Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Lipičeva 2, 1000, Ljubljana, Slovenia.
In Vitro Cell Dev Biol Anim. 2013 Mar;49(3):196-204. doi: 10.1007/s11626-013-9585-5. Epub 2013 Feb 14.
Optimizing culture conditions is known to be crucial for the differentiation of urothelial cell cultures and the formation of the permeability barrier. However, so far, no data exist to confirm if air-liquid (AL) and liquid-liquid (LL) interfaces are physiologically relevant during urothelial differentiation and barrier formation. To reveal the influence of interfaces on the proliferation, differentiation, and barrier formation of the urothelial cells (UCs) in vitro, we cultured UCs under four different conditions, i.e., at the AL or LL interfaces with physiological calcium concentration and without serum or without physiological calcium concentration and with serum. For each of the four models, the urothelial integrity was tested by measuring the transepithelial resistance (TER), and the differentiation stage was examined by immunolabeling of differentiation-related markers and ultrastructural analysis. We found that the UCs at a LL interface, regardless of the presence or absence of calcium or serum, form the urothelium with more cell layers and achieve a higher TER than UCs at an AL interface. However, UCs grown at an AL interface with physiological concentration of calcium in medium form only one- to two-layered urothelium of UCs, which are larger and express more differentiation-related proteins uroplakins than UCs in other models. These results demonstrate that the interface itself can play a major, although so-far neglected, role in urothelial physiology, particularly in the formation of the urothelial permeability barrier in vitro and the regulatory mechanisms related with urothelial differentiation. In the study, the culturing of UCs in three successive steps is proposed.
优化培养条件对于尿路上皮细胞培养的分化和渗透屏障的形成至关重要。然而，迄今为止，尚无数据证实气液（AL）和液液（LL）界面在尿路上皮分化和屏障形成过程中是否具有生理相关性。为了揭示界面对体外尿路上皮细胞（UCs）增殖、分化和屏障形成的影响，我们在四种不同条件下培养 UCs，即在具有生理钙浓度的 AL 或 LL 界面上、无血清或无生理钙浓度和有血清的情况下。对于这四种模型中的每一种，通过测量跨上皮电阻（TER）来测试尿路上皮的完整性，并通过免疫标记分化相关标志物和超微结构分析来检查分化阶段。我们发现，无论是否存在钙或血清，处于 LL 界面的 UCs 形成的尿路上皮细胞层数比处于 AL 界面的 UCs 多，并且达到的 TER 比 AL 界面的 UCs 高。然而，在具有生理钙浓度的培养基中的 AL 界面上生长的 UCs 仅形成一层至两层 UCs 尿路上皮，其比其他模型中的 UCs 更大，并表达更多的分化相关蛋白尿路上皮蛋白。这些结果表明，界面本身可以在尿路上皮生理学中发挥重要作用，尽管迄今为止这一作用被忽视了，特别是在体外形成尿路上皮渗透屏障和与尿路上皮分化相关的调节机制中。在这项研究中，提出了分三个连续步骤培养 UCs。
