125-132). 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 the micronuclear cytological studies of existing HepG2 and derived cell lines (DIAMOND L, et al, carcinogenis, 1980,1 (10): 871-875,SHAH U K,et al.MUTATION RES,2018,834 (2018): 35-41).

After positive compound MMC treatment, the 3D liver cell model can detect the statistically significant increase of micronucleus cytology indexes within a certain cytotoxicity range. The invention proves that the CBMN-cyt method can be successfully applied to the HepG2 liver sphere, and a 3D liver cell micronucleus cytology method is initially established. And comparing the MMC micronucleus cytology results of the 2D model with those of the 3D model, wherein the detection sensitivity of the 3D hepatocyte model MN and the Nbaud is higher than that of the 2D hepatocyte model. The method is further validated by using genotoxic compounds, suspected genotoxic compounds and negative compounds of different mechanisms, which further expand the compound detection range of 3D hepatocyte micronucleus cytology.

Example 2 validation of 3D liver cell micronucleus cytology assay

According to the recommendation of an in vitro mammal cell micronucleus test (OECD TG487,2016), two genotoxic compounds to be metabolically activated, namely benzopyrene (BaP) and Cyclophosphamide (CPA), an aneuploid inducer Colchicine (COL), two suspected genotoxic compounds, namely (-) -epigallocatechin gallate (EGCG) and furfuryl thioacetate, and a non-genotoxic compound, namely amiodarone, are selected, an established 3D liver cell micronucleus cytometric test method is verified, and the applicability of the method for compounds with different mechanisms is explored.

In this example, 6 representative substances with different genotoxic mechanisms were selected to perform preliminary verification on in vitro 3D hepatocyte micronucleus cytology, and experimental results were compared with micronucleus cytology results obtained by conventional cell culture methods.

Benzopyrene (BaP) is a known indirect mutagen, and is positive in MN test results. BaP, a pre-carcinogen, belongs to a polycyclic aromatic hydrocarbon compound, can be activated by CYPs (1A 1 and/or 3A 4) and microsomal epoxide hydrolysates in sequence to produce genotoxicity. When HepG2 cells are exposed to BaP, different metabolites of BaP (BaP-4, 5-diol; baP-7,8-diol; baP-9, 10-diol), some quinones and various hydroxyl metabolites are produced; the major DNA adduct, produced by the reaction of BaP-7,8-diol-9, 10-epoxide (BPDE), is the final oncogenic intermediate (KIRCHNER S, ZELLER A. Mutation Research/Genetic Toxicology and Environmental Mutagenesis,2010,702 (2): 193-198).

Colchicine (COL) is a typical aneuploidy inducer, featuring a typical non-oncogenic mutagen. Positive in vivo and in vitro micronucleus assays. It causes a distortion of chromosome number due to metaphase block caused by inhibition of tubulin polymerization. In the in vitro micronucleus assay colchicine clearly induced micronucleus increase in CHL cells and human lymphocytes, L5178Y cells (KNASMuLLER S, et al, mutation research,1998,402 (1-2): 185).

Cyclophosphamide (CPA) must be metabolically activated in vivo to exert a cytotoxic effect. About 70% to 80% of CPA is metabolized in vivo via CYP2B6, CYP3A4, CYP3A5, CYP2C9, CYP2C19 to intermediates that are readily available to produce phosphamidon and acrolein via non-enzymatically catalyzed elimination reactions, thereby producing genotoxicity (Chen Lingyan et al. Pharmaceutical journal 2014,49 (7): 971-976.).

(-) -epigallocatechin gallate (EGCG) is the main component of green tea extract, and positive results appear in L5178Y tk+/-mouse lymphocytes in vitro mammal gene mutation test by oxidative stress, and in vivo micronucleus research results are negative (UEHARA T, et al, toxicology,2008,250 (1): 15-26). According to the invention, EGCG is used as a model compound for detecting non-in-vivo genetic toxicity and in-vitro genetic toxicity, and whether an in-vitro 3D liver cell model has better prediction capability on suspicious genetic toxicity substances is further evaluated. Experimental results show that under 2D cell culture, positive results appear for MN in 62.5-500 μg/mL dose group. It has been reported that EGCG or catechin (the chemical family to which EGCG belongs) produces H under in vitro culture conditions ₂ O ₂ Thus, it can be positive in vitro genotoxicity tests (JOHNSON M K, LOO G.Mutat Res,2000,459 (3): 211-218.).

Furfuryl thioacetate is a commonly used food flavor. Furylacetate was found to be cytotoxic (< 80% relative cell density) and genotoxic in the Blue Screen assay; the Ames experiment is a negative result; in vitro human peripheral blood lymphocyte micronucleus assay 100 μg/mL (-S9, 3 h) and 43 μg/mL (-S9, 24 h) dose group positive; the micronucleus assay in mice was negative (API A M, et al food and Chemical Toxicology,2020, 144:111615.). The in vivo and in vitro test data are combined and the furfuryl thioacetate can be considered to be a non-genotoxic compound.

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

2.1 micronuclear cytological detection of benzopyrene

Micronuclear cytology of benzopyrene was tested as shown in tables 8 and 9.

MN ≡was significantly increased at each treatment concentration in the 2D hepatocyte model group compared to the solvent control group, nbud ≡was significantly increased (p.ltoreq.0.05) in the 10.09, 20.19 μg/mL concentration group, and no BaP showed significant cytotoxicity in this range (see table 8).

None of the 3D cell model groups showed significant cytotoxicity compared to the solvent control group, and the MN and Nbud were significantly increased (p.ltoreq.0.05) at 2.52, 5.05, 10.09, 20.19 μg/mL treatment concentrations, and NPB was significantly increased only in the 10.09 μg/mL concentration group, but no dose dependence was seen (see table 9). At the same dose, the MN per mill of the 3D cell model group is higher than that of the corresponding 2D cell model group, and the sensitivity of the index of the 3D cell model Nbaud per mill is higher than that of the 2D cell model group.

TABLE 8 2D cell micronucleus cytology assay results for BaP

* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass bridge

TABLE 9 3D cell micronucleus cytology assay results for BaP

* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass bridge

Both 2D and 3D cell models can detect BaP genotoxicity by MN, an indicator. Compared with the experimental result of the 2D cell model, the MN rate of the 3D cell model experiment is higher than that of the 2D cell model, and the remarkable increase of Nud can be detected at a lower concentration, so that the BaP genotoxicity related information is provided.

2.2 micronuclear cytological assays of colchicine

The results of the micronuclear cytology assay for colchicine are shown in tables 10 and 11.

RI and cytostatic rate of the 2D cell model group showed that cytotoxicity of COL increased with increasing concentration. MN thousandth and Nbud thousandth of the 2D cell model group were first increased at the treatment concentration of 0.0025 to 0.01 μg/mL and decreased at the high dose of 0.02 μg/mL compared to the solvent control group (see table 10).

Compared with the solvent control group, the 3D cell model group has 0.0025-0.02 mug/mL of MN permillage, nboud permillage and NPB permillage which are all obviously increased (p is less than or equal to 0.05), and concentration-dependent cytotoxicity is generated (see Table 11). The positive control group was significantly different in NPB%o, but was within the recommended negative range of 0-10%o (LORGE, et al, mutation Research/Genetic Toxicology and Environmental Mutagenesis,2006,607 (1): 13-36). Compared with a 2D liver cell model group, the 3D liver cell micronucleus cytology method has higher sensitivity in detecting COL, and can detect the genetic toxicity of COL in a larger concentration range through MN, nbud, NPB.

Table 10 COL 2D cell micronucleus cytology assay results

* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass bridge

TABLE 11 3D cell micronucleus cytology assay results for COL

* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass

In this example, the 2D results showed that MN milland Nbeam millincreased at concentrations of 0.0025 to 0.01. Mu.g/mL and decreased at high dose of 0.02. Mu.g/mL compared to the solvent control. This may be due to cell loss caused by mitotic arrest. In addition, since COL can induce mitotic arrest, the RI index may be overly reflective of this strong cytostatic activity (especially over a longer treatment time of 48 h) compared to acute cell death. Thus, aneuploidy inducers inhibit micronuclei formation through mitosis over a range of higher toxic concentrations.

In detecting COL, an improper concentration interval may reduce sensitivity to MN of COL, and an excessive concentration interval may miss the maximum reaction concentration or even the weaker reaction concentration, so that an appropriate concentration interval is critical for detection of COL. In the COL treatment process, RI and cell growth inhibition rate indexes show that the hepatocyte spheroid has a certain resistance to the cell proliferation inhibition toxicity of COL, and the genetic toxicity of COL can be detected through MN, nbeam and NPB within the range of 0.0025-0.02 mug/mL.

2.3 micronuclear cytological assays of cyclophosphamide

The results of the micronuclear cytology assay for cyclophosphamide are shown in tables 12 and 13.

RI and cytostatic rate of the 2D cell model group showed a concentration-dependent increase in the cytotoxicity of CPA. MN ≡mill significantly increased (p.ltoreq.0.05) at treatment concentrations of 312.5-25000 μg/mL compared to the solvent control, with significantly increased but no apparent concentration dependence seen for the 312.5 and 2500 μg/mL groups Nbud mill, NPB mill (see table 12).

Comparing micronucleus related indexes of the CPA group with those of a solvent control group, and obviously increasing MN per mill within the dosage range of 312.5-2500 mug/mL; the dose range of 625-2500. Mu.g/mL was significantly increased by Nbeam mill and no significant increase in NPB mill was seen (see Table 13). On the Nbeam millindex, the 3D cell model is sensitive compared with the 2D cell model, and has dose correlation.

TABLE 12 results of 2D cell micronucleus cytology assay for CPA

* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass bridge

TABLE 13 3D cell micronucleus cytology assay results for CPA

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* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass bridge

In this example, the MN ∈mill at the treatment concentration of 312.5 to 2500. Mu.g/mL was significantly increased (p.ltoreq.0.05). The 3D liver cell group observed a significant increase in MN ≡thousandth (p.ltoreq.0.05) at treatment concentrations of 312.5-2500. Mu.g/mL. Micronucleus induction rates were similar for the 2D and 3D HepG2 cell models, probably because the CYP2B6 and CYP3A4 metabolizing enzyme genes that induced the intermediates for CPA metabolism did not show enhanced expression in the 3D hepatocyte model. The 3D liver cell model group is 625-2500 mug/mL Nbeam per mill obviously increased, has certain dose correlation and is more sensitive than a 2D model.

2.4 Micronuclear cytological assay for (-) -epigallocatechin gallate

The results of the micronuclear cytology assay for (-) -epigallocatechin gallate are shown in tables 14 and 15.

The RI and cell growth inhibition rate of the 2D cell model group show that EGCG has obvious cytotoxicity to 2D liver cells in the concentration range of 62.5-500 mug/mL. MN per mill is significantly increased (p.ltoreq.0.05) for each concentration group compared to the solvent control group; 250. the Nbeam per mill of the 500 mu g/mL dose group is obviously increased; no significant differences were seen for each concentration group of NPB%.

RI and inhibition of cell growth of 3D cell model group showed that EGCG had obvious cytotoxicity to 3D liver cells only in 500. Mu.g/mL dose group, MN milland Nbaud millof 250, 500. Mu.g/mL dose group were significantly increased, NPB millwas not significantly increased in each concentration group (see Table 15).

TABLE 14 2D cell micronuclear cytology assay for EGCG

* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass bridge

Table 15 results of 3D cell micronucleus cytology assay for EGCG

* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass bridge

The 3D liver cell micronucleus cytology method has no obvious increase in each concentration group of MN permillage, nbou permillage and NPB permillage in the dosage range of 62.5 and 125 mug/mL. Cytotoxicity and genotoxicity occurred in the 250, 500 μg/mL dose group. Within about 20% of the same cytotoxicity range, both 2D and 3D cell model groups appeared to be genotoxic.

2.5 detection of Furfural thioacetate by micronuclear cytology

The results of the micronuclear cytology assay for 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 furyl thioacetate has no obvious cytotoxicity to 2D liver cells in the concentration range of 125-1000 mug/mL, and compared with the solvent control group, the MN mill, nbeam milland NPB millof each dose group have no obvious difference (see Table 16). No obvious cytotoxicity and genetic toxicity appear when 125-1000 mug/mL of furfuryl thioacetate acts on 3D liver cells (see Table 17).

TABLE 16 results of 2D cell micronucleus cytology assay for furfuryl thioacetate

* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass bridge

Table 17 results of 3D cell micronucleus cytology assay for furyl thioacetate

* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition ratio; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass bridge

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

2.6 micronuclear cytological detection of amiodarone

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

The 2D cell model group RI and the cell growth inhibition showed that amiodarone had a concentration-dependent cytotoxicity on 2D hepatocytes at the highest dose of 20.19 μg/mL. Compared with the solvent control group, no significant difference was seen in MN thousandth, nboud thousandth and NPB thousandth of each dose group of amiodarone (see Table 18). The cytotoxicity of the 3D cell model group was lower at the same concentration compared to the 2D cell model. No significant increase in genotoxicity was detected in both the 2D and 3D cell model groups (see table 19).

Table 18 results of 2D cell micronuclear cyto-histology of amiodarone

* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass bridge

TABLE 19 3D cell micronuclear cytology assay results for amiodarone

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* : p is less than or equal to 0.05; CBPI: cytokinesis-blocking index; RI: replication index; cytostasis: cell growth inhibition rate; MN: a micronucleus; nboud: nuclear buds; NPB: nuclear mass bridge

In this example, the concentration-dependent changes in cytotoxicity index RI, cytostatic rate of both 2D and 3D cell micronucleus cytology occurred in the 51.6. Mu.g/mL dose range. Unlike the uniform exposure of monolayer tiled growing HepG2 cells, when hepatocyte spheroids are overall exposed to amiodarone, the exposure level of internal cells is lower due to mass transport inside and outside the spheroids. In addition, the 3D cells have the functions of ECM formation and whole intercellular substance information exchange after long-term culture, and can better resist the toxic action of external substances. No genotoxicity of amiodarone was detected in both the 2D HepG2 cell model and the 3D HepG2 cell model of the present invention.

Table 20 summary of 2D and 3D hepatocyte micronucleus cytology results

Note that: the number represents the lowest detected concentration (. Mu.g.mL) ^-1 ) The method comprises the steps of carrying out a first treatment on the surface of the n.t.: no detection or biological significance was observed in the concentration range of the study.

The invention uses positive compounds BaP, COL, CPA with different mechanisms, suspicious genotoxic compounds EGCG and furyl thioacetate, and non-genotoxic compounds amiodarone to verify the 3D liver cell micronucleus cytology method. BaP, COL, CPA as positive compound, furyl thioacetate and amiodarone as negative compound, EGCG as high concentration group, and 2D liver cell micronucleus cytology results. The 3D liver cell model improves the threshold value of the EGCG in-vitro micronucleus cytology detection concentration. The detection results of all substances are integrated, and compared with the conventional micronucleus cytology, the detection sensitivity of Nud index of 3D liver cell micronucleus cytology can be doubled.

Sequence listing

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<120> a method for detecting genotoxicity using 3D hepatocyte in vitro micronuclear cytology

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Claims (8)

1. A method for detecting genotoxicity using 3D hepatocyte in vitro micronuclear cytology, comprising: the method is characterized in that the 3D cell culture adopts a bracket-free culture method combining a hanging drop method and an ultralow adsorption culture method, and the bracket-free culture method is used for inoculating cell spheres which are cultured to 3 days into an ultralow adsorption 96-well plate for continuous culture until the 7 th day; the cells to be detected in the 3D cell culture are humanized liver cell HepG2 cells; the 3D cell culture comprises:

(1) Subjecting cells in the logarithmic growth phase to pancreatin digestion;

(2) Diluting the digested cell suspension to 2.5X10 ⁵ /mL；

(3) Inoculating the cell suspension at 20 μl/drop into the inside of an inverted 100mm dish cover;

(4) Culturing the cell suspension, and forming cell spheres with initial inoculation number inside the hanging drop; the initial inoculation number is 5000 cells/drop;

the compound treatment comprises the following steps: sample adding, cleaning, digestion and centrifugation; the digestion treatment time is 6-8 min;

the tabletting and staining comprises the following steps: hypotonic treatment, fixing and dyeing; the hypotonic treatment time is 3-5 min.

2. The method as recited in claim 1, further comprising:

(5) Transferring the cell spheres in the step (4) into a U-shaped bottom 96-well plate subjected to ultralow adsorption culture for continuous culture, and changing the culture medium every other day.

3. The method of claim 1, wherein the compound-treated positive control is an adduct formed by DNA cross-linking.

4. The method of claim 3, wherein the positive control is mitomycin C with a CAS number of 50-07-7.

5. The method of claim 1, wherein the genotoxicity is a toxic effect caused by physical and chemical factors in the environment acting on organisms to cause various damages to genetic material at chromosome level, molecular level and base level.

6. The method of claim 5, wherein the genotoxicity is that of a chemical.

7. The method of claim 6, wherein the chemical substance is a genotoxic compound requiring metabolic activation, a aneuploidy inducer, a suspected genotoxic compound, or a non-genotoxic compound.

8. The method of claim 7, wherein the chemical is benzopyrene, cyclophosphamide, colchicine, (-) -epigallocatechin gallate, furfuryl thioacetate, and amiodarone.