Faculty Bibliography

Sequential changes in epithelial cells of collagenase-dissociated rat ventral prostate were studied by thin-section and freeze-fracture electron microscopy. Epithelial cells did not attach to the substrate for 48 h. Pelleted cells obtained 1, 24, and 48 h after dissociation were assigned to three categories depending on morphology and cellular associations. (a) Solitary epithelial cells degenerated as determined by extensive vacuolization in the cytoplasm and aggregation of intramembranous particles (IMP). (b) Epithelial clusters consisted of a homogeneous population of well-maintained, closely packed cells. Aggregation of IMP was minimal. Tight junctions that formed between cells at the periphery of the clusters appeared normal and provided an effective permeability barrier demonstrated by the exclusion of ruthenium red tracer. (c) Tissue fragments were comprised of varying combinations of epithelial, endothelial, and smooth muscle cells as well as fibroblasts and erythrocytes. Maintenance of tissue fragments was variable. Plasma membranes often displayed aggregated IMP and proliferated tight junctional strands. An effective permeability barrier was absent. After the 48 h "latent period," epithelial cells in the clusters lost interdependence, disassociated from one another, and attached to the substrate. These isolated cells, which did not display aggregated IMP, retained the ability to form an effective permeability barrier upon reaching confluency. During the first 48 h, epithelial cells did not tolerate solitary existence, yet as participants in clusters they were well maintained. After this interval, they no longer required interactions with neighbors in order to survive. These results indicate that under our experimental conditions, an adaptation period is required by prostatic epithelial cells. The enhanced quality of maintenance associated with epithelial clusters suggests that control over the internal microenvironment, provided by a tight junctional barrier, may be important during the initial period of adaptation in vitro.

This report describes the design, construction and operation of a photovoltic cell densitometer. The densitometer has proven to be very useful in evaluating the effect of various culture medium supplements on the growth of primary cultures

Stromal"epithelial cell interactions may be important for the regulation of normal and abnormal prostatic growth. While androgen transformation by rat ventral prostate and canine prostate has been investigated in vivo and in organ culture, little is known about metabolic pathways in cultures of epithelial cells and fibroblasts. Metabolism of radioisotope"labeled 17β"hydroxy C19"steroids was studied in primary cultures of highly"enriched rat ventral prostate and canine prostate epithelial cells and fibroblasts isolated by selective attachment procedures. The fibroblasts contained little testosterone 5α"reductase in contrast to high activity in epithelial cells. We found high levels of 5α"androstane"3α,17β"diol (3α"diol) dehydrogenase and the terminal 5α"androstane"3β,17β"diol (3β"diol) hydroxylases in both cell types; 3α"diol was a more effective precursor of 5α"dihydrotestosterone than was testosterone. For prostatic fibroblasts these pathways seem to be differentiated functions, since rat"lung fibroblasts converted 3β"diol to 5α"dihydrotestosterone and 3α"diol. We conclude that epithelial cells and fibroblasts make interactive contributions to prostatic androgen metabolism. 1982 American Society of Andrology

Epithelial-cell enriched primary cultures were established from canine prostate by a collagenase digestion and selective attachment procedure. The epithelial cells in primary culture retained differentiated structure and function. The epithelial cells were attached to one another by tight junctions and desmosomes to form "lumenlike structures" that resemble the acini of the intact organ and appeared to contain typical protein synthetic organelles (1,2). The cultures contained significant levels of acid phosphatase and ornithine decarboxylase (2) and retained the ability to metabolize testosterone to dihydrotestosterone and other 5α-reduced metabolites (2-4)

This study describes a method for establishing primary cultures in 24-well culture vessels and evaluating the effects of serum, hormones, and other factors on cell growth using densitometry. Primary cultures of rat ventral prostate epithelial cells were grown in 24-well culture vessels containing F12K culture medium supplemented with various concentrations of the following substances: fetal bovine serum (FBS), horse serum (HS), testosterone (T), dihydrotestosterone (DHT), hydrocortisone (HC), zinc (Zn), transferrin (TR), cysteine (CYS), glutamine (GLT), selenium (SEL), and ascorbic acid (AC). The effect of each supplement on cell growth was evaluated on fixed and stained cultures using a photovoltaic cell densitometer designed to read the total culture surface of a 16-mm well. Increasing concentrations of both HS and FBS resulted in an increase in cell growth. T, Zn, HC, TR, and AC each had a stimulatory effect on cell growth. CYS, GLT, and DHT had little effect on cell growth, while SEL was inhibitory to cell growth. This data compares favorably with that obtained by other methods, such as morphometric analysis and ornithine decarboxylase production. These results indicate that densitometry is a useful method for determining the effect of supplements on cell growth in primary culture.

Epithelial-cell-enriched primary cultures were established from canine prostate. Minced tissue was dissociated with 750 units/ml of collagenase in F12K tissue culture medium containing 1% fetal bovine serum. This treatment resulted in the gradual removal of stromal elements from the base of the epithelial cells. After 30 minutes of digestion, aggregates of epithelial cells free of stroma were dislodged from the minced pieces of prostate. These aggregates were washed and plated at high density in F12K plus 10% fetal bovine serum. After 12-16 hours in vitro the unattached cellular aggregates were removed from the culture dishes, washed, and reinoculated into new culture vessels containing fresh medium. After 48 hours in vitro, the aggregates had attached to the culture vessels and spread out to yield discrete patches of epithelial cells. By 120 hours in vitro the patches of cells had grown and coalesced to form a confluent monolayer of epithelial cells. Ultrastructural examination of these cultures indicated that adjacent cells were joined by desmosomes and tight junctions and had tonofilaments and microvilli, giving the cells an epithelial appearance. The cells contained rough endoplasmic reticulum, Golgi apparatus, and secretory granules similar to those of the epithelial cells in the intact organ. In addition, intracellular "blebs" containing acid phosphatase were observed in the monolayers and were found to increase in size and number with time in vitro. Differentiated function of the cultures was demonstrated by the presence of ornithine decarboxylase and acid phosphatase and the ability of the cultures to metabolize testosterone to primarily 5 alpha-reduced metabolites.