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Jillian Meadows, Jianzhong CHEN, Karen Dionne, Kelly K Nichols; Identification of lipid species produced by differentiated immortalized human meibomian gland epithelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5662.
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© ARVO (1962-2015); The Authors (2016-present)
Manipulation of immortalized human meibomian gland epithelial cells (HMGECs) has been proposed as a preclinical model to investigate cellular physiology of the meibomian glands. We aim to investigate the lipid spectra produced by HMGECs with respect to our and others’ previous reports of the human meibum lipidome. An in vitro study has been conducted to investigate lipids from HMGECs at various time points and by different extraction techniques relative to human meibum.
Immortalized HMGECs were cultured in DMEM/F12 (1:1) containing 10 ng/ml EGF and 10% FBS for 9, 16, and 23 days to promote differentiation and lipid production. Using a modified Folch method, cells were lysed with pre-mixed 2:1 chloroform-methanol solution (v/v) and agitated to complete lysis and dissolve lipids. Ammonium acetate additive (either 1 mM or 10 mM) in HPLC-grade water was used to facilitate separation of polar and non-polar phases. The lower non-polar phase was withdrawn, diluted 10-fold with methanol, and directly infused into a TripleTOF 5600 mass spectrometer (Sciex, Framingham, MA) with electrospray ionization. MS and MS/MSall spectra were acquired in both positive and negative modes. MS/MSall analysis was performed with the SWATH technology, where product ion MS/MS spectra for all precursor ions were acquired in the m/z range of interest (200 to 1200) at every one Dalton step.
MS/MS spectra were similar among the three different time points and the two different concentrations of ammonium acetate additives. Phosphatidylcholines were the most abundant species detected in positive ion mode. Phosphatidylinositols and phosphatidylserines were the most abundant species in negative ion mode. For all conditions, the non-polar cholesteryl esters and wax esters were of low abundance (<2%).
MS/MS spectra of the lipids produced by differentiated HMGECs differ from the typical spectra of human meibum previously acquired from our lab and referenced in the literature. These results suggest either poor differentiation of immortalized HMGECs or that there are additional cellular and/or physiologic processes within the glands that may modify the final contents of meibum prior to secretion onto the ocular surface.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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