| Publication number |
CLPUB00479 |
|---|
| Authors |
Ward S.L., Walker T.L., Trocme S.D., Hallberg C.K., Kruszewski F.H., DiPasquale L.C. |
|---|
| Title |
Tight epithelial barrier established by transfected human conjunctival cell lines. |
|---|
| Citation |
Invest. Ophthalmol. Vis. Sci. 39:S85-S85(1998) |
|---|
| Abstract |
Purpose: lumen conjunctival epithelial cell lines were developed for use
in models of the human ocular surface for toxicological evaluations. The
purpose of this study was to characterize the properties of the new cell
lines.
Methods. Two human conjunctival cell lines, HC0597 and HC7.08, were
prepared by transfection of primary human conjunctival cells with the
large 'T' antigen plasmid p-RSV-T. Primary cells were isolated from human.
bulbar conjunctiva (S.E. Texas Lions Eye Bank). Both cell lines grow as
monoloyers on plastic, or stratified on permeable membranes in serum-free
KGM. The morphology of the conjunctival cells in low (0.15 mM) and high (1.
13 mM) calcium media was evaluated by light microscopy. Barrier function
in stratified cultures was evaluated by measuring transepethelial
electrical resistance (TER) and transepithelial permeability to Na
fluorescein (TEP). Keratins were identified by Western blotting.
Results: Both transfected human conjunctival cell lines demonstrated
cobblestone-like morphology typical of epithelial cells, and expressed
epithelial cytokeratins. Both cell lines have an extended life span. Cell
line HC0597 was cultured to passage #30 before growth slowed, and cell
line HC7.08 Is still growing at passage #30. Both conjunctival cell lines
undergo a change in morphology in high-calcium medium. The cells become
flattened, and cell-cell junctions become less distinct in high-calcium
KGM. Both cell lines stratify and form tight junctions when cultured on
permeable membranes. At day 10 of culture (3 days submerged and 7 days at
the air-liquid interface) HC0597 had a TER of 364+/-42 omega cm2 and
retained 98+/-1% of the fluorescein applied to the apical surface: HC7.08
had a TER of 217 +- 20 omega cm2 and retained 96 +- 1% fluorescein.
Conclusions: Conjunctival cell lines with an extended life span have been
produced. The transfected cell lines retained morphology and function that
are characteristic of human conjunctival epithelium. Since these cell
lines retained the ability to stratify and differentiate in culture, they
will be useful in developing in vitro models of the conjunctival surface.
|
|---|
| Cell lines |
CVCL_UZ31; HC0597 CVCL_UZ32; HC0708 |
|---|
