1. Bladders were excised from deeply anesthetized (urethane, 1.2 gm • kg−1, ip) Sprague Dawley rats (of either sex), cut open, and gently stretched (urothelial side down).
2. Anesthesia was determined to be adequate for surgery by monthly testing for the absence of a withdrawal reflex to a strong pinch of a hind paw and absence of an eye-blink reflex to tactile stimulation of the cornea.
3. After tissue removal, all animals were killed via overdose of anesthetic.
4. The tissue was incubated overnight in primary minimal essential medium, penicillin/streptomycin/fungizone, and 2.5 mg • ml−1 dispase.
5. The urothelium was gently scraped from underlying tissue, treated with 0.25% trypsin, and resuspended in primary cell culture medium.
6. The dissociated cell suspension (0.1 ml, 50,000–150,000 cells ml−1) was plated on the surface of Collagen-coated dishes and maintained in culture for 1–3 d.
7. Because long-term maintenance of cells in culture could significantly change the properties of some types of cells, the cells in this study were examined after a short time in culture.
8. In general, all cells were used within the first 3 d after plating.
9. All cells in these cultures were cytokeratin positive and therefore were presumably of epithelial origin.
Reference
1. Birder LA, Apodaca G, de Groat WC, Kanai AJ (1998) Adrenergic- and capsaicin-evoked nitric oxide release from urothelium and afferent nerves in urinary bladder. Am J Physiol 275: F226–F229.
2. Truschel ST, Ruiz WG, Shulman T, Pilewski J, Sun TT, Zeidel ML, Apodaca G (1999) Primary uroepithelial cultures. J Biol Chem 274: 15020–15029.
3. Birder LA, Kanai AJ, de Groat WC, Kiss S, Nealen ML, Burke NE, Dineley KE, Watkins S, Reynolds IJ, Caterina MJ (2001) Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Proc Natl Acad Sci USA 98:13396–13401.
4. Bevan S, Winter J (1995) Nerve growth factor (NGF) differentially regulates the chemosensitivity of adult rat cultured sensory neurons. J Neurosci 15:4918–4926.
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