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Medi Eslani, Judy Hamouie, Hongyu Ying, Peiman Hematti, Ali R Djalilian; A Reproducible in Vitro Assay to Measure Immunomodulatory Properties of Mesenchymal Stem Cell on the Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):705.
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© ARVO (1962-2015); The Authors (2016-present)
The anti-inflammatory effects of mesenchymal stem cells (MSCs) have been recognized as one of their major modes of action and the basis for their utilization in a number of human clinical trials. However, there is no well-established reproducible in vitro assay to evaluate the effect of MSCs on inflammation of human corneal epithelial cells (HCEC). Activation of Toll-like receptor 3 (TLR3) in HCECs results in significant induction of cytokines and chemokine. We developed a reproducible in vitro assay, based on TLR3 activation, to evaluate the immunomodulatory effects of MSC derived conditioned media (CM) on HCECs.
MSCs were harvested from the human bone marrow and cultured in serum containing media. Serum-free conditioned media were collected from them at 80% to 90% confluency. Growth factor-starved telomerase-immortalized HCEC were treated with polyinosinic-polycytidylic acid (poly I:C) for 30 minutes (to activate TLR3) then washed and exposed to either conditioned media or basic medium for 6 hours. The mRNA expression of selected cytokines was determined by real-time qPCR.
The optimal concentration of poly I:C for this assay was determined to be 1 µg/ml. Of downstream TLR3 related cytokines and chemokines, IL-6, TNF- α and CCL5/RANTES demonstrated better consistency. TLR3 activation of HCECs resulted in 2.7 ± 1.1, 3.2 ± 0.9 and 27.6 ± 7.6 fold increase in IL-6, TNF-α and CCL5 compared to control, respectively (P < 0.001). BM-MSC derived CM significantly decreased inflammatory cytokine release to 1.3 ± 0.8, 1.7 ± 1.4 and 11.6 ± 5.3 fold change, respectively (P value for each comparison < 0.01). CM from low passage (passage number ≤4) BM-MSC had slightly more anti-inflammatory effect compared to high passage (passage number ≥ 8), both from one donor (P < 0.05).
We have developed a reproducible in vitro assay to evaluate the immunomodulatory properties of MSC-derived CM on HCECs. Based on this assay, low passage BM-MSC derived CM demonstrated significant anti-inflammatory effects in corneal epithelial cells.
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