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Swati Pradhan-Bhatt, Xinqiao Jia, Stephen C Pflugfelder; Isolation of Primary Human Conjunctival Cells for Use in Ocular Surface Reconstruction. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4357.
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Damage to the ocular surface due to chemical, mechanical or thermal injuries or by autoimmune disorders often leads to scarring of the conjunctiva, loss of mucin producing goblet cells and loss of the regenerating stem cells. These conditions worsen with time and lead to chronic inflammation, dry eye, and corneal opacification, which often culminate in vision impairment. Our goal is to engineer a functional, transplantable conjunctival tissue substitute with the use of a biocompatible and biodegradable hyaluronic acid (HA)-based scaffold to replace damaged ocular surface tissues and restore normal ocular functions.
Human conjunctival tissue was purchased from National Disease Research Interchange (NDRI). Conjunctival tissue was subjected to mild enzymatic dissociation prior to culture. Explants were allowed to adhere in culture plates for 7 days. Resulting cells were maintained in KSFM growth media or in Hepato-STIM media supplemented with EGF. Isolated cells were characterized phenotypically using immunohistochemistry.
Immunostaining studies with isolated conjunctival cells revealed the presence of mixed cultures of differentiated conjunctival epithelial cells expressing markers including cytokeratin 19 (K19) and EpCAM and specialized secretory goblet cells expressing the mucins, MUC5AC and MUC5B. Additionally, a modified culture protocol with the use of Hepato-STIM media led to isolation of progenitor cell populations expressing K7, nerve growth factor (NGF), K5 and K14.
We report the successful isolation and phenotypic characterization of primary human conjunctival epithelial cells, secretory goblet cells and progenitor cells. Isolation of these key cell populations represents a major stride toward the generation of a bioengineered human conjunctival substitute. Future studies will involve the use of HA-based scaffolds to construct 3D layered conjunctival epithelium.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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