The purpose of this protocol is to evaluate in an in vitro model whether artificial tear formulations can protect human corneal epithelial cells from desiccation. This method can be used to help identify dry eye formulations that can aid in ocular protection for individuals with dry eye symptoms. This assay utilizes a very sensitive measure of detecting corneal epithelial cell metabolic activity.
As a result, small changes in corneal cell health due to desiccation can be detected. Demonstrating the procedure today will be Parisa Mirzapour, Nijani Nagaarudkumaran, and Adeline Suko. Begin by growing immortalized human corneal epithelial cells in collagen-coated flasks with 20-milliliters of DMEM/F12 containing 10%fetal bovine serum and 1%penicillin/streptomycin at 37 degrees Celsius and 5%carbon dioxide, changing the media every two to three days.
Once the cells are almost confluent, remove the cell culture media, and add four to six milliliters of cell dissociation media to each flask. Incubate the cells at 37 degrees Celsius until they detach, periodically checking them under the microscope. Add two to six milliliters of DMEM/F12 with 10%FBS to each flask, and transfer the contents to a 50-milliliter centrifuge tube.
Centrifuge the cells at 450 to 500 times g for five minutes. Then aspirate the supernatant, and resuspend the cells in pre-warmed media. Determine the cell concentration with a hemocytometer, and calculate the volume that contains 100, 000 cells.
Add media to each well of a 48-well, collagen I-coated culture plate, along with the calculated volume of cells, making sure that the final volume in each well is 0.5 milliliters. Then incubate the cells for 24 hours. For the control procedure, remove the culture media from the wells, and immediately treat the cells with 150 microliters of a test formulation or media control solution.
Then incubate the cells for 30 minutes. Remove the test solution from the cells, and add 0.5 milliliters of 10%metabolic dye solution. Incubate the cells for another four hours.
After the incubation, remove 100 microliters of dye solution from each well, and transfer it to a 96-well plate. Use a plate reader to measure the fluorescence of each well, setting the excitation to 540 nanometers and emission to 590 nanometers. To perform the recovery procedure, incubate the cells with the test solutions or controls as previously described.
Then add 0.5 milliliters of DMEM/F12 media to each well. Incubate the cells for 18 hours. Then remove the media, and test for metabolic activity.
To perform the control procedure, remove the culture media from the cells in the 48-well plate, and immediately treat them with the test formulation or media control solution. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 30 minutes. After the incubation, remove the test solutions from the cells, and place them in a 37 degrees Celsius and 45%humidity chamber for five minutes to desiccate.
Next, add 0.5 milliliters of 10%metabolic dye solution, and incubate the cells for four hours at 37 degrees Celsius and 5%carbon dioxide. After the incubation, transfer 100 microliters of the metabolic dye solution from each well to a 96-well plate, and measure the fluorescence. To perform the recovery procedure, repeat the previously described protocol, and include an 18-hour incubation with DMEM/F12 medium after the desiccation step.
Then perform statistical analysis on the data as described in the text manuscript. Three dry eye formulations were compared for their effect on the viability of human corneal epithelial cells. Solutions one and two had a significant effect on the metabolic activity of the cells before desiccation.
Cells exposed to solution one showed an additional drop in cell metabolic activity after an 18-hour recovery, which means they were initially injured and the injury was not repaired. In comparison, solution three only had a mild effect on the metabolic activity of the epithelial cells. When comparing the ability of these lipid-containing dry eye formulations to protect the cells, it was found that solutions one and two did not protect cells from desiccation stress.
Solution three, however, offered some protection. When seeding the cells into collagen-coated plates, make sure that the cell suspension contains cells that are uniformly mixed so that each well is seeded with equal cell numbers. Solution three demonstrated statistically significant improvement in cell viability and protection from desiccation stress over the other two lipid-containing products.
It is possible that mineral oil with phospholipids, along with HP-guar and propylene glycol, allow solution three to provide hydration protection to the corneal cells and reduced evaporation under desiccation.
