• Co-culture of human alveolar epithelial (hAELVi) and macrophage (THP-1) cell lines.

      Kletting, Stephanie; Barthold, Sarah; Repnik, Urska; Griffiths, Gareth; Loretz, Brigitta; Schneider-Daum, Nicole; de Souza Carvalho-Wodarz, Cristiane; Lehr, Claus-Michael; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (Springer Nature, 2018-01-01)
      The air-blood barrier is mainly composed of alveolar epithelial cells and macrophages. Whereas the epithelium acts as a diffusional barrier, macrophages represent an immunological barrier, in particular for larger molecules and nanoparticles. This paper describes a new co-culture of human cell lines representing both cell types. Acquiring, culturing and maintaining primary alveolar epithelial cells presents significant logistical and technical difficulties. The recently established human alveolar epithelial lentivirus immortalized cell line, hAELVi, when grown on permeable filters, forms monolayers with high functional and morphological resemblance to alveolar type I cells. To model alveolar macrophages, the human cell line THP-1 was seeded on pre-formed hAELVi monolayers. The co-culture was characterized regarding cellular morphology, viability and barrier function. Macrophages were homogenously distributed on the epithelium and could be kept in co-culture for up to 7 days. Transmission electron microscopy showed loose contact between THP-1 and hAELVi cells. When grown at air liquid interface, both cells were covered with extracellular matrix-like structure, which was absent in THP-1 mono-culture. In co-culture with macrophages, hAELVi cells displayed similar, sometimes even higher, transepithelial electrical resistance than in mono-cultures. When exposed to silver and starch nanoparticles, hAELVi mono-cultures were more tolerant to the particles than THP-1 mono-cultures. Viability in the co-culture was similar to that of hAELVi mono-cultures. Transport studies with sodium fluorescein in the presence/absence of EDTA proved that the co-culture acts as functional diffusion barrier. These data demonstrate that hAELVi-/THP-1 co-cultures represent a promising model for safety and permeability studies of inhaled chemicals, drugs and nanoparticles.
    • Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier.

      Kuehn, Anna; Kletting, Stephanie; de Souza Carvalho-Wodarz, Cristiane; Repnik, Urska; Griffiths, Gareth; Fischer, Ulrike; Meese, Eckart; Huwer, Hanno; Wirth, Dagmar; May, Tobias; et al. (2016-03-17)
      This paper describes a new human alveolar epithelial cell line (hAELVi - human Alveolar Epithelial Lentivirus immortalized) with type I-like characteristics and functional tight junctions, suitable to model the air-blood barrier of the peripheral lung. Primary human alveolar epithelial cells were immortalized by a novel regimen, grown as monolayers on permeable filter supports and characterized morphologically, biochemically and biophysically. hAELVi cells maintain the capacity to form tight intercellular junctions, with high trans-epithelial electrical resistance (> 1000 Ω*cm²). The cells could be kept in culture over several days, up to passage 75, under liquid-liquid as well as air-liquid conditions. Ultrastructural analysis and real time PCR revealed type I-like cell properties, such as the presence of caveolae, expression of caveolin-1, and absence of surfactant protein C. Accounting for the barrier properties, inter-digitations sealed with tight junctions and desmosomes were also observed. Low permeability of the hydrophilic marker sodium fluorescein confirmed the suitability of hAELVi cells for in vitro transport studies across the alveolar epithelium. These results suggest that hAELVi cells reflect the essential features of the air-blood barrier, as needed for an alternative to animal testing to study absorption and toxicity of inhaled drugs, chemicals and nanomaterials.
    • Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

      Gordon, Sarah; Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus C23, D-66123 Saarbrücken, Germany. (2015)
    • Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology.

      Gordon, Sarah; Daneshian, Mardas; Bouwstra, Joke; Caloni, Francesca; Constant, Samuel; Davies, Donna E; Dandekar, Gudrun; Guzman, Carlos A; Fabian, Eric; Haltner, Eleonore; et al. (2015)
      Models of the outer epithelia of the human body - namely the skin, the intestine and the lung - have found valid applications in both research and industrial settings as attractive alternatives to animal testing. A variety of approaches to model these barriers are currently employed in such fields, ranging from the utilization of ex vivo tissue to reconstructed in vitro models, and further to chip-based technologies, synthetic membrane systems and, of increasing current interest, in silico modeling approaches. An international group of experts in the field of epithelial barriers was convened from academia, industry and regulatory bodies to present both the current state of the art of non-animal models of the skin, intestinal and pulmonary barriers in their various fields of application, and to discuss research-based, industry-driven and regulatory-relevant future directions for both the development of new models and the refinement of existing test methods. Issues of model relevance and preference, validation and standardization, acceptance, and the need for simplicity versus complexity were focal themes of the discussions. The outcomes of workshop presentations and discussions, in relation to both current status and future directions in the utilization and development of epithelial barrier models, are presented by the attending experts in the current report.
    • Safety assessment of excipients (SAFE) for orally inhaled drug products.

      Metz, Julia K; Scharnowske, Lara; Hans, Fabian; Schnur, Sabrina; Knoth, Katharina; Zimmer, Horst; Limberger, Markus; Groß, Henrik; Lehr, Claus Michael; Hittinger, Marius; et al. (Springer, 2020-01-29)
      The development of new orally inhaled drug products requires the demonstration of safety which must be proven in animal experiments. New in vitro methods may replace, or at least reduce, these animal experiments provided they are able to correctly predict the safety or eventual toxicity in humans. However, the challenge is to link human in vitro data to human in vivo data. We here present a new approach to the safety assessment of excipients (SAFE) for pulmonary drug delivery. The SAFE model is based on a dose response curve of 23 excipients tested on the human pulmonary epithelial cell lines A549 and Calu-3. The resulting in vitro IC50 values were correlated with the FDA-approved concentration in pharmaceutical products for either pulmonary (if available) or parenteral administration. Setting a threshold of 0.1% (1 mg/mL) for either value yielded four safety classes, allowed to link IC50 data as measured on human cell cultures in vitro with the concentrations of the same compounds in FDA-approved drug products. The necessary in vitro data for novel excipients can be easily generated while the SAFE approach allows putting them in the context for eventual use in human pulmonary drug products. Excipients, that are most likely not safe for use in humans, can be early excluded from further pharmaceutical development. The SAFE approach helps thus to avoid unnecessary animal experiments.