BACKGROUND. Three-dimensional (3D) culture of benign prostatic epithelial cell lines can recapitulate acinar morphogenesis in vitro, but the broad applicability of this approach has not been described. The present studies examine the culture conditions important for prostatic acinar morphogenesis in vitro and the role of extracellular calcium in this process. METHODS. With optimized culture conditions, RWPE-1, pRNS-1-1, PZ-HPV-7, PNT1A, BPH-1, and PrEC were analyzed for their ability to undergo acinar morphogenesis in 3D culture and by immunoblotting. RWPE-1 cells were further examined for the effects of calcium on morphology, E-cadherin membrane localization and multicellular layering in 2D culture and for acinar morphogenesis, luminal apoptosis, and luminal filling in 3D. RESULTS. Cell lines grown in low-calcium medium have the ability to form acinar structures with lumens, which correlates with E-cadherin expression, but low calcium is not required for this process. Adding CaCl 2 to the medium strongly inhibits lumen formation, luminal apoptosis and induces luminal filling, and luminal filling is blocked by an interfering antibody. CONCLUSIONS. Optimized medium composition allows nearly all seeded RWPE-1 cells to undergo acinar morphogenesis, forming consistent structures representative of normal adult prostate glands. Low-calcium-containing medium appears selective for cells capable of undergoing acinar morphogenesis in vitro, and branching and luminal space within the acini are strongly influenced by extracellular calcium levels, likely through the actions of E-cadherin. These results provide important information about a relevant in vitro model with which to study prostate development and carcinogenesis and highlight the importance of extracellular calcium in regulating 3D morphology.
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