P Saha1, K J Kim, V H Lee. 1. Department of Pharmaceutical Sciences and Ophthalmology, University of Southern California, Los Angeles, USA.
Abstract
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 0.5-1.8 x 10(6) cells/cm2 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 (Ieq = PD/TEER) estimated. RESULTS: There appears to be a critical plating density of 1.5 x 10(6) cells/cm2 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 1.5 x 10(6) cells/cm2. 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 V (apical side negative), and and Ieq of 8.0 +/- 0.4 microA/cm2 (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.
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 0.5-1.8 x 10(6) cells/cm2 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 (Ieq = PD/TEER) estimated. RESULTS: There appears to be a critical plating density of 1.5 x 10(6) cells/cm2 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 1.5 x 10(6) cells/cm2. 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 V (apical side negative), and and Ieq of 8.0 +/- 0.4 microA/cm2 (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.
Authors: Mohamed G Qaddoumi; Hideo Ueda; Johnny Yang; Jasmine Davda; Vinod Labhasetwar; Vincent H L Lee Journal: Pharm Res Date: 2004-04 Impact factor: 4.200