An engineered 3D human airway mucosa model based on an SIS scaffold

Three-dimensional respiratory tissue models have been generated using, for example, human primary airway epithelial cells (hAEC) or respective cell lines. To investigate ciliopathies, such as primary ciliary dyskinesia, the presence of functional kinocilia in vitro is an essential prerequisite. Since access to hAEC of healthy donors is limited, we aimed to identify a respiratory epithelial cell line that is capable to display functional kinocilia on at least 60% of the apical surface. Thus, we cultured four different human respiratory cell lines with human primary airway fibroblasts under airlift conditions, characterized the morphology, and analyzed ciliary function. Only one of the tested cell lines showed beating kinocilia; however, <10% of the whole surface was covered and ciliary beating was undirected. Positive control tissue models using hAEC and fibroblasts displayed expected directed ciliary beating pattern around 11 Hz. Our data show that the available cell lines are not suitable for basic and applied research questions whenever functional kinocilia are required and that, rather, hAEC-or human induced pluripotent stem cell-derived tissue models need to be generated.To study ciliopathies or Bordetella pertussis infection in vitro, three-dimensional respiratory tissue models with functional kinocilia covering at least 60% of the model's surface are mandatory. We cultured four respiratory cell lines on a fibroblastloaded biological scaffold and showed that none of them met this requirement. In contrast, primary airway cell-derived models sufficiently reflected the mucociliary phenotype. To further search for an alternative to primary respiratory cells, investigations on other cell lines should be conducted or even new cell lines have to be generated. IntroductionT hree-dimensional (3D) cell cultures of the respiratory epithelium/mucosa afford research on-for examplethe impact of airborne pollutants, host-pathogen interactions, and drug permeation. 1-5 As source for respiratory epithelial cells, primary cells, induced pluripotent stem cells (iPSC), or cell lines can be used. Both primary cells and iPSC provide the opportunity to generate personalized tissue models, for example, to study individual drug responses or drug efficacy. Moreover, they show a high in vitro-in vivo correlation. These models feature a pseudostratified epithelial morphology, barrier properties, basal cells, mucus-producing goblet cells, and ciliated cells facilitating mucociliary clearance. [6][7][8][9]

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“…S1). hAEC on the SIS differentiated well as published elsewhere 6,7 and showed directed ciliary beating ( Supplementary Video S3).…”

Section: Resultssupporting

confidence: 57%

“…These models feature a pseudostratified epithelial morphology, barrier properties, basal cells, mucus-producing goblet cells, and ciliated cells facilitating mucociliary clearance. [6][7][8][9]…”

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confidence: 99%

Investigation on Ciliary Functionality of Different Airway Epithelial Cell Lines in Three-Dimensional Cell Culture

Lodes

Seidensticker

Perniss

et al. 2020

Tissue Engineering Part A

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“…Isolation of primary human tracheal epithelial cells (hTEC) was performed as described previously [96]. Briefly, the airway mucosa was removed mechanically from human tracheobronchial biopsies, which were subsequently placed into plastic cell culture dishes and covered with Airway Epithelial Cell Growth Medium (AECG, # PB-C-MH-350-0099, PeloBiotech).…”

Section: Methodsmentioning

confidence: 99%

IntracellularStaphylococcus aureusemploys the cysteine protease staphopain A to induce host cell death in epithelial cells

Stelzner

Hertlein

Sroka

et al. 2020

Preprint

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Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Intracellularity is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death by intracellular S. aureus after translocation into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. Our study suggests that staphopain A is utilized by S. aureus to mediate escape from the host cell and thus contributes to tissue destruction and dissemination of infection. Author Summary Staphylococcus aureus is a well-known antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium asymptomatically colonizes the upper respiratory tract and skin of about one third of the human population and takes advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus is not regarded as a professional intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A.

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“…We measured with anon-destructive 785 nm laser and the maximum possible excitationpower of 80 mW for the optimal signalto-noise ratio,a sd escribed elsewhere. [15] After system calibration with silicon, single cells were pinpointed in their center and measured using accumulated scans of 3 10 s. Images were taken with the BioRam system and assembled in Adobe Photoshop CS5 [17] Raman spectra of 100 M4E4-labeled, 200 M4E-labeled, and 202 control cells were collected. Themeasurements were performed on seven different days.T og et reference Raman spectra, we measured suspensions of M4E and M4E4 particles.Background Raman spectra were acquired in the center of microwells that were filled with water.…”

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confidence: 99%

Rapid Analysis of Cell–Nanoparticle Interactions using Single‐Cell Raman Trapping Microscopy

Steinke

Zunhammer²,

Chatzopoulou

et al. 2018

Angew Chem Int Ed

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Iron oxide nanoparticles have been used in preclinical studies to label stem cells for non-invasive tracking and homing. The search continues for novel particle candidates that are suitable for clinical applications. Since standard analyses to investigate cell-particle interactions and safety are labor-intensive, an efficient procedure is required to guide future particle development and to exclude adverse health effects. The application of combined Raman trapping microscopy with fluidic chips is reported for the analysis of single cells labeled with different types of aminated iron oxide particles. Multivariate data analysis revealed Raman signal differences that could be clearly assigned to cell-particle interactions and cytotoxicity, respectively. A validation dataset verified that more than 95 % of the spectra were correctly classified. Thus, our approach enables rapid discrimination of non-hazardous from cytotoxic nanoparticles as a prerequisite for safe clinical applications.

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An engineered 3D human airway mucosa model based on an SIS scaffold

Cited by 46 publications

References 25 publications

Investigation on Ciliary Functionality of Different Airway Epithelial Cell Lines in Three-Dimensional Cell Culture

Investigation on Ciliary Functionality of Different Airway Epithelial Cell Lines in Three-Dimensional Cell Culture

IntracellularStaphylococcus aureusemploys the cysteine protease staphopain A to induce host cell death in epithelial cells

Rapid Analysis of Cell–Nanoparticle Interactions using Single‐Cell Raman Trapping Microscopy

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