Application progress of air-liquid interface exposure technology in respiratory system toxicity induced by diesel exhaust in vitro / 环境与职业医学

Diesel exhaust (DE) is an important pollution source widely existing in the living and production environment, which is closely related to the health of the public and occupational groups. The International Agency for Research on Cancer has classified DE as a Group 1 carcinogen. Considering the negative health impacts on the respiratory system due to DE exposure in vitro, it is crucial to apply reliable test systems allowing accurate assessment of the biological effects of DE. The exposure technology of respiratory system in vitro is considered as one of the feasible measures to implement the 3R (reduce, refine, and replace) principle in animal experiments. Compared with the traditional submerged culture in vitro models, the air-liquid interface (ALI) exposure technology has the advantages including fewer influencing factors, easier exposure condition control, and shorter exposure cycle. ALI has become an important tool to study molecular events associated with physiology and pathology of respiratory system, and action modes and interactions of different cell types. Also, ALI has been increasingly widely used because it can simulate the actual processes of human respiratory system cells and/or tissues to DE exposure. This review was intended to introduce the development and advantages of ALI exposure technology, and further summarized the application progress of ALI exposure technology in studying the respiratory toxicity induced by DE exposure in vitro, so as to provide new ideas and pathways for the use of ALI exposure technology in the study of biomarkers and mechanisms of respiratory toxicity associated with DE exposure, and provide basic data to screen and promote biomarkers for exposed populations.

Establishment of a culture system for human nasal mucosa organoids with controllable differentiation / 南方医科大学学报

OBJECTIVE@#To establish a culture system for human nasal mucosal organoids with controllable differentiation to reproduce the structure and function of the source tissue through staged expansion-differentiation culture.@*METHODS@#Fresh samples of surgically resected middle turbinate and nasal polyp tissues were collected, from which the nasal mucosa epithelial cells were isolated by enzymatic digestion and filtration for continuous culture at the air-liquid interface for expansion (EO group) or staged culture for expansion and differentiation (DO group). Immunohistochemical staining was used to characterize the structure, cellular composition and ciliary function of nasal mucosal organoids in the two groups. The secretion function of the differentiated nasal mucosal organoids in DO group was evaluated using PAS staining.@*RESULTS@#Both of the two organoid culture systems yielded vacuolar or solid spherical 3D organoids, and their diameters increased progressively with time. On day 16 of culture, more vacuolar organoids occurred in DO group, while more solid spherical organoids were seen in EO group, and the proportion of vacuoles was significantly greater in DO group than in EO group [(54.67±13.26)% vs (21.67±8.57)%, P < 0.05]. Short tandem repeat (STR) test of the nasal mucosal organoids and the source tissue showed a 100% match between them. On day 21 of culture, scanning and transmission electron microscopy of the nasal mucosal organoids identified ultrastructure of cilia in DO group and short villi structure in most of the organoids in EO group. Immunohistochemical staining showed positivity for P63 (basal cells), β-tubulin (ciliated columnar cells), and MUC5AC (goblet cells) in the organoids. Compared with those in EO group, the organoids in DO group showed significantly greater percentages of ciliated cells [(7.95±1.81)% vs (27.04±5.91)%, P < 0.05] and goblet cells [(14.46±0.93)% vs (39.85±5.43)%, P < 0.05) with a similar percentage of basal cells [(56.91±14.12)% vs (53.42±15.77)%, P > 0.05]. The differentiated nasal mucosal organoids in DO group were positively stained for glycogen.@*CONCLUSION@#The staged expansion-differentiation culture method allows more stable and prolonged growth of the cultured cells in vitro to produce organoids with controllable differentiation closely resembling the morphological structure and functions (ciliary function and secretory function) of the source tissue.

Assessment of Benchmark Dose in BEAS-2B Cells by Evaluating the Cell Relative Viability with Particulates in Motorcycle Exhaust / 生物医学与环境科学（英文）

Objective@#This study aimed to use an air-liquid interface (ALI) exposure system to simulate the inhalation exposure of motorcycle exhaust particulates (MEPs) and then investigate the benchmark dose (BMD) of MEPs by evaluating cell relative viability (CRV) in lung epithelial BEAS-2B cells.@*Methods@#The MEPs dose was characterized by measuring the number concentration (NC), surface area concentration (SAC), and mass concentration (MC). BEAS-2B cells were exposed to MEPs at different concentrations @*Results@#Our results reveal that BMD of NC and SAC were estimated by the best-fitting Hill model, while MC was estimated by Polynomial model. The BMDL for CRV following ALI exposure to MEPs were as follows: 364.2#/cm @*Conclusion@#These results indicate that MEPs exposure

Effects of aerosolized glycerol on human lung epithelial cells by air-liquid interface cloud exposure / 中国药理学通报

Aim To investigate the cytotoxicity of glycerol aerosol on human lung adenocarcinoma cells (A549) by air-liquid interface exposure, aimingto provide a guidance for the application of glycerol in inhalation products and e-cigarettes. Methods A549 cells cultured at air-liquid interface condition were exposed by VITROCELL cloud exposure system. Firstly, the delivery efficiency of the system was verified by fluorescein sodium, and repeatability and difference between inserts were investigated. Then, a glycerol aerosol airliquid interface exposure test was performed, and a negative control (1% PBS) and high, medium, and low dose of glycerol(10%, 6%, and 4%) were set to examine the cell viability, LDH release, as well as the expression of IL-6. Results The exposure system delivered an accurate dose. The delivery efficiency was high, the deposition rate was 53. 4%, and the characteristic parameters such as repeatability was 14. 8% and insert-insert difference was 5. 2%. After exposure to aerosolized glycerol, there was no significant difference in cell variability, LDH release, and IL-6 expression of A549 cells compared to the negative control. Conclusions A reliable and rapid in vitro model of acute exposure model of the respiratory tract is established. A single short-term exposure to aerosolized glycerol has no significant effect on cell viability and cytotoxicity, as well as inflammatory changes.

Characterization of air-liquid interface culture of A549 alveolar epithelial cells

Alveolar epithelia play an essential role in maintaining the integrity and homeostasis of lungs, in which alveolar epithelial type II cells (AECII) are a cell type with stem cell potential for epithelial injury repair and regeneration. However, mechanisms behind the physiological and pathological roles of alveolar epithelia in human lungs remain largely unknown, partially owing to the difficulty of isolation and culture of primary human AECII cells. In the present study, we aimed to characterize alveolar epithelia generated from A549 lung adenocarcinoma cells that were cultured in an air-liquid interface (ALI) state. Morphological analysis demonstrated that A549 cells could reconstitute epithelial layers in ALI cultures as evaluated by histochemistry staining and electronic microscopy. Immunofluorescent staining further revealed an expression of alveolar epithelial type I cell (AECI) markers aquaporin-5 protein (AQP-5), and AECII cell marker surfactant protein C (SPC) in subpopulations of ALI cultured cells. Importantly, molecular analysis further revealed the expression of AQP-5, SPC, thyroid transcription factor-1, zonula occludens-1 and Mucin 5B in A549 ALI cultures as determined by both immunoblotting and quantitative RT-PCR assay. These results suggest that the ALI culture of A549 cells can partially mimic the property of alveolar epithelia, which may be a feasible and alternative model for investigating roles and mechanisms of alveolar epithelia in vitro.

Evaluation of the viability of BEAS-2B cells exposed to gasoline engine exhaust with different particle sizes by air-liquid interface / 中华劳动卫生职业病杂志

Objective@#To evaluate the viability of gasoline engine exhaust (GEE) with different particle sizes on human lung cell line BEAS-2B in vitro by air-liquid interface (ALI) .@*Methods@#GEE were collected with a Tedlar bag and their particulate matter (PM) number, surface and mass concentration in three kind of GEE (filtered automobile exhaust, non-filtered automobile exhaust and motorcycle exhaust without three-way catalytic converter) were measured by two type of particle size spectrometer including TSI-3321 and SMPS-3938. Five groups were included, which divided into blank control group, clean air group, filtered automobile exhaust group, non-filtered automobile exhaust group and motorcycle exhaust without three-way catalytic converter group. Except the blank control group, BEAS-2B cells, cultured on the surface of Transwells, were treated with clean air or GEE by ALI method at a flow rate of 25 ml/min, 37 ℃ for 60 min in vitro. CCK-8 cytotoxicity test kit was used to determine the cell relative viability of BEAS-2B cells.@*Results@#In the filtered automobile exhaust, non-filtered automobile exhaust and motorcycle exhaust without three-way catalytic converter, high concentrations of fine particles can be detected, but the coarse particles only accounted for a small proportion, and the sequence of PM concentration was motorcycle exhaust without three-way catalytic converter group> non-filtered automobile exhaust group> filtered automobile exhaust group (P<0.001) . Compared with the clean air group, the cell relative viability in the 3 GEE-exposed groups were significantly lower (P<0.001) . Among the comparisons of GEE exposure groups with different particle size spectra, the sequence of the cell relative viability was filtered automobile exhaust group >non-filtered automobile exhaust group> motorcycle exhaust without three-way catalytic converter group (P<0.001) . When took the clean air control group as a reference, the mean of the cell relative viability in the filtered automobile exhaust group, non-filtered automobile exhaust group and motorcycle exhaust without three-way catalytic converter group, was decreased by 26.34%, 36.00% and 49.59%, respectively.@*Conclusion@#GEE with different particle size spectra could induce different levels of toxic effects to the human lung cells BEAS-2B by ALI. After lowering the concentration of particles in the GEE and using the three-way catalytic converter could obviously improve the survival rate of lung cells.

Ultra-Structures And 14C-mannitol Transport Study of Human Nasal Epithelial Cells using ALI Culture Technique / 결핵

BACKGROUND: The information on nasal transport and the metabolism of peptides have been obtained from pharmacokinetic investigations in experimental animals. However, there are no transport and metabolic studies of human nasal epithelial cells. In this study, the permeability characteristics and the metabolic properties of in vitro human nasal cell monolayers were investigated. Material and METHODS: Normal human inferior nasal conchal tissue samples were obtained from patients undergoing endoscopic nasal cavitary surgery. The specimens were cultured in a transwell using an air-liquid interface (ALI) culture, and the transepithelial electrical resistance (TEER) value of the blank filter and confluent cell monolayers were measured. To determine the % leakage of mannitol, 4µmol 14C-labelled mannitol was added and the % leakage was measured every 10 minute for 1 hour. RESULT: Human nasal epithelial cells in the primary culture grew to a confluent monolayer within 7 days and expressed microvilli. The tight junction between the cells was confirmed by transmission electron microscopy. The TEER value of the blank filter, fifth day and seventh day reached 108.5 ohm.cm2, 141 ohm.cm2 and 177.5 ohm.cm2, respectively. Transcellular % leakage of the 14C-mannitol at 10, 20, 30, 40, 50 and 60 minutes was 35.67±5.43, 34.42±5.60, 32.75±5.71, 31.76±4.22, 30.96±3.49 and 29.60±3.68 %, respectively. CONCLUSION: The human nasal epithelial monolayer using ALI using techniques is suitable for a transcellular permeability study. The data suggests that human nasal epithelial cells in as ALI culture technique shows some promise for a nasal transport and metabolism study.