A Method for Preparation of Freshly-Isolated Human Urinary Bladder Smooth Muscle Cells: Utility for Characterization of Whole-Cell Cation Currents

Abstract

Ion channels of the urinary bladder smooth muscle (UBSM) determine cellular excitability and contractility and, hence, regulate the two main functions of the organ, urine storage and voiding. Preparation of the viable, fresh, and single UBSM cells presents an important technical challenge for subsequent biophysical and electrophysiological studies. Here, we describe a method for successfully obtaining freshly-isolated human single UBSM cells from patient-donors undergoing open bladder surgeries and its application for the recording of voltage-step-induced cation currents. The initial step in the method involves dissection to separate the UBSM layer (also known as detrusor smooth muscle and muscularis propria) from mucosa (urothelium, lamina propria, and muscularis mucosa). The UBSM is then cut into small pieces in Ca2+-free (nominal) dissection/digestion solution (DS). UBSM pieces are next transferred to and sequentially treated separately with DS containing papain and collagenase at ∼37 °C for 30-45 min per step. Following washes with enzyme-free DS and trituration with a fire-polished pipette, the pieces release single UBSM cells. Freshly-isolated UBSM cells are ideally suited for patch-clamp electrophysiological and pharmacological characterizations. Specifically, single UBSM cells contract in response to the muscarinic agonist carbachol (3 μM). In amphotericin-B perforated patch-clamp recordings, the TRPM4 channel blocker 9-phenanthrol concentration-dependently reduces voltage-step-induced cation currents. UBSM cells can also be studied by other techniques such as: single cell RT-PCR, microarray analysis, immunocytochemistry, in situ proximity ligation assay, and Ca2+ imaging. Studies on freshly-isolated human UBSM cells have provided important insights characterizing the properties of various ion channels including cation-permeable channels in the urinary bladder and will continue as a gold standard in elucidating UBSM cellular properties and regulatory mechanisms. Funding: NIH R01DK106964 grant to Georgi V. Petkov.