Tobacco product damage evaluation method based on airway organoid

Abstract

The invention discloses a tobacco product damage evaluation method based on an airway organoid. The method comprises the following steps: contaminating the airway organoid by using a tobacco product extract; and evaluating the damage caused by the tobacco product extract according to the morphological change and the physiological index change of the airway organs. The airway organ tissue of the lung of the mouse is induced and differentiated into various airway cell types, and the tobacco product is exposed. The exposure effect comprises the growth and the cell activity of the damaged organoid and the change of the proportion of multiple cell types in the organoid, so that the complex process of different cell damages in the body is simulated. In addition, typical adverse effects, such as oxidative stress, inflammation and genetic toxicity, occurring in the two-dimensional culture are also induced.

Description

Technical Field

[0001] The present application relates to the technical field of detection equipment, and in particular to a tobacco product damage evaluation method based on airway organoids. Background Art

[0002] With the development of economy and society, the impact of tobacco products on health has received more and more attention. In real life, smoking is one of the main causes of lung damage, which can stimulate the overreaction of the immune system. In addition, lung damage may also be a trigger for other lung diseases, including serious diseases such as lung cancer.

[0003] Currently, research on tobacco product lung injury is usually conducted using in vitro cells, but traditional culture methods cause cells to gradually lose their original state in the in vitro environment. Their morphology, biological function, genetics, etc. are very different from those of in vivo cells. There are great limitations in their application in the study of tobacco product lung injury, and they cannot accurately simulate the actual impact of aerosols on the respiratory system. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a method for evaluating tobacco damage based on airway organoids; in the present application, the airway organ tissues in the lungs of mice are induced to differentiate into various airway cell types and exposed to tobacco products. The exposure effects include damage to the growth and cell viability of organoids, as well as changes in the proportion of multiple cell types in organoids, thereby simulating the complex process of damage to different cells in vivo. In addition, typical adverse consequences that occur in two-dimensional culture, such as oxidative stress, inflammation, and genotoxicity, are also induced.

[0005] The technical solution provided by the present invention is as follows: A method for evaluating tobacco product damage based on airway organoids comprises the following steps: poisoning airway organoids with tobacco product extracts; and evaluating damage caused by tobacco product extracts by observing changes in the morphology and physiological indicators of airway organoids.

[0006] Preferably, the morphological change of the airway organoid is specifically a change in diameter; The changes in physiological indicators of the airway organoids include any one or more of changes in γ-H2AX, ratio of differentiated cell types, cytokines, reactive oxygen levels, and cell viability.

[0007] Preferably, the tobacco product extract is arranged into multiple concentration gradients to poison the airway organoids.

[0008] Preferably, the tobacco product extract is obtained by collecting smoke generated by burning tobacco products through a filter and then centrifuging it.

[0009] Preferably, a high-content imaging system is used to evaluate changes in γ-H2AX, immunofluorescence staining, and morphology of airway organoids.

[0010] Preferably, airway organoids are established by the following method: S1. Obtain mouse lung tissue and digest it to obtain single cells; S2. Mix the single cells with Matrigel and culture them with conditioned medium.

[0011] Preferably, the conditioned medium contains the following components: a mixture of penicillin and streptomycin, HEPES, GlutMAX, R-spondin, Noggin, B27, Y27632, A8301, SB202190, FGF-7, FGF-10, and Primocin.

[0012] Preferably, the conditioned medium uses the following components at the following concentrations: Penicillin-streptomycin mixture 1-2×; HEPES8-10 mM; GlutMAX1-2×; R-spondin 300-500 ng / mL; Noggin volume fraction 8-12%; B271-2×; N-acetylcysteine ​​1-1.5 mM; Nicotinamide 4-6mM; Y276324-6mM; A8301 400-600 μM; SB202190400-600nM; FGF-7 20-30ng / ml; FGF-1080-120ng / ml; Primocin 40-60 μg / ml; Human heregulin-β 5-10 nM.

[0013] Preferably, airway organoids are established by the following method: S1. Obtain fresh lung tissue from mice, wash it 2-3 times with Advanced DMEM / F12, centrifuge it, mince it, add DNase I digestion solution, digest it in a 37°C incubator for 30-50 min, filter it, centrifuge it, and collect the cell clusters; then add red blood cell lysis solution and mix well, stop the lysis, centrifuge it again and discard the supernatant to obtain cell clusters; S2. The cell clusters were seeded in Matrigel and then cultured with conditioned medium.

[0014] The health hazards of tobacco products are obvious, especially in the airways. The airways are mainly composed of basal cells, secretory cells and ciliated cells, which are significantly damaged under exogenous stimulation. Traditional in vitro toxicology studies mainly use two-dimensional cells, which cannot replicate the characteristics of airway structure. In order to establish a more accurate in vitro model to study the toxicity of tobacco products to the airways, this application established a three-dimensional airway organoid tissue and used this model to evaluate the health effects of the airways. In this application, the airway organoid tissue in the lungs of mice was induced to differentiate into various airway cell types and exposed to tobacco products. The exposure effects included damage to the growth and cell viability of organoids, as well as changes in the proportion of multiple cell types in organoids, thereby simulating the complex process of different cell damage in vivo. In addition, typical adverse consequences that occur in two-dimensional culture, such as oxidative stress, inflammation and genotoxicity, were also induced. All the above results show that airway organoid tissue is an alternative to animal testing in toxicology studies and will help to gain a deeper understanding of the potential hazards of heated tobacco products to respiratory health. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1. Morphology of AOs; (A) Bright field micrographs of AOs from day 1 to day 5, scale bar, 50 μm; (B) Changes in the average diameter of organoids from day 1 to day 5, data are presented as mean ± SEM, n = 40. (C) Immunofluorescence analysis of AOs. AOs were stained with P63 (red), MUC5AC (red), and acetylation of α-tubulin (green), respectively. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm; Figure 2. Toxicity of tobacco products to AOs. (A) 3D cell viability after 24 h (black) and 48 h (grey) exposure. (B) Cytotoxicity of AOs measured by 3D cell viability assay. Data are presented as mean ± SEM, n=3. ***p<0.01, ***p<0.001 compared with control group.

[0017] Figure 3. Average diameter of AO after exposure to heated tobacco products. (A) Average diameter of AO before exposure. (B) Average diameter of AOs after exposure. Data are relative to the control group and represent mean ± SEM, n=40. **P<0.01, ***P<0.001 compared with the control group.

[0018] Figure 4. Changes in cell type-specific differentiation markers in AOs after 48 h of exposure. (A) MUC5AC mRNA expression. (B) KRT5 mRNA expression. (C) FOXJ1 mRNA expression. Data are presented as mean ± SEM, n=3. *p<0.05, **p<0.01, ***p<0.001 compared with the control group.

[0019] Figure 5. Cytokine secretion after HTPs treatment. (A) TNF-α, (B) IL-1β, (C) IL-17A, (D) KC, (E) MIP-1β, (F) Eotaxin, (G) IL-12(P40), (H) RANTES. Compared with the control group, *P<0.05, **P<0.01, ***P<0.001.

[0020] Figure 6. HTP aerosol induces cell apoptosis and oxidative stress. (A) Immunofluorescence staining of caspase-3. Scale bar, 50 μm. (B) Relative expression of caspase-3. The expression of caspase-3 in each group was compared with that in the control group (Ctrl). (C) Relative expression of ROS. The expression of ROS in each group was compared with that in the control group (Ctrl). Data were normalized to the control group and represent mean ± SEM, n = 3.

[0021] Figure 7. HTP aerosol induces the expression of γ-H2AX. (A) Immunofluorescence identification of γ-H2AX. Scale bar, 50 μm. (B) Relative expression of γ-H2AX. The expression of γ-H2AX in each group was compared with that in the control group (Ctrl). The data were normalized to those of the control group and represent the mean ± SEM, n = 3. DETAILED DESCRIPTION

[0022] In order to enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0023] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or are publicly available.

[0024] 1. Experimental part 1. Experimental Animals C57BL / 6J mice (male, 4-6 weeks, 20-25 g) were purchased and kept in a well-ventilated animal breeding room with a circadian rhythm of 12 / 12 h light-dark cycle (8:00 am-8:00 pm), temperature controlled at 20-24°C, and relative humidity of 50-70%. All animals were acclimatized in the breeding room for one week before the experiment. All experimental procedures and processes were carried out in strict compliance with the requirements of the Regulations on the Administration of Laboratory Animals of the People's Republic of China.

[0025] 2. Preparation of tobacco product extracts Tobacco products were purchased from the market and were coded as HTP1 and HTP2 in this experiment.

[0026] Aerosol samples were collected by the solvent-free method. Specifically, aerosol samples were generated by an automatic smoking machine (ESM100L-S01, Qingdao, China), using the Health Canada enhanced (HCI) smoking regime (puff volume 55 ml, puff time 2 seconds, puff interval 30 seconds) recommended by the World Health Organization Tobacco Product Regulation Research Group (Health Canada, 2000), and aerosols were collected using Cambridge filters.

[0027] After collecting the total particulate matter (TPM), the entire sample was placed in a centrifuge tube and centrifuged at 4°C, 12,000 rpm for 10 minutes before collection. After centrifugation, the liquid was collected and filtered through a 0.22 μm sterile filter membrane for use.

[0028] 3. Cultivation of airway organoids (AOs) 3.1 Mice were anesthetized and killed by cervical dislocation. After soaking in 75% ethanol, the mice were quickly transferred to a biosafety cabinet to obtain fresh lung tissue. Normal lung tissue of mice was obtained and placed in sterile PBS in an ice box.

[0029] 3.2 Transfer the tissue to the clean bench and wash the lung tissue 2-3 times with 3 mL Advanced DMEM / F12 (Gibco, catalog number: 12634010) to remove blood cells.

[0030] 3.3 Transfer the washed and centrifuged lung tissue to a 35 mm culture dish and use scissors and tweezers to remove excess blood clots and tissue; discard excess liquid on the lung tissue and mince the lung tissue with a blade.

[0031] 3.4 Use a sterile Pasteur pipette to add 2 mL of DNase I digestion solution to the culture dish, rinse the chopped tissue and transfer all of it into the digestion solution.

[0032] 3.5 Place in a 37℃ incubator for digestion for 15 min, observe the digestion progress and shake manually; digest for another 15 min.

[0033] 3.6 Add 50 μL DNase I digestion solution and digest at 37℃ for 15 min.

[0034] 3.7 Then filter using a 40 μm cell sieve. Add Advanced DMEM / F12 medium was added and the cells were centrifuged at 500 rpm for 10 min to collect the cell pellet.

[0035] 3.9 Add 2 mL of red blood cell lysis buffer and mix well, then add 9 mL of Advanced DMEM / F12 to terminate lysis. Centrifuge at 500 rpm for 10 min, discard the supernatant, obtain cell clusters, and inoculate them in 60% matrigel. Then, Advanced DMEM / F12 medium containing 1× penicillin-streptomycin mixture, 10 mM HEPES, 1× GlutaMAX, 500 ng / mL R-spondin, 10% Noggin, 1× B27, 1.5 mM N-acetylcysteine, 5 mM nicotinamide, 6 mM Y27632, 600 μM A8301, 600 nM SB202190, 30 ng / ml FGF-7 (recombinant human fibroblast growth factor-7), 120 ng / ml FGF-10 (recombinant human fibroblast growth factor-10), 60 μg / ml primocin, and 8 nM human heregulin-β was added in sequence. The lung organ tissue was transferred to a fresh drop of the mixture of Matrigel and conditioned medium every 3-5 days.

[0036] 4. Tobacco Product Exposure Methods for Airway Organoids 4.1 Remove mature airway organoids from the incubator and photograph them on a high-content microscope.

[0037] 4.2 Discard the culture medium and add tobacco smoke extract to the airway organoid culture medium at different concentration gradients, with 200 μL added to each well of the airway organoid.

[0038] 4.3 After all the ingredients are added, place the 48-well plate containing the airway organoids in a 37°C incubator until the airway organoids mature.

[0039] 5. Immunofluorescence staining After the airway organoids were infected using the method in Section 4, they were rinsed three times with PBS and then cell recovery solution (500 μL) was added. After rinsing with PBS, 4% PFA was added for 30 minutes. The primary antibody was incubated overnight at 4°C, and the secondary antibody was incubated in the dark at room temperature for 2 hours, and then rinsed three times with PBS. The cell nuclei were stained for 20 minutes, and then the organ tissue was observed and analyzed using a high-content screening instrument.

[0040] 6. Cell Viability Analysis Airway organoids were seeded at a density of approximately 100 per well in a 96-well plate. After 24 hours of culture, the airway organoids were infected using the method described in Section 4. After 24 or 48 hours of continuous culture, 3D cell viability assay solution (Promega reagent, 10% advanced DMEM / F12 solution, 100 μL) was added and shaken for 5 minutes to lyse the organoids. After 25 minutes at room temperature, the absorbance value was measured using a microplate reader.

[0041] 7. Organoid Diameter Measurement After the airway organoids were infected using the method described in Section 4, 40 organoids were randomly selected from all experimental wells of the 48-well plate for organ diameter statistics. Before and after the extraction treatment, the plate was examined using a high-content screener to obtain images, and all images were analyzed using Image J.

[0042] 8. Reactive oxygen species (ROS) detection Using ROS-Glo™ H 2 O 2 The assay measures hydrogen peroxide (a ROS) in airway organoids exposed to HTP aerosol. After pretreatment, 20 μL of H 2 O 2Substrate solution (125 μM), after exposure, pre-configured ROS-GloTM detection solution (100 μL) was added to each well, incubated with shaking at room temperature for 20 min, and the luminescence value was immediately detected using a microplate reader (SPARK; 200 Tecan, CH).

[0043] 9. Detection of γ-H2AX γ-H2AX is an indicator of DNA double-strand breaks. Its presence indicates that DNA is severely damaged. The expression of p-γ-H2AX was detected using a DNA damage detection kit (C2035S, Biotech, China). Organoids were seeded evenly in 96-well plates at a density of 100 per well and incubated at 37°C, 5% CO 2 The cells were cultured under the conditions of 5% PBS for 24 hours and then exposed to the test samples for 48 hours. The treated AOs were rinsed with PBS for 5 minutes and then fixed with 4% PFA for 10 minutes. After the blocking solution was reacted for 15 minutes, the cells were washed three times with PBS. Finally, γ-H2AX monoclonal antibody and Alexa Fluor 488 secondary antibody were added and incubated. The cell nuclei were stained for 5 minutes at room temperature and then washed for 3 minutes. Images were collected and analyzed using a high-content screening instrument (PerkinElmer; Waltham, US).

[0044] 10. Quantitative analysis of cytokines Using Bio-Plex TM The pro mouse cytokine assay (Cat. No. M60009RDPD, Bio-Rad, USA) was used to detect potential inflammation. Briefly, sample and standard solutions were prepared and diluted. 50 μL of beads (1×) were added to each well of a 96-well plate and washed twice. Then, an equal volume of standard solution and sample was added, incubated at room temperature in the dark for 1 h, and washed three times. Then, 25 μL of diluted antibody detection solution was added, incubated at room temperature for 30 min, and washed three times. Then, 50 μL of streptavidin-phycoerythrin was added to each well and incubated at room temperature for 10 min at 180 g. Finally, 125 μL of detection buffer was added to each well, incubated at room temperature for 30 s at 180 g, and data were detected using the Bio-Plex Suspension Array System (Bio-Rad; California, USA).

[0045] 2. Experimental Results Analysis 1. Establishment of airway organoids After organoid differentiation, the growth of organoids was observed every day. The morphological changes of organoids are shown in Figure 1A. On the first day, AOs were irregular cell clusters (Figure 1A). From the second day of induction, AOs gradually became rounded and reached a morphological maximum on the fifth day of induction, with an average diameter of approximately 160 μm (Figure 1B). From the sixth day, the growth of organoids showed obvious changes, manifested as degeneration and death from the inside, so in our study, all AOs pretreatment should be within 5 days. In order to verify the different cell types in airway organoids (Lawrence, M. L et al., 2022), we performed immunofluorescence, and the results showed that basal cells (P63), secretory cells (MUC5AC), and ciliated cells (acetylated α-tubulin) were all identified (Figure 1C), indicating that airway organoids were successfully established.

[0046] 2. Effects of tobacco products on airway organoid cell viability After incubating AOs with HTP aerosol for 48 h, the results are shown in Figure 2, indicating that tobacco products reduced cell viability in a dose-dependent manner. The IC50 values ​​of HTP1 and HTP2 were calculated to be 8140 μg / mL and 10116 μg / mL, respectively.

[0047] 3. Exposure to heated tobacco products can inhibit the growth of AOs The TPM of both HTPs was observed to be dose-dependently cytotoxic, so we investigated the potential effects of HTPs on AO growth. Before exposure, we statistically analyzed the average diameter of AOs in different groups to be approximately 80 μm (Figure 3A). After 48 h of exposure, the average diameter of AOs in the control group increased to approximately 120 μm (Figure 3B, right). As shown in Figure 3B, exposure to TPM of both HTPs reduced AO diameter in a concentration-dependent manner. Starting from 1000 μg / mL, AO diameter was significantly reduced, indicating that this morphological parameter is more sensitive to TPM of HTPs than to cytotoxicity assays.

[0048] 4. Heated tobacco products cause changes in the proportion of differentiated cell types Our study explored the mRNA expression of differentiated cell types such as secretory cells (MUC5AC), basal cells (KRT5), and ciliated cells (FOXJ1), and the results showed that after 48 h of exposure to HTPs, the mRNA expression of MUC5AC and KRT5 increased significantly, indicating that different heated tobacco products induced the number of secretory cells and basal cells (Figure 4A-B), while the number of ciliated cells decreased significantly, as shown by the decrease in FOXJ1 mRNA expression (Figure 4C). It has been reported that airway damage and changes in cell differentiation and cell type ratios, including a decrease in ciliated cells and an increase in mucus-secreting secretory cells and proliferating basal cells, occur after exposure to cigarette smoke (Goldfarbmuren KC et al., 2020), suggesting that AOs can mimic the effects of tobacco products on different cell types in vivo in vitro.

[0049] 5. Contact with heated tobacco products induces inflammation The protein levels of cytokines were evaluated, and HTPs induced the secretion of pro-inflammatory cytokines (Figure 5 AC). Meanwhile, anti-inflammatory cytokines (Figure 5 DG) and adaptive immune factors with anti-inflammatory effects (Figure 5 H) were significantly suppressed. Taken together, these results indicate that both HTPs induce significant inflammation under high-concentration exposure.

[0050] 6. Exposure to heated tobacco products induces apoptosis and oxidative stress We found that exposure of AOs to HTP2 significantly induced apoptosis, and the apoptotic rate increased in a dose-dependent manner. In contrast, HTP1 induced apoptosis only at high concentrations (Figure 6 AB). These findings suggest that heated tobacco products have a potential effect on cell fate.

[0051] Oxidative stress is characterized by elevated levels of ROS, which disrupt the redox balance in cells and lead to oxidative stress. Our results showed that both HTPs induced significant oxidative stress after exposure to different concentrations of TPM in HTP aerosol. Notably, the ROS content of HTPs was significantly increased, ranging from 6 to 36 times higher than that of the control group, highlighting the obvious differences between different tobacco products in inducing ROS (Figure 6C).

[0052] 7. Heated tobacco products cause DNA damage DNA damage can lead to H2AX phosphorylation, which in turn forms phosphorylated histone H2AX--γ-H2AX. Therefore, the presence and amount of γ-H2AX can be used as a powerful indicator to assess the degree of DNA damage and cellular genotoxicity. Our results showed that the level of γ-H2AX induced by HTPs increased significantly at the IC50 concentration, and this trend persisted at 0.5 times the IC50 and 0.25 times the IC50 concentration, indicating that HTPs had a significant effect on H2AX phosphorylation and DNA damage within this concentration range ( Figure 7 AB), provides more detailed and comprehensive information for studying the effects of heated tobacco products on organ tissues.

[0053] 8. Summary Heated tobacco products (HTPs) are a new type of tobacco product. The content of harmful and potentially harmful components in its aerosol is lower than that in tobacco smoke, but it is not completely risk-free. This application explores the toxicology of HTPs in vitro by culturing airway organoids.

[0054] The study in this application found that the diameter of cultured airway organoids was about 100 μm, while the diameter of two-dimensional cells, such as H292 cells and BEAS-2B cells, which are commonly used for in vitro evaluation of tobacco product toxicology, was about 16 μm, indicating that AOs are larger than two-dimensional cells. In general, its diameter is a direct indicator of its growth state. Therefore, we measured the diameter after exposure, and the results showed that exposure to HTPs significantly inhibited the growth and development of AOs. In addition, it was observed that the secretion of mucin was induced after exposure, the number of basal cells and secretory cells increased, and the number of ciliated cells decreased, indicating that HTP exposure caused changes in the proportion of differentiated cells, reflecting the characteristics and advantages of organoids in in vitro toxicology studies.

[0055] The results showed that pro-inflammatory cytokines were promoted, while anti-inflammatory cytokines and adaptive immune cytokines were suppressed. From the perspective of inflammatory response, high-concentration HTP aerosol exposure can induce intracellular inflammation, which may subsequently induce further damage such as cell apoptosis.

[0056] The increase in ROS levels can lead to the disruption of redox balance. Both novel tobacco products induced the massive production of reactive oxygen species in a dose-dependent manner, and the effective concentration of HTP1 tended to be lower than that of HTP2.

[0057] Caspase 3 is one of the important signaling pathways involved in cell apoptosis. Our experiments showed that HTP can induce the expression of Caspase 3.

[0058] Due to the multicellular structure, interactions between different cell types can also induce potential effects. These studies showed that exposure to cigarette smoke induces changes in oxidative stress and inflammation, which is consistent with the results of two-dimensional cell in vitro experiments and animal in vivo experiments, indicating that the results obtained from organoid-based toxicological evaluation systems are comparable.

[0059] Our data show that exposure to high concentrations of HTPs can also cause DNA damage. Combined with the results of other cytotoxic endpoints, the in vitro findings suggest that high concentrations of HTPs pose a potential risk to human health, which supports the applicability of the AO model in the health risk analysis of HTPs.

[0060] In summary, we have successfully established an organoid evaluation system. However, unlike traditional two-dimensional (monolayer) cell culture models, the three-dimensional structure of our organoid model is formed by cell self-assembly rather than by cell stacking. Therefore, the AO model described here is closer to the target tissue and its functions, such as ciliary beating and mucus secretion. At the same time, the measurement of organ diameter and differentiation into organotypic cell types shows potential toxic effects, which is also not possible with two-dimensional cell models, which will provide the possibility of evaluating specific indicators in whole animals in vitro.

[0061] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A tobacco product damage assessment method based on airway organoids, characterized in that: The following steps are involved: Tobacco product extracts were used to poison airway organoids; the damage caused by tobacco product extracts was evaluated by observing the morphological changes and physiological changes in airway organoids.

2. The tobacco product damage evaluation method according to claim 1, characterized in that: The morphological changes in airway organoids are specifically changes in diameter; The changes in physiological indicators of the airway organoids include any one or more of changes in γ-H2AX, ratio of differentiated cell types, cytokines, reactive oxygen levels, and cell viability.

3. The tobacco product damage evaluation method according to claim 1, characterized in that: Tobacco product extracts were set into multiple concentration gradients to poison airway organoids.

4. The tobacco product damage evaluation method according to any one of claims 1 to 3, characterized in that: The tobacco product extract is obtained by collecting smoke generated by burning tobacco products through a filter and then centrifuging it.

5. The tobacco product damage evaluation method according to claim 1, characterized in that: A high-content imaging system was used to evaluate changes in γ-H2AX, immunofluorescence staining, and morphology of airway organoids.

6. The tobacco product damage evaluation method according to claim 1, characterized in that: Airway organoids were established by the following method: S1. Take mouse lung tissue and digest it to get single cells; S2. Mix the single cells with Matrigel and culture them with conditioned medium.

7. The tobacco product damage evaluation method according to claim 6, characterized in that: The conditioned medium contains the following components: penicillin-streptomycin mixture, HEPES, GlutMAX, R-spondin, Noggin, B27, N-acetylcysteine, nicotinamide, Y27632, A8301, SB202190, FGF-7, FGF-10, Primocin, and human heregulin-β.

8. The tobacco product damage evaluation method according to claim 7, characterized in that: Conditioned medium was prepared with the following components at the following concentrations: Penicillin-streptomycin mixture 1-2×; HEPES8-10 mM; GlutMAX1-2×; R-spondin 300-500 ng / mL; Noggin volume fraction 8-12%; B271-2×； N-acetylcysteine ​​1-1.5 mM; Nicotinamide 4-6mM; Y276324-6mM; A8301 400-600 μM; SB202190400-600nM; FGF-7 20-30ng / ml; FGF-1080-120ng / ml; Primocin 40-60 μg / ml; Human heregulin-β 5-10 nM.

9. The tobacco product damage evaluation method according to claim 6, characterized in that: Airway organoids were established by the following method: S1. Obtain fresh lung tissue from mice, wash it 2-3 times with Advanced DMEM / F12, centrifuge it, mince it, add DNase I digestion solution, digest it in a 37°C incubator for 30-50 min, filter it, centrifuge it, and collect the cell clusters; then add red blood cell lysis solution and mix well, stop the lysis, centrifuge it again and discard the supernatant to obtain cell clusters; S2. The cell clusters were seeded in Matrigel and then cultured with conditioned medium.

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