Abstract
Purpose:
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.
Methods:
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.
Results:
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).
Conclusions:
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.