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. Intracellular Ca2+ concentration ([Ca2+]i) was measured using fura2/AM in cultured cells stimulated by exogenous stimuli. Immunofluorescence microscopy on fura2/AM labeled cells was used to assign cell phenotype to individual intracellular calcium measurements post-hoc.
Results :
Human conjunctival explants were grown using IOBA media for 14 days and were stained by immunohistochemistry for 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 individuals. Both goblet and stratified squamous cells significantly increased [Ca2+]i when stimulated by the α2 adrenergic agonist isoproterenol (10-5M), the α1 adrenergic agonist phenylephrine (10-4M), the nonspecific adrenergic agonist epinephrine (10-6M), the purinergic agonist ATP (10-5M), the cholinergic agonist carbachol (10-4M), and the parasympathetic agonist vasoactive intestinal peptide (VIP) (10-8M).
Conclusions :
We can reliably and reproducibly grow mixed cultures containing the three phenotypically distinct cell types comprising the conjunctival epithelium. Furthermore, cells cultured according to this method exhibit functional responses to neurotransmitters.
This is a 2020 ARVO Annual Meeting abstract.