Isolation of normal mammary epithelial cells
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.
