Characteristics of separated epithelial and stromal subfractions of prostate: I. Rat ventral prostate.

Abstract

These studies were initiated with the objective of isolating epithelial and stromal cells of human prostatic tissue in undamaged state, in order to study the cellular distribution of steroid receptors in benign prostatic hyperplasia (BPH) relative to normal prostate. Initial experiments showed that when BPH tissue immersed in tissue culture media was progressively fragmented by various cutting procedures, epithelial elements were selectively released as clumps of variable size and individual cells, but that a large percentage of these cells were damaged, as evidenced by their failure to exclude trypan blue (TB). These observations suggested that if tissue fragmentation were carried out under defined conditions that minimize cell damage, BPH subfractions might be obtained containing a large percentage of undamaged cells. To determine conditions of tissue fragmentation which result in maximal recovery of epithelial cells which exclude TB, rat ventral prostate (RVP) was chosen as a model system. Experiments with RVP revealed that maximal yields of such cells were obtained in "large" epithelial clumps (greater than 30 cells per clump) released under the following conditions: (1) chopping the tissue with razor blades in a large volume (2 ml/100 mg RVP) of a Ca2+-free tissue culture medium ( Joklik 's-MEM) containing 1% casein, (2) carrying out the entire fractionation procedure in the cold, and (3) maintaining a 1% casein concentration in the medium during chopping, as well as in subsequent washing procedures, to protect cells from proteolytic activity. In large epithelial clumps, cells in the interior of the clump were not stained by TB but the cells at the periphery of the clump were freely permeable to TB. Single epithelial cells and small epithelial clumps (3-10 cells) released by razor blade fragmentation were also permeable to TB. When large epithelial clumps were incubated at 20 degrees C for 90 min, the clumps disaggregated into smaller clumps and morphologically intact single cells, which did not exclude TB. The residual tissue fragments remaining after chopping contained the bulk of stromal cells plus some epithelial elements. The latter could be removed by gentle rubbing of the fragments on a sieve in the presence of medium. The stromal fraction thus obtained consisted of stromal cells, embedded in mesenchymal matrix, which were not stained by TB and appeared normal when examined histologically by light microscopy.(ABSTRACT TRUNCATED AT 400 WORDS)