Tyson Darren R, Inokuchi Junichi, Tsunoda Toshiyuki, Lau Alice, Ornstein David K
Department of Urology, University of California Irvine, Irvine medical Center, Irvine, CA 92868-2656, USA.
Prostate. 2007 Nov 1;67(15):1601-13. doi: 10.1002/pros.20628.
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.
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.
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.
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.
良性前列腺上皮细胞系的三维（3D）培养可在体外重现腺泡形态发生，但该方法的广泛适用性尚未见报道。本研究探讨了体外前列腺腺泡形态发生的重要培养条件以及细胞外钙在此过程中的作用。
在优化的培养条件下，通过免疫印迹分析RWPE-1、pRNS-1-1、PZ-HPV-7、PNT1A、BPH-1和PrEC在3D培养中进行腺泡形态发生的能力。进一步检测RWPE-1细胞在二维培养中钙对形态、E-钙黏蛋白膜定位和多细胞分层的影响，以及在三维培养中对腺泡形态发生、管腔凋亡和管腔填充的影响。
在低钙培养基中生长的细胞系有能力形成带管腔的腺泡结构，这与E-钙黏蛋白表达相关，但该过程并不需要低钙。向培养基中添加CaCl₂强烈抑制管腔形成、管腔凋亡并诱导管腔填充，且管腔填充被干扰抗体阻断。
优化的培养基成分可使几乎所有接种的RWPE-1细胞发生腺泡形态发生，形成代表正常成年前列腺的一致结构。含低钙的培养基似乎对能够在体外进行腺泡形态发生的细胞具有选择性，腺泡内的分支和管腔空间受细胞外钙水平的强烈影响，可能是通过E-钙黏蛋白的作用。这些结果提供了有关用于研究前列腺发育和致癌作用的相关体外模型的重要信息，并突出了细胞外钙在调节三维形态中的重要性。
