Isolation and Characterization of Smooth Muscle Cells from Rat Corpus Cavernosum Tissue for the Study of Erectile Dysfunction

ABSTRACT

PURPOSE: Primary culture of the cavernous smooth muscle cells from corpus cavernous tissues is known to be difficult, mainly because of contamination with fibroblasts. We applied a new method for better isolation of rat penile smooth muscle cells (RPSMCs) from rat corpus cavernosum tissue for reliable ex vivo research on erectile dysfunction. MATERIALS AND METHODS: With the use of 8-week-old adult male Sprague-Dawley rats, ex vivo migrations of rat cavernous tissue were measured by penis and aortic ring assay by use of a Matrigel-based D-valine-modified culture method. The expression of alpha-smooth muscle actin (alpha-SMA) and platelet/endothelial cell adhesion molecule (PECAM)-1 in the RPSMCs was determined by standard immunofluorescent staining and immunoblotting. The expression patterns of phosphodiesterase (PDE) family mRNA in RPSMCs were compared with patterns in rat aortic smooth muscle cells (RASMCs) by use of quantitative real-time reverse transcription polymerase chain reaction. RESULTS: Immunocytochemical staining showed greater alpha-SMA-positive and PCAM-1-negative fluorescence. Moreover, whereas the expression of alpha-SMA was detected in the RPSMCs, that of PECAM-1 was not. The levels of PDE1A, PDE1B, PDE1C, PDE2A, PDE3A, PDE4A, PDE4B, PDE4C, PDE4D, and PDE5A mRNA in the RPSMCs were about 3.2-, 4.4-, 3.4-, 29.0-, 3.5-, 2.8-, 2.9-, 6.1-, 45.0-, and 6.0-fold the corresponding expression in RASMCs. CONCLUSIONS: We developed a two-stage tissue culture method utilizing a Matrigel-based sprouting culture system to facilitate stromal cell sprouting and an adherent culture system using D-valine to eliminate the contamination of fibroblasts into the smooth muscle cells.