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Enrique Salero, Maitee Urbieta, Alfonso L. Sabater, Maria del Carmen Piqueras, Sanjoy K Bhattacharya, Victor L Perez; Proteomic analysis of secreted factors produced by human limbal epithelial cell cultures during in-vitro growth and expansion. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2603.
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In-vitro growth and expansion of human limbal epithelium using various culture modalities has been extensively reported in the literature. However, little is known about the secreted factors released by these cells during their growth and expansion in culture or the impact of such factors on cell limbal epithelial cell differentiation and function. We hypothesize that, in-vitro cultured limbal epithelial cells may produce secreted factors involved in regulation of their phenotype and function irrespective of the use exogenous supplements
Limbal epithelial tissue of cadaveric origin was obtained from the Florida Lions Eye Bank, processed, and digested overnight in collagenase at 37°C. Cell cultures were started at a density of 1x105 cells/mL/well using stem cell media supplemented with 2% BSA. Culture supernatant was collected at days 7, 15, 20, and 27 of culture and a proteomic profile was generated by mass spectroscopy. Data was analyzed using functional protein association network software (STRING) and a protein classification system based on protein analysis through evolutionary relationships (PANTHER)
We found that in-vitro cultured limbal epithelial cells release a plethora of factors within the first 27 days of growth/expansion. Overlap of secreted factors is observed across all time points analyzed; however, day 7 cultures appeared to be the most metabolically active, secreting over 300 gene products involved in dozens of biological processes including; extracellular matrix organization and disassembly, cell activation and adhesion, and wound healing among others. Data analysis suggests that at this level of enrichment proteins are at least partially biologically connected as a group
In-vitro cultured limbal epithelial cells release a multitude of biologically relevant factors into their culture medium. These factors may be produced as part of their normal physiology or as a response to stress. Negative or positive regulation exerted by these factors, such as changes in concentration and availability to cells, may determine the fate, phenotype and function(s) of these cells after culture in-vitro. Addition or removal of secreted factors may enable manipulation of cultured cell phenotype for potential use in the clinic, particularly in restoring ocular surface defects
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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