June 2021
Volume 62, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2021
Identification of Human Epithelial Cell Type by Analysis of Cell Function in Cultured Conjunctival Epithelium
Author Affiliations & Notes
  • Jeffrey Bair
    Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Robin Hodges
    Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Darlene A Dartt
    Schepens Eye Research Institute of Massachusetts Eye and Ear, Boston, Massachusetts, United States
  • Footnotes
    Commercial Relationships   Jeffrey Bair, None; Robin Hodges, None; Darlene Dartt, None
  • Footnotes
    Support  Grant Support: NIH NEI R01EY029789
Investigative Ophthalmology & Visual Science June 2021, Vol.62, 867. doi:
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      Jeffrey Bair, Robin Hodges, Darlene A Dartt; Identification of Human Epithelial Cell Type by Analysis of Cell Function in Cultured Conjunctival Epithelium. Invest. Ophthalmol. Vis. Sci. 2021;62(8):867.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : We previously developed a method to culture human goblet cells using RPMI medium. Our purpose is to culture and characterize three types of cells found in the conjunctival epithelium: goblet, stratified squamous, and undifferentiated.

Methods : A co-culture of goblet cells, stratified squamous cells, and undifferentiated cells was established from pieces of human conjunctiva in IOBA medium. Immunofluorescence was used to determine the amount and phenotype of cells grown. To determine cell function intracellular Ca2+ concentration ([Ca2+]i) was measured using fura2/AM in cultured cells stimulated by exogenous stimuli. Additionally immunofluorescence microscopy on fura2/AM labeled cells was used to assign cell phenotype to individual intracellular Ca2+ measurements post hoc.

Results : Explants of human conjunctiva were grown in IOBA media for 14 days and stained by immunohistochemistry using two cell markers for each type: cytokeratin (CK) 7 and the lectin Ulex Europeaus Agglutin 1 (UEA1) for goblet cells, CK4 and Bandeiraea Simplicifolia Lectin 1 for stratified squamous cells, and p63 and PAX6 for undifferentiated cells. All three cell types were present in cultures from twelve different individuals. [Ca2+]i was significantly increased in both goblet and stratified squamous cells (identified post hoc) when stimulated by the b adrenergic agonist isoproterenol (10-5M), the α1 adrenergic agonist phenylephrine (10-4M), and the nonspecific adrenergic agonist epinephrine (10-6M). Increases in [Ca2+]i elucidated by the purinergic agonist ATP (10-5M) and the cholinergic agonist carbachol (10-4M) were significantly greater in goblet cells than stratified squamous cells.

Conclusions : In mixed cultures containing the three phenotypically distinct human cell types that comprise the conjunctival epithelium, stratified squamous and goblet epithelial cells can be differentially identified by their [Ca2+]i response to purinergic and cholinergic agonists. Furthermore, the activity of goblet cells is regulated by systems that utilize adrenergic, purinergic, and cholinergic agonists.

Grant Support: NIH NEI R01EY029789

This is a 2021 ARVO Annual Meeting abstract.

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