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Building up a co-culture and surfactant model for routine use in ali exposures

: Javala, Pasi; Ritter, Detlef; Knebel, Jan


Basic & clinical pharmacology & toxicology 115 (2014), Nr.1, S.156
ISSN: 1742-7835
ISSN: 1742-7843
Nordic Workshop "Frontiers in Cell Toxicity Testing" <2013, Charlottenlund>
Scandinavian Society for Cell Toxicology (Workshop) <29, 2013, Charlottenlund>
Fraunhofer ITEM ()

In recent years there has been lot of effort to develop more relevant and real life resembling exposure methods for in vitro inhalation studies. Meanwhile several exposure concepts are available; all of them are based on the air-liquid-interface (ALI-) technique, which is generally agreed as a standard to expose target cells of the respiratory tract to airborne substances. Currently there is a large need for cell culture models for routine and screening purposes of airborne substances beside more high sophisticated models, which are widely used for mechanistic studies. Therefore we composed a cell culture model from human A549 epithelial cells and THP-1 monocytes using the ALItechnique. Moreover, an artificial lung surfactant was included in the model. A549 were cultured on 0.4 lm pore size cell culture membranes on the apical side to form a monolayer and thereafter THP-1 monocytes were differentiated to macrophages and added to the A-549 culture within the surfactant. Macrophages were then allowed to adhere for 24 h before excessive surfactant material was removed from the top of the cell layer to form air lifted culture conditions. Several compositions and concentrations of the surfactant, different seeding days of the macrophages as well as several ratios between the epithelial cells and macrophages were tested. Surfactant was composed from phospholipids, free fatty acids, cholesterol, proteins and antioxidants. Different concentrations were tested ending up in a setup that is near to the natural surfactant concentration as described in the literature. Macrophages were seeded on top of the A549 layer using different concentrations (10%, 20% and 30% of the calculated A549 cell amount). Co-culture started 72 h after seeding the A549 cells on the membranes. Current co-culture models ignore to determine the final cell ratio in the resulting co-culture. Using surface antigens CD14 and CD45 and FACS analysis the proportion of the macrophages in the co-culture was detected. A 30% addition of the THP-1 cells resulted in approximately 10-15% in the ready co-culture. In a first step the resulting co-culture model was tested with model substances (particulate samples and H2O2) in order to describe the reactivity of the model. Ongoing experiments evaluate this model under the air liquid interface exposure conditions to airborne substances exposed via a simulated inhalation route.