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% CO2/95% O2 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% CO2/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.