[Serial cultivation of canine smooth muscle cells from urinary bladders]

To provide an ideal seed cell for tissue engineered urinary bladder and urethra by serially culturing canine smooth muscle cells from urinary bladder in vitro and compare biological characteristics of different passages of cells.

Bladder smooth muscle cells of 12-month-old male dogs weighing 10-12 kg were isolated from adult dogs' urinary bladders by collagenase and trypsin digestion and serially cultured in DMEM medium supplemented with 10% serum of newborn bovines. Morphology and proliferation of the cells were observed and the serially-cultured cells were identified with the transmission electron microscope and immunohistochemistry.

The cells appeared spindle in parallel rows when they grew to the degree of subconfluency, and showed the "peak-valley" structure under the inverted phase contrast microscope. The cells could be proliferated serially to the 12th passage in vitro. The growth curve showed the cells before the 7th passage had the similar proliferation characteristics and the growth cycle was about 40 hours. The TEM showed myofilament and the dense body in cytoplasm of smooth muscle cells. Smooth muscle actin was positive by immunohistochemical staining. After the 7th passage, the cells' growth became slow, and myofilament and the dense body in cytoplasm vanished.

The canine smooth muscle cells from urinary bladder can be serially cultured in vitro and highly purified and largely proliferated by the appropriate method. The cells before the 7th passage can be used as optimal seed cells for tissue engineered urinary bladder and urethra.