Air-liquid Interface Cell Culture Research Articles

Comparing the Toxicological Responses of Pulmonary Air-Liquid Interface Models upon Exposure to Differentially Treated Carbon Fibers.

In recent years, the use of carbon fibers (CFs) in various sectors of industry has been increasing. Despite the similarity of CF degradation products to other toxicologically relevant materials such as asbestos fibers and carbon nanotubes, a detailed toxicological evaluation of this class of material has yet to be performed. In this work, we exposed advanced air-liquid interface cell culture models of the human lung to CF. To simulate different stresses applied to CF throughout their life cycle, they were either mechanically (mCF) or thermo-mechanically pre-treated (tmCF). Different aspects of inhalation toxicity as well as their possible time-dependency were monitored. mCFs were found to induce a moderate inflammatory response, whereas tmCF elicited stronger inflammatory as well as apoptotic effects. Furthermore, thermal treatment changed the surface properties of the CF resulting in a presumed adhesion of the cells to the fiber fragments and subsequent cell loss. Triple-cultures encompassing epithelial, macrophage, and fibroblast cells stood out with an exceptionally high inflammatory response. Only a weak genotoxic effect was detected in the form of DNA strand breaks in mono- and co-cultures, with triple-cultures presenting a possible secondary genotoxicity. This work establishes CF fragments as a potentially harmful material and emphasizes the necessity of further toxicological assessment of existing and upcoming advanced CF-containing materials.

Gene transfection using branched cationic amphiphilic compounds for an aerosol administration in cystic fibrosis context

For cystic fibrosis gene therapy, the aerosolization of genetic materials is the most relevant delivery strategy to reach the airway epithelium. However, aerosolized formulations have to resist shear forces while maintaining the integrity of plasmid DNA (pDNA) during its journey from the nebulization to the epithelial cells. Herein, we compared the efficiency of gene delivery by aerosolization of two types of formulations: (i) BSV163, a branched cationic amphiphilic compound, co-formulated with different DOPE ratios (mol/mol) and DMPE-PEG5000 and (ii) 25 KDa branched polyethylenimine (b-PEI)-based formulation used as control. This study also aims to determine whether BSV163-based formulations possess the ability to resist the nebulization mechanisms and protect the nucleic acids (pDNA) cargo. Therefore, two CpG free plasmids (pGM144 or pGM169) encoding either the luciferase reporter gene or hCFTR respectively were used. Air-Liquid Interface (ALI) cell-culture was selected as an in-vitro model for aerosol experiments due to its closer analogy with in vivo morphology. Results highlighted that DOPE ratio influences the capacity of the BSV163 based-formulations to mediate high transfection efficacies. Furthermore, we proved that addition of DMPE-PEG5000 upon the formation of the BSV163/DOPE (1/1) lipid film instead of post-insertion led to a higher transgene expression. The aerosolization of this formulation on ALI cell-culture was more efficient than the use of b-PEI-based formulation.

Staphylococcus aureus Fibronectin-Binding Proteins Contribute to Colonization of the Female Reproductive Tract.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen and frequent colonizer of human skin and mucosal membranes, including the vagina, with vaginal colonization reaching nearly 25% in some pregnant populations. MRSA vaginal colonization can lead to aerobic vaginitis (AV), and during pregnancy, bacterial ascension into the upper reproductive tract can lead to adverse birth outcomes. USA300, the most prominent MRSA lineage to colonize pregnant individuals, is a robust biofilm former and causative agent of invasive infections; however, little is known about how it colonizes and ascends in the female reproductive tract (FRT). Our previous studies showed that a MRSA mutant of seven fibrinogen-binding adhesins was deficient in FRT epithelial attachment and colonization. Using both monolayer and multilayer air-liquid interface cell culture models, we determine that one class of these adhesins, the fibronectin binding proteins (FnBPA and FnBPB), are critical for association with human vaginal epithelial cells (hVECs) and hVEC invasion through interactions with α5β1 integrin. We observe that both FnBPs are important for biofilm formation as single and double fnbAB mutants exhibit reduced biofilm formation on hVECs. Using heterologous expression of fnbA and fnbB in Staphylococcus carnosus, FnBPs are also found to be sufficient for hVEC cellular association, invasion, and biofilm formation. In addition, we found that an ΔfnbAB mutant displays attenuated ascension in our murine vaginal colonization model. Better understanding of MRSA FRT colonization and ascension can ultimately inform treatment strategies to limit MRSA vaginal burden or prevent ascension, especially during pregnancy and in those prone to AV.

Novel 3D-Printed Cell Culture Inserts for Air-Liquid Interface Cell Culture.

In skin research, widely used in vitro 2D monolayer models do not sufficiently mimic physiological properties. To replace, reduce, and refine animal experimentation in the spirit of ‘3Rs’, new approaches such as 3D skin equivalents (SE) are needed to close the in vitro/in vivo gap. Cell culture inserts to culture SE are commercially available, however, these inserts are expensive and of limited versatility regarding experimental settings. This study aimed to design novel cell culture inserts fabricated on commercially available 3D printers for the generation of full-thickness SE. A computer-aided design model was realized by extrusion-based 3D printing of polylactic acid filaments (PLA). Improvements in the design of the inserts for easier and more efficient handling were confirmed in cell culture experiments. Cytotoxic effects of the final product were excluded by testing the inserts in accordance with ISO-norm procedures. The final versions of the inserts were tested to generate skin-like 3D scaffolds cultured at an air–liquid interface. Stratification of the epidermal component was demonstrated by histological analyses. In conclusion, here we demonstrate a fast and cost-effective method for 3D-printed inserts suitable for the generation of 3D cell cultures. The system can be set-up with common 3D printers and allows high flexibility for generating customer-tailored cell culture plastics.

Bioactive Cell-Derived ECM Scaffold Forms a Unique Cellular Microenvironment for Lung Tissue Engineering.

Chronic lung diseases are one of the leading causes of death worldwide. Lung transplantation is currently the only causal therapeutic for lung diseases, which is restricted to end-stage disease and limited by low access to donor lungs. Lung tissue engineering (LTE) is a promising approach to regenerating a replacement for at least a part of the damaged lung tissue. Currently, lung regeneration is limited to a simplified local level (e.g., alveolar–capillary barrier) due to the sophisticated and complex structure and physiology of the lung. Here, we introduce an extracellular matrix (ECM)-integrated scaffold using a cellularization–decellularization–recellularization technique. This ECM-integrated scaffold was developed on our artificial co-polymeric BETA (biphasic elastic thin for air–liquid interface cell culture conditions) scaffold, which were initially populated with human lung fibroblasts (IMR90 cell line), as the main generator of ECM proteins. Due to the interconnected porous structure of the thin (<5 µm) BETA scaffold, the cells can grow on and infiltrate into the scaffold and deposit their own ECM. After a mild decellularization procedure, the ECM proteins remained on the scaffold, which now closely mimicked the cellular microenvironment of pulmonary cells more realistically than the plain artificial scaffolds. We assessed several decellularization methods and found that 20 mM NH4OH and 0.1% Triton X100 with subsequent DNase treatment completely removed the fibroblasts (from the first cellularization) and maintains collagen I and IV as the key ECM proteins on the scaffold. We also showed the repopulation of the primary fibroblast from human (without chronic lung disease (non-CLD) donors) and human bronchial epithelial (16HBE14o−) cells on the ECM-integrated BETA scaffold. With this technique, we developed a biomimetic scaffold that can mimic both the physico-mechanical properties and the native microenvironment of the lung ECM. The results indicate the potential of the presented bioactive scaffold for LTE application.

Gene Expression Profiling of Mono- and Co-Culture Models of the Respiratory Tract Exposed to Crystalline Quartz under Submerged and Air-Liquid Interface Conditions.

In vitro lung cell models like air-liquid interface (ALI) and 3D cell cultures have advanced greatly in recent years, being especially valuable for testing advanced materials (e.g., nanomaterials, fibrous substances) when considering inhalative exposure. Within this study, we established submerged and ALI cell culture models utilizing A549 cells as mono-cultures and co-cultures with differentiated THP-1 (dTHP-1), as well as mono-cultures of dTHP-1. After ALI and submerged exposures towards α-quartz particles (Min-U-Sil5), with depositions ranging from 15 to 60 µg/cm2, comparison was made with respect to their transcriptional cellular responses employing high-throughput RT-qPCR. A significant dose- and time-dependent induction of genes coding for inflammatory proteins, e.g., IL-1A, IL-1B, IL-6, IL-8, and CCL22, as well as genes associated with oxidative stress response such as SOD2, was observed, even more pronounced in co-cultures. Changes in the expression of similar genes were more pronounced under submerged conditions when compared to ALI exposure in the case of A549 mono-cultures. Hereby, the activation of the NF-κB signaling pathway and the NLRP3 inflammasome seem to play an important role. Regarding genotoxicity, neither DNA strand breaks in ALI cultivated cells nor a transcriptional response to DNA damage were observed. Altogether, the toxicological responses depended considerably on the cell culture model and exposure scenario, relevant to be considered to improve toxicological risk assessment.

Designing Self-floating Anisotropic Macroporous Hydrogel by Step Emulsification and Buoyancy-assisted Microfluidics

Macroporous hydrogel with self-floating properties shows promising applications in gastric drug delivery, water purification and air–liquid interface cell culture. Microfluidic bubble templating is an emerging routine to produce macroporous hydrogel. However, current methods are in lack of the control over the anisotropy and porosity. Here, we describe new developments for the buoyancy-assisted microfluidic fabrication of anisotropic hydrogel products by templating from bubbles formed using step emulsification. The step emulsification method generates bubbles with exceptional precision which enables convenient tailoring the internal porosity of the hydrogel particles. Further design over the device structure and the buoyancy during the drop formation produces irregular hydrogel products such as macroporous fibers and core/shell beads. The anisotropic hydrogel beads provide pressure-responsive swimming behaviors and controllable release of the carried cargos. They are proved to be competent as carriers for culture of bronchial epithelial cells on air–liquid interfaces.

Study of the ciliary function in adult and pediatric patients with cystic fibrosis

Mucociliary clearance MCC) results in an effective interaction between the mucus layer and the normal coordinated ciliary beating. In cystic fibrosis (CF), MCC is impaired due to dehydrated mucus, but ciliary function is poorly studied. Recent data suggested that abnormal ciliary beating may contribute to poor MCC in CF patients. However, results are controversial: ciliary beat frequency (CBF) might be either decreased or increased, compared with healthy subjects. We aimed to evaluate if ciliary function may have a role in impaired mucociliary clearance in CF patients and if this ciliary dyskinesia was present in childhood. Ciliated epithelial samples were obtained by nasal brushing from 22 pediatric (0-17 years) and 15 adult CF patients (18-78 years). Beating cilia were recorded using digital high-speed videomicroscopy at 37 °C. Ciliary functional analysis (CFA) was assessed by CBF and the percentage of abnormal ciliary beat pattern (CBP) and was compared with 14 healthy subjects (HS) (22–54 years). There was a significant difference in CBF only between adult CF patients and HS. Abnormal CBP was significantly higher in CF patients, regardless of the age of patients ( Table 1 ). This study suggested that an abnormal ciliary function, present in childhood, might contribute to impaired MCC in CF patients as shown by lower CBF or higher dyskinetic CBP compared with HS. To evaluate if ciliary dyskinesia is primary or secondary to chronic inflammation, CFA should be repeated after air-liquid interface cell culture.

Air-liquid interphase culture confers SARS-CoV-2 susceptibility to A549 alveolar epithelial cells

The human lung cell A549 is susceptible to infection with a number of respiratory viruses. However, A549 cells are resistant to Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) infection in conventional submerged culture, and this would appear to be due to low expression levels of the SARS-CoV-2 entry receptor: angiotensin-converting enzyme-2 (ACE2). Here, we examined SARS-CoV-2 susceptibility to A549 cells after adaptation to air-liquid interface (ALI) culture. A549 cells in ALI culture yielded a layer of mucus on their apical surface, exhibited decreased expression levels of the proliferation marker KI-67 and intriguingly became susceptible to SARS-CoV-2 infection. We found that A549 cells increased the endogenous expression levels of ACE2 and TMPRSS2 following adaptation to ALI culture conditions. Camostat, a TMPRSS2 inhibitor, reduced SARS-CoV-2 infection in ALI-cultured A549 cells. These findings indicate that ALI culture switches the phenotype of A549 cells from resistance to susceptibility to SARS-CoV-2 infection through upregulation of ACE2 and TMPRSS2.

A primary nasopharyngeal three-dimensional air-liquid interface cell culture model of the pseudostratified epithelium reveals differential donor- and cell type-specific susceptibility to Epstein-Barr virus infection.

Epstein-Barr virus (EBV) is a ubiquitous γ-herpesvirus with latent and lytic cycles. EBV replicates in the stratified epithelium but the nasopharynx is also composed of pseudostratified epithelium with distinct cell types. Latent infection is associated with nasopharyngeal carcinoma (NPC). Here, we show with nasopharyngeal conditionally reprogrammed cells cultured at the air-liquid interface that pseudostratified epithelial cells are susceptible to EBV infection. Donors varied in susceptibility to de novo EBV infection, but susceptible cultures also displayed differences with respect to pathogenesis. The cultures from one donor yielded lytic infection but cells from two other donors were positive for EBV-encoded EBERs and negative for other lytic infection markers. All cultures stained positive for the pseudostratified markers CK7, MUC5AC, α-tubulin in cilia, and the EBV epithelial cell receptor Ephrin receptor A2. To define EBV transcriptional programs by cell type and to elucidate latent/lytic infection-differential changes, we performed single cell RNA-sequencing on one EBV-infected culture that resulted in alignment with many EBV transcripts. EBV transcripts represented a small portion of the total transcriptome (~0.17%). All cell types in the pseudostratified epithelium had detectable EBV transcripts with suprabasal cells showing the highest number of reads aligning to many EBV genes. Several restriction factors (IRF1, MX1, STAT1, C18orf25) known to limit lytic infection were expressed at lower levels in the lytic subcluster. A third of the differentially-expressed genes in NPC tumors compared to an uninfected pseudostratified ALI culture overlapped with the differentially-expressed genes in the latent subcluster. A third of these commonly perturbed genes were specific to EBV infection and changed in the same direction. Collectively, these findings suggest that the pseudostratified epithelium could harbor EBV infection and that the pseudostratified infection model mirrors many of the transcriptional changes imposed by EBV infection in NPC.

Transwell Insert-Embedded Microfluidic Devices for Time-Lapse Monitoring of Alveolar Epithelium Barrier Function under Various Stimulations.

This paper reports a transwell insert-embedded microfluidic device capable of culturing cells at an air-liquid interface (ALI), mimicking the in vivo alveolar epithelium microenvironment. Integration of a commercially available transwell insert makes the device fabrication straightforward and eliminates the tedious device assembly processes. The transwell insert can later be detached from the device for high-resolution imaging of the cells. In the experiments, the cells showing type-I pneumocyte markers are exploited to construct an in vitro alveolar epithelium model, and four culture conditions including conventional liquid/liquid culture (LLC) and air–liquid interface (ALI) cell culture in normal growth medium, and ALI cell culture with inflammatory cytokine (TNF-α) stimulation and ethanol vapor exposure are applied to investigate their effects on the alveolar epithelium barrier function. The barrier permeability is time-lapse monitored using trans-epithelial electrical resistance (TEER) measurement and immunofluorescence staining of the tight junction protein (ZO-1). The results demonstrate the functionalities of the device, and further show the applications and advantages of the constructed in vitro cell models for the lung studies.

Bone morphogenetic protein-2 as a novel biomarker for refractory chronic rhinosinusitis with nasal polyps

Bone morphogenetic proteins (BMPs), which are members of the TGF-β superfamily, regulate bone remodeling by stimulating osteoblasts and osteoclasts. Although the association between osteitis and poor surgical outcomes is well known in patients with chronic rhinosinusitis (CRS), BMPs have not been fully investigated as potential biomarkers for the prognosis of CRS. Our aim was to investigate the role of BMPs in osteitis in patients with CRS with nasal polyps (NPs) (CRSwNPs), as well as associations between BMPs and inflammatory markers in sinonasal tissues from patients with CRSwNP. We investigated the expression of 6 BMPs (BMP-2, BMP-4, BMP-6, BMP-7, BMP-9, and BMP-10) and their cellular origins in NPs of human subjects by using immunohistochemistry and ELISA of NP tissues. Exploratory factor analysis was performed to identify associations between BMPs and inflammatory markers. Air-liquid interface cell culture of human nasal epithelial cells was performed to evaluate the induction of the epithelial-mesenchymal transition by BMPs. Of the 6 BMPs studied, BMP-2 and BMP-7 were associated with refractoriness. Only BMP-2 concentrations were higher in patients with severe osteitis and advanced disease extent according to the computed tomography findings. Eosinophils and some macrophages were identified as cellular sources of BMP-2 in immunofluorescence analysis. An invitro experiment revealed that BMP-2 induced epithelial-mesenchymal transition in air-liquid interface-cultured human nasal epithelial cells, particularly in a TH2 milieu. BMP-2 could reflect the pathophysiology of mucosa and bone remodeling and may be a novel biomarker for refractory CRSwNP.

A human surfactant B deficiency air-liquid interface cell culture model suitable for gene therapy applications

Surfactant protein B (SPB) deficiency is a severe monogenic interstitial lung disorder that leads to loss of life in infants as a result of alveolar collapse and respiratory distress syndrome. The development and assessment of curative therapies for the deficiency are limited by the general lack of well-characterized and physiologically relevant in vitro models of human lung parenchyma. Here, we describe a new human surfactant air-liquid interface (SALI) culture model based on H441 cells, which successfully recapitulates the key characteristics of human alveolar cells in primary culture as evidenced by RNA and protein expression of alveolar cell markers. SALI cultures were able to develop stratified cellular layers with functional barrier properties that are stable for at least 28 days after air-lift. A SFTPB knockout model of SPB deficiency was generated via gene editing of SALI cultures. The SFTPB-edited SALI cultures lost expression of SPB completely and showed weaker functional barrier properties. We were able to correct this phenotype via delivery of a lentiviral vector pseudotyped with Sendai virus glycoproteins F/HN expressing SPB. We believe that SALI cultures can serve as an important in vitro research tool to study human alveolar epithelium, especially for the development of advanced therapy medicinal products targeting monogenic disorders.

Toxicity and Gene Expression Profiling of Copper- and Titanium-Based Nanoparticles Using Air-Liquid Interface Exposure.

To assess the toxicity of nanomaterials, most in vitro studies have been performed under submerged conditions, which do not reflect physiological conditions upon inhalation. An air-liquid interface (ALI) exposure may provide more reliable data on dosimetry and prevent interactions with cell culture media components. Therefore, an ALI exposure was combined with a high-throughput RT-qPCR approach to evaluate the toxicological potential of CuO and TiO2 nanoparticles (NP) in A549 cells. While TiO2 NP did not show any cytotoxicity or other effects compromising genomic stability up to 25.8 μg/cm2, CuO NP revealed a dose-dependent cytotoxicity, starting at 4.9 μg/cm2. Furthermore, CuO NP altered distinct gene expression patterns indicative for disturbed metal homeostasis, stress response, and DNA damage induction. Thus, induction of metal homeostasis associated genes (MT1X, MT2A) at 0.4 μg/cm2 and higher suggested uptake and intracellular dissolution of CuO NP, which was verified by a dose-dependent increase in intracellular copper concentration. Starting at 4.9 μg/cm2, oxidative stress markers (HMOX1, HSPA1A) were induced dose-dependently, supported by elevated ROS levels. Furthermore, a dose-dependent induction of genes associated with DNA damage response (DDIT3, GADD45A) was observed, in concordance with an increase in DNA strand breaks. Finally, transcriptional data suggested the induction of apoptosis at high doses, while flow cytometric analysis revealed increased numbers of either late apoptotic or necrotic cells and clearly necrotic cells at the highest concentrations. Thus, an ALI cell culture system was successfully combined with a comprehensive high-throughput RT-qPCR system, allowing the quantification of NP deposition and their impact on genomic stability. For CuO NP, in principle the data confirm observations made under submerged conditions with respect to intracellular copper ion release, as well as oxidative and genotoxic stress response. However, the results derived from ALI exposure allow the assessment of dose-response-relationships as well as the comparison of relative toxic potencies of different NP.

Mimicking the human respiratory system: Online in vitro cell exposure for toxicity assessment of welding fume aerosol

In assessing the biological impact of airborne particles in vitro, air-liquid interface (ALI) exposure chambers are increasingly preferred over classical submerged exposure techniques, albeit historically limited by their inability to deliver sufficient aerosolized dose. A novel ALI system, the Dosimetric Aerosol in Vitro Inhalation Device (DAVID), bioinspired by the human respiratory system, uses water-based condensation for highly efficient aerosol deposition to ALI cell culture. Here, welding fumes (well-studied and inherently toxic ultrafine particles) were used to assess the ability of DAVID to generate toxicological responses between differing welding conditions. After fume exposure, ALI-cultured cells showed reductions in viability that were both distinct between welding conditions and linearly dose-dependent with respect to exposure time; comparatively, submerged cell cultures ran in parallel did not show these trends across exposure levels. DAVID delivers a substantial dose in minutes (> 100 μg/cm2), making it preferable over previous ALI systems, which require hours of exposure to deliver sufficient dose, and over submerged techniques, which lack comparable physiological relevance. DAVID has the potential to provide the most accurate assessment of in vitro toxicity yet from the perspectives of physiological relevance to the human respiratory system and efficiency in collecting ultrafine aerosol common to hazardous exposure conditions.

Hypertonic saline and seawater solutions damage sinonasal epithelial cell air-liquid interface cultures.

Nasal irrigation (NI) is commonly used to treat several sinonasal diseases, including chronic rhinosinusitis with nasal polyps (CRSwNP); however, the effects of NI on the sinonasal epithelium are not fully known. The aim of this study was to investigate the effects of commonly used NI solutions on epithelial mucociliary and barrier functionality in primary cultured human nasal epithelial cells (HNECs). HNECs from control subjects and patients with CRSwNP were established as air-liquid interface (ALI) cultures. Differentiated cultures were treated with different NI solutions, including isotonic 0.9% and hypertonic 3.0% saline, isotonic and hypertonic seawater, and Ringer lactate solution. The changes in ciliary beat frequency (CBF), numbers of ciliated and goblet cells, and cytotoxicity were measured. Epithelial barrier functionality was assessed by measuring the transepithelial electric resistance (TER), paracellular flux, and expression of tight junction protein zonula occludens-1 (ZO-1) and occludin. Isotonic saline, isotonic seawater, and Ringer lactate solutions did not affect epithelial mucociliary and barrier function in either control or CRSwNP-derived ALI cultures; however, hypertonic saline induced a significant disruption of these cell functions in both cultures. Hypertonic seawater caused a transient decrease of CBF and TER in CRSwNP-derived ALI cultures, in contrast to inducing an obvious mucociliary and barrier dysfunction and cytotoxicity in control ALI cultures. Although isotonic NI solutions appear to not affect epithelial mucociliary and barrier function in control and CRSwNP-derived ALI cultures, hypertonic saline and seawater solutions damaged sinonasal epithelial cells in ALI cultures. The safety and efficacy of these solutions requires further investigation.

MicroRNA Let-7f-1-3p attenuates smoke-induced apoptosis in bronchial and alveolar epithelial cells in vitro by targeting FOXO1

Bronchial and alveolar epithelial cell apoptosis is a vital step in smoke-induced lung injury. We investigated whether and how microRNA (miRNA) Let-7f-1-3p would regulate smoke-induced apoptosis in bronchial and alveolar epithelial cells. Human small airway epithelial cells (HSAEC) and human pulmonary alveolar epithelial cells (HPAEpiC) were cultured using an air-liquid interface cell culture system. These cells were treated with Let-7f-1-3p agomir or antagomir for 24 h before smoke exposure or sham operation, after which the cells were rinsed and cultured for 24 h before cell viability, apoptosis, cytolysis, Caspase-9/8/3 activity assays, quantitative real-time polymerase chain reaction and Western blot. Bioinformatic and luciferase reporter assays were performed to predict or verify the target gene of Let-7f-1-3p. We found that smoke exposure significantly reduced Let-7f-1-3p expression level in HSAEC and HPAEpiC. Let-7f-1-3p agomir significantly attenuated cell apoptosis, cytolysis and Caspase-3, -8 and -9 activation while rescuing cell viability of smoke-exposed HSAEC and HPAEpiC. Let-7f-1-3p agomir downregulated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), Fas ligand (FasL) and B-cell lymphoma-2 (Bcl2)-like protein 11 (Bim) protein level in HSAEC and HPAEpiC. Forkhead box-O1 (FOXO1) was verified as a putative regulatory target of Let-7f-1-3p. Smoke exposure increased FOXO1 mRNA and protein level in HSAEC and HPAEpiC, which was attenuated by Let-7f-1-3p agomir treatment. FOXO1 inhibition by small-molecule drug partially attenuated the increase in smoke-exposed HSAEC and HPAEpiC apoptosis, cytolysis and the decrease in cell viability caused by Let-7f-1-3p antagomir treatment. We concluded Let-7f-1-3p attenuated smoke-induced apoptosis in HSAEC and HPAEpiC by targeting FOXO1.

Air-liquid interface culture changes surface properties of A549 cells

A549 cells are common models in the assessment of respiratory cytotoxicity. To provide physiologically more representative exposure conditions and increase the differentiation state, respiratory cells, for instance Calu-3 bronchial epithelial cells, are cultured at an air-liquid interface (ALI). There are indications that A549 cells also change their phenotype upon culture in ALI. The influence of culture in two variations of transwell cultures compared to conventional culture in plastic wells on the phenotype of A549 cells was studied. Cells were characterized by morphology, proliferation and transepithelial electrical resistance, whole genome transcription analysis, Western blot and immunocytochemical detection of pro-surfactant proteins. Furthermore, lipid staining, surface morphology, cell elasticity, surface tension and reaction to quartz particles were performed. Relatively small changes were noted in the expression of differentiation markers for alveolar cells but A549 cells cultured in ALI showed marked differences in lipid staining and surface morphology, surface tension and cytotoxicity of quartz particles. Data show that changes in physiological reactions of A549 cells in ALI culture were rather caused by change of surface properties than by increased expression of surfactant proteins.

Carbendazim induces death in alveolar epithelial cells: A comparison between submerged and at the air-liquid interface cell culture

The fungicide Carbendazim is widely used in agriculture and preservation of films and fibers. In mammals, it can promote germ cell mutagenicity, carcinogenicity, and reproductive toxicity. However, few data about the effects of this toxicant upon the respiratory system are available. In this work, we evaluated Carbendazim toxicity upon A549 alveolar cells both in monolayer and upon air-liquid interface cell system. Monolayer cell exposed to non-cytotoxic concentrations of this fungicide showed cell arrest at G2/M phase, and did not show additional alterations. On the other hand, alveolar 3D reconstructed epithelial model (air-liquid interface cell system) was characterized and exposed to IC25 of Carbendazim using the Vitrocell® Cloud 12 chamber. Expression of Active Caspase-3, α-tubulin and ROS was significantly increased after such exposure. Mitochondrial activity was also reduced after exposed to Carbendazim. The obtained results indicate that besides the environmental and reproductive toxicity concerns regarding Carbendazim exposure, pulmonary toxicity must be considered for this fungicide. In addition, we observed that the way of exposure impacts considerably on the cell response for in vitro assessment of chemicals inhalation toxicity profile.

Development of a tissue-engineered skin substitute on a base of human amniotic membrane

Allogenic graft material and tissue engineering have recently shown promising results for the improvement of both esthetic and functional outcomes in the treatment of large skin defects. We chose human amniotic membrane as a cellular scaffold in order to develop a skin substitute for later in vivo uses. Various methods of de-epithelialization of the human amniotic membrane were evaluated by histological analysis including hematoxylin–eosin and laminin staining, optic coherence tomography, and scanning electron microscopy with 0.25/0.02% trypsin/ethylenediaminetetraacetic acid treatment and mechanical cell removal showing an almost complete loss of the epithelium and a mainly intact basement membrane. Novel examination of human amniotic membrane by optic coherence tomography was feasible, but difficulties were experienced in handling and interpretation of the tissue as no comparable data exist. Subsequently, we developed an air–liquid interface cell culture to cultivate keratinocytes and fibroblasts on the de-epithelialized human amniotic membrane. We achieved a mostly keratinized surface on the epidermal side with a confluent fibroblast network on the chorion side.