Grainger Christopher I, Greenwell Leona L, Lockley David J, Martin Gary P, Forbes Ben
Pharmaceutical Science Research Division, King's College London, Franklin Williams Building, London, SE1 9NH, UK.
Pharm Res. 2006 Jul;23(7):1482-90. doi: 10.1007/s11095-006-0255-0. Epub 2006 Jun 21.
The aim of this study was to compare the effect of liquid-covered culture (LCC) and air-interfaced culture (AIC) on Calu-3 cell layer morphology and permeability, thus assessing the fitness of these culture systems as models of airway epithelium barrier function.
Cell layers were grown on 0.33 cm2 Transwell polyester cell culture supports. Cell layers grown using LCC and AIC were evaluated by using light and electron microscopy, transepithelial electrical resistance (TER), and permeability to the transepithelial flux of fluorescein sodium (flu-Na), and by varying molecular weight dextrans labeled with fluorescein isothiocyanate (FITC-dex). The tight junction protein, zona occludens protein-1 (ZO-1), was visualized by confocal microscopy and apical glycoprotein secretions were identified by using alcian blue.
Cells grown via AIC produced a more columnar epithelium with a more rugged apical topography and greater glycoprotein secretion compared to cells grown via LCC. Apical protrusions appearing to be cilia-like structures were observed on occasional cells using AIC, but typical airway ciliated cell phenotypes were not produced under either condition. Secretory granules were observed in cells cultured under both conditions. Cells cultured using LCC exhibited higher levels of ZO-1 protein than the AIC counterpart. The maximal TER of cells using LCC, 1,086 +/- 113 ohms cm2 at 11-16 days, was significantly greater than the TER of cells cultured using AIC, 306 +/- 53 ohms cm2 at 11-13 days. Apparent permeability (P(app)) values for the transport of flu-Na using LCC and AIC were 1.48 +/- 0.19x10(-7) and 3.36 +/- 0.47x10(-7) cm s(-1), respectively. Transport rates of flu-Na and FITC-dex were inversely proportional to molecular weight, and were significantly lower (p < 0.05) in cell layers grown using LCC than AIC. Renkin analysis fitted the data to single pore populations of radii 7.7 and 11.0 nm for LCC and AIC, respectively.
Distinct differences in morphology and permeability result when Calu-3 cells are grown using AIC or LCC. Cells cultured using AIC generate a model more morphologically representative of the airway epithelium than cells cultured using LCC.
本研究旨在比较液体覆盖培养（LCC）和空气界面培养（AIC）对Calu-3细胞层形态和通透性的影响，从而评估这些培养系统作为气道上皮屏障功能模型的适用性。
细胞层生长在0.33平方厘米的Transwell聚酯细胞培养支持物上。使用光学显微镜和电子显微镜、跨上皮电阻（TER）、对荧光素钠（flu-Na）跨上皮通量的通透性以及通过用异硫氰酸荧光素（FITC-葡聚糖）标记的不同分子量葡聚糖来评估使用LCC和AIC培养的细胞层。通过共聚焦显微镜观察紧密连接蛋白闭合蛋白-1（ZO-1），并使用阿尔辛蓝鉴定顶端糖蛋白分泌物。
与通过LCC培养的细胞相比，通过AIC培养产生的细胞形成更柱状的上皮，顶端形貌更粗糙，糖蛋白分泌更多。使用AIC时，偶尔在细胞上观察到似乎是纤毛样结构的顶端突起，但在两种条件下均未产生典型的气道纤毛细胞表型。在两种条件下培养的细胞中均观察到分泌颗粒。使用LCC培养的细胞中ZO-1蛋白水平高于AIC培养的细胞。使用LCC培养的细胞在11至16天时的最大TER为1,086±113欧姆·平方厘米，显著高于使用AIC培养的细胞在11至13天时的TER（306±53欧姆·平方厘米）。使用LCC和AIC时flu-Na转运的表观通透性（P(app)）值分别为1.48±0.19×10⁻⁷和3.36±0.47×10⁻⁷厘米·秒⁻¹。flu-Na和FITC-葡聚糖的转运速率与分子量成反比，并且在使用LCC培养的细胞层中显著低于AIC（p<0.05）。Renkin分析分别将LCC和AIC的数据拟合为半径为7.7和11.0纳米的单孔群体。
使用AIC或LCC培养Calu-3细胞时，形态和通透性存在明显差异。与使用LCC培养的细胞相比，使用AIC培养的细胞生成的模型在形态上更能代表气道上皮。
