Extensive experience with flow cytometry analysis and sorting of human and mouse lung, skin, hair follicle and thymus. I learned FACS during my PhD in the US, at University of Pittsburgh and at Duke University. Flow cytometry was the entry point into the single cell world. Since then, I implemented single cell technologies both at EPFL (a Biomark, Fluidigm instrument) as well as in Giessen. In Giessen, we purchased several single cell instruments, we trained users and provided opportunities for continual training in the form of workshops, and single cell clubs.
To study epithelial cell regenerative capacity in vitro, I set-up a co-culture system comprising of lung epithelial cells and lung fibroblast. This was based on the experience gained during my PhD, when I developed an organoid assay for mouse epithelial cells. Defining and refining the culture conditions was a team effort. After many tries, we understood that organoid culture has three phases, each with its own culturing conditions; initiation, expansion and differentiation. This dual co-culture system was used to study both epithelial and fibroblast cells in normal and diseased lung, as well as their interaction. The system is amenable to medium to high through-put upscaling for compound testing and drug discovery.
PCLS represent very thin slices (200-500 microns) of lung tissue that can be maintained in culture. They offer the advantage of having a quasi normal cellular environment that can be manipulated for the purpose of pharmaceutical drug discovery and development. This assay is very instrumental also in studying cell behavior during injury and repair, in healthy and diseased tissues. To confer the solid consistency necessary for sectioning, the lung has to be filled with low melt agarose, cooled on ice, and then sectioned. While not at all difficult to perform, organ procurement is a major concern. The technique needs to be adjusted for different diseases, according to the changes in tissue structure. Other challenges that we had to overcome were to establish culturing conditions and understand the cellular dynamics during the time of culture. This work led to several scientific collaborations and a patent.
Roxana Wasnick MD PhD
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