Presentation Authors: Ming Lu*, Kejia Zhu, Peter Schulam, Toby Chai, New Haven, CT
Introduction: The urothelium plays an important role in urinary storage/emptying and innate immune responses. Current methods of studying urothelial biology utilize either freshly isolated or primary cultured cells, both of which involve enzymatic processes to remove the urothelial cells from the bladder and do not maintain the unique distribution of cells in the multilayered urothelium. We present a new method for obtaining a pure urothelium devoid of lamina propria and detrusor from mice bladders.
Methods: C57BL6 mice were anesthetized and perfused with PBS solution. The bladder was excised and cut into four pieces (Fig. A). Under magnification, the mucosa was separated from the detrusor using forceps (Fig. B). Next, a corner of the mucosa was held with forceps. Then a modified forceps was used to strip apart the urothelium from the lamina propria. The stripping motion was a longitudinal sliding of the jaws clamped around the lamina propria, thus separating off the urothelium intact without disrupting the urothelial cells, resulting in 3 tissues of urothelium, detrusor, and lamina propria (Fig. C). The total time to complete the process was 15-20 minutes.
Results: To demonstrate the purity of urothelial tissue, RT-PCRs were performed from the dissected urothelium, lamina propria, and detrusor. Uroplakin, a specific marker of urothelial cells, was expressed only in the harvested urothelial tissue, not in the lamina propria or detrusor. Tissue H&E staining was also performed to confirm the urothelial purity. We have performed several experiments from the urothelial tissues including isolation of individual basal, intermediate cells, umbrella cell in situ patch clamp electrophysiology, and urothelial Western immunoblotting.
Conclusions: This non-enzymatic method is simple and time efficient. The major advantages of this method are: 1. Maintenance of urothelial cellular orientation into the 3 layers: apical (umbrella), intermediate and basal; 2. Acquisition of pure urothelial tissue for molecular biological experiments to reflect truly only urothelium; 3. Acquisition of pure lamina propria; 4. Ability to perform single cell functional experiments such as patch-clamp electrophysiology in situ of umbrella cells. We also recently adapted this method to dissect out pure urothelial tissues from human bladders.