The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Commemorative Booklet for CAAT 20th Anniversary Symposium

Isolation and Characterization of Human Goblet Cells in Culture for Toxicity Testing

M. A. Shatos1, Darlene A. Dartt, and Peter Rubin2
1Schepens Eye Research Institute, Boston and 2Massachusetts Eye and Ear Infirmary, Boston

The development of a human goblet cell line with an expanded life span that secretes mucin and other antibacterial proteins is a useful model that potentially could be used in place of the Draize test.

This study sought to isolate, characterize and compare goblet cells from normal human conjunctival tissue, derived from male and female patients of various ages, for use in toxicity testing. This is the first phase of our long-term effort to develop a human goblet cell line whose life span is extended by the insertion of telomerase.

Goblet cells were isolated using explant cultures established from normal conjunctival tissue harvested from patients who had undergone ocular surgery. Cells derived from the explants were grown in RPMI culture medium supplemented with 10% fetal bovine serum and characterized using morphology, histochemistry, indirect immunofluorescence microscopy and RT-PCR. Cellular function was assessed by evaluating the ability of human goblet cells to proliferate in response to growth factors, as well as their ability to secrete the goblet cell-specific mucin MUC 5AC.

Goblet cells were successfully isolated and grown from conjunctival explants of all patients, irrespective of age. All human goblet cells isolated to date exhibited positive immunocytochemical reactivity with alcian blue Periodic Acid/Schiff's reagent; goblet cell specific cytokeratin-7; HPA lectin, goblet cell specific mucin MUC 5AC and negative reactivity to the stratified squamous epithelial cell cytokeratin-4. In addition, the mucin MUC 5AC was secreted into the growth media by goblet cells and MUC 5AC mRNA was detected in cultured human goblet cells using RT-PCR. EGF elicited a concentration-dependent increase in goblet cell proliferation of 205, 277 and 541% control with 10, 20 and 40 ng/ml, respectively.

These results indicate that human conjunctival goblet cells retaining characteristics of goblet cells in vivo can be cultured from patients of various ages. Cultured human goblet cells are suitable candidates for telomerase insertion to extend their proliferative life span, which will allow them to be used in toxicity testing.