Role of Mesenchymal Stem Cell-Derived Conditioned Medium in Modulating the Benzalkonium Chloride-Induced Cytotoxic Effects in Cultured Corneal Epithelial Cells In Vitro

Abstract

Purpose

Benzalkonium chloride (BAK) is a common preservative in ophthalmic formulations that causes cytotoxic damage to the corneal epithelial cells. This study aims to explore the role of mesenchymal stem cell (MSC)-derived conditioned medium in modulating the BAK-induced cytotoxic effects in cultured human corneal epithelial cells (HCECs) as a cell-free therapeutic agent.

Methods

The in vitro cultured HCECs derived from a HCE cell line were treated with BAK (0.001% and 0.005%, diluted in DMEM/F12, v/v) for 15 min, washed with 1xPBS, and allowed to recover for 24 h in human bone marrow MSC-derived conditioned medium (MSC-CM: undiluted (100%) and diluted (50%, v/v)). On the other hand, HCECs were co-incubated with BAK (0.005%, v/v) and MSC-CM (100% and 50%, v/v) for 24 h. The HCEC-derived conditioned medium (HCE-CM) was used as an optimal control for MSC-CM, whereas HCECs cultured in DMEM/F12 were used as a control. The DMEM/F12 was used as the base medium for the culture of HCECs and preparation of HCE- and MSC-CM. The role of MSC-CM in modulating the metabolic activity, cell death, epithelial repair, and proliferation, in BAK-treated HCECs was evaluated using MTT assay, Propidium iodide staining, scratch assay, and Ki-67 staining, respectively.

Results

Compared to the control, recovery of BAK-treated (0.001% and 0.005%, for 15 min) HCECs in MSC-CM showed significantly reduced cell death with enhanced metabolic activity, epithelial repair, and proliferation. However, in comparison with HCE-CM, the beneficial effects of MSC-CM were predominantly observed at lower BAK concentration (0.001%, for 15 min). Whereas the co-incubation of BAK (0.005%) and MSC-CM for a longer duration (24 h) was marginally beneficial.

Conclusions

Our results suggest that the MSC-CM is effective in modulating the BAK-induced cell death, retardation of metabolic activity and proliferation in cultured HCECs, particularly at lower concentration (0.001%) and shorter exposure (15 min) of BAK.

Keywords:

• Mesenchymal stem cells
• conditioned medium
• benzalkonium chloride
• corneal epithelium
• injury

Acknowledgements

We are grateful to Md. Hasnat Ali, Senior Statistician, L V Prasad Eye Institute for his help in statistical analysis of the data and Udayachandrika M. for her help in flow cytometry.

Author contributions

Sreya Mitra performed the experiments, analyzed the data, performed statistical analysis, and wrote the manuscript. Vasudeva Tati performed the experiments, analyzed the data, performed statistical analysis and helped in revision and writing of the manuscript. Sayan Basu supervised the study and helped in designing experiments. Sachin Shukla designed the study, planned the experiments, supervised the study, analyzed the data, and wrote the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability

The data that support the findings of this study are available from the corresponding author [Sachin Shukla], upon reasonable request.

Additional information

Funding

Science and Engineering Research Board (SRG/2020/000842) and Indian Council of Medical Research (5/4/6/18/OPH/2020-II-NCD), Govt. of India, for extramural funding support to Sachin Shukla; Hyderabad Eye Research Foundation for Intramural Support.