Category: Assay Development and Screening
One of the goals of the “3R principle” is to replace in vivo testing methodologies with in vitro procedures. To help realize this vision, Philip Morris International R&D have invested in the development of a systems biology approach which combines different omics technologies with high-content screening (HCS). A large battery of HCS assays were previously established for different 2D cellular models to investigate how specific substances alter the cellular phenotype. However, the requirement for more physiologically relevant in vitro systems, closely mimicking the characteristics and functionalities of the part of the human body they model, has led to the development of more complex cellular models which range from 2D co-cultures to 3D organotypic tissues with one or more cell types. Today, 3D models are widely used for different applications and are now also part of our in vitro model portfolio. To further evolve our HCS assay portfolio and leverage the work performed in 2D models, we sought to establish high-content investigations of 3D bronchial tissues. When grown to confluence on a Transwell™ system and cultured at the air-liquid interface, normal human bronchial epithelial cells form a polarized, pseudostratified epithelium composed of basal, ciliated and goblet cells. With its architecture closely mimicking that of the human large airways, this culture system not only provides a useful tool for the in vitro study of airway epithelial biology, it also models the lung response more accurately than cells grown in 2D monolayers. In particular, we investigated phenotypic markers which are known to characterize this 3D model. Cell type-specific markers such as β-tubulin 4, Muc5AC and p63 were selected to identify ciliated, goblet and basal cells, respectively. In addition, ZO-1, a member of the family of tight junction proteins, was included. As a proof-of-concept an in vitro model of IL-13-driven goblet cell hyperplasia was evaluated by apically treating the tissues in a repeated fashion for two weeks. An evaluation of the phenotype was then performed using the Muc5AC-specific antibody staining. Using our developed method we demonstrated that IL-13 induced an almost 4-fold increase in goblet cell density as shown by the total area of the spot detection. In addition, looking at the average intensity, we also noted an increase of nearly 40% of Muc5AC production at single cellular lever. This approach demonstrates that high-content imaging can be used to evaluate more complex 3D in vitro models. In fact, it can be exploited either as a tool for fast and accurate quality control of the tissue, evaluating both the expression and the distribution of tissue maturation markers, or as experimental endpoint in experiments looking at specific phenotypic changes upon treatment.
Diego Marescotti– Manager High Content SCreening, Philip Morris International R&D, Neuchatel, Neuchatel, Switzerland
Manager High Content SCreening
Philip Morris International R&D
Neuchatel, Neuchatel, Switzerland
After completing his PhD in Oncology and Molecular Pharmacology, at the University of Ferrara (Italy) I continued my research investigating the mechanism of tumor escape from immune surveillance. As a scientist I joined Philip Morris International in 2012 bringing my knowledge and interest for flow cytometry and imaging. I am now leading a team of researchers which operate a high-throughput system for compound profiling.