An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods.It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm²) was higher than in A549 (172 ± 59 Ω cm²) but similar to 16HBE14o- cells (1469 ± 156 Ω cm²). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm²) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm²) and 16HBE14o- (558 ± 267 Ω cm²). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549.The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.     

Pulmonary cell culture models to study the safety and efficacy of innovative aerosol medicines

Background: Pulmonary cell culture models for the development of new aerosol medicines are attracting increasing interest. Ease of handling, ethical acceptance and high explanatory power are the main advantages of cell culture systems in pulmonary drug research. Objective: Pulmonary cell culture models are described and evaluated regarding their suitability for the biopharmaceutical characterisation of innovative aerosol medicines. Methods: The review focuses on the peculiarities of the pulmonary cell culture models arising from the specialised pulmonary epithelia, the clearance systems in the lung and the limited but functionalised lung fluid layers. Additional aerosol deposition systems suitable for the close to in vivo simulation of aerosol delivery on pulmonary cell cultures are described. Results: Suitable cell culture models of the cellular part of the human air–blood barrier are established and well characterised. However, the physical barriers on top of the cellular barriers – surfactant, mucus and so on – and their influence on the safety and efficacy of aerosol medicines, are still underestimated.     

The influence of chitosan content in cationic chitosan/PLGA nanoparticles on the delivery efficiency of antisense 2′-O-methyl-RNA directed against telomerase in lung cancer cells

Tailorable cationic chitosan/PLGA nanoparticles (CPNP) were used for the delivery of an antisense 2′-O-methyl-RNA (2OMR) directed against RNA template of human telomerase. Here, we describe the influence of the chitosan content on binding efficiency, complex stability, uptake in different human lung cell types and finally demonstrate the efficacy of this nanoplex system.CPNPs were prepared by the emulsion-solvent evaporation method using different amounts of chitosan and purified by preparative size exclusion chromatography. The characterization by photon correlation spectroscopy and zeta potential measurements showed a small increase in size and an increase of zeta potential with increasing amounts of chitosan. Binding efficiency and complex stability with 2OMR was high in water and correlated well with the chitosan content of particles but was weak in physiologically relevant media (PBS and RPMI cell culture medium). However, flow cytometry analysis showed that the uptake of 2OMR into A549 lung cancer cells was considerably higher in combination with nanoparticles and dependent on the amount of chitosan when compared to 2OMR alone. Confocal laser scanning microscopy revealed that the uptake into A549 cells is mediated via complexes of 2OMR and chitosan/PLGA nanoparticles despite the weak binding in cell culture medium. The nanoparticles were well tolerated and efficient in inhibiting telomerase activity.