Tissue culture of rabbit ciliary body epithelial cells on permeable supports.
The aqueous humor is produced by the epithelium of the ciliary body, a complex structure encircling the anterior segment of the eye. Aqueous humor production occurs by active transport, but the mechanism of this process is not understood. To produce a preparation in which active transport can be investigated, we have attempted to prepare cultures suitable for measurements of ion and water flux. We have grown rabbit ciliary body epithelial cells on permeable supports, coated with several extracellular matrix proteins. We then examined the ability of these proteins to promote the growth of a differentiated layer of epithelial cells. Non-pigmented and pigmented cells formed sheets of contiguous cells when grown on a variety of support media. The most successful substrate was a permeable support produced by Falcon/Becton Dickinson coated with a mixture of collagen IV, laminin and heparan sulfate. Under these conditions, cultures could be maintained for several months, but pigmented cell cultures did not develop a measurable transepithelial resistance (TER), and the TER of non-pigmented cell cultures was typically only 20-30 omega cm2. Much higher TERs exceeding 200 omega cm2 could be measured from non-pigmented cell cultures 3-5 days after plating, but these high values were unstable. Examination of the cultures with electron microscopy revealed that the cells were partially differentiated with the formation of a basal lamina and intercellular junctions. Labelling with a specific monoclonal antibody marker for tight junction protein (ZO-1) suggested that non-pigmented cell cultures showed extensive tight junction formation. The low TER of the non-pigmented cell cultures appears therefore not to be due to the lack of tight junctions but rather to the presence of spaces between cells.
Cilluffo
M C
MC
Jules Stein Eye Institute, UCLA 90024-7008.
Fain
M J
MJ
Fain
G L
GL
D000818:Animals; D002924:Ciliary Body / Q000502:physiology / Q000648:ultrastructure*; D046508:Culture Techniques / Q000379:methods*; D004847:Epithelial Cells; D016326:Extracellular Matrix Proteins; D007365:Intercellular Junctions / Q000648:ultrastructure; D008564:Membrane Potentials; D008854:Microscopy, Electron; D010858:Pigmentation; D011817:Rabbits; D013194:Staining and Labeling; D013997:Time Factors; D016610:Tissue Embedding
D016326:Extracellular Matrix Proteins