A primary culture model of rabbit conjunctival epithelial cells exhibiting tight barrier properties

Purpose. The present study was conducted to develop and characterize a functional primary culture of pigmented rabbit conjunctival epithelial cells on permeable support exhibiting tight barrier properties. Methods. Conjunctival epithelial cells were isolated by 0.2% protease treatment, cultured at 0.5-1.8x10(6) cells/cm(2) onto collagen-treated Transwell filters, and were maintained either in the presence of 1% fetal bovine serum throughout or serum-free media from day 3 onwards. Transepithelial potential difference (PD) and transepithelial electrical resistance (TEER) were measured and equivalent short-circuit current (I-eq=PD/TEER) estimated. Results. There appears to be a critical plating density of 1.5x10(6) cells/cm(2) for functional development of tight epithelial cell cultures. The culture conditions as noted above did not affect either the time when peak bioelectric parameters were attained (days 8-10) or the magnitude of these parameters at a plating density of 1.5x10(6) cells/cm(2). Specifically, cells grown in a serum-free media showed a peak TEER of 1.9+/-0.2 k Omega.cm2(,) a PD of 14.2+/-1.6 mV (apical side negative), and an I-eq of 8.0+/-0.4 mu A/cm(2) (mean+/-SEM, n=45). Electron microscopy of serum-weaned cultures revealed a multilayered epithelium with numerous microvilli on the outermost layer of cells, while sporadic positive Periodic Acid Schiff (PAS) staining under light microscopy suggested the presence of mucin-secretory goblet cells. Conclusions. A functional, tight, epithelial barrier of the pigmented rabbit conjunctiva on a permeable support has been developed, which may be useful for mechanistic studies of ion and drug transport at the cellular level.