Generation of a human urinary bladder smooth muscle cell line Yongmu Zheng & Shaohua Chang & Ettickan Boopathi & Sandra Burkett & Mary John & S. Bruce Malkowicz & Samuel Chacko Received: 19 July 2011 /Accepted: 14 November 2011 /Published online: 19 January 2012 / Editor: T. Okamoto # The Society for In Vitro Biology 2012 Abstract We report a cell line (hBSM) established from human urinary bladder wall smooth muscle that maintains most of the phenotypic characteristics of smooth muscle cells. Cells were dissociated from the muscular layer with collagenase (1 mg/ml) and collected and grown in M199 supplemented with 10% fetal calf serum and 1% antibiotic– antimycotic. Primary cultures were grown for 2 d and small colonies were isolated by placing glass rings around the colonies. These colonies were picked up with a fine-tipped Pasteur pipette and subcultured. This procedure was repeated several times until a culture with a uniform stable morphology was obtained. hBSM cells are elongated with tapered ends, and in high density cultures, they form swirls of cells arranged in parallel. These cells have a doubling time of approximately 72 h. Western blotting and immunofluorescence microscopy revealed stable expression of smooth muscle-specific proteins, including myosin isoforms (N-terminal isoforms SM-A/B and C-terminal isoforms SM1/2), SM22, α-smooth muscle actin, h-caldesmon, Ca 2+ -dependent myosin light chain kinase, and protein kinase G. These cells contract upon exposure to 10 μM bethanechol and this contraction is reversible by washing away the drug. Karyotyping showed tetraploidy with a modal chromosome number of 87, with multiple rearrangements. To our knowledge, the hBSM cell line is the first human cell line established from bladder wall smooth muscle that expresses both N- and C-terminal smooth muscle myosin isoforms. This cell line will provide a valuable tool for studying transcriptional regulation of smooth muscle myosin isoforms and effects of drugs on cellular function. Keywords Human smooth muscle . Myosin isoform . Contractility . Stable phenotype Introduction Smooth muscle (SM) cells are the major component cells of the walls of viscous organs and the vascular wall, and their ability to contract and relax is the hallmark of the physiologic function of these organs. These mesoderm-derived (mesen- chymal) cells have common characteristics, although cells in various regions of the body are heterogeneous with respect to their ability to express contractile proteins and contract. These cells contain contractile proteins (SM-specific myosin and actin) and proteins that regulate actin–myosin interactions (tropomyosin, myosin light chain kinase, SM phosphatase, SM-specific caldesmon, calponin, etc.), and they contract in response to neuro-endocrine stimulation (Somlyo and Somlyo 1968). These cells are also secretory, synthesizing most of the extracellular matrix that surrounds the SM cells in these tissues (Burke et al. 1977). In adults, SM cells in the wall of the organs do not divide under basal conditions, although they proliferate in response to injury and regenerate. In Y. Zheng : S. Chang : E. Boopathi : M. John : S. B. Malkowicz : S. Chacko Division of Urology, University of Pennsylvania, Philadelphia, PA, USA S. Chacko Department of Pathobiology, University of Pennsylvania, Philadelphia, PA, USA S. Burkett Cytogenetics Core Facility, National Cancer Institute, Frederick, MD, USA S. Chacko (*) Glenolden Research Laboratory, University of Pennsylvania, 500 S Ridgeway Ave, Glenolden, PA 19036, USA e-mail: samuel.chacko@uphs.upenn.edu In Vitro Cell.Dev.Biol.—Animal (2012) 48:84–96 DOI 10.1007/s11626-011-9473-9