A primary culture model of rabbit conjunctival epithelial cells exhibiting tight barrier properties.
OBJECTIVE
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.
Saha
P
P
Department of Pharmaceutical Sciences and Ophthalmology, University of Southern California, Los Angeles, USA.
Kim
K J
KJ
Lee
V H
VH
D000818:Animals; D002452:Cell Count; D018929:Cell Culture Techniques; D002469:Cell Separation; D003228:Conjunctiva / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure; D016895:Culture Media, Serum-Free; D017097:Electric Impedance; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology / Q000648:ultrastructure; D017136:Ion Transport; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D008854:Microscopy, Electron; D011817:Rabbits; D019108:Tight Junctions / Q000502:physiology*
D016895:Culture Media, Serum-Free