This method can be used to evaluate in vitro the toxicity of new ophthalmic product formulations. By refining the concentrations of chemicals and product formulations using ocular cells in vitro, the use of animals for assessing new product safety can be minimized. In vitro assays evaluate toxicity endpoints that cannot be assessed in vivo, such as determining the effect of a chemical on mitochondrial function, cell membrane integrity, and enzyme activity.
Evaluate cell toxicity in vitro is key to understanding potential mechanisms of toxicity. This is essential for establishing a maximum tolerable dose of therapeutic chemicals in the eye. Demonstrating the procedure will be Nijani Nagaarudkumaran, a researcher from the Jones Laboratory.
Begin by seating both pHCECs and iHCECs onto collagen coated Petri dishes with glass bottom cover slips at a concentration of 1 x 10 to the 5th with 1 milliliter of human ocular epithelium medium, or HOEM. Grow pHCECs and iHCECs 1 set of cultures for 24 hours and another set for 48 hours in a 37-degree-Celsius incubator with 5%carbon dioxide. After incubation, stain the cells with 500 microliters of annexin staining buffer solution containing 4 micromolar calcein, 8 micromolar ethidium homodimer 1, and annexin 5 for 20 minutes at 37 degrees Celsius.
After staining the cells, adjust the confocal laser scanning microscope to capture the excitation and emission wavelengths, as described in the text manuscript. Obtain the two-dimensional and three-dimensional images following the instructions in the manuscript. Seed the cells at 5 x 10 to the 4th per milliliter of HOEM in each well of a 24-well collagen 1 coated culture plate and incubate at 37 degrees Celsius with 5%carbon dioxide for 3 hours.
After incubation, reduce the volume of the medium in the wells to 300 microliters. Next, expose the cells to UVA radiation at 6.48 watts per square meter, and UVB radiation at 1.82 watts per square meter at 37 degrees Celsius for 5 and 20 minutes in separate experiments. After UV radiation, add 200 microliters of fresh HOEM to each well and incubate for 20 hours at 37 degrees Celsius with 5%carbon dioxide.
The next day, collect the cell supernatant in each well and transfer it into sterile 2-milliliter polypropylene tubes. Freeze the supernatant at minus 80 degrees Celsius. To determine the cytokine levels released by the pHCECs and iHCECs after UV exposure, use a multiplex cytokine assay and follow the kit's instructions to quantify interleukin 6, interleukin 8, interleukin-1 beta, and TNF-alpha.
Prepare 10%metabolic assay reagent in DMEM and F12. Replace the culture medium in each well with 1 milliliter of the 10%metabolic assay solution and incubate at 37 degrees Celsius with 5%carbon dioxide for 4 hours. Measure the fluorescence of each solution using a fluorescent plate reader at 530 nanometers excitation and 590 nanometers emission wavelengths.
Seed cells at 5 x 10 to the fourth per milliliter of HOEM in each well of a 24-well collagen 1 coated culture plate. Incubate at 37 degrees Celsius with 5%carbon dioxide for 3 hours. After incubation, remove the medium and expose the cells to 1 milliliter of chemical toxins, which include 0.001%benzalkonium chloride, or BAK, 0.01%hydrogen peroxide, and 0.0025%sodium dodecyl sulfate in PBS for 5 and 15 minutes.
After exposure to the chemical toxins, remove the toxins from the wells, rinse with 1 milliliter of PBS, and add 1 milliliter of HOEM to each well. After 20 hours of incubation, perform a metabolic assay by replacing the medium with 1 milliliter of a 10%metabolic assay solution and incubating at 37 degrees Celsius with 5%carbon dioxide for 4 hours. Measure the fluorescence of each solution using a fluorescent plate reader at 530 nanometers excitation, and 590 nanometers emission wavelengths.
Transfer the cell supernatants from the wells following the 20-hour incubation into separate sterile 2-milliliter polypropylene tubes and freeze them at minus 80 degrees Celsius. Use the same multiplex platform used to assess the cytokines from the UV-treated cells following the kit's instructions to quantify interleukin 6, interleukin 8, interleukin-1 beta and TNF-alpha. Seed cells at 1 x 10 to the 5th per milliliter of HOEM in each well of a 24-well collagen 1 coated culture plate and incubate at 37 degrees Celsius with 5%carbon dioxide for 24 hours.
After incubation, remove the medium and expose the cells to 1 milliliter of chemical toxins, including 0.001%BAK, 0.005%BAK, and 0.01%BAK in PBS for 5 minutes. After exposure, remove the chemical toxins, rinse the wells with 1 milliliter of PBS and add 1 milliliter of HOEM to each well. After 20 hours of incubation, stain the cells with 500 microliters of annexin staining buffer solution containing calcein, ethidium homodimer, and annexin 5 for 20 minutes at 37 degrees Celsius.
Adjust the microscope to measure intensities at excitation and emission wavelengths of 630 and 675 nanometers for annexin 5, 495 and 515 nanometers for calcein AM, and 528 and 617 nanometers for ethidium 1 staining. Then acquire images of the cells using the fluorescence microscope. The iHCECs were found to be small cells that range from 10 to 20 micrometers and the pHCECs were in the range of 20 to 50 micrometers after 24 hours of growth.
Similar differences in size range were observed for iHCECs and pHCECs after 48 hours of growth. Compared to the non-UV exposed cells, the metabolic activity of irradiated pHCECs was significantly reduced at 20 minutes of exposure. For iHCECs, the metabolic activity decreased for cells irradiated at both 5 and 20 minutes.
Therefore, it took a longer exposure time to reduce the metabolic activity of the pHCECs than the iHCECs. The maximum cytokine release by the iHCECs was at 5 minutes of UV exposure. The maximum cytokine release for pHCECs occurred at 20 minutes of UV exposure.
In terms of total amounts of inflammatory cytokines released the pHCECs released substantially more interleukin-1 beta, interleukin 8, and TNF-alpha than the iHCECs, whereas the iHCECs released more interleukin 6. Both pHCECs and iHCECs showed a change in the release of interleukin 6 after exposure to all three chemicals. BAK caused a decrease in the release of interleukin 6 compared to the control for both primary and immortalized HCECs.
Hydrogen peroxide caused an increase in the release of interleukin 6 from iHCECs, and a decrease in the release from pHCECs. Both pHCECs and iHCECs were significantly damaged at 0.005%and 0.1%of BAK. After performing in vitro assessments of ocular cell toxicity, further animal models of ocular toxicity could be undertaken to determine the effect of an ocular product on corneal nerves or the ocular immune system.
These techniques are essential for determining the mechanisms of chemical and product formulation toxicity on the eye and will help minimize animal use for product development testing.
