June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Engineering of a tear-secreting system by coculturing lacrimal gland and conjunctival epithelial cells
Author Affiliations & Notes
  • Nicole Qiaozhi Lu
    Materials Science and Engineering, Johns Hopkins University, Baltimore, MD
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD
  • Hongbo Yin
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD
    Ophthalmology, West China Hospital, Sichuan University, Chengdu, China
  • Osama Al-Sheikh
    Oculoplastics and Orbit Division,, King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia
  • Jennifer Elisseeff
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD
    Biomedical Engineering, Johns Hopkins University, Baltimore, MD
  • Michael Peter Grant
    Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD
  • Footnotes
    Commercial Relationships Nicole Qiaozhi Lu, None; Hongbo Yin, None; Osama Al-Sheikh, None; Jennifer Elisseeff, Collagen Vitrigel (P); Michael Grant, Stryker CMF (C), Synthes CMF (C)
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 2263. doi:
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    • Get Citation

      Nicole Qiaozhi Lu, Hongbo Yin, Osama Al-Sheikh, Jennifer Elisseeff, Michael Peter Grant; Engineering of a tear-secreting system by coculturing lacrimal gland and conjunctival epithelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2263.

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

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Abstract
 
Purpose
 

Dry eye is a multi-factorial disease, and an in vitro ocular surface model is in need for dry eye study and therapeutic screening purposes. In our study, a coculture system composed of lacrimal gland (LG) and conjunctival epithelial cells (CECs) was established.

 
Methods
 

Rabbit LGs were digested with collagenase I, hyaluronidase and DNase I at 37 °C. Cell spheroids were formed in a shaking flask on an orbital shaker. CECs were isolated by dispase II. Coculture was established in a transwell insert system as illustrated in figure 1a. The morphology of LG cell spheroids was visualized by phalloidin staining. Cells under coculture conditions were harvested and compared to non-coculture samples. Gene expression was analyzed by qPCR. Mucin secreted by conjunctival goblet cells was stained with texas-red conjugated dextran, and imaged under a confocal microscope. LG secretion was quantified by β-hexosaminidase assay after carbachol stimulation.

 
Results
 

LG cells aggregated into spheroids within 2 days, with an average diameter of 108 μm. The spheroids formed 3-dimensional organoids inside Matrigel, as revealed by F-actin staining (FITC, figure 1b). Gene expression analysis of LG spheroids showed that cocultured cells expressed significant higher level of E-cadherin, EpCAM and lactoferrin compared to non-cultured samples. After carbachol stimulation, cocultured LG cell spheroids had more hexosaminidase in both supernatants and cell lysates. For CECs, qPCR results indicated that mucin 5AC and cytokeratin 4 was upregulated, while cytokeratin 7 was downregulated in cocultured cells. After 10 days, cocultured CECs secreted a thinker, more continuous mucin layer than non-cocultured ones (figure 1c).

 
Conclusions
 

LG cell spheroids have the ability to develop into lacrimal organoids when embedded in Matrigel, which highly resembles the structure of LG acini. Coculture is beneficial for both LG cell spheroids and CECs, as proven by gene expression and protein secretion results. This coculture system could be further engineered into an in vitro model for dry eye study and therapeutic screenings.  

 
(a) Schematics of the coculture system. (b) F-actin staining (FITC) of LG cell spheroids in Matrigel at day 5. Blue: nuclei stained with DAPI. Scale bar: 100 μm. (c) Confocal microscopic images (xz scanning) of the mucin layer secreted by CECs, stained with texas red conjugated dextran. Scale bar: 50 μm.
 
(a) Schematics of the coculture system. (b) F-actin staining (FITC) of LG cell spheroids in Matrigel at day 5. Blue: nuclei stained with DAPI. Scale bar: 100 μm. (c) Confocal microscopic images (xz scanning) of the mucin layer secreted by CECs, stained with texas red conjugated dextran. Scale bar: 50 μm.

 
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