1. A small piece of tissue from each specimen was removed and minced.
2. The tissue was digested with collagenase overnight.
3. To remove the collagenase and the majority of the stromal cells, the tissue was rinsed and centrifuged.
4. The
tissue was inoculated into a 60-mm tissue culture dish coated with
collagen type I and grown in primary cell system supplemented with
growth factors and hormones.
5. Prostatic stromal cells do not grow
in the serum-free conditions used in this study, yet these conditions
maintain the growth and differentiation of prostatic epithelial cells.
6. Morphological characteristics of the cultures used in the present study were consistent with the growth of epithelial cells.
7. The cells that grew out from the tissue were aliquoted and stored in liquid nitrogen.
8. The
histology of each specimen was verified by inking and fixing the
prostate after dissection and serially sectioning the marked area.
9. The histology of sections immediately adjacent to the area of the dissection was reviewed.
10. The
frozen aliquots were thawed to produce secondary cultures, which were
grown in primary cell system supplemented with growth factors and
hormones.
11. Nomenclature for epithelial cell strains is “E”
followed by the histology of origin (peripheral zone) and then the
strain number.
12. Stromal cell cultures were established by
inoculating collagenase-digested tissues into primary cell system with
10% fetal bovine serum and 100 μg/ml gentamicin in 60-mm tissue culture
dishes.
13. Although epithelial cells attach in this medium, they do not grow well and are lost after the first passage.
14. Passaged cultures were aliquoted and stored frozen in liquid nitrogen until use.
15. Stromal cell cultures do not express keratin but express vimentin and fibronectin.
16. Nomenclature
for stromal cell strains is“ F” followed by the histology of origin
(peripheral zone) and then the strain number.
17. Strain numbers for stromal cells do not necessarily correlate with strain numbers for epithelial cells.
Reference
1. Peehl D. M. Culture of human prostatic epithelial
cells Freshney I. A. eds. . Culture of Epithelial Cells, : 159-180,
Wiley Liss, Inc New York 1992.
2. Kabalin J., Peehl D., Stamey T.
Clonal growth of human prostatic epithelial cells is stimulated by
fibroblasts. Prostate, 14: 251-263, 1989.
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