Abstract
Purpose :
An intact epithelium is key to maintaining corneal integrity and barrier function. Bacterial keratitis and diabetes are among the conditions that can compromise the corneal epithelium leading to impaired wound healing and sight-threatening opacity. Electrical cell-substrate impedance sensing or ECIS® is a non-invasive method to measure real-time cellular behaviors such as barrier function and cell migration. The current study optimizes human corneal epithelial cells as assessed by ECIS® to generate quantifiable measurements that more accurately reflect changes in cell behavior and prove as a more advanced assay to monitor cells in vitro.
Methods :
Human telomerase-immortalized corneal epithelial cells (HUCLs) were used to optimize the ECIS® system. Cells were placed in either K-SFM or DMEM+F12 media (37 degrees Celsius, 5% CO2). Five cell densities (30,000 – 500,000 cells/well of a 96-well plate) were used to determine the optimal seeding density for ECIS® monitoring of cellular behavior over time. Parameters of assessment included: overall impedance (Z), barrier resistance (R), and cell capacitance (C). Mathematical modeling of the R data further generated Rb, α, and Cm values. ARPE-19 cells were used as a control.
Results :
Impedance, resistance, and cell capacitance measurements revealed DMEM+F12 at 60,000 cells as the optimal condition for ECIS® assessment. This was further supported by logarithmic curves that reached a cell migration plateau (time to confluency) at 8h with DMEM+F12 compared to 14h with K-SFM; and resistance and capacitance ratios with optimal cell migration slope and plateau achieved with DMEM+F12 at 60,000 cells compared to all other groups. Additionally, ECIS® modeling analysis indicated the contribution of α (basolateral adhesion), Rb (paracellular junctional space), and Cm (membrane capacitance) to overall barrier function were significantly improved for DMEM+F12 compared to K-SFM.
Conclusions :
Our results consistently demonstrated that 60,000 cells are an optimal seeding density for ECIS® analysis of HUCLs. Further, DMEM+F12 media provided better growing conditions, allowing for enhanced barrier function compared to widely used K-SFM. This work highlights the sensitivity of the ECIS® biosensor technology and the importance of optimizing not only cell density but media used for in vitro culturing.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.