Poster D5 - Measurement of Chloride Secretion to Study How Osmotic Pressures Regulate 3D Epithelial Spheroid Homeostasis

Friday, October 25, 2024

3:30 PM - 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Kevin Wheat

Undergraduate Researcher
The Ohio State University
CLAYTON, Ohio, United States

Co-Author(s)

Bradley Sweeney, MS

PhD Candidate
The Ohio State University
Fort Myers, Florida, United States

Primary Investigator(s)

DC

Daniel Conway, PhD

Professor
The Ohio State University, United States

Introduction: Epithelial structures exist in many systems across the body and are responsible for the protection and function of many organ systems. One common cell type for studying epithelial systems, Madin-Darby Canine Kidney (MDCK) cells, is known to form 3D cyst structures when cultured in MatrigelTM. The center of the cysts is commonly referred to as the lumen space. It is known that when the cysts are in homeostasis, they maintain a relatively fixed lumen pressure [Narayanan Current Biology 2020]. Lumen pressure is a key component in developing and maintaining homeostasis, and one factor that can affect lumen pressure is Cl secretion. Lumen composition is not well defined in the MDCK cyst structure and the relative flux of chloride ions in this system is not well characterized. Our goal was to visualize and quantify the relative levels of Cl- concentration in the lumen space to better define the chloride component of lumen pressure for understanding the factors that drive lumen growth and homeostasis.

Materials and

Methods: Three MDCK cell lines were established with genetically encoded Chloron-1, a biosensor for chloride [Tutol ACS Central Science 2023]. These lines consist of Chloron-1 expressed in the cytoplasm, apically secreted Chloron-1, and Chloron-1 apically attached to gp135. Tests were conducted in 2D with chloride addition and depletion utilizing gluconate as a substitute and through CFTR activation via 10uM Forskolin addition. For 3D experiments, the MDCK cells were cultured in MatrigelTM for 7 days to allow for cyst formation. 3D experiments were conducted with 10uM Forskolin and CFTRinh172 to fluctuate chloride levels in the lumen through CFTR activity.

Results, Conclusions, and Discussions: We show validation of the intracellular and apically attached sensors in 2D. From the Forskolin tests, it was seen that adding Forskolin secreted Cl- , and the cells expressing Chloron-1 intracellularly fluoresced dimmer due to loss of intracellular chloride. Cells expressing Chloron-1 that were apically attached showed changes in intensity upon media exchange between chloride-depleted saline and chloride-containing media. The 3 cell lines were validated in 3D to show the proper expression of the sensor in the cellular system of interest.
From the Forskolin and gluconate exchange tests, the sensor was validated to work as intended and work extracellularly. This means the Chloron-1 biosensor may be used to visualize changes in Cl- concentration in the lumen space, giving an idea of how concentration affects lumen properties. The next step would be to gather a dynamic range of Cl- in the lumen and form a spectrum of lumen properties when compared to Cl- concentration.
From the results of the tests, we show the ability of the Chloron-1 sensor to react to physiologically relevant chloride concentrations in epithelial cells, showing the opportunity of utilizing chloride biosensors to study osmotic regulation of lumen dynamics. We anticipate that this approach will allow us to determine if chloride levels vary during lumen formation and homeostasis. This will allow us to determine if chloride, or other factors, are important in the generation and regulation of lumen pressure. are continuing to further validate the usefulness of this sensor in 3D epithelia. Our long-term goal is to better understand how osmotic pressures are regulated in 3D epithelial tissues, which may include a mechanosensation feedback mechanism that helps to increase or decrease osmotic pressures to achieve a stable setpoint.

Acknowledgements (Optional): Sheel Dodani, Department of Chemistry and Biochemistry at The University of Texas at Dallas