Evaluation of Cell Harvesting Techniques to Optimize Lipidomic Analysis from Human Meibomian Gland Epithelial Cells in Culture

Abstract

The lipidomic analysis of immortalized human meibomian gland epithelial cells (HMGECs) has been proposed as a preclinical model to study meibomian gland dysfunction. An in vitro study was conducted to evaluate neutral lipid recovery following three harvesting techniques and to identify candidate lipid biomarkers of HMGECs. HMGECs were cultured in serum-containing media for two days to promote lipid production. Cells were either harvested by 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA), harvested by 10 mM EDTA, or simultaneously harvested and extracted by 2:1 chloroform–methanol (CM). After extraction by a modified Folch technique, the nonpolar phase was processed and infused into a TripleTOF 5600 mass spectrometer (Sciex, Framingham, MA, USA) with electrospray ionization. MS and MS/MSall spectra were acquired. Nonpolar cholesteryl esters (CEs) were consistently detected in all samples, while wax esters were not. Only small differences in two out of twenty CEs were detected between harvesting methods. CM yielded less CE18:1 than the other methods but greater CE20:4 than the trypsin–EDTA method (p < 0.05 for all). Similar to human meibum, very long-chain CEs with carbon number (nc) ≥ 24 were detected in all samples and may serve as HMGEC lipid biomarkers. Further work is needed to address the absence of wax esters. Overall, the three harvesting methods are reasonably equivalent, though CM promotes much better efficiency and is recommended for higher throughput.

Candidate Molecular Compounds as Potential Indicators for Meibomian Gland Dysfunction

“…Due to the increase in saturation (five times) of cholesterol esters relative to wax esters ( 53 ), a decrease in cholesterol esters may inversely affect the meibum’s melting point, thereby increasing its viscosity. This assertion has been confirmed biophysically, biochemically, and clinically in patients with MGD ( 2 , 49 ).…”

“…There are around 34 cholesterol esters with carbon numbers ranging from 14 to 34 detected in human meibomian gland epithelial cells. Notwithstanding prevailing conditions, human meibomian gland epithelial cells exhibit a cholesterol ester profile that is around 14.0% saturated, 60.6% monounsaturated, and 25.4% polyunsaturated ( 49 ). At the ocular surface in healthy people, the quantity of cholesterol esters is lesser than wax esters ( 50 ).…”

“…Cholesterol esters with the number of carbons ≥24 are not present in culture media, serum, epithelial cells, or other tissues except the sebaceous glands making them promising molecules with the potential for distinguishing disease states as diagnostic biomarkers ( 49 ). One study has intimated that cholesterol esters with the number of carbons ≥24 are potential lipid biomarkers of human meibomian gland epithelial cells ( 49 ). Meibomian glands epithelial cells are less differentiated and regenerated in MGD ( 3 , 49 ).…”

“…One study has intimated that cholesterol esters with the number of carbons ≥24 are potential lipid biomarkers of human meibomian gland epithelial cells ( 49 ). Meibomian glands epithelial cells are less differentiated and regenerated in MGD ( 3 , 49 ).…”

“…It is evident that cholesterol esters as a functional group of molecules are not the preview of any specific tissue; however, cholesterol esters with ultra-long-chain fatty acids are limited to or present only in meibomian and sebaceous glands ( 49 ). To further support the argument that cholesterol esters are potential biomarkers, recent studies by Borchman et al ( 50 ) and Shrestha et al ( 38 ) demonstrated that cholesterol esters (exact species not specified) are reduced in adults with meibomian gland dysfunction ( 54 ).…”