Second-generation lung-on-a-chip array with a stretchable biological membrane

AbstractThe complex architecture of the lung parenchyma and the air-blood barrier is difficult to mimic in-vitro. Recently reported lung-on-a-chips used a thin, porous and stretchable PDMS membrane, to mimic the air-blood barrier and the rhythmic breathing motions. However, the nature, the properties and the size of this PDMS membrane differ from the extracellular matrix of the distal airways. Here, we present a second-generation lung-on-a-chip with an array of in vivo-like sized alveoli and a stretchable biological membrane. This nearly absorption free membrane allows mimicking in vivo functionality of the lung parenchyma at an unprecedented level. The air-blood barrier is constituted by human primary lung alveolar epithelial cells from several patients and co-cultured with primary lung endothelial cells. Typical markers of lung alveolar epithelial cells could be observed in the model, while barrier properties were preserved for up to three weeks. This advanced lung alveolar model reproduces some key features of the lung alveolar environment in terms of composition, alveolar size, mechanical forces and biological functions, which makes this model a more analogous tool for drug discovery, diseases modeling and precision medicine applications.

Download Full-text

Second-generation lung-on-a-chip with an array of stretchable alveoli made with a biological membrane

Communications Biology ◽

10.1038/s42003-021-01695-0 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Pauline Zamprogno ◽

Simon Wüthrich ◽

Sven Achenbach ◽

Giuditta Thoma ◽

Janick D. Stucki ◽

...

Keyword(s):

Alveolar Epithelial Cell ◽

Biological Membrane ◽

Dynamic Environment ◽

Basal Membrane ◽

Barrier Properties ◽

Alveolar Epithelial Cells ◽

Simple Method ◽

Alveolar Epithelial

AbstractThe air-blood barrier with its complex architecture and dynamic environment is difficult to mimic in vitro. Lung-on-a-chips enable mimicking the breathing movements using a thin, stretchable PDMS membrane. However, they fail to reproduce the characteristic alveoli network as well as the biochemical and physical properties of the alveolar basal membrane. Here, we present a lung-on-a-chip, based on a biological, stretchable and biodegradable membrane made of collagen and elastin, that emulates an array of tiny alveoli with in vivo-like dimensions. This membrane outperforms PDMS in many ways: it does not absorb rhodamine-B, is biodegradable, is created by a simple method, and can easily be tuned to modify its thickness, composition and stiffness. The air-blood barrier is reconstituted using primary lung alveolar epithelial cells from patients and primary lung endothelial cells. Typical alveolar epithelial cell markers are expressed, while the barrier properties are preserved for up to 3 weeks.

Download Full-text

TRIM72 is required for effective repair of alveolar epithelial cell wounding

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00172.2014 ◽

2014 ◽

Vol 307 (6) ◽

pp. L449-L459 ◽

Cited By ~ 15

Author(s):

Seong Chul Kim ◽

Thomas Kellett ◽

Shaohua Wang ◽

Miyuki Nishi ◽

Nagaraja Nagre ◽

...

Keyword(s):

Epithelial Cells ◽

Molecular Mechanisms ◽

Striated Muscle ◽

Alveolar Epithelial Cell ◽

Alveolar Epithelial Cells ◽

Lung Cells ◽

Alveolar Epithelial ◽

High Tidal Volume Ventilation

The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure.

Download Full-text

C3 Promotes Clearance of Klebsiella pneumoniae by A549 Epithelial Cells

Infection and Immunity ◽

10.1128/iai.72.3.1767-1774.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1767-1774 ◽

Cited By ~ 26

Author(s):

Beatriz de Astorza ◽

Guadalupe Cortés ◽

Catalina Crespí ◽

Carles Saus ◽

José María Rojo ◽

...

Keyword(s):

Epithelial Cells ◽

Klebsiella Pneumoniae ◽

Alveolar Epithelial Cells ◽

Clinical Isolates ◽

Complement Components ◽

Innate Defense ◽

Alveolar Epithelial

ABSTRACT The airway epithelium represents a primary site for contact between microbes and their hosts. To assess the role of complement in this event, we studied the interaction between the A549 cell line derived from human alveolar epithelial cells and a major nosocomial pathogen, Klebsiella pneumoniae, in the presence of serum. In vitro, we found that C3 opsonization of poorly encapsulated K. pneumoniae clinical isolates and an unencapsulated mutant enhanced dramatically bacterial internalization by A549 epithelial cells compared to highly encapsulated clinical isolates. Local complement components (either present in the human bronchoalveolar lavage or produced by A549 epithelial cells) were sufficient to opsonize K. pneumoniae. CD46 could competitively inhibit the internalization of K. pneumoniae by the epithelial cells, suggesting that CD46 is a receptor for the binding of complement-opsonized K. pneumoniae to these cells. We observed that poorly encapsulated strains appeared into the alveolar epithelial cells in vivo but that (by contrast) they were completely avirulent in a mouse model of pneumonia compared to the highly encapsulated strains. Our results show that bacterial opsonization by complement enhances the internalization of the avirulent microorganisms by nonphagocytic cells such as A549 epithelial cells and allows an efficient innate defense.

Download Full-text

Flagellin/TLR5 Stimulate Myeloid Progenitors to Enter Lung Tissue and to Locally Differentiate Into Macrophages

Frontiers in Immunology ◽

10.3389/fimmu.2021.621665 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xin Lei ◽

Jara Palomero ◽

Iris de Rink ◽

Tom de Wit ◽

Martijn van Baalen ◽

...

Keyword(s):

Epithelial Cells ◽

Low Frequency ◽

Lavage Fluid ◽

Lung Lavage ◽

Alveolar Epithelial Cells ◽

Mouse Bone Marrow ◽

Alveolar Epithelial ◽

Toll Like Receptor 5

Toll-like receptor 5 (TLR5) is the receptor of bacterial Flagellin. Reportedly, TLR5 engagement helps to combat infections, especially at mucosal sites, by evoking responses from epithelial cells and immune cells. Here we report that TLR5 is expressed on a previously defined bipotent progenitor of macrophages (MΦs) and osteoclasts (OCs) that resides in the mouse bone marrow (BM) and circulates at low frequency in the blood. In vitro, Flagellin promoted the generation of MΦs, but not OCs from this progenitor. In vivo, MΦ/OC progenitors were recruited from the blood into the lung upon intranasal inoculation of Flagellin, where they rapidly differentiated into MΦs. Recruitment of the MΦ/OC progenitors into the lung was likely promoted by the CCL2/CCR2 axis, since the progenitors expressed CCR2 and type 2 alveolar epithelial cells (AECs) produced CCL2 upon stimulation by Flagellin. Moreover, CCR2 blockade reduced migration of the MΦ/OC progenitors toward lung lavage fluid (LLF) from Flagellin-inoculated mice. Our study points to a novel role of the Flagellin/TLR5 axis in recruiting circulating MΦ/OC progenitors into infected tissue and stimulating these progenitors to locally differentiate into MΦs. The progenitor pathway to produce MΦs may act, next to monocyte recruitment, to fortify host protection against bacterial infection at mucosal sites.

Download Full-text

Murine Alveolar Epithelial Cells and Their Lentivirus-mediated Immortalisation

Alternatives to Laboratory Animals ◽

10.1177/026119291804600207 ◽

2018 ◽

Vol 46 (2) ◽

pp. 73-89

Author(s):

Sandra Sapich ◽

Marius Hittinger ◽

Remi Hendrix-Jastrzebski ◽

Urska Repnik ◽

Gareth Griffiths ◽

...

Keyword(s):

Epithelial Cells ◽

Cell Lines ◽

Experimental Testing ◽

Barrier Properties ◽

Alveolar Epithelial Cells ◽

Mouse Strains ◽

Alveolar Type ◽

Inhalation Toxicity ◽

Alveolar Epithelial

In this study, we describe the isolation and immortalisation of primary murine alveolar epithelial cells (mAEpC), as well as their epithelial differentiation and barrier properties when grown on Transwell® inserts. Like human alveolar epithelial cells (hAEpC), mAEpC transdifferentiate in vitro from an alveolar type II (ATII) phenotype to an ATI-like phenotype and exhibit features of the air–blood barrier, such as the establishment of a thin monolayer with functional tight junctions (TJs). This is demonstrated by the expression of TJ proteins (ZO-1 and occludin) and the development of high transepithelial electrical resistance (TEER), peaking at 1800ω•cm2. Transport across the air–blood barrier, for general toxicity assessments or preclinical drug development, is typically studied in mice. The aim of this work was the generation of novel immortalised murine lung cell lines, to help meet Three Rs requirements in experimental testing and research. To achieve this goal, we lentivirally transduced mAEpC of two different mouse strains with a library of 33 proliferation-promoting genes. With this immortalisation approach, we obtained two murine alveolar epithelial lentivirus-immortalised (mAELVi) cell lines. Both showed similar TJ protein localisation, but exhibited less prominent barrier properties (TEERmax ~250Ω•cm2) when compared to their primary counterparts. While mAEpC demonstrated their suitability for use in the assessment of paracellular transport rates, mAELVi cells could potentially replace mice for the prediction of acute inhalation toxicity during early ADMET studies.

Download Full-text

Cross-talk between LAM cells and fibroblasts may influence alveolar epithelial cell behavior in Lymphangioleiomyomatosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00351.2021 ◽

2021 ◽

Author(s):

Debbie Clements ◽

Suzanne Miller ◽

Roya Babaei-Jadidi ◽

Mike Adam ◽

S. Steven Potter ◽

...

Keyword(s):

Epithelial Cells ◽

Cross Talk ◽

Ex Vivo ◽

Alveolar Epithelial Cell ◽

Alveolar Epithelial Cells ◽

Dependent Manner ◽

Epithelial Migration ◽

Alveolar Epithelial

Lymphangioleiomyomatosis (LAM) is a female specific cystic lung disease in which TSC2 deficient LAM cells, LAM-Associated Fibroblasts (LAFs) and other cell types infiltrate the lungs. LAM lesions can be associated with type II alveolar epithelial cells (AT2 cells). We hypothesised that the behaviour of AT2 cells in LAM is influenced locally by LAFs. We tested this hypothesis in patient samples and in vitro. In human LAM lung, nodular AT2 cells show enhanced proliferation when compared to parenchymal AT2 cells, demonstrated by increased Ki67 expression. Further, nodular AT2 cells express proteins associated with epithelial activation in other disease states including Matrix Metalloproteinase 7, and Fibroblast Growth Factor 7 (FGF7). In vitro, LAF conditioned medium is mitogenic and positively chemotactic for epithelial cells, increases the rate of epithelial repair and protects against apoptosis. In vitro, LAM patient-derived TSC2 null cells cocultured with LAFs upregulate LAF expression of the epithelial chemokine and mitogen FGF7, which is a potential mediator of fibroblast-epithelial crosstalk, in an mTOR dependent manner. In a novel in vitro model of LAM, ex vivo cultured LAM lung-derived microtissues promote both epithelial migration and adhesion. Our findings suggest that AT2 cells in LAM display a proliferative, activated phenotype and that fibroblast accumulation following LAM cell infiltration into the parenchyma contributes to this change in AT2 cell behaviour. Fibroblast-derived FGF7 may contribute to the cross-talk between LAFs and hyperplastic epithelium in vivo, but does not appear to be the main driver of the effects of LAFs on epithelial cells in vitro.

Download Full-text

Nanomaterials induce different levels of oxidative stress, depending on the used model system: Comparison of in vitro and in vivo effects

10.21203/rs.3.rs-57664/v1 ◽

2020 ◽

Author(s):

Isabel Karkossa ◽

Anne Bannuscher ◽

Bryan Hellack ◽

Wendel Wohlleben ◽

Julie Laloy ◽

...

Keyword(s):

Oxidative Stress ◽

Epithelial Cells ◽

Alveolar Macrophages ◽

Model System ◽

Alveolar Epithelial Cells ◽

Model Systems ◽

Alveolar Epithelial ◽

Different Levels

Abstract Background The immense variety and constant development of nanomaterials (NMs) raise the demand for a facilitated risk assessment, for which knowledge on NMs mode of actions (MoAs) is required. For this purpose, a comprehensive data basis is of paramountcy that can be obtained using omics. Furthermore, the establishment of suitable in vitro test systems is indispensable to follow the 3R concept and to master the high number of NMs. In the present study, we aimed at comparing NM effects in vitro and in vivo using a multi-omics approach. We applied an integrated data evaluation strategy based on proteomics and metabolomics to four silica NMs and one titanium dioxide-based NM. For in vitro investigations, alveolar epithelial cells and alveolar macrophages were treated with different doses of NMs, and the results were compared to effects on rat lungs after short-term inhalations and instillations at varying doses with and without a recovery period.Results Since the production of reactive oxygen species (ROS) is described to be a critical biological effect of NMs, and enrichment analyses confirmed oxidative stress as a significant effect upon NM treatment in vitro in the present study, we focused on different levels of oxidative stress. Thus, we found opposite changes for proteins and metabolites that are related to the production of reduced glutathione in alveolar epithelial cells and alveolar macrophages, illustrating that NMs MoAs depend on the used model system. Interestingly, in vivo, pathways related to inflammation were affected to a greater extent than oxidative stress responses. Hence, the assignment of the observed effects to the levels of oxidative stress was different in vitro and in vivo as well. However, the overall classification of “active” and “passive” NMs was consistent in vitro and in vivo.Conclusions The consistent classification indicates both tested cell lines to be suitable for NM toxicity assessment even though the induced levels of oxidative stress strongly depend on the used model systems. Thus, the here presented results highlight that model systems need to be carefully revised to decipher the extent to which they can replace in vivo testing.

Download Full-text

ER Stress is Involved in Epithelial-To-Mesenchymal Transition of Alveolar Epithelial Cells Exposed to a Hypoxic Microenvironment

International Journal of Molecular Sciences ◽

10.3390/ijms20061299 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1299 ◽

Cited By ~ 4

Author(s):

Eva Delbrel ◽

Yurdagül Uzunhan ◽

Abdoulaye Soumare ◽

Thomas Gille ◽

Dominique Marchant ◽

...

Keyword(s):

Epithelial Cells ◽

Er Stress ◽

Epithelial To Mesenchymal Transition ◽

Alveolar Epithelial Cells ◽

Mesenchymal Transition ◽

Alveolar Epithelial ◽

Rat Lungs ◽

Fibrotic Process

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal interstitial lung disease of unknown origin. Alveolar epithelial cells (AECs) play an important role in the fibrotic process as they undergo sustained endoplasmic reticulum (ER) stress, and may acquire a mesenchymal phenotype through epithelial-to-mesenchymal transition (EMT), two phenomena that could be induced by localized alveolar hypoxia. Here we investigated the potential links between hypoxia, ER stress and EMT in AECs. Methods: ER stress and EMT markers were assessed by immunohistochemistry, western blot and qPCR analysis, both in vivo in rat lungs exposed to normoxia or hypoxia (equivalent to 8% O2) for 48 h, and in vitro in primary rat AECs exposed to normoxia or hypoxia (1.5% O2) for 2–6 days. Results: Hypoxia induced expression of mesenchymal markers, pro-EMT transcription factors, and the activation of ER stress markers both in vivo in rat lungs, and in vitro in AECs. In vitro, pharmacological inhibition of ER stress by 4-PBA limited hypoxia-induced EMT. Calcium chelation or hypoxia-inducible factor (HIF) inhibition also prevented EMT induction under hypoxic condition. Conclusions: Hypoxia and intracellular calcium are both involved in EMT induction of AECs, mainly through the activation of ER stress and HIF signaling pathways.

Download Full-text