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

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.

journal_name

Curr Eye Res

journal_title

Current eye research

authors

Saha P,Kim KJ,Lee VH

doi

10.3109/02713689608995151

subject

Has Abstract

pub_date

1996-12-01 00:00:00

pages

1163-9

issue

12

eissn

0271-3683

issn

1460-2202

journal_volume

15

pub_type

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