Isolation of human prostatic smooth muscle cells

                      Isolation of human prostatic smooth muscle cells    

Human prostate tissues
1.Human hyperplastic prostates were obtained during surgery from four men through transurethral resection of the prostate.
2.All these patients had histories of prostatism and were diagnosed as having benign prostatic hyperplasia on the basis of the combination of rectal digital examinations, transrectal sonography of the prostate, and urodynamic studies (including uroflowmetry, urethral pressure profile, and cystometry).

Tissue explants and subcultures
1.Tissue specimens were immediately placed into a sterile Petri dish and minced into small pieces of ~2 × 2 × 2 mm under a laminar flow hood.
2.The minced tissue pieces then were transferred into a 15-ml polypropylene centrifuge tube containing 3 ml of 0.1% collagenase type 1 in HBSS, pH 7.4, and bubbled with a mixture of 5% CO^₂/95% O^₂ at 37° for 40 min.
3.The tube was subjected to a rotator at a speed of 300 rpm for 3 min. After a brief settlement, the supernatant fraction was discarded, and the tube was filled with 3 ml of 0.1% trypsin in PBS in the above condition for 10 min.
4.The tissue pieces were centrifuged at a speed of 1000 rpm for 3 min, and then the pieces were washed twice with HBSS and transferred to sterile flasks, which were precoated with 10 μg/ml collagen type 1, containing RPMI-1640 medium supplemented with 10% FCS (v/v), penicillin (100 units/ml)/streptomycin (100 μg/ml), amphotericin B (2.5 mg/ml), and testosterone (10 nM).
5.Cultures were maintained in a humidified incubator at 37° in 5% CO^₂/air.
6.Usually with 5–7 days, successfully attached explants would have cells emanating around the tissue.
7.After 1 week, the medium was changed and thereafter changed every 3 days.
8.Once the cells in the flask reached confluence (~2–3 weeks), the explant was transferred to another flask and cultured to reach confluence.
9.After this cultivation procedure was followed for five times, the cells that grew out of the explant were trypsinized from the fifth culture flask for subculture and split into a 1:3 ratio as soon as monolayers became confluent.

Immunofluorescence
1.Isolated human prostatic cells were plated onto a chamber slide, cultured for 48 hr, and fixed with 100% methanol for 5 min.
2.After washing (three times) with PBS, fixed cells were incubated (37°C) with anti-vimentin, anti-cytokeratin, anti-smooth muscle α-actin, or anti-desmin IgG (dilution, 1:50–100) for 40 min.
3.Cells were again washed (three times) with PBS and incubated with fluorescein-conjugated goat anti-mouse IgG (dilution, 1:100) for 40 min.
4.Green fluorescence was evaluated using a fluorescence microscope.

Characterization of cultured human prostatic cells
Cells were plated onto a chamber slide and cultured for 48 hr. Cultured cells were stained with antibodies to vimentin, cytokeratine, smooth muscle α-actin, and desmin, as described in Experimental Procedures. Prostatic cells (A, C, E, and G) were immunostained with anti-vimentin (B), anti-cytokeratin (D), anti-smooth muscle α-actin (F), and anti-desmin (H), respectively.