用于预测眼部药物吸收的人角膜上皮细胞培养模型。

Culture model of human corneal epithelium for prediction of ocular drug absorption.

作者信息

Toropainen E, Ranta V P, Talvitie A, Suhonen P, Urtti A

机构信息

Department of Pharmaceutics, University of Kuopio, Finland.

出版信息

Invest Ophthalmol Vis Sci. 2001 Nov;42(12):2942-8.

PMID:11687540

Abstract

PURPOSE

The main purpose of this study was to develop a cell culture model of immortalized epithelium from the human cornea for drug permeability testing.

METHODS

Immortalized human corneal epithelial (HCE) cells were grown on filters, with various filter materials and coating procedures. In the optimal case, HCE cells were grown on polyester filters coated with rat tail collagen gel containing fibroblast cells. Transepithelial electrical resistance (TER) was measured during the growth of the cells to evaluate the epithelial differentiation and tightness of the epithelial cell layers. Transmission electron microscopy (TEM) was used to show the formation of tight junctions, desmosomes, and microvilli. Cellular morphology was characterized by light microscopy. Permeabilities of (3)H-mannitol and 6-carboxyfluorescein were determined, to evaluate the intercellular spaces of the epithelium. Rhodamine B was used as a lipophilic marker of transcellular permeability. Permeabilities of the excised rabbit corneas were determined in side-by-side diffusion chambers.

RESULTS

The TER values of the corneal epithelial cultures were 200 to 800 Omega x cm(2), depending on the culture conditions. In optimal conditions, cultured corneal epithelium consisted of five to eight cell layers, TER was at least 400 Omega x cm(2), and the most apical cells were flat, with tight junctions, microvilli, and desmosomes. The permeability coefficients (P(cell), 10(-6) cm/sec) for (3)H-mannitol, 6-carboxyfluorescein, and rhodamine B were 1.42 +/- 0.36, 0.77 +/- 0.40, and 16.3 +/- 4.0, respectively. Corresponding values (at 10(-6) cm/sec) for the isolated rabbit corneas were 0.38 +/- 0.16, 0.46 +/- 0.27, and 18.1 +/- 4.0, respectively.

CONCLUSIONS

The TER, morphology, and permeability of the cultured corneal epithelial cells resemble those of the intact cornea. This cell culture model may be useful in evaluation of corneal drug permeation and its mechanisms.

摘要

目的

本研究的主要目的是建立一种用于药物渗透性测试的人角膜永生化上皮细胞培养模型。

方法

将永生化人角膜上皮（HCE）细胞接种于具有不同滤膜材料和包被程序的滤膜上。在最佳条件下，将HCE细胞接种于涂有含成纤维细胞的大鼠尾胶原凝胶的聚酯滤膜上。在细胞生长过程中测量跨上皮电阻（TER），以评估上皮细胞分化及上皮细胞层的紧密性。采用透射电子显微镜（TEM）观察紧密连接、桥粒和微绒毛的形成。通过光学显微镜对细胞形态进行表征。测定³H-甘露醇和6-羧基荧光素的渗透性，以评估上皮细胞的细胞间隙。罗丹明B用作跨细胞渗透性的亲脂性标志物。在并排扩散室中测定离体兔角膜的渗透性。

结果

角膜上皮培养物的TER值在200至800Ω×cm²之间，具体取决于培养条件。在最佳条件下，培养的角膜上皮由五至八层细胞组成，TER至少为400Ω×cm²，最顶端的细胞扁平，具有紧密连接、微绒毛和桥粒。³H-甘露醇、6-羧基荧光素和罗丹明B的渗透系数（P(cell)，10⁻⁶cm/秒）分别为1.42±0.36、0.77±0.40和16.3±4.0。离体兔角膜的相应值（10⁻⁶cm/秒）分别为0.38±0.16、0.46±0.27和18.1±4.0。