Research Grants 2003-2004

Development and Use of an Immortalized Human Goblet Cell Line for the Use in Ocular Toxicity Testing

Darlene Dartt, PhD
The Schepens Eye Research Institute, Harvard University, Boston, Massachusetts

The conjunctival epithelium along with the cornea makes up the ocular surface. The conjunctiva has long been used as a component of the Draize test and mucin secretion from this tissue as one of its endpoints. To replace the Draize test, many laboratories have developed cell lines and primary cultures of corneal epithelial cells and conjunctival stratified squamous cells. No one, however, has been able to culture conjunctival goblet cells. The longterm objective of the present application is to develop a cell line of human conjunctival goblet cells with an extended life span and subsequently to use these cells for ocular irritancy testing. The specific aims are:

Develop a cell line with and extended life span from human goblet cells in primary culture;
Characterize the new cell line; and
Determine the effect of neurotransmitters and growth factors on mucin and protein secretion from the cell line and the effect of ocular irritants on this secretion.

We recently established and characterized primary cultures of human conjunctival goblet cells from explants of surgically removed conjunctiva. For specific aim 1, we will attempt to transfect these cells with SV40 and telomerase. We will select the surviving colonies. After selection of telomerase positive cells, they will be expanded into cell line(s) and frozen down.

For specific aim 2, we will characterize the colonies of cells. We will first determine if the surviving cells retain their goblet cell characteristics by western blotting and immunofluorescence, microscopy. We will investigate whether these cells contain the intermediate filament keratin 7, the secretory mucin muc 5ac, terminal sugars on the secretory mucins that recognize the lectin Helix pornatia agglutinnin (HPA), and secretory product that stains with alcian blue/periodic acid Schiffs reagent. These are all specific markers of goblet cells. We will next determine if these cells contain keratin 4 and terminal sugars on the mucin secretory product that recognize the lectin Banderia, both of which are specific for the stratified squamous cells of the conjunctiva. We will select colonies that contain goblet cell markers. Colonies of cells will also be analyzed by electron microscopy to investigate if they contain large numbers of secretory vesicles, also indicative of goblet cells. Finally, we will determine the population doubling time of these cells with the goal of obtaining a 20 fold or more doubling capacity. For specific aim 3, we will use the newly created cell line of conjunctival goblet cells to determine the effect of neurotransmitters and growth factors on their secretion of mucin. Cells will be grown on glass coverslips in 6 well tissue culture plates. To measure secretion coverslips containing cells will be incubated with agonists for varying times from 15 to 120 minutes. The medium will be removed and the cells scraped and homogenized. Medium and cell homogenate will be analyzed for the amount of secretory product using an enzyme linked lectin assay with HPA lectin. We will test the neurotransmitters and growth factors that we have previously found to stimulate rat conjunctival goblet cell secretion. These results will be used as a baseline to then determine if known ocular irritants effect basal, neurotransmitter, or growth factor stimulated secretion.

The development of a goblet cell line with an expanded life span that secretes mucins and other antibacterial proteins is a useful model that could be potentially used as an alternative to the Draize test.