Kreft Mateja Erdani, Jerman Urška Dragin, Lasič Eva, Lanišnik Rižner Tea, Hevir-Kene Neli, Peternel Luka, Kristan Katja
Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Pharm Res. 2015 Feb;32(2):665-79. doi: 10.1007/s11095-014-1494-0. Epub 2014 Aug 22.
The further characterization of the cell line RPMI 2650 and the evaluation of different culture conditions for an in vitro model for nasal mucosa.
Cells were cultured in media MEM or A-MEM at air-liquid (A-L) or liquid-liquid (L-L) interfaces for 1 or 3 weeks. Different cryopreservation methods and cell culture techniques were evaluated with immunolabelling of junctional proteins, ultrastructural analysis using electron microscopy, transepithelial electrical resistance (TEER) measurements, permeation studies with dextran and jacalin, and gene expression profiling of 84 drug transporters.
Cell proliferation and differentiation depended on the used medium. The established epithelia expressed occludin, claudin-1, and E-cadherin under all conditions. Cells grown at the A-L interface formed more layers and exhibited a higher TEER and lower dextran and jacalin permeability than at the L-L interface, where cells morphologically exhibited a more differentiated phenotype. The expression of ABC and SLC transporters depended on culture duration and interface.
The RPMI 2650 cells form a polarized epithelium resembling nasal mucosa. However, different culture conditions have a significant effect on cell ultrastructure, barrier integrity, and gene expression, and should be considered when using this cell line as an in vitro model for drug permeability studies and screening of nasal drug candidates.
进一步鉴定RPMI 2650细胞系，并评估用于鼻黏膜体外模型的不同培养条件。
将细胞在MEM或A-MEM培养基中于气液（A-L）或液液（L-L）界面培养1或3周。通过连接蛋白的免疫标记、电子显微镜超微结构分析、跨上皮电阻（TEER）测量、右旋糖酐和刀豆球蛋白A的渗透研究以及84种药物转运体的基因表达谱分析，评估不同的冷冻保存方法和细胞培养技术。
细胞增殖和分化取决于所用培养基。在所有条件下，所建立的上皮细胞均表达闭合蛋白、紧密连接蛋白-1和E-钙黏蛋白。与在L-L界面生长的细胞相比，在A-L界面生长的细胞形成更多层，表现出更高的TEER以及更低的右旋糖酐和刀豆球蛋白A渗透性，在L-L界面生长的细胞在形态上表现出更分化的表型。ABC和SLC转运体的表达取决于培养持续时间和界面。
RPMI 2650细胞形成类似鼻黏膜的极化上皮。然而，不同的培养条件对细胞超微结构、屏障完整性和基因表达有显著影响，在将该细胞系用作药物渗透性研究和鼻用候选药物筛选的体外模型时应予以考虑。
