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Isolation, characterization, and propagation of rat conjunctival goblet cells in vitro
M A Shatos1, J D Rios, V Tepavcevic
1Schepens Eye Research Institute, Boston, Massachusetts 02114, USA. shatos@vision.eri.harvard.edu
Purpose:
To isolate, culture, and characterize goblet cells from the conjunctiva of rats.
Methods:
Conjunctival tissue was surgically removed from Sprague-Dawley rats. Goblet cells were then isolated from the nictitating membrane and fornix using explant cultures. Cells derived from the explants were grown and propagated in RPMI medium supplemented with 10% fetal bovine serum. They were characterized using an enzyme-linked lectin assay (ELLA) with the lectin Ulex europaeus agglutinin-1 (UEA-1), Western blot analysis, PCR, light and electron microscopy, specialized histochemistry and indirect immunofluorescence microscopy.
Results:
Goblet cells were successfully isolated from conjunctival explants by scraping nongoblet cells from the culture vessel. To date, cultures have been passaged a minimum of three times without the loss of their specific cellular markers. Cells identified as goblet cells fulfilled the following criteria: positive staining for alcian blue/periodic acid Schiff reagent, cytokeratin (CK)-7, the lectins UEA-I and Helix pomatia agglutinin (HPA), MUC5AC, and M(3) muscarinic receptor; detection of MUC5AC mRNA using RT-PCR; and negative staining for CK-4, M(1) muscarinic receptor, and Banderia simplicifolia lectin. The authors also measured, using the ELLA, substantial amounts of UEA-I-detectable high-molecular-weight glycoproteins and MUC5AC released into the medium.
Conclusions:
Cultured goblet cells retain many characteristics of goblet cells in vivo and thus may serve as a useful tool in delineating the pathobiology of the ocular surface.