Abstract: FR-PO742
Microinjection-Based Analyses in a 3D Cyst Model
Session Information
- Cystic Kidney Diseases: Clinical/Translational
November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1001 Genetic Diseases of the Kidneys: Cystic
Authors
- Kraus, Andre, University Hospital Erlangen, Erlangen, BY, Germany
- Schiffer, Mario, University Hospital Erlangen, Erlangen, Germany
- Buchholz, Bjoern, University of Erlangen-Nuremberg, Erlangen, Germany
Background
ADPKD is characterized by continuous cyst growth. Hereby fluid secretion into the cyst lumen plays a major role. We could show that in addition to cAMP-mediated chloride secretion also ATP- and Ca2+-dependent chloride secretion contributes to cyst enlargement. Hereby the binding of luminal ATP to P2Y2-receptors leads to activation of the apical chloride channel Anoctamin 1. Functional studies in our established in vitro cyst model are partly restricted, since applied pharmaceuticals and substances only reach the basolateral side of the cysts. In addition, the quantification of the apical secretion rate of various molecules such as ATP is not possible. Our goal was to establish a micro puncture technique for in vitro cysts, which would allow us to inject or withdraw substances into or out of the cyst lumen.
Methods
In our in vitro 3D-cyst model, principal-like MDCK collecting duct cells (plMDCK) form cysts within a collagen matrix. Those cysts get punctured and substances injected utilizing a microinjector in combination with a micromanipulator and a high-resolution macroscope. Successful injection can be monitored by co-injection of a dye into the cyst’s lumen. Injected cysts can then be tracked over time in a live cell imaging chamber.
Results
With this new technique we are able to puncture cysts and inject substances into in vitro cysts starting at a size of >100µM. Correct application can be visualized by applying a coloured dye in addition and visualizing the cyst thereafter in a live cell imaging chamber. Therefore, we are able to apply various substances of interest at the apical side of the cysts and analysing their direct effect on cyst growth. In addition, small amounts of fluid can be extracted from the cysts and further analysed. This technique will be used to test for luminal ATP concentrations under different culture conditions.
Conclusion
We have established a new method, allowing us to puncture in vitro cysts and applying substances luminally or measuring concentrations of different molecules in the cyst lumen such as ATP by extracting cyst’s fluid. In addition, the punctured cysts can be imaged over time in our live cell imaging setup.