Bai Shuhua, Yang Tianzhi, Abbruscato Thomas J, Ahsan Fakhrul
Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 Coulter Drive, Amarillo, TX 79106, USA.
J Pharm Sci. 2008 Mar;97(3):1165-78. doi: 10.1002/jps.21031.
This study tests the hypothesis that human nasal RPMI 2650 cells grown at an air-liquid interface is a feasible model for drug transport studies via the nasal route. RPMI 2650 cells were cultured in Eagle's minimal essential medium (MEM) at both air-liquid and liquid-liquid interfaces. For each culture regimen, monolayer integrity was tested by measuring the transepithelial resistance (TEER) as well as the transport of paracellular and transcellular markers across the monolayer. The expression of tight junction proteins-differentiation markers-in cells of the different monolayers was studied by western blot analysis and confocal microscopy. The highest TEER values (192 +/- 3 Omega . cm2) were observed for RPMI 2650 cells seeded onto collagen-coated permeable polytetrafluoroethylene inserts and grown at an air-liquid interface for 10 days; a seeding density of 4 x 10(5)/cm2 generated and maintained a cell monolayer with suitable barrier properties at days 9-12. Microscopic examination showed that RPMI 2650 cells grown on filter inserts formed a fully confluent monolayer. The apparent permeability coefficients of the paracellular marker, [14C] mannitol, and the transcellular marker, [3H] propranolol, were 5.07 +/- 0.01 x 10(-6) cm/s and 16.1 +/- 0.1 x 10(-6) cm/s, respectively. Western blot analysis indicated the presence of four tight junction proteins: ZO-1, occludin, claudin-1 and E-cadherin; and the quantities of ZO-1, occludin, and E-cadherin were significantly higher in cells grown at an air-liquid interface than in cells grown at a liquid-liquid interface. Confocal microscopic studies showed ZO-1, F-actin, occludin and claudin-1 proteins at cell-cell contacts and revealed significant differences in the distributions and densities of ZO-1 protein in cells grown at the two types of interface. The data indicate that RPMI 2650 cells grown at an air-liquid interface form polarized monolayers with the cells interconnected by tight junction proteins. This human nasal cell line model could provide a useful tool for in vitro screening of nasal drug candidates.
在气液界面培养的人鼻RPMI 2650细胞是通过鼻腔途径进行药物转运研究的可行模型。RPMI 2650细胞在气液界面和液液界面的伊格尔最低限度基本培养基（MEM）中培养。对于每种培养方案，通过测量跨上皮电阻（TEER）以及细胞旁和跨细胞标志物跨单层的转运来测试单层完整性。通过蛋白质印迹分析和共聚焦显微镜研究不同单层细胞中紧密连接蛋白（分化标志物）的表达。将RPMI 2650细胞接种到胶原包被的可渗透聚四氟乙烯插入物上并在气液界面培养10天，观察到最高的TEER值（192±3Ω.cm2）；接种密度为4×10(5)/cm2在第9 - 12天产生并维持了具有合适屏障特性的细胞单层。显微镜检查显示，在滤膜插入物上生长的RPMI 2650细胞形成了完全汇合的单层。细胞旁标志物[14C]甘露醇和跨细胞标志物[3H]普萘洛尔的表观渗透系数分别为5.07±0.01×10(-6) cm/s和16.1±0.1×10(-6) cm/s。蛋白质印迹分析表明存在四种紧密连接蛋白：ZO - 1、闭合蛋白、Claudin - 1和E - 钙黏蛋白；在气液界面生长的细胞中，ZO - 1、闭合蛋白和E - 钙黏蛋白的量显著高于在液液界面生长的细胞。共聚焦显微镜研究显示，在细胞 - 细胞接触处有ZO - 1、F - 肌动蛋白、闭合蛋白和Claudin - 1蛋白，并揭示了在两种界面生长的细胞中ZO - 1蛋白分布和密度的显著差异。数据表明，在气液界面生长的RPMI 2650细胞形成了极化单层，细胞通过紧密连接蛋白相互连接。这种人鼻细胞系模型可为鼻腔候选药物的体外筛选提供有用工具。
